THE NOVEL GEMCITABINE PHOSPHOLIPID CONJUGATE KPC34 HAS ACTIVITY

AGAINST MULTIPLE MODELS OF RESISTANT ACUTE LYMPHOBLASTIC

LEUKEMIA

BY

PETER ALEXANDER

A Dissertation Submitted to the Graduate Faculty of

WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES

in Partial Fulfillment of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

Cancer Biology

May, 2015

Winston-Salem, North Carolina

Approved By:

Dr. Timothy S. Pardee, M.D., Ph.D., Advisor

Dr. Graca Almeida-Porada, M.D., Ph.D., Chair

Dr. William H. Gmeiner, Ph.D, M.B.A.

Dr. Gregory L. Kucera, Ph.D.

Dr. Frank C. Marini, Ph.D.

Dr. Thaddeus J. Wadas, M.S., Ph.D.

ii

DEDICATION and ACKNOWLEDGMENTS

I dedicate this dissertation to my family – past, present and future. I dedicate my

work to the memory of my grandparents Tsilya Shusterman, Alexander Silayev,

Adrienne Simon, and Richard Goldman and to my great-grandparents Lillian Wolff,

Miriam Goldman, and Nathan Goldman. I would like to express my immense gratitude to

my parents Lori and Victor Alexander for all that they have done for me throughout my

life. They are both incredibly loving and caring people who constantly sacrificed

themselves for me and for my education. I appreciate the constant love, support and

advice that they have provided me over the years. I would especially like to acknowledge

my father Victor for sharing his love of science with me and helping start me on the path

to a career in science. Lastly, I would like to thank my girlfriend Amie Severino for her

support, encouragement, help, and love. I look forward to providing the same generous

support when she writes her dissertation and to the many years together that the future

holds for us.

I would like to acknowledge by advisor Dr. Timothy Pardee for his excellent

mentoring and for providing me with the opportunity to continue my doctorate studies. I

appreciate his endless patience and availability for answering questions despite his busy

schedule. I would like to thank my committee members Drs. Graca Almeida-Porada,

Gregory Kucera, Thaddeus Wadas, William Gmeiner, and Frank Marini for their

guidance, support and time, and specifically thank Dr. Kucera for his advice and

collaboration on this project. I would like to thank the other members of the Pardee lab,

Kristi Pladna, Guerry Jeanne Cook, and Mike Murphy, for their help and daily

companionship in lab. I greatly appreciate their assistance whenever I went out of town

and for troubleshooting day-to-day issues. I would like to thank my friends and

colleagues over the years at Wake Forest, particularly Drs. Andrew Burke and Paul Cao

iii

for guiding me during my first few years as a graduate student and for Dr. Daniel Stovall

for his assistance in writing my dissertation.

I would like to acknowledge the technical help at Wake Forest from the many

members of the Animal Resource Program, Pathology Department and Mass

Spectrometry Core, Dr. James Wood of the Flow Cytometry Core, Tiffany Walker-West

and Ruth Gregory of the Tumor Procurement Core, and Dr. Purnima Dubey and Yelena

Karapova of the Cell and Viral Vector Core. I would like to thank Dr. David Caudell from

the Pathology Department for his assistance in analyzing and processing tissue samples

and for providing experimental advice. I would like to thank Dr. Ravi Singh for his

ongoing professional advice and aid in editing my presentations and dissertation. I would

like to thank the many faculty and administrative staff members of the Cancer Biology

Department who have assisted me over the years, and to the Department for supporting

me through the NCI training grant. I would also like to thank the Wake Forest

Translational Science Institute for their financial support of this project.

Finally, I would like to thank my many previous mentors at various research

institutions, including Drs. Frank and Suzy Torti (Wake Forest), Drs. Russel Kaufman

and Tao Wang (Wistar Institute), Drs. Andres-Klein-Szanto, Daniel Bassi and Ricardo

López de Cicco; Drs. Jose and Irma Russo and Gabriela Balogh; and Dr. Xiangxi Xu

(Fox Chase Cancer Center), Drs. Alexander Nikitin and Andrea Flesken-Nikitin (Cornell

University) and Dr. Barbara Solow (USDA) for their mentorship and for providing

excellent research opportunities. I would like to express my heartfelt thanks to Mr.

Dennis Erlick, the former mentor of the Mental-Gifted Support Program at Central High

School for his tireless devotion to his students and passion for science. I sincerely doubt

that I would be where I am today without his tireless dedication, along with the countless

other students whom he mentored.

iv

TABLE OF CONTENTS List of Illustrations and Figures………………………………………………………………..vi

List of Abbreviations……………………………………………………………………………viii

Abstract…………………………………………………………………………………………..xii

CHAPTER 1

General Introduction

I.1 Significance and Subtypes of Acute Leukemia…………………………………………...1

I.2 Existing Therapies for Acute Leukemias………………………………………………..…2

I.3 Mechanisms of Chemoresistance in Acute Leukemias…………………………….……6

I.4 Lipid Conjugates of Nucleoside Analogs…………………………………………………10

CHAPTER 2

Improving Nucleoside Analogs via Lipid Conjugation; Is fatter any better?

II.1

Abstract……………………………………………………………………………………........18

II.2 Introduction…………………………………………………………………………………18

II.3 Lipid-Drug Conjugate Chemistry…………………………………………………………20

II.4 Lipid-conjugated Drugs and the Blood-Brain Barrier…………………………………..20

II.5 Clinically Approved Cytarabine Conjugates…………………………………………….22

II.6 Drug Duplex Conjugates …………………………………………………………………24

II.9 Myristic Acid and 12-Thioethyldodecanoic Acid Conjugated Analogs……….………27

II.8 Phosphoramidate Conjugated Analogs………………………………………….…..…27

v

II.9 Phospholipid Gemcitabine Conjugate ………………………………………………….29

II.10 5-Elaidic Acid Ester Conjugated Analogs……………………………………………..30

II.11 Self-Emulsifying Compounds ………………………………………………………….33

II.12 Discussion………………………………………………………………………………...34

CHAPTER 3

KPC34 is Active Against Multiple Models of Resistant Acute Lymphoblastic

Leukemia

III.1 Abstract………………………………………………………………………………….…41

III.2 Introduction………………………………………………………………………………...43

III.3 Methods………………………………………………………………………………….…47

III.4 Results…………………………………………………………………………………...…52

III.5 Discussion and Future Directions…………………………………………………….…79

References……………………………………………………………………………….……..87

Curriculum Vitae ……………………………………………………………………………...104

vi

LIST OF ILLUSTRATIONS AND FIGURES

Chapter 1

Figure 1 Nucleoside Analog Metabolism……………………………………………………13

Figure 2 Factors Influencing Environmental Chemoresistance…………………………..14

Figure 3 PKC Signaling Pathway……………………………………………………………15

Figure 4 Structure and Activity of KPC34…………………………………………………..16

Chapter 2

Figure 5 Chemical structures of conventional nucleoside analogs………………………36

Figure 6 Chemical structures of clinically approved cytarabine conjugates……………..37

Figure 7 Chemical structures of duplex drug conjugate…………………………………...38

Figure 8 Chemical structures of fatty acyl cytarabine prodrugs…………………………..39

Figure 9 Chemical structures of phosphoramidate-conjugated prodrugs………………..39

Figure 10 Chemical structure of KPC34…………………………………………………….40

Figure 11 Chemical structures of lipid-conjugated nucleoside analogs developed by

Clavis Pharma …………………………………………………………………………………40

Chapter 3

Table 1 IC50 values of human and murine cell lines treated with KPC34…………….….61

Figure 12 KPC34 induces apoptosis in a dose-dependent fashion…………………..…62

Figure 13 KPC34 inhibits PKC signaling in vitro in a time-dependent fashion………...63

Figure 14 KPC34 overcomes stroma-mediated chemoresistance in vitro…………..….64

Fig 15 KPC34 is active in vivo against a syngeneic model of murine ALL……………..66

Figure 16 KPC34 reduces CNS burden in mice bearing ALL………………………....…67

Figure 17 KPC34 is more effective than Ara-C when combined with Doxorubicin……70

Figure 18 KPC34 is active against an Ara-C resistant model of murine ALL…………..72

vii

Figure 19 Oral administration of KPC34 induces more DNA damage than i.p.

administration in vivo…………………………………………………………………………..74

Figure 20 KPC34 overcomes in vivo inhibition of hENT1………………………………...77

Figure 21 KPC34 is tolerated by mice in vivo………………………………………………79

Figure 22 Dietary fat composition influences survival after KPC34 treatment…………..81

viii

LIST OF ABBREVIATIONS

Akt – protein kinase B

ALL – acute lymphoblastic leukemia

AML – acute myeloid leukemia

AML1-ETO – acute myeloid leukemia 1 and eight-twenty one fusion protein

APL – acute promyelocytic leukemia

AnxV – annexin V

Ara-C – cytarabine

ARF – cyclin dependent kinase inhibitor 2A

Axl – AXL receptor tyrosine kinase

BBB – blood brain barrier

BCR-Abl – fusion protein composed of breakpoint cluster region (BCR) and Abelson

murine leukemia viral oncogene homolog 1 (Abl)

Bim – Bcl2-like 11 apoptosis facilitator

Bcl2 – B-cell lymphoma 2

Bcl-xL – B-cell lymphoma extra large or Bcl2-like isoform 1

Bv8 – prokinectin-2

CBF – core binding factor

CBFβ-MYH11 – core binding factor subunit β and myosin-11 fusion protein

CD34 – hematopoietic progenitor cell antigen CD34

CD44 – receptor for hyaluronic acid

CMPK – cytidine monophosphate kinase

CNS – central nervous system

CSF – cerebrospinal fluid

CXCL12 – C-X-C chemokine motif 12 or stromal cell-derived factor 1

ix

CXCR4 – C-X-C chemokine receptor type 4

DAG – diacylglycerol

dCK – deoxycytidine kinase

DNMT3A – DNA (cytosine-5)-methyltransferase 3 alpha

Dox – doxorubicin

ECM – extracellular matrix

ERK1/2 – extracellular signal related kinases 1 and 2 or mitogen-activated protein

kinase (MAPK3 and 1)

FLT3-ITD – internal tandem duplication of Fms-like tyrosine kinase 3

H+E – hematoxylin and eosin

H2AFX – H2A histone family, member X

HDM2 – MDM2 oncogene, E3 ubiquitin ligase (human)

hENT1 – human equilibrative transporter 1

HIF-1α – hypoxia-inducible factor 1-alpha

i.p. – intraperitoneal

i.v. – intravenous

IAP – inhibitor of apoptosis protein

IC50 – half maximal inhibitory concentration

IκBα – nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha

IL-6 – interleukin 6

IP3 – inositol 1,4,5-triphosphate

JAK – Janus kinase

KIT – v-Kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog

MDR – multi-drug resistance protein or p-glycoprotein 1

MEK1/2 – mitogen-activated protein 1/2

MLL – mixed lineage leukemia

x

MLL-ENL – fused transcript composed of mixed lineage leukemia (MLL) and

myeloid/lymphoid mixed-lineage leukemia [trithorax homolog, Drosophila]; translocated

to, 1 (ENL)

mTOR – mammalian target of rapamycin

NDP – nucleoside diphosphate kinase

NF-κB – nuclear factor κB

NPM1 – nucleophosmin

NRAS – neuroblastoma RAS viral oncogene homolog

NSG – NOD Scid gamma

p.o. – per os or oral administration

p21WAF/CIP – cyclin-dependent kinase inhibitor 1 or CDK-interacting protein 1

p27Kip1 – cyclin-dependent kinase inhibitor 1B

p65 – nuclear factor NF-kappa-B p65 subunit or RELA

Ph Cr – Philadelphia Chromosome

PI – propidium iodide

PI3K – phosphoinositide 3-kinase

PIP2 – phosphatidylinositol 4,5-bisphosphate

PKC – protein kinase C

PLC-γ – phospholipase C γ

PML-RARα – promyelocytic leukemia and retinoic acid receptor-alpha fusion protein

PPAR-γ – peroxisome proliferator-activated receptor gamma

RACK – receptor for activated C kinase 1

Raf1 – v-raf-1 murine leukemia viral oncogene homolog 1

STAT5 – signal transducer and activator of transcription 5

TGF-β – transforming growth factor beta

TKI – tyrosine kinase inhibitor

xi

VCAM1 – vascular cell adhesion molecule-1

VEGF – vascular endothelial growth factor

VLA4 – very late antigen-4

Wnt – Wingless-related integration site

xii

ABSTRACT

Acute leukemias are cancers of the blood and bone marrow that affect thousands

of Americans annually. These diseases can be myeloid or lymphoid in origin. Common

components of therapy for both types of disease involve cytarabine and an

anthracycline. After initially responding to treatment, most patients eventually relapse

with disease that is resistant to conventional modalities. There is a pressing need for

novel therapeutic agents to treat the acute leukemias, especially in the relapsed setting.

An ideal drug to treat relapsed leukemia would bypass common uptake and

activation resistance mechanisms, be orally available, and could effectively cross the

blood-brain barrier to treat central nervous system (CNS) disease. We tested the

cytotoxicity of KPC34, a novel phospholipid conjugate of gemcitabine rationally designed

to overcome multiple mechanisms of leukemic resistance. We found this drug to be

active against human and murine acute leukemia cells in vitro and effectively treated a

syngeneic, orthotopic model of acute lymphoblastic leukemia in immune competent

mice.

KPC34 was cytotoxic to all leukemic cell lines tested with IC50 values in the

nanomolar range, inhibited classical PKC signaling, and overcame a model of stroma-

mediated chemoresistance in vitro. This drug treated models of naïve and

chemoresistant ALL more effectively than cytarabine or gemcitabine. Treatment of mice

with KPC34 and doxorubicin was more effective than doxorubicin and cytarabine, which

mimics existing components of therapy for patients with ALL. Mice re-treated with

KPC34 that relapsed after initial treatment with cytarabine and doxorubicin saw dramatic

improvements in hind limb paralysis, suggesting that this drug is active against CNS

disease.

xiii

KPC34 was statistically superior over cytarabine and its parent drug,

gemcitabine, in improving survival in mice bearing naïve and chemoresistant acute

lymphoblastic leukemia. KPC34 was tolerated by mice with minimal toxicity. These

promising pre-clinical results justify the continued development of KPC34 for the

treatment of relapsed ALL.

1

CHAPTER I

GENERAL INTRODUCTION

I.1 SIGNIFICANCE AND SUBTYPES OF ACUTE LEUKEMIA

Acute leukemias are diseases of the blood and bone marrow that lead to an

accumulation of immature hematopoietic progenitor cells, eventually leading to bone

marrow failure and death. These cancers can be subdivided into acute lymphoblastic

leukemia (ALL) or acute myeloid leukemia (AML), depending to their cell of origin. [1, 2].

In 2015, there will be an estimated 27,000 new cases of ALL and AML and 11,000

combined deaths in the United States [3]. Despite some improvement in the treatment of

acute leukemias, the adult 5-year survival rate remains at 30-40% for both myeloid and

lymphoblastic leukemias [4, 5]. AML primarily affects the elderly (median age of

diagnosis is over the age of 60), while ALL mainly occurs in children or the elderly [6, 7].

Approximately one quarter of adult ALL patients and 5% of pediatric patients are

positive for Philadelphia chromosome (Ph+), a reciprocal translocation occurring

between chromosomes 9 and 22 – t(9;22)(q34;q11) – that produces a fusion protein

known as BCR-Abl [8]. BCR-Abl is a constitutively active tyrosine kinase that can

promote tumor growth and proliferation via activation of the PI3K pathway [9, 10].

AML is a heterogeneous disease with a multitude of genetic abnormalities, which

can often overlap within the same cell. These genetic aberrations affect clinical response

and can have prognostic value [11]. Common fusion proteins as a result of chromosomal

alterations include AML1-ETO t(8;21), PML-RARα t(15;17), CBFβ-MYH11 inv(16), and

various MLL fusion proteins del(11q23) [12]. However approximately 40-50% of AML

patients have a normal karyotype [13]. Many patients with normal karyotypes still have

2

mutations in KIT, FLT3, NRAS, KRAS, and DNMT3A [12]. The most common FLT3

mutation is internal tandem duplications (FLT3-ITD). The number of duplications varies,

but mutations result in auto-dimerization and auto-phosphorylation, leading to over-

activation of Akt and STAT5 [14]. These mutations occur in 15-35% of adults with AML

[15] and are associated with poor clinical outcome [16], as FLT3-ITD status was one of

the most significant prognostic factors in this disease based on one multivariate analysis

[17]. A smaller percent of patients (5-10%) exhibit mutations in the tyrosine kinase

domain of FLT3, including point mutations, insertions and deletions [14, 18]. These

patients rarely have overlapping FLT3-ITD mutations and display differentially activated

downstream targets of FLT3 [19]. c-Kit mutations activate the PI3K and MAP kinase

pathways and are also associated with a poor prognosis in patients with CBF AML [20].

