first-in-human rna polymerase i transcription inhibitor cx ... · transcription inhibitor cx-5461...

1036 | CANCER DISCOVERY AUGUST 2019 www.aacrjournals.org

First-in-Human RNA Polymerase I Transcription Inhibitor CX-5461 in Patients with Advanced Hematologic Cancers: Results of a Phase I Dose-Escalation Study Amit Khot 1 , Natalie Brajanovski 2 , Donald P. Cameron 3 , 4 , Nadine Hein 3 , Kylee H. Maclachlan 1 , 2 , 4 , Elaine Sanij 2 , 4 , 5 , John Lim 6 , John Soong 6 , Emma Link 4 , 7 , Piers Blombery 1 , 4 , 8 , Ella R. Thompson 4 , 8 , Andrew Fellowes 2 , 8 , Karen E. Sheppard 2 , 4 , 9 , Grant A. McArthur 2 , 4 , 10 , Richard B. Pearson 2 , 4 , 9 , 11 , Ross D. Hannan 2 , 3 , 4 , 9 , 11 , 12 , Gretchen Poortinga 2 , 4 , 10 , and Simon J. Harrison 1 , 4

RESEARCH ARTICLE

ABSTRACT RNA polymerase I (Pol I) transcription of ribosomal RNA genes (rDNA) is tightly regulated downstream of oncogenic pathways, and its dysregulation is a common

feature in cancer. We evaluated CX-5461, the fi rst-in-class selective rDNA transcription inhibitor, in a fi rst-in-human, phase I dose-escalation study in advanced hematologic cancers. Administration of CX-5461 intravenously once every 3 weeks to 5 cohorts determined an MTD of 170 mg/m 2 , with a pre-dictable pharmacokinetic profi le. The dose-limiting toxicity was palmar–plantar erythrodysesthesia; photosensitivity was a dose-independent adverse event (AE), manageable by preventive measures. CX-5461 induced rapid on-target inhibition of rDNA transcription, with p53 activation detected in tumor cells from one patient achieving a clinical response. One patient with anaplastic large cell lym-phoma attained a prolonged partial response and 5 patients with myeloma and diffuse large B-cell lymphoma achieved stable disease as best response. CX-5461 is safe at doses associated with clinical benefi t and dermatologic AEs are manageable.

SIGNIFICANCE: CX-5461 is a fi rst-in-class selective inhibitor of rDNA transcription. This fi rst-in-human study establishes the feasibility of targeting this process, demonstrating single-agent antitu-mor activity against advanced hematologic cancers with predictable pharmacokinetics and a safety profi le allowing prolonged dosing. Consistent with preclinical data, antitumor activity was observed in TP53 wild-type and mutant malignancies.

1 Clinical Haematology, Peter MacCallum Cancer Centre and Royal Mel-bourne Hospital, Melbourne, Victoria, Australia. 2 Cancer Research Divi-sion, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. 3 The ACRF Department of Cancer Biology and Therapeutics, The John Curtin School of Medical Research, Australian National University, Australian Capital Territory, Australia. 4 Sir Peter MacCallum Department of Oncol-ogy, University of Melbourne, Parkville, Victoria, Australia. 5 Department of Pathology, University of Melbourne, Parkville, Victoria, Australia. 6 Senhwa Biosciences, Inc., New Taipei City, Taiwan, Republic of China. 7 Centre for Biostatistics and Clinical Trials, Peter MacCallum Cancer Centre, Mel-bourne, Victoria, Australia. 8 Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. 9 Department of Biochem-istry and Molecular Biology, University of Melbourne, Parkville, Victoria, Australia. 10 Department of Medicine, St. Vincent’s Hospital, University of Melbourne, Parkville, Victoria, Australia. 11 Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia. 12 School of Biomedical Sciences, University of Queensland, Brisbane, Queensland, Australia.

Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/).

A. Khot and N. Brajanovski contributed equally to this research.

R.B. Pearson, R.D. Hannan, G. Poortinga, and S.J. Harrison are the co–senior authors of this article.

Current address for D.P. Cameron: Department of Cell and Molecular Biol-ogy, Karolinska Institutet, Stockholm, Sweden; current address for J. Lim: TP Therapeutics, Inc., San Diego, California.

Corresponding Authors: Gretchen Poortinga, Cancer Research Division, Peter MacCallum Cancer Centre, 305 Grattan Street, Melbourne, Victoria 3000, Australia. Phone: 385595290; E-mail: [email protected] ; and Simon Harrison, [email protected]

Cancer Discov 2019;9:1036–49

doi: 10.1158/2159-8290.CD-18-1455

©2019 American Association for Cancer Research.

