HIF Activation by Hypoxia

histone acetyltransferase

activity

VEGF-A

Epo

Von-Hippel-Lindau tumor suppressor

• tumors require an oxygen supply – biallelic inactivation of VHL leads to a variety of tumors

• congenital mutations in VHL may also lead to polythycemia

cis-acting sequence – hypoxia response element

prolyl hydroxylaseE3 ubiquitin

ligase

erythropoietin a cytokine that promotes red cell production

under hypoxic conditions PHD has reduced activity

Vascular Endothelial Growth Factor (VEGF) Family

• 7 separate genes encoding VEGF family members – VEGF (A-F) and placental growth factor

• splice variants produced from individual genes further increasing diversity

• VEGF-A a prototype

• 165 amino acids• multiple isoforms expressed in different tissues

6 amino acids found at the C-terminus coming

from exon 8a or 8b determines whether factor is pro- or anti-

angiogenic

choice between exons

Differential Splicing Gives Rise to Pro- and Anti-Angiogenic Forms of VEGF-A

favored downstream of HIF activation

3’ proximal splice site

3’ distal splice site

basal expression or promoted by a signaling pathway opposing angiogenesis

exon 7

VEGF-A Structure

ER secretory pathway

VEGF functions as a dimer VEGF-A can bind to one of two receptors

The GAG heparin can influence

VEGF-A/receptor interactions

6 amino acids at the C-terminus determines whether NP-1 is engaged

and whether VEGF-A isoform is pro- or anti-angiogenic

Anti - SLTRKDPro - CDKPRR

major form expressed at basal levels

in many adult tissues

VEGF in purple and yellow

VEGF Receptor Signaling

VEGF receptor is a member of the receptor tyrosine kinase family

Ligand binding to extracellular domain induces intracellular kinase activity and subsequent downstream signaling pathways

neuropilin 1

C-terminus

C-terminus

not all of these signaling pathways occur in the same cell at the same time - context

VEGF Signaling to Endothelial Cells Promoting Angiogenesis: Migration of Tip Cells

before initiating any substantive movement toward VEGF gradient endothelial cells have to deal with

the basal laminaphenotypic change in endothelial

cells, enhanced expression of matrix degrading proteases and

enhanced migratory behavior

epithelial mesenchymal

tip cells undergo a

epithelial to mesenchymal transition to

initiate angiogenesis

Tip cells are highly motile, but non-proliferative; there is roughly one tip cell per spout

matrix-metalloproteases

Some of the Phenotypic Characteristics of Cells Involved in Angiogenesis are Shared by Metastatic

Tumor Cells

blood vessel lining

1 2

3

cells undergo a epithelial to

mesenchymal transition to

promote metastasis

broad front of migration

slender projections extending from leading edge of lamellipodia, driven

by actin polymerization

cytoskeletal rearrangements needed for directional migration regulated by small

GTPase Rho/Rac

integrins found at tips of filopodia help extensions

adhere and pull cells forward

matrix metalloproteases help tips move through barriers

integrins at trailing edge need to dissociate

Migratory Behavior of Tip Cells

gene expression needed for motility driven by

transcription factors like SRF

anchoring points to substratum, focal adhesions

Endothelial Cells of the Stalk have Phenotypes Distinct from Endothelial Tip Cells and Quiescent

Endothelial Cells

stalk cells are proliferative, but

lack sprouts

Interaction of Dll4 with notch represses VEGFR2 expression in stalk cells

suppressing the formation of new

spouts

tip cells have sprouts but much

reduced proliferation

Dll = delta-like ligand

Notch can also repress expression

Steps in Cancer Metastasis

blood vessel lining1 2

3

colon adenocarcinoma metastasized to the lung

central areas of necrosis caused by anoxia as tumor

outgrows blood supply

Metastases may Retain Characteristics of Original Tumor and also Require a Blood Supply as Tumors Grow

Normal Angiogenesis is a Careful Balance of Pro- and Anti-Angiogenic Stimuli

This is not the case for tumor vasculature where pro-angiogenic signals predominate

creating vasculature that is anything but normal

angiostatin – inhibitor of

angiogenesis

Angiogenesis as a Target of Cancer Chemotherapy

M. Judah Folkman major proponent of using this approach to treat cancer

bevacizumab – antibody that recognizes and binds VEGF

• used alone to treat glioblastoma refractory to other treatments

• used in combination with other drugs to metastatic colorectal cancer, some non-small cell lung cancers, and metastatic renal cell cancer

Strategies for Anti-Angiogenesis Therapy

sorafenib – small molecule inhibitor of VEGFR-2 – tyrosine kinase inhibitor

Suffixes in drug development

mab – antibody-based therapy

nib – small molecule kinase inhibitors

(u)

humanized antibodies

A Notorious Angiogenesis Inhibitor

thalidomide lenalidomide

currently approved for use in multiple myeloma and myelodysplastic

syndromes

tumor vessels lack normal arterial venous hierarchy, and are leaky and often

insufficient

this can impede drug delivery

anti-angiogenic therapy can

normalize tumor vasculature and improve delivery of other drugs

Effects of Normalizing Tumor Vasculature in Anti-Angiogenesis Therapy

normalized vasculature may reduce metastasis

A Bevacizumab “Success” Story

J. Clin. Onc. (2007) 25, 1539-1544

829 patients, metastatic colon cancer, treated with standard therapy or standard therapy + bevacizumab