I.2 EXISTING THERAPIES FOR ACUTE LEUKEMIAS

Despite various genetic backgrounds, the historic standard of care for AML has

been induction chemotherapy using the nucleoside analog cytarabine (Ara-C) and an

anthracycline (daunorubicin or idarubicin) followed by consolidation therapy. Nucleoside

analogs are modified nucleosides that incorporate into the DNA of replicating cells and

anthracyclines are topoisomerase II poisons that intercalate between DNA and RNA

base pairs independent of cell cycle. Both of these drugs inhibit DNA synthesis and

replication, causing cell death. Induction therapy is given to induce remission by killing

the bulk leukemic population (defined as the presence of less than 5% of leukemic blasts

in the bone marrow) and the goal of consolidation therapy is to prevent relapse by

eliminating non-detectable leukemic cells.

For patients with AML, induction therapy involves 7 days of Ara-C (100-200

mg/m2 I.V.) combined with 3 days of anthracycline treatment (daunorubicin at least 60

mg/m2 by continuous I.V. or bolus idarubicin 12 mg/m2), also known as the 7+3 regimen.

3

Approximately 70% of patients achieve an initial remission with this protocol [21]. This is

followed by consolidation therapy of high-dose Ara-C (2-3 g/m2) [4, 22, 23]. Alternative

consolidation therapy options include further rounds of chemotherapy or

allogenic/autologous bone marrow transplantation, but these treatments have not been

particularly successful in patients with a poor prognosis [22].

The standard of care for ALL is induction therapy containing the microtubule

inhibitor vincristine, daunorubicin, a steroid glucocorticoid agonist (prednisolone or

dexamethasone), and L-asparaginase (catalyzer of the hydrolysis of L-asparagine)

followed by treatment with Ara-C and the nitrogen mustard alkylating agent

cyclophosphamide. The remission rate following induction therapy in adults with ALL is

60-80% [24]. After induction therapy, patients can undergo allogenic bone marrow

transplantation or consolidation chemotherapy with various protocols, most of which

contain methotrexate. This is followed by maintenance therapy involving daily

administration of the thiopurine 6-mercaptopurine, weekly administration of the antifolate

drug methotrexate, monthly administration of vincristine, and a once monthly 5 day

course of prednisone to further eliminate any residual cells [1, 25].

Acute leukemia patients with specific genetic abnormalities often receive targeted

treatments tailored to match their alterations. Patients who are Ph+ respond poorly to

standard treatments alone. They often receive allogeneic bone marrow transplantation

and inclusion of one of the tyrosine kinase inhibitors (TKIs) imatinib, dasatinib or nilotinib

is now a standard addition to existing chemotherapeutic regimens for these patients [9,

26]. Imatininb mesylate was the first TKI to gain approval for the treatment of Ph+

disease [27], but nilotinib [28] and dasatinib [29] both inhibit BCR-ABL more effectively

by orders of magnitude and can successfully treat patients with imatinib-resistant

disease, leading to their increased use in the clinical setting [28, 30].

4

Sorafenib is a first-generation TKI that inhibits FLT3 and is effective in patients

with naïve and relapsed AML [31]. However, interactions with the stroma have been

shown to induce leukemic resistance [32], and efforts are currently underway to test

second-generation TKIs, including quizartinib [33], pacritinib [34] and midostaurin

(PKC412) [35]. Some patients with c-Kit mutations respond to imatinib [36], but clinical

response may depend on the type of mutation. Single-agent and combination studies

with imatinib are currently ongoing [37, 38], as well as studies with dasatinib and

PKC412. Attempts to inhibit Ras activity using the farnesyl transferase inhibitors

tipifarnib [39, 40] and lonafarnib [41] have so far failed in patients. About 10% of AML

cases are sub-classified as acute promyelocytic leukemia (APL) and 95% of patients

express the t(15;17) fusion protein caused by reciprocal translocation of the PML and

RARA genes [42]. All of these patients have all-trans-retinoic acid added to their

induction therapy, which causes the immature promyelocytic cells to differentiate into

functional myeloid cells [43].

The standard and targeted therapies described above for ALL and AML generally

induce a complete but transient remission and relapsed disease becomes much more

difficult to treat with existing therapeutic modalities [4]. The 5-year survival rate for

patients with relapsed ALL is 7% and the 3-year survival rate in AML is 8-29% [5, 44].

While children diagnosed with ALL have an initial 80% 5-year survival rate, upon relapse

this rate drops to 15-50% [45, 46]. In elderly ALL and AML patients the 5 year survival is

very poor – less than 10% for both diseases [47, 48].The low survival rate of elderly

patients is due to many factors, such as existing medical conditions that limit tolerance

for chemotherapy, biological differences in disease, and complications during and after

treatment [49]. This includes a high risk of infection due to neutropenia, increased

toxicity from treatment with vincristine, L-asparaginase and steroids, limited

anthracycline treatment due to cardiotoxicity, coagulation abnormalities, hepatotoxicity,

5

avascular necrosis, complications from drug interactions, and metabolic disturbance [1].

Another possible explanation for the poor response rate is that very few elderly patients

are enrolled on prospective clinical trials, in large part due to ineligibility caused by the

factors described above, and the scarcity of clinical trials designed specifically for elderly

patients [49].

CNS involvement occurs in 8% of ALL patients at diagnosis and increases to 10-

15% in relapsed patients [50, 51], while under 5% of AML patients have CNS involved

disease [4]. CNS involvement often manifests through neoplastic meningitis, which is

usually fatal within 4-6 weeks if left untreated [52] or intracranial hemorrhaging [53]. CNS

involvement is a poor prognostic factor, as adult patients with CNS involved disease

have a significantly lower survival rate compared to patients without CNS involvement

[54] and CNS recurrence is highly linked to bone marrow recurrence and low survival

[55]. Rates of CNS involvement in ALL were previously higher until CNS prophylaxis

became a routine component of treatment, as 30% of adult patients who achieved

complete response would relapse with CNS involved disease [56, 57]. CNS prophylaxis

includes combinations of intrathecal chemotherapy using methotrexate, Ara-C and

steroids [58], high-dose systemic chemotherapy using Ara-C (1-7.5 g/m2) or

methotrexate (5-8 g/m2), and radiation therapy [1, 59], as standard-dosed chemotherapy

has poor blood-brain barrier (BBB) penetration [60]. However, these treatments have

undesirable complications, including increased toxicity, cognitive impairment, infection,

inaccurate lumbar puncture, and chemical meningitis [59]. Some progress has been

made with the use of a liposomal formulation of cytarabine (DepoCyt). This drug has

been shown to treat CNS disease more effectively than parental cytarabine, but it

caused neurotoxicity in 15% of patients tested and still requires intrathecal

administration [58].

6

The backbone of consolidation therapy for both ALL and AML and for induction

therapy of AML is the nucleoside analog Ara-C. Nucleoside analogs are a class of drugs

that resemble endogenous nucleosides and are used to treat many cancers, especially

leukemias and lymphomas. Ara-C and other nucleoside analogs require active transport

into the cell, mediated by the human equilibrative nucleoside transporter (hENT1) [61].

Once inside the cell, Ara-C undergoes three phosphorylation steps to become active.

The first phosphorylation step is mediated by deoxycytidine kinase (dCK) and is the rate-

limiting step of activation. After further phosphorylation by nucleoside diphosphate

kinase (NDP) and cytidine monophosphate kinase (CMPK), the tri-phosphate form of the

drug is transported across the nucleus, where it incorporates into replicating strands of

DNA. DNA polymerases are unable to add nucleotides after the incorporated nucleoside

analog, resulting in DNA strand breaks and chain termination and ultimately cell death

via the induction of apoptosis [62] (Figure 1). However, these drugs are non-specifically

taken up by rapidly-dividing cell populations, and as a result the clinical efficacy of Ara-C

and other nucleoside analogs is often limited by off-target toxicities to the bone marrow

and gastrointestinal tract, particularly myelosuppression and mucositis [63].

I.3 MECHANISMS OF CHEMORESISTANCE IN ACUTE LEUKEMIAS

Patients with acute leukemia commonly develop several mechanisms of

resistance against nucleoside analogs. This includes cell-autonomous mutations or

dysregulation of proteins and signaling pathways that can reduce drug uptake and

activation or confer apoptotic resistance, as well as chemoresistance mediated by

physical and soluble interactions with the environment. Cell-autonomous resistance can

involve genetic alterations leading to reduced activity of hENT1 and dCK [64-66] and

increased activity of the p-glycoprotein drug efflux pump multidrug resistance protein 1

(MDR1) [67]. Clinically, reduced dCK and hENT1 expression and increased MDR1

7

expression correlate with poor response to Ara-C therapy and ultimately poor prognosis

in both ALL and AML [65, 68-70]. As this class of drugs requires cells to be entering the

S phase of mitosis for nucleic acid incorporation, disruptions to normal cell cycle

progression in leukemic cells also correlate with poor clinical outcome in AML [71]. In

addition, Ara-C has poor in vivo pharmacokinetics, with a low initial plasma half-life of 7-

20 minutes at least partially due to inactivation via deamination and rapid elimination by

the body [72]. Genetic differences in the cytidine deaminase gene have been linked to

variations in Ara-C sensitivity in AML patients [73]. Poor oral availability of the drug

necessitates repetitive intravenous administration to achieve clinically effective doses in

patients [74], as these drugs do not efficiently diffuse across the gastrointestinal tract

[48]. These multiple mechanisms of resistance and poor pharmacokinetics ultimately

limit the clinical effectiveness of Ara-C and lead to relapsed disease.

Besides drug-induced cell-autonomous chemoresistance, leukemic cells can

avoid apoptosis by interacting with the bone marrow stroma [75] (Figure 2). In vitro

studies have shown that ALL and AML cells are protected from apoptosis induced by

treatment with the cytotoxic agents Ara-C, gemcitabine, etoposide, daunorubicin,

epirubicin, and sorafenib upon co-culture with normal bone marrow stroma cells [32, 76-

79]. Physical interactions between leukemic cells and stromal cells are mediated via

binding of VLA-4 (α4β1 integrin) to the cell adhesion protein VCAM1, the chemokine

receptor CXCR4 to its ligand SDF-1 (also known as CXCL12), and the cell-surface

glycoprotein CD44. These three pathways have all been implicated in the migration and

homing of AML cells to the bone marrow niche [80-82], and VLA-4 binding to VCAM1

has been shown to mediate attachment of myeloid leukemia cells to blood vessel walls

[83]. Increased CXCR4 [84-87] and CD44 [88, 89] expression correlate with poor patient

prognosis, while reports on the association of VLA-4 expression with clinical outcome

have been conflicting [90-92].

8

Disrupting the chemo-protective effect of stroma on leukemic cells could

theoretically be accomplished in two ways: 1) by reducing retention of leukemic cells in

the bone marrow niche or 2) by developing drugs that are capable of inducing apoptosis

despite the protection provided by bone marrow. Multiple studies have attempted the

first approach, as experimental suppression of leukemic cell homing to the bone marrow

by inducing peripheral mobilization has been demonstrated via CXCR4 [93-96] and

CD44 [97] inhibition in vivo. The CXCR4 inhibitor AMD3100 (Plerixafor) is approved for

treatment of lymphoma and multiple myeloma [98] and is currently being tested for the

treatment of relapsed AML in combination with chemotherapy [99]. Inhibition of

VCAM/VLA-4 interaction restored sensitivity to Ara-C or daunorubicin in patient AML

cells co-cultured with bone marrow stroma cells and to Ara-C in vivo, representing an

example of the second approach [90].

An important mechanism of stroma-mediated leukemic survival is the activation

of NF-κB signaling. Integrin-mediated activation of this pathway in leukemia inhibits

apoptosis by decreasing transcription of the pro-apoptotic protein Bim [100], increasing

transcription of the anti-apoptotic proteins Bcl2 [101], Bcl-xL [100] and IAP [102], and by

inducing growth arrest via activation of p27 [103]. Interactions between VCAM/VLA-4

have been shown to induce NF-κB activation in both ALL and AML cells co-cultured with

stroma cells [104]. The Notch pathway has also been shown to activate NF-κB in

leukemia [100] via its ligand Jagged, which is commonly expressed in osteoblasts and

has been shown to induce leukemogenesis in normal myeloid and lymphoid progenitor

cells [105]. Notch signaling activated by bone marrow stroma has also been implicated

in drug resistance in T cell ALL [106, 107], at least partially mediated by p21-induced

growth arrest [108]. These factors reduce the efficacy of most traditional

chemotherapeutics, which often depend on the induction of apoptosis in actively

proliferating cells.

9

Other elements of stroma-induced chemo-protection include reduced hENT1

activity [76] and activation of the Akt [109] and Wnt [105, 110] signaling pathways. The

Wnt pathway is activated by VLA-4 [111] and mutant receptor tyrosine kinases, including

FLT3, AML1-ETO, and PML-RARα [112]. Activation of this pathway has been shown to

promote leukemic drug resistance via stromal interactions [113], possibly by transcription

of the anti-apoptotic proteins survivin [114] and galectin-3 [115]. Notch [107], NFκB [116]

and VLA-4 [90] signaling have been shown to induce PI3K/Akt activity, which is linked to

survival and chemoresistance in ALL [107] and AML [116]. Soluble factors produced by

stromal cells can also mediate chemoresistance in leukemia cells. The cytokine IL-6 is

produced by bone marrow stroma [117] and has been shown to inhibit chemotherapy-

induced apoptosis in AML [118]. However, multiple studies suggest that physical

interactions seem to provide a greater protective effect than soluble factors against

cytotoxic agents [76, 77, 95]. Overcoming stroma-mediated chemoresistance, be it by

inhibition of leukemic cell migration or the development of novel therapeutics, appears to

be a vital step in effectively killing leukemic cells harbored in this chemo-protective niche

and in preventing disease recurrence due to residual leukemic cells.

Another common mechanism of chemoresistance in ALL and AML is

overexpression of members of the PKC signal transduction regulator family [119]. There

are many subtypes of PKC members, and most are activated by calcium and/or

diacylglycerol (DAG) signaling (Figure 3). Upon activation of G protein coupled

receptors by external ligands, phospholipase C-γ (PLC-γ) cleaves phosphatidylinositol

4,5-bisphosphate (PIP2) into diacylgycerol and inositol triphosphate (IP3), which releases

Ca2+ ions from the endoplasmic reticulum. These signaling molecules activate PKC and

receptor for activated C kinase 1 (RACK1) localizes PKC to the plasma membrane [120].

Phorbol esters can also activate PKC independently of PLC-γ by mimicking DAG [121].

Upon localization to the plasma membrane, PKC activates the NF-κB pathway by

10

phosphorylation-mediated degradation of the inhibitory IκBα [122] or by activating p65, a

component of the NF-κB signaling dimer [123]. PKC can also phosphorylate and activate

Bcl2 [124] and the MAP kinase pathway (ERK1/2) via Raf1 [125]. Downstream activation

of these pathways results in increased survival, proliferation and inhibition of apoptosis,

which can aid cancer cells in avoiding drug-induced apoptosis [101, 126, 127].

Up-regulation of the conventional PKC isoforms α, βI and βII has been shown to

increase leukemic chemoresistance in ALL [128, 129] and are associated with poor

patient prognosis in AML [130, 131]. PKC-induced protection from chemotherapy is

believed to be mediated at least partially by activation of the anti-apoptotic protein Bcl2

via the Nf-κB signaling pathway, as increased Bcl2 expression also correlates with poor

patient outcome [128, 130, 132]. Pharmacological inhibition of PKC isoforms is toxic to

AML cells [124, 133, 134] and ALL cells [134, 135] in vitro, but there have been few

clinical studies of clinical inhibition of PKC in patients with acute leukemia. This signaling

pathway remains an attractive therapeutic target in acute leukemia due to its inhibition of

drug-induced cytotoxicity.

I.4 LIPID CONJUGATES OF NUCLEOSIDE ANALOGS

Despite knowing many clinically-relevant mediators of chemoresistance in acute

leukemias, minimal progress has been made in treating chemoresistant disease. There

is a desperate need for new therapeutic agents to effectively treat leukemia, particularly

in the relapsed and CNS-involved setting, while sparing patients from unwanted

deleterious side effects. An ideal drug for the treatment of acute leukemias could be

administered orally, efficiently cross the BBB, and would have improved

pharmacokinetics compared to existing intravenously-administered nucleoside analogs,

alleviating the need for spinal injection or high-dose intravenous therapy. A drug

designed to effectively treat resistant disease would be able to overcome multiple

11

mechanisms of leukemic chemoresistance, including hENT1-free uptake, dCK-free

activation, and neutralizing environment-mediated chemoresistance (possibly by

inhibiting the PKC/NF-κB/Bcl2 signaling axis).

A drug development strategy that incorporates many of these useful properties is

lipid conjugation of nucleoside analogs. This approach has recently resulted in a

promising new class of chemotherapeutics. The imparted chemical modifications

improve pharmacokinetic properties, including solubility, stability, circulation time, BBB

penetration, oral availability, and reduced drug efflux [60, 136-138]. Several lipid

conjugates of cytarabine have already been approved for clinical use in Japan but are

limited by uptake and activation resistance mechanisms and the need for intravenous

administration [139-141]. Multiple novel lipid-conjugated nucleoside analogs are

currently undergoing early clinical trials in the United States, but many are limited due to

the need for hENT1 for intracellular uptake and dCK for activation.

One recent example of this technology is elacytarabine, a lipid conjugate of

cytarabine developed by Clavis Pharmaceuticals. It was shown to bypass hENT1-

mediated uptake and increased exposure time in vivo compared to cytarabine [142].

Based on these promising properties and early phase clinical trials, elacytarabine

entered a Phase III clinical trial in 2010 to treat patients with relapsed AML. However,

the study failed and was discontinued in 2013 as elacytarabine did not demonstrate a

statistical benefit in survival over investigator’s drug of choice [143]. The disappointing

failure of this once promising drug highlights the need to overcome multiple mechanisms

of chemoresistance in patients, as clearly just bypassing hENT1 was insufficient to

provide a benefit for relapsed AML patients.

Gemcitabine is a cytidine analog similar to Ara-C, but is more effective at causing

DNA damage due to inhibition of ribonucleotide reductase and more efficacious chain

termination that is less susceptible to proofreading by DNA repair enzymes [144, 145].

12

However, its potential as a treatment for leukemia has been hampered by the conditions

in which it has been clinically tested. Gemcitabine has mostly been used as a treatment

for patients with relapsed leukemia. This is a poor demonstration of its clinical potential

as Ara-C and gemcitabine share many common mechanisms of chemoresistance, and

patients previously treated with Ara-C exhibit reduced response rates compared to

patients who had not received prior therapy [146]. Both drugs require active uptake into

the cell by hENT1 and dCK for the rate-limiting activation step.

KPC34 is a novel lipid conjugate of gemcitabine attached to an amido-containing

phospholipid group (Figure 4A) rationally designed to overcome multiple mechanisms of

leukemic chemoresistance. It is orally bioavailable with improved pharmacokinetics

relative to gemcitabine and is predicted to cross the BBB [147]. Cleavage of the amido-

containing phospholipid group by phospholipase C is predicted to yield a lipid moiety that

can act as a diacylglycerol mimetic capable of inhibiting classical forms of PKC and

releases the gemcitabine monophosphate molecule for further activation (Figure 4B).

Lipid drugs conjugated using similar chemistry were shown to bypass hENT1 inhibition

and overcome MDR1 efflux [148, 149]. KPC34 may be able to enter cells by endocytosis

or passive diffusion across the cell membrane, as water-soluble small lipid molecules

(400-600 Da) have been shown to cross the BBB via diffusion through the endothelial

plasma membranes [147]. Utilizing gemcitabine with an existing monophosphate group

is predicted to overcome resistance due to decreased dCK activity. Based on these

promising characteristics, we tested the activity of KPC34 against ALL models,

specifically against chemoresistant disease.

13

Fig 1. Nucleoside Analog Metabolism. Entry of the drug into the cell is facilitated by

hENT1. The drug undergoes three consecutive phosphorylation steps by dCK, CMPK

and NDP before the active triphosphate form can enter the nucleus. Upon incorporation

into DNA, the drug causes DNA polymerase to stall, resulting in apoptosis.

14

Figure 2 Factors Influencing Environmental Chemoresistance. Leukemic cells receive

anti-apoptotic and proliferative signals via soluble and physical interactions with the bone

marrow stroma.

15

Figure 3 PKC Signaling Pathway. Receptor-mediated activation of PLC-γ cleaves PIP2

into DAG and Ca2+, which then result in the activation of PKC by association with the

plasma membrane and RACK. PKC phosphorylation can further activate ERK1/2 via

Raf1 and Bcl2 and induce the activity of NF-κB.

16

Figure 4 Structure and Activity of KPC34. A) Chemical structure of KPC34. B) Proposed

mechanism of intracellular activation of KPC34.

17

CHAPTER 2

IMPROVING NUCLEOSIDE ANALOGS VIA LIPID CONJUGATION; IS FATTER ANY

BETTER?

Peter Alexander2, Gregory L. Kucera1,2, Timothy S. Pardee1,2

Authors’ Affiliations:

1 Internal Medicine, Wake Forest Baptist Health, Winston-Salem, NC; 2 Cancer Biology,

Comprehensive Cancer Center of Wake Forest University, Winston-Salem, NC

Corresponding Author:

Timothy S. Pardee, Comprehensive Cancer Center of Wake Forest University, Medical

Center Blvd, Winston-Salem, NC, 27157; Phone (336) 716-5847; Fax (336) 716-5687; e-

mail: tspardee@wakehealth.edu

PA wrote the manuscript, TSP and GK assisted with conceptual guidance and editing of

the manuscript.

This chapter, “Improving Nucleoside Analogs via Lipid Conjugation; Is fatter any better?,”

is in preparation for submission to Critical Reviews in Oncology/Hematology.

18

II.1 Abstract

In the past few decades, nucleoside analog drugs have been used to treat a

large variety of cancers. These anti-metabolite drugs mimic nucleosides and interfere

with chain lengthening upon incorporation into the DNA or RNA of actively replicating

cells. However, efficient delivery of these drugs is limited due to their pharmacokinetic

properties, and tumors often develop drug resistance. In addition, nucleoside analogs

are generally hydrophilic, resulting in poor bioavailability and blood-brain barrier

penetration. Conjugating these drugs to lipids modifies their pharmacokinetic properties

and may improve in vivo efficacy. This review will cover recent advances in the field of

conjugation of phospholipids to nucleoside analogs. This includes conjugation of myristic

acid, 12-thioethyldodecanoic acid, 5-elaidic acid esters, phosphoramidate, and self-

emulsifying formulations. Relevant in vitro and in vivo data will be discussed for each

drug, as well as any available data from clinical trials.

II.2 Introduction

Nucleoside analogs are a class of synthetic cytotoxic antimetabolite drugs

commonly used as primary treatment for a variety of cancers, particularly leukemias and

lymphomas (Figure 5). These drugs closely resemble endogenous purine and

pyrimidine nucleosides, and their mechanism of action includes causing chain

termination on incorporation into nucleic acid strands via inhibition of DNA or RNA

polymerases.

Nucleoside analogs require transport by the human equilibrative nucleoside

transporter 1 (hENT1) to enter cells [62]. Once inside the cell, phosphorylation to the

monophosphate form of the drug is mediated by cellular kinases, which is often the rate-

limiting step of activation [150]. Two further phosphorylation steps are required to

produce the active triphosphate form, which can then be incorporated into growing

19

nucleic acid chains. Once incorporated, these drugs cause DNA chain termination and

DNA strand breaks, leading to apoptosis [151]. One nucleoside analog, gemcitabine,

also can inhibit ribonucleotide reductase [145], and two others, decitabine and

azacytidine, can inhibit DNA methyltransferases [152]. The activity of these drugs

depends on their incorporation into replicating DNA during S phase of the cell cycle and

is not specific to cancer cells; rapidly dividing normal cells (including those in the bone

marrow, gastrointestinal tract, and hair follicles) are often damaged as well. This results

in the unwanted side effects that limit clinical administration of nucleoside analogs

including myelosuppression, mucositis, and hair loss.

Many patients develop resistance to nucleoside analog agents, ultimately

reducing their clinical benefit. Resistance mechanisms include reduced drug uptake due

to decreased expression of transport proteins such as hENT1, increased activity of the

P-glycoprotein drug efflux pump, lower rates of drug activation due to loss of

deoxycytidine kinase (dCK) expression, and inactivation due to deamination by cytidine

deaminase for Ara-C and gemcitabine or ribonucleotide reductase and nuclear

exonucleases for fludarabine [153]. Conventional nucleoside analogs exhibit poor

passive diffusion across the gastrointestinal tract and require active transport by either

concentrative or equilibrative transporters [48]. All these factors limit oral bioavailability

of these drugs, so they must be given intravenously [62]. Researchers began modifying

nucleoside analogs shortly after they were first approved for clinical use. The

development of novel pro-drugs or conjugates by attaching various lipid moieties to the

parental drugs has improved their pharmacokinetic properties, including uptake, plasma

half-life, and activity in vivo. This review will focus on recent advances in nucleoside

analogs specifically conjugated to phospholipid groups.

20

II.3 Lipid-Drug Conjugate Chemistry

Most lipid-drug conjugate chemistry utilizes liposomal incorporation, salt

formation with a fatty acid, or covalent linkage of esters, ethers, glycerides, or

phospholipids to create novel pro-drugs [154-156]. Fatty acid conjugates generally

attach a drug with an amino or alcohol function to the carboxylate group of the fatty acid

or attach the drug to the ω-position of a modified fatty acid. In phospholipid conjugates,

the drug is linked via the phosphate group or glycerol backbone of the phospholipid.

Alternatively, one or two fatty acids of the phospholipid can be replaced by the drug.

Glycerides can form drug conjugates via an ester bond with carboxylate-containing

drugs [156]. Unilamellar liposomes are primarily composed of cholesterol and

phospholipids (either phosphoglycerides or sphingolipids). Liposomal drugs are well-

studied and have altered tissue distribution compared to their parental drugs, as the

conjugated drugs tend to take on the pharmacokinetic properties of the liposomal carrier

[157]. Liposome-encapsulated drugs exhibit reduced elimination by the body [157] and

generally have milder toxicity profiles [158, 159], as well as increased solubility, stability,

and circulation [136-138] and can improve BBB penetration of the active drug [60].

These effects can vary depending on liposomal size, charge, rigidity, and dose [157].

II.4 Lipid-conjugated Drugs and the Blood-Brain Barrier

The BBB protects the brain and central nervous system (CNS) from circulating

substances and is composed of several types of barriers, including the vascular BBB

and blood-cerebrospinal fluid barrier (primarily the choroid plexus) [160]. The various

barriers include the brain and choroid plexus endothelium (endothelial and capillary

cells), astrocytes, pericytes, and microglia. Endothelial cells form tight and adherens

junctions strengthened by astrocytes and pericytes and also highly express various

transporter and efflux proteins. The BBB utilizes the ATP-binding cassette p-glycoprotein

21

efflux pumps, including the multidrug resistant protein (MRP) family members, to

transport biologically active molecules away from the brain [161]. In particular,

hydrophilic drugs have difficulty crossing the BBB due to the lack of active transport and

drug efflux pumps. These features act as a physical and transport barrier that effectively

block most drugs (98% of all small-molecule drugs) from entering the brain and CNS

[162].

Drugs can cross the BBB passively by transmembrane diffusion, especially if

they are lipid soluble and have a molecular weight under 600 Da. To a lesser degree,

other factors that influence drug solubility across the BBB include tertiary structure,

degree of protein binding, and charge [160]. Small lipid and amphipathic molecules

including phosphatidylcholine and phosphatidylethanolamine are known to be effectively

effluxed by p-glycoprotein pumps in an ATP-dependent fashion [163, 164]. An estimated

50% of clinically used anti-cancer therapeutic agents are effluxed by this system

spanning a variety of drug types including taxanes, vinca alkaloids, campothecin,

mitomycins, and anthracyclines [165]. Despite the fact that nucleoside analogs are

generally not considered to be substrates of p-glycoprotein for efflux [166], the activity of

these transporters must be taken into consideration when administering lipid-conjugated

nucleoside analogs. Studies have found increased cellular concentrations of drug-lipid

conjugates containing amphiphilic glycerides, polysorbates, and polyethoxylated castor

oil. The increase in accumulation is believed to be due to inhibition of the activity of P-

glycoprotein efflux pumps by the anionic amphiphilic lipid groups, resulting in reduced

drug efflux [167]. Changes in uptake mechanisms and inhibition of P-glycoproteins are

the most likely explanations for the increase in BBB penetration of lipid-conjugated

drugs. The size and degree of lipid solubility are factors that must be taken into careful

consideration for lipid-containing drugs to successfully deliver their payload with the

brain and CNS.

22

II.5 Clinically Approved Cytarabine Conjugates

Based on advances in drug delivery, several lipid conjugates of cytarabine have

been used clinically in Japan for the treatment of acute leukemias, including enocitabine,

ancitabine and cytarabine ocfosfate (Figure 6). Enocitabine and ancitabine require dCK

for activation and must be infused intravenously.

After demonstrating efficacy as a single agent or synergy in combination with

cyclophosphamide, daunorubicin or vinblastine in a mouse model of leukemia [168],

ancitabine was tested as a single agent in patients with melanoma [169] or combined

with daunorubicin [170] or amsacrine [171] in children with refractory acute

nonlymphocytic leukemia. This drug had virtually no effect in patients with melanoma

and did not demonstrate superiority when combined with daunorubicin over patients

treated with Ara-C and daunorubicin. Both studies also found significant toxicity,

including thrombocytopenia and cardiotoxicity. Combination with amsacrine produced

complete remission in almost half of evaluated patients, but about 10% of patients did

not survive therapy.

Enocitabine was shown to significantly increase survival compared to Ara-C in a

mouse model of leukemia [172, 173] and demonstrated prolonged release when

administered i.v. in patients with acute leukemia [174, 175]. Despite the drug’s

lipophilicity, poor CSF penetration was observed, while a significant increase in bone

marrow fluid availability was seen compared to plasma [175]. In a Phase II study in

patients with naïve AML, 36% of patients reached complete remission and 24%

achieved partial remission, and increased response was correlated with higher doses

[176]. Toxicity was found to be mild and acceptable. Enocitabine combined with

mitoxantrone, 6-mercaptopurine and prednisolone was shown to be an effective

treatment for naïve patients with acute leukemia [177], and combination with aclarubicin

23

and prednisolone was an effective salvage therapy in patients with AML [178]. However,

a more recent study found reduced remission and survival rates in patients with naïve

AML treated with enocitabine compared to cytarabine [179]. These studies suggest that

enocitabine has limited efficacy as a single agent, particularly in the relapsed setting, but

can be highly effective when administered in combination with other therapeutics.

Cytarabine ocfosfate, a prodrug of cytarabine 5′-monophosphate conjugated to a

long-chain fatty alcohol group, can be delivered orally and is resistant to deamination. It

first demonstrated efficacy in a mouse model of leukemia [180, 181] and colorectal

adenocarcinoma [182], and lipophilicity was found to be improved by conjugation of the

long-chain fatty acyl group. Phase II trials testing subcutaneous or oral administration of

cytarabine ocfosfate with interferon-α2b and found some success in improving survival

and response rate in patients with hematological malignancies. Patients were unable to

continue treatment due to side effects, and a modified dosing regimen might hold

promise in the future [183-185]. Oral administration of cytarabine ocfosfate in AML

patients with relapsed disease induced complete remission in ~10% of patients and

partial remission and stable disease in some of the other patients studied.

Pharmacokinetic analysis suggested intestinal and hepatic absorption of the drug,

resulting in prolonged release mimicking continuous i.v. infusion. Off-target toxicities

limited dose-escalation in these studies as well [139, 186]. A small combination study of

orally administered cytarabine ocfosfate and etoposide showed higher induction of

complete remission in treating patients with hematological malignancies compared to

previous single agent or combination trials [187], and two recent follow-up studies

supported these findings [188, 189].

Ancitabine, enocitabine and cytarabine ocfosfate have not been approved for

clinical use in the United States. Their clinical efficacy appears to be ultimately limited by

dCK dependence for activation and various toxic side effects [141]. They may have

24

potential to treat hematological malignancies when used in combination with other drugs,

but there appear to be few clinical trials still interested in testing these drugs.

DepoCyt (DTC 101) is an extended release formulation of cytarabine (8,9,11,17),

produced by Sigma-Tau [190]. Cytarabine is encapsulated inside vesicles within a lipid

foam composed mainly of cholesterol and dioleoylphosphatidylcholine [191]. This drug

was developed to treat neoplastic and lymphomatous meningitis, fairly common

symptoms of CNS infiltration by solid and hematologic malignancies [192, 193] that can

quickly become fatal if left untreated [52]. Previous treatment options involved

combinations of intrathecal chemotherapy using Ara-C, methotrexate, and steroids [58],

high-dose systemic chemotherapy using methotrexate (5-8 g/m2) or Ara-C (1-7.5 g/m2),

and radiation therapy [1, 59, 194]. These therapeutic options come with undesirable side

effects, including increased toxicity, cognitive deficiency, infection, chemical meningitis,

and inaccurate lumbar puncture [59]. Depocyt was shown to be more effective than

intrathecally administered methotrexate [195, 196] or cytarabine [58, 197] in multiple

tumor types. However, Depocyt still requires intrathecal administration and caused

neurotoxicity in approximately 15% of patients tested [58]. The T1/2 of Depocyt was 141

hours in the brain and 277 hours in the lumbar space, compared to 3.4 and 2 hours for

cytarabine, respectively [198-200]. This large difference in half-life is the most likely

explanation for the increase in toxicity. Ideally, CNS involved cancer would be treated by

a drug that does not require intrathecal administration and has reduced neurotoxic side

effects.