Standard therapy is FOLFOX

• Fluorouracil• Leucovorin• Oxaliplatin

OH H

OCH2

N

N

O

O

+

OH H

OCH2

N

N

O

O

H3C

+

O – P - O

O

O-

O – P - O

O

O-

N

N

H2NN

N5

O H2C

CH2

H

N10

R

N5,N10-methylene tetrahydrofolate

N

N

H2NN

N5

OCH2

N10

R

dihydrofolate

dUMP

dTMP

thymidylate synthase

Thymidylate Synthesis

• C6 is in the β position of an αβ unsaturated ketone and so is susceptible to a nucleophilic attack

6

H

• H5 of uracil exchanges with solvent• H6 of N5N10 –THF transfers to the C5 methyl group

Thymidylate Synthase: Reaction Mechanism

N

CN

C

C

C

12

34

5

6

O

O

H

U

dRib

F

H+

Fluorine is the most electronegative atom and will not be released as F+ consequently the enzyme is trapped in a covalent ternary complex with substrate and cofactor

Fluorodeoxyuracil is used clinically as a anticancer drug. It selectively kills rapidly dividing cells making thymidine.

suicide inhibitor

Side effects include anemia, infections, nausea, and hair loss

N

CN

C

C

C

O

O

HN

CN

C

C

C

O

O

H F

HH

H

F

Hdihydropyrimidine dehydrogenase

or uracil /thymidine

Defects in Pyrimidine Catabolism can Cause Life-Threatening Toxicities when Using 5-Fluoruracil

pharmacogenomics

Platinum Drugs

The addition of bevacizumab to oxaliplatin, fluorouracil, and leucovorin improves survival duration for patients with previously treated metastatic colorectal cancer.

A Bevacizumab “Success” Story

J. Clin. Onc. (2007) 25, 1539-1544

829 patients, metastatic colon cancer, treated with standard therapy or standard therapy + bevacizumab

The median duration of survival for the group treated with FOLFOX4 and bevacizumab was 12.9 months compared with 10.8 months for the group treated with FOLFOX4 alone (corresponding hazard ratio for death 0.75; P .0011), and 10.2 months for those treated with bevacizumab alone.

1 year survival SC+B, 56%; SC, 43%

Limitations of Anti-Angiogenic Therapy for Cancer

• Many tumors overcome effects of targeted anti-angiogenic therapies by up-regulating alternative pathways

other growth factors unrelated to VEGF can also promote angiogenesis, eg fibroblast derived growth factor and platelet derived growth factor

• Hypoxia may promote more invasive phenotypes in tumor cells

Biological Agents Targeting the Same Molecule

Chimeric Antibody Fully Human Antibody

Soluble TNF receptor, normally involved in

down regulating inflammatory responses

Fc region only present for stability in serum

Intercept: obstruct (someone or something) so as to prevent them from continuing to a

destination

So what is Etanercept Intercepting?

TNF, tumor necrosis factor

• Name derived from an experiment performed in 1896 demonstrating that bacterial extracts could shrink tumors

• Extracts contained bacterial lipopolysaccharide which stimulated inflammation and induced TNF

Fully Human Monoclonal Antibodies (The Adalimumab Story)

Goal: develop an antibody to treat inflammatory diseases

• ~ 1% of population

• average age of onset 40 years

• female/male 3:1

• joint erosion within 2 years of diagnosis, disabilities after 5 years

• average medical costs $23,00/year

• chronic pain, stiffness of joints; tiredness and lethargy

Tumor Necrosis Factor (TNF): A Cytokine Target for Treating Rheumatoid Arthritis

Anti-TNF

Early studies suggested TNF might be a master regulator of the inflammatory process

Encouraging Data from First Phase I Trail for Infliximab

CRP: C reactive protein, a marker for inflammation

Phase II Trial Indicates Infliximab Synergizes with Methotrexate

Loss of response correlates with patient antibodies to Infliximab

Immunosuppressive effects of methotrexate likely reduce patients ability to raise antibodies to Infleximab

HUMA: human anti-mouse antibodies

In the setting of Crohn’s disease ~ 60% of patients raise antibodies to Infliximab by the 5th injection

HUMA Responses to Different Anti-TNF Drugs

Infliximab – 51, 21, and 7% of patients raised antibodies in response to 1, 3, and 10 mg/kg dosing

Fewer antibodies and higher doses of drug suggest tolerance mechanisms may come into play

Adalimumab – less immunogenic 6% of patients raised antibodies at the lowest dose

Etanercept – less immunogenic 2% of patients raised antibodies at the standard dose

Antibodies will always have novel sequences in their antigen binding regions – antibodies against these regions are called anti-idiotype antibodies

Approaches to Isolation of Fully Human Antibodies: Phage Display

Each individual phage particle embodies both phenotype (expressed antibody) and genotype (the gene encoding the antibody)

Select for a phage particle that binds to a particular antigen and you immediately have the gene encoding the antibody responsible for binding