II.6 Drug Duplex Conjugates

One strategy of modifying the pharmacokinetic properties of nucleoside analogs

is to conjugate two drug molecules together. The lipophilicity of these conjugates can be

increased by using a lipid backbone or by using a drug with lipid moieties attached.

25

Utilizing drug duplexes is expected to improve cellular uptake and alter distribution

profiles in vivo due to the conjugated drug’s lipophilic and amphiphilic properties.

Previous studies suggest that the conjugated drugs act similarly to free drugs upon

uptake and cleavage inside the cell. Delivering nucleoside analogs in this fashion may

protect the drugs from inactivation or degradation due to lack of substrate recognition,

resulting in improved intracellular delivery [201].

Teams led by Drs. Herbert Schott and Reto Schwendener created two novel drug

duplexes by conjugating the 5-Fluorouracil (5-FU) derivative 2’-deoxy-5-fluorouridine (5-

FdU) and a 3-ethynyl nucleoside (ECyd). These nucleoside analogs were either coupled

directly via a phosphodiester bond – producing 2’-deoxy-5-fluorouridylyl-(3’-5’)-3’-C-

ethynylcytidine (5-FdU(3’-5’)ECyd) – or indirectly via a lipophilic

octadecylglycerophospholipid backbone, producing 3’-C-ethynylcytidinylyl-(5’→1-O)-2-

O-octadecyl-sn-glycerylyl-(3’-O→5’)-2’-deoxy-5-fluorouridine (ECyd-lipid-5-FdU) (Figure

7). Combining these two drugs should inhibit both DNA and RNA synthesis and may

reduce chemoresistance. Both complexes are believed to require phosphodiesterase

cleavage for activation. Cleavage of ECyd-lipid-5-FdU results in the monophosphate

form of both drugs, while cleavage of 5-FdU(3’-5’)ECyd produces one drug in the

monophosphate form and one that requires phosphorylation, depending on which side of

the phosphate group is cleaved.

5-FdU(3’-5’)ECyd and ECyd-lipid-5-FdU were screened against the NCI-60

cancer cell line panel. 5-FdU(3’-5’)ECyd acted additively, synergistically, or

antagonistically in different cell lines compared to individual drug treatment of 5-FdU or

ECyd [202]. These differences in cytotoxicity are hypothesized to be due to the chemical

properties of the duplexes, including cytostatic potential and the direction of the cleaved

phosphodiester bond [202]. These results highlight the importance of comparing the

toxicity of multidrug complexes to simultaneous administration of their component drugs.

26

5-FdU(3’-5’)ECyd and ECyd-lipid-5-FdU were tested in various pre-clinical

cancer models. Both drugs did not significantly affect survival in DBA/2J mice bearing

L1210 murine leukemia cells. ECyd-lipid-5-FdU did have greater anti-leukemic activity

compared to 5-FdU(3’-5’)ECyd treatment, most likely due to a combination of increased

lipophilicity and intracellular release of two monophosphate nucleoside analogs. The

duplexes had more off-target toxicity than 5-FdU alone but less than ECyd [203]. 5-

FdU(3’-5’)ECyd was effective against in vivo melanoma models, but efficacy depended

on dose and schedule [204]. 5-FdU(3’-5’)ECyd was found to be cleaved extracellularly

and required nucleoside transporters for transport, unlike ECyd-lipid-5-FdU. Treatment

with 5-FdU(3’-5’)ECyd, caused rapid nucleotide accumulation in the tumor site followed

by the liver, whereas ECyd-lipid-5-FdU resulted in gradual nucleotide accumulation in

the tumor and more rapid nucleoside uptake by the liver. However, these

pharmacokinetic differences did not affect the anti-tumor response [205].

In vitro studies of these duplex drugs may underestimate their clinical benefit due

to the benefit of their unique pharmacokinetic properties in vivo. Association with the lipid

membrane by duplex drugs may delay their cytoplasmic release and active metabolism,

resulting in prolonged intracellular exposure compared to unmodified parental drugs.

Delivering these modified drugs in liposomal formulations may increase drug delivery by

increasing solubility and stability, decreasing toxicity, and enhancing receptor-free tumor

accumulation compared to the parental drugs [204]. More in vivo studies are required to

determine if 5-FdU(3’-5’)ECyd and ECyd-lipid-5-FdU can be successfully delivered orally

in other tumor models and to determine optimal dosing schedules. Rational

combinations of drugs linked by lipid complexes may be a promising avenue for

increasing anti-tumor efficacy of treatments and preventing chemoresistance.

27

II.7 Myristic Acid and 12-Thioethyldodecanoic Acid Conjugated Analogs

Previous work by the Parang group has shown that conjugation of fatty acid

derivatives to antiviral agents changes their biological properties compared to the parent

drugs. They recently applied this approach against cancer cells in vitro using several

cytarabine conjugates, with the expectation of sustained release and improved cellular

uptake of the drugs based on previous studies [206-208]. Chhikara et al. created

cytarabine prodrugs by synthesizing three classes of lipophilic fatty acyl conjugates – 5’-

O-substituted, 2’-O-substituted, and 2’,5’-disubstituted using myristic acid or 12-

thioethyldodecanoic acid (Figure 8). These conjugates displayed varying antitumor

effects in vitro against a human T cell leukemia line. The 5’-O-substituted compounds

did not inhibit growth, suggesting that the location of substitution may prevent the

release of cytarabine. The 2’,5’-dimyristoyl derivative was as effective as cytarabine in a

time-dependent fashion, while 2’-fatty acyl derivatives of cytarabine demonstrated lower

degrees of inhibition. These results show a clear dependence on chemical conformation

of cytarabine derivatives for biological activity, and time-dependent cytotoxicity suggests

a gradual intracellular release of the active drug. Further in vivo testing of these drugs is

required to determine if the lipophilic formulation also increases bioavailability and

cellular uptake, and the potential benefit of conjugating the monophosphate form of

cytarabine to avoid dependency on dCK activation [209].

II.8 Phosphoramidate Conjugated Analogs

NuCana has developed several nucleoside analog pro-drugs by adding a

phosphoramidate moiety (ProTide) to the monophosphate form of the drug (Figure 9)

[210]. This moiety has been shown to protect the phosphate group, reducing metabolic

degradation [211], as well as potentially increasing intracellular delivery of the active

drug. NUC-1031 (Acelarin) is a gemcitabine monophosphate analog designed to bypass

28

hENT1 uptake, dCK activation, and deactivation by cytidine deaminase [212, 213] and

was found to be active against gemcitabine-resistant human pancreatic cancer cell lines

in a xenograft model [214]. NUC-1031 is currently being tested in an ongoing Phase I/II

trial in patients with various refractory solid cancers. Patients received multiple four-week

cycles of NUC-1031 with two different schedules. The drug could be safely administered

at four times the maximum tolerated dose (MTD) of gemcitabine, and over half the

patients treated had stable disease. Five out of 36 patients were even able to achieve

tumor shrinkage of over 30%.

Pharmacokinetic data revealed a 13-fold increase in the intracellular active form

of gemcitabine (dFdCTP) compared to the parental form of gemcitabine, suggesting

greater activity independent of increased dose [215]. NuCana plans on beginning Phase

III trials in 2015 for patients with pancreatic, ovarian, non-small cell lung, and biliary

cancers. The safety and efficacy of NUC-1031 in combination with carboplatin is

currently being tested in a Phase IB trial for patients with recurrent ovarian cancer and

will also be tested in patients with biliary cancers mid 2015.

NuCana is also modifying the pyrimidine analogs 5-FU and 5-fluoro-2'-

deoxyuridine (FUdR) by attaching a phosphoramidate moiety to the active form of these

drugs, fluorodeoxyuridine monophosphate (FdUMP). These modified drugs, NUC-3373

and NUC-3641, respectively, prevent degradation by phosphorolytic enzymes, bypass

thymidine kinase-mediated activation, and are taken up passively. A screen of 39 FUDR

conjugates against several human tumor cell lines found these two drugs to be much

more cytotoxic in vitro than 5-FU, independent of thymidine kinase and hENT1 [216].

NuCana plans to begin testing NUC-3373 in clinical trials for patients with colorectal and

non-small cell lung cancers mid 2015. The company is also in the early stages of

applying the same lipid conjugate technology to fludarabine (NUC-4545), clofarabine

(NUC-5435), and cladribine.

29

II.9 Phospholipid Gemcitabine Conjugate

Kucera et al. has conjugated gemcitabine monophosphate to an amido-

containing phospholipid moiety [148], producing the drug KPC34 (Figure 10). It was

rationally designed to overcome multiple forms of chemoresistance, including dCK-

independent activation and hENT1-free uptake, improve pharmacokinetics, increase

BBB penetration, and inhibit PKC. KPC34 has been shown to be as effective or superior

compared to gemcitabine treating several types of cancer in vitro and in vivo. In the

human promyelocytic cell line HL60, pharmacological inhibition of hENT1 by

dipyridamole decreased the IC50 of gemcitabine 35-fold, compared to only 4-fold for

KPC34. In the same study, KPC34’s cytotoxicity was tested in the human breast cancer

cell line MCF-7 and a clone over-expressing the multidrug resistance protein 1 (MDR1)

efflux pump, BC-19. KPC34 maintained much of its cytotoxicity when compared to

doxorubicin, a known substrate of MDR1. [148] Based on drugs previously synthesized

with similar chemistry [149], KPC34’s phospholipid group is predicted to act as a

diacyglycerol mimetic upon cleavage by a phospholipase C-like enzyme. Diacylglycerol

is necessary for the activation of classical members of the PKC family by recruiting them

to the plasma membrane. Increased PKC signaling is associated with chemoresistance

in acute leukemia [119]. KPC34’s cleaved moiety may antagonize PKC signaling by

sequestering PKC away from the plasma membrane and reducing its activation.

Treatment of acute lymphoblastic leukemic cells in vitro with KPC34 inhibited PKC

signaling by over 50% as assessed by immunoblotting for p-PKC α and βII (Alexander et

al., manuscript in preparation).

In the Lewis lung mouse tumor model 50 mg/kg of KPC34 prolonged survival as

effectively as 120 mg/kg of gemcitabine. KPC34 could be administered orally with similar

survival benefit, was well-tolerated, and had a greatly improved plasma half-life

compared to gemcitabine [148]. In a syngeneic immunocompetent model of murine

30

acute lymphoblastic leukemia, KPC34 was also well-tolerated and oral dosing provided a

greater survival benefit compared to intraperitoneal administration of cytarabine,

gemcitabine, or KPC34 (Alexander et al., manuscript in preparation). KPC34 overcame

induced cytarabine chemoresistance in vivo and was more effective than gemcitabine.

Cytarabine and gemcitabine share similar mechanisms of resistance. The efficacy of

KPC34 in this model of chemoresistance suggests the value of phospholipid conjugation

for improving outcomes.

The amphipathic nature of KPC34 may result in the formation of water-soluble

lipid aggregates. In one report, KPC34 formed spherical particles in aqueous media with

an average size of 115 nm [148]. The hENT-1 independent uptake suggests that KPC34

may enter the cell by endocytosis or passive diffusion across the cell membrane.

Previous studies have shown that water-soluble small lipid molecules (400-600 Da) can

cross the BBB via diffusion through the endothelial plasma membranes [147]. Improved

survival seen with KPC34 versus gemcitabine in a naïve model of acute lymphoid

leukemia may be due to increased clearance of CNS-involved disease, as hind limb

paralysis occurred later in the KPC34 treated animals (studies ongoing). Further, KPC34

successfully restored motor function in mice that developed hind limb paralysis after

treatment with cytarabine and doxorubicin, indirect evidence of its ability to cross the

BBB (Alexander et al., unpublished data). KPC34 appears to be a promising new option

for patients with relapsed acute lymphoblastic leukemia, and this drug is scheduled to

enter early phase clinical trials in the next few years.

II.10 5-Elaidic Acid Ester Conjugated Analogs

Clavis Pharmaceuticals developed and tested two novel lipid-conjugated

nucleoside analogs, CP-4055 and CP-4126, in an attempt to improve the

pharmacokinetics of gemcitabine. CP-4055 (elacytarabine) is cytarabine covalently

31

conjugated to elaidic acid ester at the 5’-OH of the ribose sugar, and CP-4126 is

gemcitabine similarly attached to the same hydrophobic lipid moiety (Figure 11). The

rationale for this conjugation is that linkage of fatty acids to the nucleoside analogs is

predicted to increase receptor-independent drug uptake by passive diffusion across the

plasma membrane, resulting in increased levels of active intracellular drug. The lipid

formulation is also believed to increase plasma half-life and prevent intracellular

deamination. These compounds are hydrolytically cleaved into their respective parent

nucleoside analog, possibly by carboxylesterases, and require phosphorylation by dCK

to reach the active triphosphate form [217]. In vitro studies demonstrated that the lipid

formulation increased uptake of the prodrugs in a hENT-1- independent fashion

compared to the parental forms of the drugs [142, 218]. Both novel drugs demonstrated

gradual increased nuclear accumulation of the active drug compared to the parental

drugs, even after drug removal. The gradual intracellular release of drug suggests that

the lipid formulation may trap conjugates in an intracellular compartment after uptake

[142]. Both CP-4055 and CP-4126 overcame resistance to cytarabine and gemcitabine

in a human T-cell leukemia cell line lacking hENT1 [219, 220]. In mouse models using a

variety of human cancer xenografts, CP-4126 was as effective as gemcitabine when

administered intraperitoneally. When administered orally, CP-4126 was well tolerated

and as effective as intraperitoneal delivery [218]. A significant increase in survival after

CP-4055 treatment was seen compared to cytarabine in a variety of human xenograft

models in mice [221].

These promising results led to clinical trials of CP-4055 and CP-4126 as orphan

drugs in Europe and America, with fast-track designation by the FDA. Both drugs have

undergone various Phase I and II clinical trials for solid and hematologic malignancies. A

Phase I study of CP-4126 administered orally to patients with solid tumors once a week

found few adverse side effects, with a maximum tolerated dose of 3,000 mg/day.

32

However, the best anti-tumor outcome was stable disease, and no patients achieved

partial or complete response. Low plasma levels of active gemcitabine were attributed to

high intestinal absorption and deamination by the liver [217]. These preliminary results

led Clavis to abandon testing oral delivery of CP-4126 for intravenous delivery. CP-4126

was tested in a Phase II study with pancreatic cancer patients who expressed low levels

of hENT1. No difference was found in overall survival compared to gemcitabine

treatment [222]. As a result, Clavis suspended all further developmental work on CP-

4126.

A Phase I dose escalation study of CP-4055 in patients with solid tumors was

well-tolerated and produced few significant adverse effects. The major dose-limiting

toxicity (DLT) was myelosuppression [223]. CP-4055 was then tested in a Phase I study

of 77 patients with refractory leukemia starting at 2000 mg/m2 on days 1-5 with various

infusion times. Patient deaths were not attributed to CP-4055 or affected by dose, and

11% of patients displayed a complete remission. The DLT was hyperbilirubinemia, which

lasted up to 13 days and was reversible once treatment was discontinued. Optimal

scheduling of CP-4055 appeared to be 2,000 mg/m2 per day for 5 days by continuous

intravenous administration (CIV), as minimal toxicities were seen at this dose [224].

CP-4055 was tested as a single agent therapy in a Phase II trial for patients with

refractory acute myeloid leukemia (AML) in whom two other therapies were not effective.

When CP-4055 was given (2,000 mg/m2/d for 5 d by CIV) a remission rate of 18% and

an overall survival rate of 5.3 months were observed. The adverse events profile was

similar to that of cytarabine [225]. A randomized Phase III clinical trial in AML patients

found no significant difference in overall survival or adverse events between patients

given CP-4055 (same dose as the Phase II study) and investigator’s choice of treatment

[143]. However, a lower dose of CP-4055 (1,000 mg/m2/d CIV on days 1-5) combined

33

with idarubicin (12 mg/m2/d IV on days 1-3) produced a 40% complete remission rate

[226].

Despite discouraging results as single-agent therapy, CP-4055 may be useful in

combination with other drugs, or as an option for patients with refractory disease. In vitro

studies found synergy between CP-4055 and gemcitabine, irinotecan, and topotecan in

a promyelocytic cell line, while co-treatment with cloretazine or idarubicin were additive.

Further consideration must be given to the scheduling of combination therapies, as 24-

hour pre-treatment with topoisomerase inhibitors was antagonistic when combined with

CP-4055 in vitro [227].

II.11 Self-Emulsifying Compounds

InnoPharmax has developed an orally available self-microemulsifying formulation

of gemcitabine, known as D07001-F4 using proprietary OralPAS® technology. Self-

microemulsifying formulation of poorly absorbed lipophilic drugs has been shown to

improve oral delivery of these poorly water-soluble molecules by incorporation of oils,

solvents, surfactants, and co-solvents/surfactants [228]. D07001-F4 was found to be

more bioavailable, 3-fold less susceptible to deamination, and more effective at inhibiting

growth in a panel of tumor cell lines compared to gemcitabine. In mouse models of

pancreatic and colon cancer, oral delivery of D07001-F4 was well-tolerated and more

effective than intravenous gemcitabine [229]. InnoPharmax has started a Phase I dose-

escalation trial in patients with advanced solid tumors or lymphoma. So far, no dose-

limiting toxicity has been observed and the drug has shown some signs of anti-tumor

activity against solid tumors. Further clinical testing is planned upon completion of this

Phase I trial.

34

II.12 Discussion

The past few years have produced great advances in the development of lipid-

conjugated nucleoside analogs. The various chemical modifications described above

have been shown to increase drug circulation and stability in the body, circumvented

hENT1 uptake and dCK activation, and overcome chemoresistance in vitro and in vivo.

The observed change in uptake from carrier-mediated to passive diffusion may be

efficacious for the treatment of relapsed disease, where reduced active drug uptake is a

common mechanism of resistance. Continued incremental modifications can help

elucidate the effects of choice and position of phospholipid group conjugation on

biological activity of nucleoside analogs, as not every modification studied has proven to

be successful pre-clinically or in clinical trials. Further research is needed to better

understand the precise effects of such modifications on drug pharmacokinetics, uptake,

and intracellular activation. Still, conjugation of fatty acids or the use of liposomes show

great promise as methods to deliver drugs to the lymphatic system or to avoid the first-

pass effect by the liver when delivered orally.

The failure of CP-4055 highlights the need for better pre-clinical models of

chemoresistance in testing novel drug conjugates. Given that many drugs will be first

tested clinically in patients resistant to traditional therapy, pre-clinical testing should have

a strong focusing on relapsed disease. Most studies have tested overcoming common

mechanisms of drug resistance in vitro, which while still useful is limited as

pharmacokinetic properties can not be tested in a dish. Further, most in vivo efficacy

testing is performed against previously untreated tumors, often using

immunocompromised mouse models. A more relevant animal model would include

chemoresistant disease in an immunocompetent model, ideally arising within the same

animal to better mimic clinical conditions. There are benefits and drawbacks of testing

35

both human and murine cancer models, but ideally drugs would be tested in both types

to fully account for the effects of the immune system and differences in species.

Despite the poor outcomes of clinical trials testing the efficacy CP-4055 and CP-

4126, many other phospholipid nucleoside analog conjugates are currently undergoing

or are poised to enter clinical trials, and many more are entering the early stages of pre-

clinical development. As is currently ongoing with CP-4055, switching treatment to a

different type of cancer may help salvage drugs that initially fail Phase III trials. Potential

mechanisms to improve the efficacy of phospholipid drug conjugates involve increasing

cytotoxicity and reducing unwanted side effects. This may be accomplished by

incorporating active tumor targeting mechanisms via conjugation of antibodies or tumor-

specific substrates, by creating multi-drug complexes, or by incorporating moieties that

have additional anti-carcinogenic effects. Once the efficacy, safety profiles, and

mechanisms of action of these novel drugs are better understood, rational combinations

with other clinically approved anti-neoplastic agents can be further explored in clinical

trials. Such combinations could test anthracyclines, other nucleoside analogs, anti-tumor

antibodies, and kinase inhibitors with nucleoside analog conjugates. Continued rational

modification of lipid-conjugated nucleoside analogs, combinations with other anti-

neoplastic agents, and application to other types of cancer will hopefully lead to

improved outcomes for patients suffering from advanced cancer.

36

Figure 5 Chemical structures of conventional nucleoside analogs. A) Cytarabine, B)

Gemcitabine, C) Clofarabine, D) Cladribine, and E) 5-Fluorouracil.

37

Figure 6 Chemical structures of clinically approved cytarabine conjugates. A)

Ancitabine, B) Enocitabine, and C) Cytarabine ocfosfate.

38

Figure 7 Chemical structures of duplex drug conjugates. A) ECyd-lipid-5-FdU and B) 5-

FdU(3’-5’)ECyd.

39

Figure 8 Chemical structures of fatty acyl cytarabine prodrugs. A) 5’-O-Monosubstituted

dimyristoyl/thioethyl dodecanoyl, B) 2’-O-Monosubstituted dimyristoyl/thioethyl

dodecanoyl, and C) 2’,5’-Disubstituted dimyristoyl.

Figure 9 Chemical structures of phosphoramidate-conjugated prodrugs. A) NUC-1031

(Gemcitabine monophosphate) and B) NUC-3373 (5-FU).

40

Figure 10 Chemical structure of KPC34.

Figure 11 Chemical structures of lipid-conjugated nucleoside analogs developed by

Clavis Pharma. A) CP-4055 (cytarabine) and B) CP-4126 (gemcitabine).

41

CHAPTER 3

KPC34 IS ACTIVE AGAINST MULTIPLE MODELS OF RESISTANT ACUTE

LYMPHOBLASTIC LEUKEMIA

III.1 ABSTRACT

Acute lymphoblastic leukemia is a cancer of the white blood cells that affects

thousands of Americans annually. Common components of therapeutic regimens include

cytarabine and an anthracycline. Most patients relapse with chemoresistant disease

after initially responding to treatment. There is a desperate need for new therapeutic

agents for the treatment of ALL, particularly relapsed disease.

An ideal novel drug to treat relapsed leukemia would be orally available,

effectively treat CNS disease, bypass common uptake and activation resistance

mechanisms, and overcome stroma-mediated and cell-autonomous chemoresistance.

We tested the cytotoxicity of a novel phospholipid conjugate of gemcitabine, KPC34,

rationally designed to overcome various mechanisms of chemoresistance in leukemia.

KPC34 was found to be cytotoxic against human and murine ALL cell lines and was able

to effectively treat a syngeneic, orthotopic model of ALL.

KPC34 was cytotoxic against the ALL cell lines tested with IC50 values in the

nanomolar range, inhibited classical PKC signaling, and overcame in vitro stroma-

mediated chemoresistance. This drug was more effective at treating mouse models of

naïve and chemoresistant ALL than cytarabine or gemcitabine. Mice treated with

42

doxorubicin and cytarabine (mimicking existing components of therapy for patients with

ALL) had reduced survival compared to mice treated with KPC34 and doxorubicin. Mice

that relapsed after initial treatment with cytarabine and doxorubicin saw striking

improvements in hind limb paralysis upon retreatment with KPC34, suggesting that this

drug effectively treats CNS disease.

KPC34 was found to be statistically superior over its parent drug gemcitabine

and cytarabine in increasing survival in mouse models of naïve and chemoresistant ALL.

Mice tolerated KPC34 treatment with minimal toxicity. These encouraging pre-clinical

findings justify further development of KPC34 for the treatment of ALL, particularly in the

relapsed setting.

43

III.2 INTRODUCTION

Acute lymphoblastic leukemia is a cancer of the blood and bone marrow that

leads to an accumulation of immature lymphoid progenitor cells, causing bone marrow

failure and ultimately death. This disease predominantly affects children and the elderly

[230]. In 2015, there will be an estimated 6,000 new cases of ALL and 1,500 deaths in

the United States [231]. The overall 5-year survival rate among adults with this disease

is 30-40%, and is even worse in elderly patients over the age of 60 at 10% [232].

Children diagnosed with ALL initially have an 80% 5-year survival rate, but upon relapse

this rate drops to 15-50% [45, 46].

For many years, the standard of care for patients with ALL has been induction

therapy containing an anthracycline followed by consolidation chemotherapy or a stem

cell transplant [1]. This strategy usually produces a complete but transient remission in

most patients, and relapsed disease becomes much more difficult to treat. ALL patients

with relapsed disease have a dismal 7% 5-year survival rate [5]. CNS involvement is a

relatively common occurrence in ALL, and is seen in 8% of patients at diagnosis. This

rate increases to 10-15% in patients with relapsed disease [50, 51]. Because standard

chemotherapeutic agents have poor blood-brain barrier (BBB) penetration, patients

require high-dose systemic chemotherapy and intrathecal injections, both of which can

lead to undesirable side effects [1]. Clearly, there is a pressing need for novel

therapeutic agents for improved treatment of ALL, particularly for patients with recurrent

disease.

An important component of consolidation therapy for ALL is the nucleoside

analog cytarabine (Ara-C). Nucleoside analogs are synthetic cytotoxic antimetabolites

that mimic naturally occurring nucleosides and are used to treat various cancers,

44

particularly leukemias and lymphomas. Upon active transport into the cell, Ara-C

undergoes three consecutive phosphorylation steps in order to produce the active form

of the drug. The rate-limiting step of activation is the initial phosphorylation step by

deoxycytidine kinase (dCK). The active tri-phosphate form of the drug is able to be

incorporated into replicating strands of DNA. DNA polymerases are unable to

incorporate nucleotides after incorporation of nucleoside analogs, causing DNA strand

breaks and chain termination, ultimately resulting in the induction of apoptosis [62]. The

clinical usefulness of Ara-C and other similar nucleoside analogs is limited by

development of chemoresistance and off-target toxicity to the gastrointestinal tract and

bone marrow [63].

Gemcitabine is a cytidine analog that acts similarly to Ara-C, but induces DNA

damage more effectively and is less vulnerable to removal by DNA repair enzymes [144,

145]. Its potential for treating leukemia has been limited by the conditions of its clinical

testing, as it has mostly primarily been tested in patients with relapsed disease. This

setting is a poor choice for testing its clinical potential as gemcitabine and Ara-C share

many similar mechanisms of chemoresistance. Both nucleoside analogs require hENT1

for active uptake into the cell and activation is rate-limited by dCK phosphorylation.

Clinically, low levels dCK and hENT1 are correlated with poor response to Ara-C therapy

and poor prognosis [65]. Studies in relapsed patients treated with gemcitabine have

shown that patients previously treated with Ara-C displayed lower response rates than

patients who had not received prior chemotherapy [146].

Another common mechanism of chemoresistance in ALL is increased expression

of isoforms of the PKC signaling family [119]. Conventional members of the PKC family

are activated by calcium ions and diacylglycerol signaling. Up-regulation of the

conventional α or β isoforms has been linked to leukemic chemoresistance in ALL [128,

45

129]. PKC-mediated chemoprotection is at least partially mediated by activation of the

NF-κB signaling pathway and the anti-apoptotic protein Bcl2 [128, 132].

Despite knowing many mediators of chemoresistance in ALL, minimal progress

has been made in successfully treating resistant disease. There is a clear need for the

development of new therapies to treat ALL, particularly in the relapsed setting. An ideal

drug for treating relapsed disease would avoid uptake and activation resistance

mechanisms by bypassing hENT1 and dCK, overcome cell-autonomous and

environment-mediated chemoresistance (possibly by inhibiting PKC), effectively cross

the BBB to treat CNS-involved disease, and would not require intravenous

administration.

A drug development strategy incorporating many of these useful features is the

conjugation of lipid groups to nucleoside analogs. This approach has generated a

promising new class of chemotherapeutics. The chemical modifications imparted by this

technology have been shown to improve many pharmacokinetic properties, such as

stability, circulation time, solubility, BBB penetration, and reduced drug efflux [60, 136-

138]. Several cytarabine lipid conjugates have been clinically approved in Japan but

their efficacy is limited by dependence on dCK for activation and the necessity of

intravenous administration [139-141]. Various lipid-conjugated nucleoside analogs are

undergoing clinical trials in the United States, but many of these drugs still require

hENT1 for intracellular transport and dCK for activation, which will most likely limit their

efficacy in relapsed patients.

Elacytarabine is a lipid conjugate of cytarabine with increased in vivo exposure

compared to cytarabine and hENT1-free uptake [142]. Based on these encouraging pre-

clinical results and early clinical trials, it was tested in a Phase III clinical trial in 2010 for

patients with relapsed AML. The study was discontinued in 2013 as elacytarabine was

unable to demonstrate significant benefit over investigator’s drug of choice [143]. The

46

failure of this promising drug underscores the importance of overcoming multiple forms

of chemoresistance in patients when rationally designing new drugs.

KPC34 is a novel drug conjugate of gemcitabine monophosphate and an amido-

containing phospholipid. This drug was rationally designed to overcome various

mechanisms of leukemic chemoresistance common to ALL. KCP34 is orally available, is

predicted to penetrate the BBB, and has demonstrated improved pharmacokinetics

compared to gemcitabine. The amido-containing phospholipid group is predicted to be

cleaved by phospholipase C intracellularly and this cleaved product is believed to mimic

diacylglycerol, inhibiting classical forms of PKC.

Based on these promising characteristics, we tested the activity of KPC34

against ALL in vitro and in vivo. To mimic physiological conditions in vitro, we treated

leukemic cells co-cultured with normal bone marrow stroma cells with KPC34. We

utilized a syngeneic orthotopic immunocompetent mouse model of ALL to test the

efficacy of KPC34 in treating leukemia in vivo. This type of model more accurately

represents the development of leukemia, as these mice have a fully intact immune

system and a more complete bone marrow niche compared to a human xenograft

mouse model. We used this mouse model to further test multiple forms of

chemoresistance in ALL in an in vivo setting.

47

III.3 METHODS

Reagents:

KPC34 was synthesized as previously published in [148]. Gemcitabine Hydrochloride

(#G6423) and dipyridamole (#D9766) were purchased from Sigma-Aldrich (St. Louis.

MO). Cytarabine was purchased from NOVAPLUS and Doxorubicin HCl was purchased

from Pfizer (New York, NY). For animal studies, all chemotherapeutic agents were

dissolved in saline prior to treatment.

Cell Culture:

The mouse leukemia cell line B6 ALL was maintained in 45% DMEM, 45% IMDM, and

10% FBS supplemented with L-glutamine, penicillin, and streptomycin. The human

leukemia cell lines CCRF-CEM and MOLT4, and Jurkat were maintained in RPMI media

(Gibco, Carlsbad, CA) supplemented with 10% FBS, penicillin and streptomycin. The

human leukemia cell line SUPB15 was maintained in IMDM media with 20% FBS, L-

glutamine, penicillin, streptomycin and 50 uM 2-mercaptoethanol. The murine bone

marrow stromal M2 cell line was grown in DMEM supplemented with 10% FBS,

penicillin, and streptomycin. All cell lines were maintained in an incubator at 37o C and

5% CO2.

Cell Viability:

Viability assays were performed in 96 well plates using the Cell Titer-Glo assay

(Promega, Madison, WI) according to the manufacturer's protocol after 72 hour exposure

to indicated drug concentration. Murine cell lines were plated at 50,000 cells/ml and

48

human cell lines were plated at 100,000 cells/ml prior to drug exposure. Both types of

cells were plated at 500 μl volume in triplicate for each treatment.

Flow Cytometry Assays:

1. Cells were seeded in 24-well plates at 100,000 cells/ml in 0.5 ml, grown for 2

days and treated with drug for 48 hours. After centrifugation and washing in cold

PBS, cells were stained with propidium iodide (Sigma Aldrich, St. Louis, MO) and

allophycocyanin (APC)-conjugated annexin V in a binding buffer (0.1 M Hepes

(pH 7.4), 1.4 M NaCl, and 25 mM CaCl2 solution) (BD Pharmingen, San Jose,

CA) according to the manufacturer’s protocol. Flow cytometric analysis was

conducted on an Accuri C6 cytometer (BD Pharmingen, San Jose, CA) with the

FCS Express software (De Novo Software, Los Angeles, CA).

2. For stromal co-culture assays, B6-ALL leukemic cells were plated at 50,000

cells/ml alone in 0.5 ml or in 0.25 ml with 0.25 ml of M2 mouse bone marrow

stromal cells at 50,000 cells/ml each. The cells were incubated for 48 hours and

then treated with indicated drugs for 48 hours. Non-adherent cells were collected

96 hours after initial plating and annexin V/propidium iodide staining was

performed as described above.

3. Dual staining of γ-H2AX and B220R was performed on bilateral femur cells

harvested from mice treated with one dose of KPC34 at 20 mg/kg i.p. or orally for

8 or 12 hours or orally dosed with an equivalent volume of saline at 8 hours.

Cells from each mouse were stained with: a) Rat anti-mouse CD45R/B220R

FITC Ab (#553087, BD Biosciences, San Jose, CA) b) γ-H2AX S139 Rabbit mAb

AlexaFluor 647 (#9720, Cell Signaling, Beverly, MA), c) B220R + γ-H2AX, d) Rat

IgG2a k Isotype Control FITC (#553929, BD Biosciences, San Jose, CA), or e)

Rabbit IgG Isotype Control Rabbit Ab AlexaFluor 647 (#3452, Cell Signaling,

49

Beverly, MA). Cells were gated by size and B220R positivity. Dual-labeled

populations were then counted for each treatment group.

Western Blotting:

Cells were lysed in Laemmli buffer(1.6 ml 10% SDS, 500 µL 1 M Tris-HCl [pH 6.8], 800

µl glycerol, 400 µl 2-mercaptoethanol, 4.7 ml H2O), and samples separated by SDS-

PAGE before transfer to an Immobilon polyvinylidene difluoride membrane (Millipore,

Billerica, MA). Primary antibodies against caspase 3 (#9662 Cell Signaling, Beverly,

MA), actin (AC-15, 1 10000; Abcam, Cambridge, MA), anti-pH2AX (#2577, 1 1000; Cell

Signaling, Beverly, MA) and a secondary antibody anti-mouse (#7076, 1 5000; Cell

Signaling, Beverly, MA or anti-rabbit (#7074, 1 5000; Cell Signaling, Beverly, MA) were

used. ImageJ software (http://imagej.nih.gov/ij/) was used to quantify band intensity.

Mice:

The Comprehensive Cancer Center of Wake Forest University Institutional Animal Care

and Use Committee approved all mouse experiments. Luciferase-tagged B6-ALL murine

leukemia cells were transplanted into 8 week old recipient C57/Bl6 mice by tail-vein

injection of 1 x 106 viable cells. Mice were monitored by bioluminescent imaging on day

6 using an IVIS100 imaging system (Caliper LifeSciences, Hopkinton, MA). Mice were

injected with 150 mg/kg D-Luciferin (Gold Biotechnology, St. Louis, MO), anesthetized

with isoflurane, and imaged for 2 min.

1. For single-agent studies, chemotherapy was initiated upon detection of clear

leukemic signal. Mice were randomly assigned to treatment groups and treated

once a day for four days with saline, 20 mg/kg KPC34 by oral gavage or

equimolar concentrations of Ara-C (7.1 mg/kg) or gemcitabine (8.8 mg/kg)

intraperitoneally (i.p.). Control animals received equivalent volumes of saline. For

50

combination studies, mice were treated with indicated concentrations of KPC34

or Ara-C once a day for four days and doxorubicin at 3 mg/kg once a day for

three days. For the duration of treatment, drinking water was supplemented with

1 mg/ml Neomycin. Animals were euthanized upon onset of hind limb paralysis,

hunched posture, or difficulty breathing.

2. To determine CNS involvement, mice were injected with leukemia and treated as

described previously in 1. Mice imaged and sacrificed upon the onset of hind limb

paralysis in saline treated animals. Mice were injected with luciferin, sacrificed 5

minutes later and imaged both dorsally and ventrally by IVIS. The brains were

exposed during dorsal imaging and later harvested. Brains were fixed in 10%

neutral-buffered formalin for 72 hours and then transferred to Surgipath

Decalifier-I solution (10% v/v formaldehyde-formic acid, Leica, Buffalo Grove, IL)

for 96 hours. Samples underwent routine tissue processing and sectioning for

H&E staining. Slides were then reviewed by a veterinary pathologist with the use

of a Nikon Eclipse 50i light microscope. Photographs of tissues were taken with

the NIS Elements D3.10 camera and software system.

3. To generate Ara-C resistant cells, mice were injected with B6-ALL leukemic cells

as described above. Upon detection of luminescent signal, mice were treated

with 100 mg/kg Ara-C daily until euthanasia was required. Mice were then

sacrificed, bilateral femur cells were harvested and the cells were cultured in

45% DMEM, 45% IMDM, and 10% FBS supplemented with L-glutamine,

penicillin, and streptomycin. Naïve C57/Bl6 mice were then injected with 500,000

cells/mouse by tail vein injection. Prior to treatment, leukemic engraftment was

confirmed by IVIS imaging 10 days post-injection as described earlier.

4. To test the effect of hENT1 inhibition on uptake of gemcitabine and KPC34,

naïve mice were injected with B6-ALL cells and engraftment was confirmed as

51

described previously. For four days, mice were pre-treated with 200 mg/kg

dipyridamole dissolved in 12.5% EtOH in D5 water (5% dextrose) or with vehicle

(12.5% EtOH in D5 water) one hour prior to treatment with either saline, KPC34

orally at 20 mg/kg, or gemcitabine at 8.8 mg/kg i.p.

5. To test the effect of dietary fat on efficacy of KPC34, naïve mice were injected

with B6-ALL cells and engraftment was confirmed as described previously. Upon

initiation of treatment by gemcitabine at 8.8 mg/kg i.p., oral gavage of KPC34 at

20 mg/kg, and i.p. or oral gavage of saline, mice were fed a high-fat diet

(D12492, Research Diets) or a calorically equivalent low-fat diet (D12492 Match

7% Sucrose) for the four days of treatment. After completion of treatment, mice

were returned to their normal diet.

Toxicology Studies:

Normal C57/Bl6 mice were treated with identical dose, schedule, and route of each drug

as in the efficacy studies (days 1-4). Seventy-two hours after the last dose, animals were

sacrificed and organs were fixed in 10% neutral-buffered formalin followed by routine

tissue processing and sectioning for H&E staining. Slides were then reviewed by a

veterinary pathologist with the use of a Nikon Eclipse 50i light microscope. Photographs

of tissues were taken with the NIS Elements D3.10 camera and software system.

Statistical Analysis:

Groups of 3 or more were analyzed using a one way ANOVA. All means were compared

by a student's 2-tailed t test. The in vivo survival graphs were generated with the Kaplan-

Meier method, with p values determined by the log-rank test. Repeated measures were

analyzed using a two way ANOVA All analyses were performed using GraphPad Prism

Version 5.02 (GraphPad Software). A p value ≤ 0.05 was considered to be significant.

52

III.4 RESULTS

KPC34 is active against ALL and AML cells and induces apoptosis in vitro.

We initially tested the effect of exposure to increasing concentrations of KPC34

on a panel of human and murine ALL and AML cell lines in vitro. The IC50 values for all

cell lines treated were all in the nanomolar range (Table I). To determine whether the

observed decrease in cell number was due to cytostatic or cytotoxic effects, the B6-ALL

cell line was incubated with 25, 50 or 100 nM KPC34 for 48 hours and apoptosis was

assessed by annexin V/propidium iodide (AnxV/PI) staining. Staining of annexin V

detects cells expressing the phosphatidylserine on their surface and is a marker of early

cell death as this phospholipid is normally found on the cytosolic surface of the plasma

membrane. PI binds to nucleic acids and positive staining indicates late cell death, as it

is membrane impermeable in normal cells. A dose-dependent increase in dual-stained

populations was seen (Figure 12A-B), demonstrating induction of apoptosis by KPC34.

Induction of apoptosis was further confirmed by immunoblotting for cleaved caspase 3 in

B6-ALL cells treated with KPC34 for 48 hours (Figure 12C). These findings suggest that

KCP34 is a potent anti-leukemic agent that inhibits leukemic cell growth by inducing

apoptosis in a dose-dependent fashion.

KPC34 inhibits PKC signaling in vitro.

Cleavage of the phospholipid group on KPC34 by phospholipase C [148] is

predicted to produce a diacylglycerol mimetic capable of inhibiting signaling by the

conventional PKC isoforms α, βI, βII and γ (Figure 3). Inhibition of this signaling pathway

may reduce leukemic chemoresistance by sensitizing cells to apoptosis. To test

KPC34’s ability to inhibit PKC signaling, we incubated B6-ALL cells with 200 nM KPC34

53

for times ranging from 0.5-4 hours and collected cell lysates for Western blotting. We

observed a decrease in phosphorylated PKC α/βII in cells treated with KPC34 as

assessed by immunoblotting (Figure 13A). A statistically significant difference was seen

between all timepoints (p = 0.0292 by one-way ANOVA), and significant differences in

normalized phosphorylation were seen between the vehicle-treated cells and cells

treated with KPC34 at 2 and 4 hours (Figure 13B). These findings demonstrate

inhibition of the PKC signaling pathway by KPC34. Combining the cytotoxicity of a

nucleoside analog with inhibition of an anti-apoptotic signaling pathway within the same

drug is a novel anti-leukemic therapy that may sensitize resistant cells to chemotherapy.

KPC34 overcomes stroma-mediated chemoresistance in vitro

To determine KPC34’s ability to overcome stroma-mediated chemoresistance,

we developed an in vitro co-culture model using leukemic cells and bone marrow stromal

cells (Figure 14A) by modifying model systems developed by other laboratories [76, 77,

81, 233]. Culture of leukemic cells with bone marrow stroma cells provides a more

accurate model of the bone marrow niche preferred by leukemic cells compared to

leukemic cells grown alone. Murine B6-ALL (Figure 14B) or human SUP-B15 ALL

(Figure 14C) cell lines were plated with or without murine M2 bone marrow stromal cells

for 48 hours, and then treated with equimolar concentrations of Ara-C or KPC34 for an

additional 48 hours. We found a significant protective effect (p < 0.0001) when co-

cultured leukemic cells were treated with Ara-C compared to KPC34 (Figure 14D).

These results demonstrate KPC34’s ability to overcome stroma-mediated

chemoresistance in vitro, warranting in vivo testing. The exact mechanism for the

restoration of chemo-sensitivity by KPC34 is currently unknown.

54

KPC34 is efficacious as a single agent against ALL in vivo

The in vivo efficacy of KPC34 was tested in a syngeneic immunocompetent

orthotopic mouse model of Ph+ pre-B cell ALL [234] (Figure 15A). These studies are the

first to test KPC34 in leukemia, as the only other in vivo testing performed with this drug

was in a Lewis lung cancer model [148]. Mice were injected with 106 cells and leukemic

engraftment was confirmed six days later by bioluminescent imaging. Four days of

treatment with KPC34 by oral gavage resulted in a significant survival advantage (p =

0.0062) compared to saline or equimolar gemcitabine treatment, with a median survival

benefit of 32 days compared to 14.5 and 15.5, respectively (Figure 15C). The increase

in survival appeared to be due to reduced or delayed leukemic infiltration of the CNS, as

little difference in total leukemic burden was seen between gemcitabine and KPC34-

treated mice post-treatment by bioluminescent imaging (Figure 15D). KPC34 was found

to be efficacious as a single agent against Ph+ ALL and could be safely administered

orally.

KPC34 reduces CNS burden in mice bearing ALL

To further confirm that the difference in survival between mice treated with

gemcitabine and KPC34 was due to CNS-involved disease, we repeated the single

agent study in naïve mice as described previously (Figure 16A). However, instead of

imaging mice 24 hours following the last treatment, all mice were sacrificed and imaged

upon the onset of hind limb paralysis in saline treated animals. Mice were injected with

luciferin, sacrificed 5 minutes later and imaged both dorsally and ventrally by IVIS.

Brains were exposed during dorsal imaging. Total luminescence was quantified within

the brain and the whole body ventrally (Figure 16B-D). Significant differences were

found between total luminescence in the brain and whole body ventrally in mice treated

with gemcitabine or KPC34 (Figure 16C-D). Leukemic infiltration was also assessed by

55

H+E staining of the harvested brains (Figure 16E). A blinded veterinary pathologist

observed expansion and infiltration of the meninges by neoplastic monomorphic round

cells in saline treated mice, with neoplastic cells extending into the bone marrow,

calvaria, muscle, and a lymph node. Gemcitabine treated mice displayed infiltration of

monomorphic cells towards the meninges and calvaria, although to a lesser extent than

in control mice. In all three brains of mice treated with KPC34, no leukemic infiltration

was observed. These studies suggest that KPC34 reduces CNS infiltration by leukemic

cells more effectively than treatment with gemcitabine. This may be due to KPC34

crossing the BBB or by reducing the number of leukemic cells able to infiltrate the brain.

Further studies are required to fully understand the mechanism of reduced leukemic

burden in the CNS. Interestingly, mice treated with KPC34 also had a lower overall

leukemic burden than mice treated with gemcitabine, potentially suggesting enhanced

remission following KPC34 treatment.

KPC34 is efficacious in combination with doxorubicin against ALL in vivo

To evaluate the possibility of using KPC34 as part of a combination regimen to

treat naïve leukemia, leukemia-bearing mice were concurrently treated with doxorubicin

for three days and either Ara-C or KPC34 for four days upon confirmation of engraftment

by bioluminescent imaging (Figure 17A). The treatment combination of Ara-C and

doxorubicin more closely resembles the current ALL treatment regimen for patients.

Combination treatment with KPC34 significantly increased median survival (p < 0.0001)

by over 13 days, with one mouse surviving 196 days (Figure 17C). These studies

suggest that KPC34 is efficacious as part of a combination treatment against Ph ALL

and that this treatment regimen is capable of inducing long-term remission in some of

the animals treated.

56

KPC34 overcomes Ara-C resistance in vivo

Given KPC34’s efficacy in overcoming environment-mediated chemoresistance

in vitro, we extended these studies into mice. To test the ability of KPC34 to overcome

chemoresistant disease in vivo, we generated an Ara-C resistant murine ALL mouse

model (Figure 18A). Mice were injected with B6-ALL cells, and upon disease

engraftment were treated with Ara-C until the mice succumbed to disease. Leukemic

cells were harvested from bilateral femurs of sacrificed mice and cultured in vitro prior to

injection into naïve recipient mice. Upon engraftment as confirmed by bioluminescent

imaging, mice were treated with saline, or equimolar doses of Ara-C i.p., gemcitabine

i.p., KPC34 i.p., or KPC34 orally. Oral administration of KPC34 greatly increased

survival compared to all other treatments (p < 0.0001, Figure 18D). This model of ALL

showed KPC34 to still be efficacious in treatment of chemoresistant disease, while Ara-

C and to a lesser extent, gemcitabine, were not. This is an important finding, as

gemcitabine and Ara-C share many common mechanisms of resistance and suggests

that the phospholipid conjugation and/or improved pharmacokinetics due to oral delivery

of KPC34 may be responsible for successfully overcoming resistance to Ara-C in vivo.

Route of administration affects KPC34-induced DNA Damage

Interestingly, significant differences in survival (Figure 18D) and leukemic burden

(Figure 18B-C) were seen between Ara-C resistant mice treated with KPC34 i.p. and

orally, suggesting that route of administration might have a meaningful impact on

therapeutic outcome. We tested the effect of KPC34 route administration on DNA

damage induction in vivo as assessed by γ-H2AX staining. γ-H2AX refers to the histone

H2AX after it undergoes phosphorylation at serine 139 in response to DNA double-

strand breaks, making it a useful marker for DNA damage. Mice were treated once with

saline orally for 8 hours or with KPC34 i.p. or orally for 8 or 12 hours upon leukemic

57

engraftment before being sacrificed (Figure 19A). Bone marrow was harvested from

bilateral femurs and stained with fluorescent antibodies for B220R, γ-H2AX or both.

Cells were then analyzed by FACS and leukemic cells were gated by size and B220R

positivity (Figure 19A). Induction of γ-H2AX was then analyzed in B220R+ cell

populations (Figure 19B). γ-H2AX induction decreased over time in mice dosed with

KPC34 i.p., while oral dosing caused a slight increase in DNA damage over time. A

significant difference in γ-H2AX induction was seen at 12 hours between orally and i.p.

treated mice (Figure 19C). These findings highlight the impact that the route of

administration has on drug activity, as oral administration of KPC34 caused greater

induction of γ-H2AX than i.p. administration of KPC34. Administration of KPC34 i.p. most

likely mimics a bolus dose of drug, while oral administration results in sustained delivery.

KPC34 successfully treats relapsed CNS disease

To assess the ability of KPC34 to treat relapsed ALL in the same animal, we

developed another model of chemoresistance by initially treating leukemia-bearing mice

with Ara-C and doxorubicin and then re-treating mice with KPC34 upon onset of hind

limb paralysis. Re-treatment with KPC34 repeatedly reversed hind limb paralysis in 24-

48 hours and was well-tolerated by mice. Mice were successfully re-treated multiple

times, with one mouse receiving 13 treatments of KCP34. Improvement in hind limb

paralysis was recorded by video (data not shown). This model more closely resembles

the chemotherapeutic regimen received by patients with ALL, and KPC34 was

efficacious in the setting of relapsed leukemia and at treating CNS disease. These

studies also suggest crossing of the BBB by KPC34.

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KPC34 overcomes in vivo inhibition of hENT1

KPC34’s ability to overcome clinically relevant cell-autonomous chemoresistance

mechanisms was assessed by pharmacological inhibition of hENT1 in vivo. Mice bearing

naïve leukemia were pre-treated for four days with the hENT1 inhibitor dipyridamole or

vehicle one hour prior to treatment with either gemcitabine or KPC34 (Figure 20A).

Dipyridamole treatment significantly reduced median survival in gemcitabine-treated

mice by 1 day, while no significant difference in median survival was seen in KPC34-

treated mice (study ongoing, Figure 20C). KPC34 treated mice pre-treated with

dipyridamole actually had an eight day increase in median overall survival compared to

mice pre-treated with vehicle, although this difference was not significant. This benefit in

survival despite inhibition of ENT1 may be due to blockade of the nucleotide salvage

pathway. These data suggest that hENT1 activity is not needed for uptake of KPC34 in

vivo.

KPC34 is well tolerated in vivo

Oral administration of KPC34 was tolerated in various mouse models of ALL. To

further test potential toxicity, healthy C57/Bl6 mice were treated once a day for four days

with saline or equimolar doses of KPC34 orally or gemcitabine i.p. as described in the

efficacy studies. Three days after the last treatment, mice were euthanized and sterna,

spleens, livers, pancreas, intestines, and kidneys were harvested, sectioned and

stained. Mice treated with KPC34 displayed mild inflammation in the intestine with some

neutrophil infiltration, occasional autolysis in the spleen and pancreas, and depletion of

various hematopoietic precursor cells. Gemcitabine-treated mice displayed some

hypercellularity and hyperplasia in the spleen, and both groups of treated mice displayed

autolysis in the intestine (Figure 21A). The increased GI toxicity observed in mice

59

treated with KPC34 may be due to increased drug exposure in vivo from oral

administration compared to i.p. administration of gemcitabine (unpublished data, [148]).

To further study potential GI toxicity, we examined the effect of route

administration on weight in mice. Mice were weighed daily prior to receiving treatment

for four days. A significant difference in relative weight was seen in mice treated with

KPC34 by oral gavage compared to mice treated with saline or gemcitabine i.p. only on

the fourth day of treatment. No significant difference was seen compared to mice treated

with saline by oral gavage, suggesting that oral delivery is at least partially responsible

for the loss in weight observed (Figure 21B). Even though mice treated with KPC34

exhibited mild symptoms of toxicity and weight loss, these effects were transient and did

not meaningfully affect survival.

Dietary fatty acids compete with KPC34 uptake in vivo.

We hypothesized that the 16 carbon chain attached to KPC34 may cause the

drug to be packaged into lipid structures in the GI tract for uptake. To test this idea, we

treated mice with gemcitabine or KPC34 as described previously but placed the mice on

either a high-fat (60% kcal, 18.5% C16 palmitic Acid) or calorically equivalent low-fat

(10% kcal) diet during the four days of treatment (Figure 22A). We found a 4.5 day

decrease in median overall survival in KPC34-treated mice that were placed on the high-

fat diet compared to treated mice placed on a low-fat diet, but this difference is not

significant with the current number of animals tested (Figure 22C). The difference in diet

did not appear to have an effect on mice treated with saline or gemcitabine. Further

studies are ongoing to determine if the differences in survival seen in KPC34-treated

mice are significant.

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These findings suggest that excess lipids from increased dietary fat compete with

KPC34 for uptake by the GI tract. One possible mechanism for KPC34 uptake involves

the formation of chylomicrons, which are lipoprotein particles mainly composed of

triglycerides and to a lesser extent, phospholipids and cholesterol. Chylomicrons are

produced in the intestinal mucosa and transport dietary fat absorbed from the intestine to

adipose and skeletal muscle tissue as well as to the liver [235]. We hypothesize that

KPC34 may be packaged into these lipoproteins upon oral administration, and that these

drug-loaded chylomicrons then compete with drug-free blood borne lipids for cellular

uptake. We are currently testing the effect of these high-fat or low-fat diets on survival in

mice treated with gemcitabine or KPC34 to indirectly demonstrate a role in lipid

packaging of KPC34, and lipid profiling will be further required to definitively determine

whether KPC34 is indeed incorporated into chylomicrons.

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Cell Line Properties nM KPC34 IC50 (95% CI)

B6-ALL Murine pre-B ALL p53+, BCR-Abl+, ARF- 10.36 (8.448-12.7)

SUPB15 Human B cell lymphoblastic ALL, p53+, BCR-Abl+

7.39 (6.25-8.73)

Jurkat Human T cell lymphoblastic ALL 27.94 (22.88-34.12)

MOLT4 Human T cell lymphoblastic ALL, heterozygous p53 (point missense mutation)

42.71 (32.59-55.96)

CCRF-CEM Human T cell lymphoblastic ALL, compound heterozygote with two p53 point mutations

175.6 (146.8-210.0)

HL60 Human promyelocytic leukemia, p53-, myc+ 144.9 (85.0-247.0)

MFL2 Murine MLL-ENL AML, FLT3 ITD+ 54.36 (18.83-157.0)

KG1a Human AML, CD34+, MDR+ 195.8 (163.5-234.4)

OCI-AML3 Human AML, mutated NPM1 and point mutation in DNMT3A

22.44 (17.94-28.07)

Table 1 IC50 values of human and murine cell lines treated with KPC34.

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Figure 12 KPC34 induces apoptosis in a dose-dependent fashion. A) Western blot

analyses of apoptotic induction. B6-ALL cells were treated with drug as indicated and

blotted with the indicated antibody. B) Flow cytometry of annexin V assay. B6-ALL cells

were treated with the indicated drug for 48 hours and then labeled with annexin V and

propidium iodide (PI). C) Quantified viable fractions of treated cells. Studies were

performed in triplicate and errors bars represent the SEM.

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Figure 13 KPC34 inhibits PKC signaling in vitro in a time-dependent fashion. A) Western

blots of p-PKCα and βII. B6-ALL cells were treated with 200 nM KPC34 for various

timepoints and were blotted with the indicated antibody. B) Quantified p-PKC expression

of B6-ALL cells treated with vehicle or KPC34 200 nM. Band intensities from blots were

calculated using ImageJ software and p-PKC values were normalized to actin for each

replicate. Studies were performed in triplicate and errors bars represent the SEM.

64

65

Figure 14 KPC34 overcomes stroma-mediated chemoresistance in vitro. A) Schema of

flow cytometry annexin V assay. Leukemia were cultured with or without M2 stroma cells

for 48 hours and then treated with the indicated drug for an additional 48 hours. B) Flow

cytometry of annexin V assay. B6-ALL (B) or SUP-B15 (C) cells were labeled with

annexin V and PI. D) Quantified viable fractions of treated cells. Errors bars represent

the SE. Tukey’s post t test was performed following one-way ANOVA. For p values, * = ≤

0.05, ** = ≤ 0.01, *** = ≤ 0.001.

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Figure 15 KPC34 is active in vivo against a syngeneic model of murine ALL. A) Schema

of treatment trial. C57/Bl6 mice were injected with B6-ALL syngeneic leukemia. Upon

engraftment as confirmed by bioluminescent imaging, mice were treated with saline by

oral gavage (Co), saline i.p. (Ci), Gemcitabine at 8.8 mg/kg i.p. (G), or KPC34 at 20

mg/kg by oral gavage (K). B) Bioluminescent image of mice on Day 10 after treatment.

C) Kaplan-Meier curves for animals treated with saline, gemcitabine, or KPC34.

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68

69

Figure 16 KPC34 reduces CNS burden in mice bearing ALL. A) Schema of treatment

trial. B) C57/Bl6 mice were injected with B6-ALL syngeneic leukemia. Upon engraftment

as confirmed by bioluminescent imaging, mice were treated with saline by oral gavage

(So), saline i.p. (Si), Gemcitabine at 8.8 mg/kg i.p. (G), or KPC34 at 20 mg/kg by oral

gavage (K). Upon onset of hind limb paralysis in control mice, all mice were imaged and

euthanized. B) Bioluminescent image of mice on Day 14. Red squares were used to

gate leukemic infiltration within the brain. C) Quantified total luminescence in treated

mice on Day 14, dorsal view. Values represent Mean + SEM. D) Quantified total

luminescence in brains of treated mice on Day 14. Values represent Mean + SEM. E)

H+E stained brains of treated mice.

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71

Figure 17 KPC34 is more effective than Ara-C when combined with doxorubicin. A)

Schema of treatment trial. C57/Bl6 mice were injected with B6-ALL syngeneic leukemia.

Upon engraftment as confirmed by bioluminescent imaging, mice were treated with

saline by oral gavage and i.p. (C), Ara-C at 7.1 mg/kg i.p. for four days and Dox at 3

mg/kg i.p. for three days (AD), or KPC34 at 20 mg/kg by oral gavage for four days and

Dox at 3 mg/kg i.p. for three days (KD). B) Bioluminescent image of mice on Day 10

after treatment. C) Kaplan-Meier curves for animals treated with saline, Ara-C + Dox, or

KPC34 + Dox.

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Figure 18 KPC34 is active against an Ara-C resistant model of murine ALL. A) Schema

of treatment trial. C57/Bl6 mice were injected with B6-ALL syngeneic leukemia. Upon

engraftment as confirmed by bioluminescent imaging, mice were treated with 100 mg/kg

Ara-C daily until succumbing to disease. Mice were sacrificed and bilateral femur cells

harvested. Ara-C resistant leukemic cells were cultured in vitro and injected into naïve

C57/Bl6 mice. Upon engraftment, mice were treated with saline by oral gavage or i.p.

(C), Ara-C at 7.1 mg/kg i.p. (A), Gemcitabine at 8.8 mg/kg i.p. (G), KPC34 at 20 mg/kg

by oral gavage (Ko), or KPC34 at 20 mg/kg i.p. (Ki) for four days. B) Bioluminescent

image of mice on Day 10 after treatment. C) Quantified total luminescence in treated

mice on Day 10 after treatment. Values represent Mean + SEM. D) Kaplan-Meier curves

for animals treated with saline, Ara-C, gemcitabine, KPC34 i.p., or KPC34 p.o.

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Figure 19 Oral administration of KPC34 induces more DNA damage than i.p.

administration in vivo. A) Schema of treatment trial. C57/Bl6 mice were injected with B6-

ALL syngeneic leukemia. Upon engraftment as confirmed by bioluminescent imaging,

mice were treated once with saline, 20 mg/kg KPC34 i.p., or 20 mg/kg KPC34 by oral

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gavage. Bilateral femur cells were harvested from saline treated mice at 8 hours and

from KPC34 treated mice at either 8 or 12 hours. Harvested cells were dual stained with

fluorescent antibodies for B220R and γ-H2AX and analyzed by flow cytometry. Cells

were initially gated by size, followed by B220R positivity. Dual-stained populations were

then measured after gating saline treated cells. B) Flow cytometry analyses of harvested

bone marrow cells stained with B220R and γ-H2AX. C) Quantified dual-stained

populations. Values represent Mean + SEM. Tukey’s post t test was performed following

one-way ANOVA.

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Figure 20 KPC34 overcomes in vivo inhibition of hENT1. A) Schema of treatment trial.

C57/Bl6 mice were injected with B6-ALL syngeneic leukemia. Upon engraftment as

confirmed by bioluminescent imaging, mice were divided into two groups. One hour prior

to receiving chemotherapy, one group was pre-treated with 200 mg/kg Dipyridamole by

oral gavage (D) while the other group was pre-treated with vehicle (V) by oral gavage.

Within each group, mice were then treated with saline i.p. (Si), Saline by oral gavage

(So), Gemcitabine at 8.8 mg/kg i.p. (G), or KPC34 at 20 mg/kg by oral gavage (K). B)

Bioluminescent image of mice on Day 10 after treatment. C) Kaplan-Meier curves for

animals treated with saline or gemcitabine and pre-treated with vehicle or Dipyridamole.

D) Kaplan-Meier curves for animals treated with saline or KPC34 and pre-treated with

vehicle or Dipyridamole.

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Figure 21 KPC34 is tolerated by mice in vivo. A) Images of organs from mice treated

with gemcitabine or KCP34. Mice were treated with saline (S) or equimolar doses of

gemcitabine i.p. (G) or KPC34 (K) by oral gavage for 4 days. 72 hours after the last

treatment, mice were sacrificed and the following organs were harvested: sterna, liver,

kidneys, spleen, small and large intestines, and pancreas. Samples were processed and

stained with hematoxylin and eosin. A blinded veterinary pathologist analyzed the

samples. All images are taken at 20X zoom, except for bone marrow taken at 60X. B)

Quantified relative weights of mice treated with saline or gemcitabine i.p. and saline or

KPC34 by oral gavage. Mice were weighed prior to each day of treatment for four days.

Relative weights were calculated from the first day of treatment. Values represent Mean

+ SEM. The Bonferroni method of multiple comparisons was performed following two-

way ANOVA.

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Figure 22 Dietary fat composition influences survival after KPC34 treatment. A) Schema

of treatment trial. C57/Bl6 mice were injected with B6-ALL syngeneic leukemia. Upon

engraftment as confirmed by bioluminescent imaging, mice were divided into two groups

and received either a high-fat (HF) or low-fat (LF) diet during days 1-4 of treatment.

Within each diet group, mice were treated by oral gavage with either saline (S) or KPC34

at 20 mg/kg (K). B) Bioluminescent image of mice on Day 10 after treatment. C) Kaplan-

Meier curves for animals treated with saline or KPC34 and low-fat or high-fat diets.

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III.5 DISCUSSION AND FUTURE DIRECTIONS

We found the phospholipid gemcitabine conjugate KPC34 to be a potent anti-

leukemic agent both in vitro and in vivo. In vitro, this drug was cytotoxic against ALL and

AML cell lines in the nanomolar range and multiple assays demonstrated its ability to

induce apoptosis in a dose-dependent fashion. Importantly, KPC34 was able to inhibit

conventional PKC signaling in ALL cells. PKC signaling is an important therapeutic

target in both ALL and AML as it inhibits drug-induced cytotoxicity by activation of the

NF-κB and Bcl2 signaling axis. Further studies should be performed to test inhibition of

the other conventional family member PKCγ, as well as the novel isoforms PKC δ, ε, η,

and θ which require DAG but not Ca2+ for activation [236].

The combination of PKC inhibition to the existing nucleoside analog-induced

cytotoxicity of gemcitabine is a novel mechanism that may increase the anti-leukemic

efficacy of KPC34. The only other known nucleoside analog with comparable effects is

sangivamycin, a purine analog isolated from Streptomyces rimosus capable of inducing

apoptosis and inhibiting PKC and VEGF activity [237]. This drug was shown to be

tolerated in humans [238] but had no therapeutic response in patients with neoplastic

diseases and dose-escalation resulted in cardiotoxicity [239, 240]. Cardiotoxicity was

further confirmed by pre-clinical testing in animals [237]. As a result, no further follow-up

clinical studies in humans have ever been conducted. However, sangivamycin-like

molecules are currently being evaluated for potential clinical efficacy if safety can be

demonstrated. Cardiotoxicity was not observed as a cause of death in mice treated with

KCP34, but further pre-clinical animal models must be thoroughly tested to ensure that

similar toxic events do not occur if KPC34 is administered to humans.

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In a syngeneic immunocompetent orthotopic Ph+ mouse ALL model, oral

administration of KPC34 demonstrated significantly greater therapeutic efficacy than its

parental drug gemcitabine administered i.p. KPC34 was also more effective when

combined with doxorubicin than Ara-C in a model of naïve ALL. This mouse model

closely mimics human Ph+ ALL both phenotypically and genetically [234], and the

treatment regimens administered mimic current therapeutic strategies used to treat

patients with naïve ALL. KPC34’s efficacy as either a single agent or in combination

supports the potential replacement of Ara-C as the nucleoside analog used in treatment

regimens for ALL.

In the first paper published using this mouse model, continuous daily or bi-daily

treatment of mice with dasatinib initially reduced leukemic burden and improved survival,

but 32/34 mice succumbed to disease while still on treatment and relapse was

characterized by strong CNS involvement. Combination of dasatinib with L-asparaginase

or dexamethasone increased survival compared to any treatment alone, and treatment

with all three agents produced long-term survival in over half the mice treated (over 160

days). It is somewhat difficult to directly compare these studies to ours with KPC34, as

there were significant differences in the types and schedule of drugs administered

(nucleoside analog conjugate alone versus TKI alone or in combination), and the study

by Boulos et al. did not calculate median survival as an endpoint. Further studies of this

mouse model treated with KPC34 and a combination of other clinically used drugs such

as dasatinib may provide for a more rational and useful comparison [234]. Other

experiments using this mouse model have not involved the testing of nucleoside analogs

and have instead focused on improving the efficacy of dasatinib treatment [241], testing

the effect of thiopurine methyltransferase status on 6-mercaptopurine treatment [242],

improving therapeutic response by combination with cyclosporine [243], or inhibition of

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the tyrosine kinase JAK by ruxolitinib combined with dasatinib and dexamethasone

treatment [244].

In addition to efficacy in a model of naïve murine ALL, KPC34 was able to

overcome multiple models of spontaneous and induced leukemic chemoresistance. This

drug was more effective than cytarabine in treating environment-mediated

chemoresistance generated by an in vitro model of leukemic cells co-cultured with

normal bone marrow stromal cells. The ability to overcome this protective effect of

stroma makes KPC34 a promising agent for successful eradication of the disease, as

previous studies have shown stroma to protect leukemia from cytarabine and

gemcitabine in vitro [76, 245] and may be responsible for minimal residue disease, the

major cause of relapse in patients [246].

Early in vitro studies focused on demonstrating stroma-induced acute leukemic

chemoresistance to approved nucleoside analogs [76, 78, 245, 247] or TKIs [32, 248].

More recently, several lab groups (primarily Drs. Michael Andreeff and Marina

Konopleva at MD Anderson) have tested the effect of inhibition of FLT3 [249], NF-κB

[104], VLA-4 [90], Wnt signaling [110], PI3K [250-252], TGF-β [253, 254], PI3K and

FLT3-ITD [255], mTOR [256], HIF-1α [257], FLT3 and HDM2 [258], Axl [259], VEGF

[260] and CXCR4 [95, 96, 261] on overcoming stroma-mediated chemoresistance. Most

of these studies focused on chemoresistance in in vitro models and did not translate

these findings to in vivo studies. Despite some recent advances, there is still substantial

room for progress in the development of novel therapeutics to overcome stroma-

mediated chemoresistance.

KPC34 was also more effective than cytarabine or gemcitabine in treating

multiple mouse models of clinically relevant chemoresistant ALL. Mice treated with

gemcitabine in an in vivo-generated model of cytarabine resistance had only a two day

median survival advantage over mice treated with Ara-C, compared to a 10 day

86

advantage for mice treated with KPC34 orally. The dramatic benefit in survival seen in

mice treated with KPC34 compared to mice treated with Ara-C or gemcitabine suggest

that the novel chemical properties of KPC34 enable it to overcome common

mechanisms of chemoresistance shared by latter two drugs. To further mimic the onset

of relapsed disease, we used a model of spontaneous recurrent disease within the same

animal treated with components of the standard of care for the treatment of ALL. Re-

treatment with KPC34 in mice with hind limb paralysis successfully reversed these

symptoms multiple times. In vivo blockade of nucleoside analog uptake by

pharmacological inhibition of ENT1 significantly reduced survival in mice treated with

gemcitabine but actually increased median survival in mice treated with KPC34

(although not statistically significant). These results further support the potential use of

KPC34 in the clinical setting for treatment of relapsed ALL.

Other in vivo models of chemoresistance in acute leukemia have focused on

retinoic acid-resistant APL [262, 263], glucocorticoid-resistant ALL [264, 265], and

dasatinib-resistant ALL [266]. Of particular note, one group generated in vivo resistant

ALL using patient xenografts after treatment with an induction-type regimen of

vincristine, dexamethasone, L-asparaginase, and daunorubicin. Biological signatures

were identified in resistant cells, and they were treated ex vivo with rationally selected

drugs – a histone deacetylase inhibitor, a membrane glucocorticoid receptor inhibitor, a

fatty acid β-oxidation inhibitor, and a PPAR-γ agonist. Based on observed synergy with

dexamethasone, the membrane glucocorticoid receptor inhibitor was tested using one of

the resistant xenograft samples in vivo. Unfortunately, treatment with the membrane

glucocorticoid receptor inhibitor, the induction-type regimen, or a combination of the two

did not result in significant improvement in event-free survival [267]. This may have been

due to reduced drug activity due to different routes of administration [268]. Despite the

failure of treating resistant cells with the chosen drugs, this study generated a very

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useful and clinically relevant model of relapsed ALL following induction therapy. This

model may be applied to the testing of other drugs to overcome leukemic

chemoresistance. A possible future study using this model could test the efficacy of

KPC34 versus Ara-C as the choice of consolidation therapy following induction therapy.

Interestingly, we noted a significant difference in survival and leukemic burden in

mice treated with KPC34 orally versus i.p. Further examination found a difference in

induction of DNA damage in vivo as measured by γ-H2AX staining in mice receiving one

dose of KPC34 administered orally or i.p. [138]. Lipid conjugation has been shown to

increase in vivo stability of drugs [269-271]. Our findings suggest that oral delivery of

KPC34 enhances its anti-leukemic effect, possibly by increasing exposure time. Initial

pharmacokinetic studies (unpublished data, [148]) found over a 7-fold increase in

plasma half-life of KPC34 administered orally compared to gemcitabine administered i.v.

(11 hours compared to 1.5 hours), supporting this hypothesis.

Preclinical models of pancreatic cancer have shown the effect of metronomic

gemcitabine therapy as a single agent [272, 273] or when combined with sunitinib [274]

or an anti Bv8-antibody [275]. Lowering the concentration of chemotherapy may spare

non-cancerous cells from toxicity [276, 277], while removing rest periods may improve

eradication of resistant cell populations [278]. The efficacy of KPC34 used in ALL mouse

studies may reflect this effect, as previous animal studies in our laboratory have used

Ara-C at 100 [279, 280] or 125 mg/kg [281] for the treatment of AML. For equimolar

comparison, Ara-C concentration was reduced by over 14-fold in our ALL studies

described, but treatment increased median survival only minimally compared to control

treated mice. Pharmacokinetic testing of gemcitabine and KPC34 at the experimental

doses used in these mouse studies is required to further explore this potential

metronomic effect, as well as testing metronomic schedules of continuous KPC34

treatment.

88

Oral delivery of KPC34 was tolerated by mice despite mild toxicity observed in

the intestines, bone marrow, spleen, and pancreas. Similar toxicity events were seen in

mice treated with gemcitabine but to a lesser extent. A possible explanation for the

observed toxicity of KPC34 in vivo is due to altered pharmacokinetics upon oral

administration, as orally administered KPC34 had over a 7 fold increase in half-life

compared to gemcitabine administered i.v. (unpublished data). Increased exposure in

vivo by oral administration may therefore be the cause of toxicity and transient weight

loss observed in mice treated with KPC34.

The oral availability of KPC34 and other lipid-conjugated nucleoside analogs

most likely influences their processing inside the body and may cause incorporation into

chylomicrons in the intestine. Incorporation of drugs into these lipid bodies would alter

their biodistribution and uptake by cells. The relationship between the field of lipid

metabolism and cancer is still emerging and therapeutic agents targeting fatty acid

availability have been undergoing clinical trials [282]. Recent studies have shown obesity

to be clinically associated with increased risk of recurrence in childhood ALL patients

[283]. Studies using in vivo mouse models have shown that obesity accelerates onset

[284] and recurrence [285] of ALL. Co-culture of leukemic cells with adipocytes was also

shown to result in leukemic chemo-protection mediated at least partially by IL-6 [285,

286]. These findings point to a potentially important role for lipids and fatty acids in the

treatment of acute leukemia. The chemo-protective effect of obesity on leukemic cells

should be further examined in relation to lipid-conjugated nucleoside analogs and may

even provide a targetable mechanism for overcoming chemoresistance.

KPC34 was able to delay the onset of CNS disease in multiple models of ALL

and successfully treat CNS-involved relapsed disease in a model mimicking the current

therapy for ALL patients. CNS involvement is a fairly common occurrence in patients

89

with relapsed ALL. The existing therapeutic options for patients carry undesirable side

effects and complications, so an orally available drug that can effectively treat CNS-

involved disease would be a welcome option for patients. Despite similarly low levels of

total leukemic burden as analyzed by bioluminescent imaging post-treatment, mice

treated with KPC34 have significantly less leukemic burden in the CNS, explaining the

dramatic difference in median overall survival seen between mice treated with

gemcitabine and KCP34. There has been relatively little published progress in research

focusing on the treatment of CNS-involved ALL in mouse models, as many studies have

instead focused on recapitulating CNS disease in vivo. Research groups have shown

some success in treating CNS disease using dasatinib but not imatinib [287], liposomal

Ara-C [288], and B43 (anti-CD19)-pokeweed antiviral protein immunotoxin [289, 290].

Further demonstration of KPC34’s efficacy in treating CNS-involved ALL would

represent a novel finding in the field of mouse models of ALL and strengthen the drug’s

translational value.

Ongoing studies in our lab include further elucidating the mechanism of KPC34’s

inhibition of PKC in vitro and confirming inhibition in vivo. We are interested in

determining mechanistically how KPC34 overcomes various forms of chemoresistance.

We plan on using mass spectrometry to compare the amount of KPC34 or gemcitabine

able to cross the BBB by analyzing CSF samples and will also determine leukemic

infiltration in the brains of mice treated with these drugs by histopathological staining.

We are currently expanding animal studies to determine the effect of dietary fat on the

efficacy of gemcitabine and KPC34. In addition to these ongoing studies in ALL, we will

test PKC inhibition in vitro using AML cell lines. We are also testing the efficacy of

KPC34 in treatment of mouse models of AML and in NSG mice bearing xenografts of

human AML patient samples (including naïve and relapsed patients).

90

Overall, our findings point to KPC34 as being useful as a single or combined

therapeutic agent for the treatment of both naïve leukemia and chemoresistant leukemia,

including CNS-involved disease. Orally available lipid conjugates of nucleoside analogs

are a promising emerging class of chemotherapeutics with distinct advantages

compared to their parental drugs, including improved pharmacokinetics, uptake and

activity in vivo. Lipid conjugation appears to impart unique benefits onto KPC34 in

overcoming multiple forms of chemoresistance compared to existing nucleoside analogs.

Oral availability of this drug eases the burden of receiving treatment, while also

improving pharmacokinetics and BBB penetration, and the attached phospholipid group

is believed to aid in hENT1-free cellular uptake. Cleavage of the phospholipid moiety is

believed to be responsible for the observed inhibition of PKC in vitro. The rationally

designed properties of KPC34 seem to aid in overcoming leukemic chemoresistance.

The strategy of rationally incorporating multiple chemical modifications to

overcome facets of clinically relevant mechanisms of resistance is potentially a

promising one for improving outcome in patients with recurrent disease in other types of

cancer. Careful thought must be put into deciding how to overcome resistance

mechanisms in order to reduce the possibility of minimal residual disease as much as

possible. Based on our studies, KPC34 appears to be an ideal drug candidate for the

treatment of patients with CNS-involved ALL, particularly given the dearth of effective

options in the relapsed setting. However, the true potential for patients can only be

realized once KPC34 undergoes clinical trials.

91

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Curriculum Vitae PETER ALEXANDER

Ph.D Candidate Department of Cancer Biology

Wake Forest School of Medicine 215-292-6340

palexand@wakehealth.edu

Campus Address: Department of Cancer Biology Hanes 3019 Wake Forest University School of Medicine Medical Center Blvd.

Winston-Salem, NC 27157 Home Address: 217 N. Spring St. Apt. B Winston-Salem, NC 27101 EDUCATION: 2008-Present Ph.D., Cancer Biology Wake Forest University School of Medicine Winston-Salem, North Carolina 27157

Thesis: The Novel Phospholipid Gemcitabine Conjugate KPC34 has Activity in Acute Leukemia

Mentor: Dr. Timothy S. Pardee Funding: NIH/NCI T32CA079448 Training Grant Wake Forest Innovations Spark Award 2004-2008 B.A., Biology University of Pennsylvania Philadelphia, Pennsylvania. 19104 RESEARCH EXPERIENCE: 2009-2012 Graduate School Wake Forest University School of Medicine Winston-Salem, North Carolina 27157

Project: Conjugation of an anti-angiogenic peptide fragment of HKa to carbon nanotubes.

Mentors: Drs. Frank and Suzy Torti Funding: NIH/NCI T32CA079448 Training Grant

2006-2008 Undergraduate Research The Wistar Institute Philadelphia, Pennsylvania 19104

Project: The role of the CD7 protein in relation to the immune system and cancer development.

Mentor: Dr. Tao Wang 2002-2004 High School Research

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Fox Chase Cancer Center Philadelphia, PA 19111

Project: The correlation between expression of the pro-protein convertase furin with malignancy in human ovarian tumors.

Mentor: Dr. Andres Klein Szanto 2003 Research Apprenticeship in the Biological Sciences Cornell University Ithaca, NY 14850

Project: Examining genetic changes in ovarian tumors of conditionally inactivated p53 and Rb mice.

Mentor: Dr. Alexander Nikitin 2001-2002 High School Research Fox Chase Cancer Center Philadelphia, PA 19111

Project: Examining differential growth between normal and cancerous human breast epithelial cells.

Mentors: Drs. Jose and Irma Russo 2000-2002 High School Research Fox Chase Cancer Center Philadelphia, PA 19111

Project: Microarray analysis of benign and malignant human ovarian tumors.

Mentor: Dr. Xiangxi Xu PROFESSIONAL MEMBERSHIP: 2013-Present American Society of Hematology 2010-2013 American Association for Cancer Research HONORS AND AWARDS: 2012-Present, 2009-2010 NIH/NCI T32CA079448 Training Grant 2003 Research Apprenticeship in the Biological Sciences Program.

Cornell University 2001-2002 Howard Hughes Medical Institute Student Scientist (High School).

Fox Chase Cancer Center BIBLIOGRAPHY: Book Chapters

1. Singh RN, Alexander P, Burke AR, Torti FM, Torti SV. Carbon Nanotubes for Therapy. In SH Cho and S Krishnan, Cancer Nanotechnology: Principles and Applications in Radiation Oncology. Boca Raton (FL): CRC Press/Taylor & Francis;2013. 205-228

Research Articles

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1. Wang T, Ge Y, Xiao M, Lopez-Coral A, Li L, Roesch A, Huang C, Alexander P, Vogt T, Xu X, Hwang WT, Lieu M, Belser E, Liu R, Somasundaram R, Herlyn M, Kaufman RE. SECTM1 produced by tumor cells chemoattracts human monocytes via CD7-mediated activation of the PI3K pathway. Journal of Investigative Dermatology.

2. Page RE, Klein-Szanto AJ, Litwin S, Nicolas E, Al-Jumaily R, Alexander P, Godwin AK, Ross EA, Schilder RJ, Bassi DE. Increased expression of the pro-protein convertase furin predicts decreased survival in ovarian cancer. Cell Oncol. 2007;29(4):289-99.

CONFERENCE PROCEEDINGS:

1. Pardee T, Jennings-Gee J, Alexander P, Gmeiner W. Thymidylate Synthase Inhibition with the Novel Fluoropyrimidine FdUMP[10] Is Highly Effective Against Acute Lymphoblastic Leukemia. ASH Annual Meeting Abstracts 2012 120: 1505.

INVITED LECTURES AND PRESENTATIONS: 2015: Poster Presentation: The Novel Phospholipid Gemcitabine Conjugate

KPC34 has Activity in Acute Leukemia. Molecular and Cellular Biosciences Recruitment. Wake Forest University.

2013: Poster Presentation: The Novel Phospholipid Gemcitabine Conjugate KPC34 has Activity in Acute Leukemia. ASH 2013 Annual Meeting. New Orleans, LA.

2013: Oral Presentation: The Novel Phospholipid Gemcitabine Conjugate KPC34 has Activity in Acute Leukemia. 3 Minute Thesis Competition. Wake Forest University.

2012: Poster Presentation: The Novel Phospholipid Gemcitabine Conjugate KPC34 has Activity in Acute Leukemia. Graduate School Research Day. Wake Forest University.

2012: Poster Presentation: The Novel Phospholipid Gemcitabine Conjugate KPC34 has Activity in Acute Leukemia. Molecular and Cellular Biosciences Recruitment. Wake Forest University.

2009-2012: Cancer Biology Department Retreat (annual poster presentation) 2008-2014: Cancer Biology Department Seminar (annual seminar) OTHER PROFESSIONAL APPOINTMENTS AND INSTITUTIONAL SERVICE: 2010-2012 Executive Board Member of Graduate Student Association. Wake Forest

University School of Medicine. 2010-2012 Yearbook Representative for Graduate School. Wake Forest University

School of Medicine. 2009-2014 Matching Matriculants and Return Students (MMARS). Wake Forest

University. Assisted incoming students with logistics of moving to Wake Forest.

PUBLIC OUTREACH: 2015 Volunteered at 2015 Healthcare Strategy Conference & Case

Competition. Biotech Place, Winston-Salem NC. 2008-2009 Participated in school visits with Wake Forest University Brain Awareness

Council

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CONFERENCES ATTENDED: 2013: ASH Annual Meeting, New Orleans, LA 2012: AACR Annual Meeting. Chicago, IL 2011: NCL Lessons Learned Workshop. Rockville, MD 2011: AACR Nano in Cancer Conference. Miami, FL UNDERGRADUATE STUDENTS ADVISED: 2013: Kristen Plantz. Wake Forest University 2007: Thomas Biel. Wistar Institute