Research. on September 7, 2020. © 2019 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from

Published OnlineFirst May 15, 2019; DOI: 10.1158/2159-8290.CD-18-1455

http://cancerdiscovery.aacrjournals.org/

First-in-Human Study of CX-5461 in Hematologic Cancers RESEARCH ARTICLE

AUGUST 2019 CANCER DISCOVERY | 1037

INTRODUCTIONDespite significant progress in the treatment of hemato-

logic malignancies with chemotherapy, mAbs, and cellular therapies over the last 40 years, with corresponding improve-ments in survival outcomes, there remain many patients who are not cured with existing therapies, necessitating the inves-tigation of agents with novel modes of action (1–4).

The availability of functional ribosomes is a fundamen-tal requirement for growth and proliferation in mamma-lian cells. The uncontrolled growth of cancer cells correlates with elevated ribosome biogenesis and also morphologically abnormal nucleoli, the sites of ribosome biogenesis; in fact, increased nucleolar size and number has been used as a marker of aggressive malignancies for more than 100 years (5, 6). RNA polymerase I (Pol I) transcription of the riboso-mal RNA (rRNA) genes (rDNA) is rate-limiting for ribosome biogenesis, a high-energy consumption process that is signifi-cantly elevated in rapidly dividing tumor cells (7, 8). As rDNA transcription underpins this process, it requires precise regu-lation; Pol I–mediated transcription is tightly controlled by oncogenes such as MYC, RAS, and PI3K, which when activated

in cancer cells contribute to hyperactivation of rDNA tran-scription (9–13). Furthermore, perturbation of rDNA tran-scription is known to elicit a nucleolar stress response (NSR), which is heightened in cancer cells, and leads to activation of both p53-dependent and p53-independent stress-response pathways (10, 14–17). Thus, rDNA transcription represents a key hub of coordinated regulation by oncogenic and tumor suppressor signaling pathways and offers novel opportunities for therapeutic targeting to treat the broad range and large numbers of human malignancies, including those driven by these oncogenes. Furthermore, a ribosome biogenesis stress response elicited by the indirect and/or nonspecific targeting of rDNA transcription is associated with the efficacy of many standard chemotherapeutics, including actinomycin D and some platinum-based agents, thus supporting a rationale for advancing this clinically effective concept (18, 19).

CX-5461 is the first-in-class selective small-molecule inhib-itor of Pol I–mediated transcription, which inhibits rDNA transcription in the low nanomolar range by preventing the association of the Pol I–specific transcription initiation selectivity factor SL1 with the rDNA promoter, exhibiting greater than 200-fold selectivity relative to the inhibition




Khot et al.RESEARCH ARTICLE


of Pol II–driven transcription ( 20, 21 ). Preclinical studies show that inhibition of Pol I transcription by CX-5461 leads to cell-cycle arrest and cell death by both a canonical p53-dependent NSR and a noncanonical p53-independ-ent nucleolar-localized DNA damage response (DDR) that requires activation of the ATM and ATR kinase signal-ing pathways ( 10, 14, 15 ). Importantly, as a single agent, CX-5461 shows a robust survival benefi t in murine models of a range of hematologic cancers including MYC-driven B-cell lymphoma ( 10, 22 ), acute myeloid leukemia (AML; ref. 16 ), and multiple myeloma ( 17, 23 ), leading to rapid tumor cell clearance and/or disease reduction with minimal toxicity.

On the basis of this encouraging preclinical data, we initi-ated a fi rst-in-human dose-escalation study of CX-5461 in patients with relapsed and refractory hematologic malig-nancies (Australia and New Zealand Clinical Trials Registry, #12613001061729). The primary objective was to determine the safety and tolerability of CX-5461 when administered by intravenous infusion once every 3 weeks. The secondary objectives were to assess the pharmacokinetic and pharmaco-dynamic profi le of CX-5461 and the preliminary antitumor activity, and to investigate the impact of TP53 mutational sta-tus as well as mutations in other potential CX-5461 response factors including ATM/ATR pathway members, as predictive biomarkers of effi cacy. Dose escalations were planned in 7 cohorts (from 25 to 450 mg/m 2 ), in an accelerated design, with change to a 3+3 design based on predefi ned toxicity cri-teria. We report here on the fi ndings of this fi rst-in-human, fi rst-in-class study.

RESULTS Patient Demographics and Disease Characteristics

Between July 27, 2013, and May 4, 2016, 17 patients with advanced hematologic malignancies were recruited, of whom 16 received CX-5461 at the Peter MacCallum Cancer Cen-tre (Melbourne, Australia). The demographic features and baseline characteristics of treated patients are summarized in Table 1 . All patients had measurable progressive disease at the time of enrollment and were representative of a heav-ily pretreated population with a median number of 7 prior therapies (range, 1–14 therapies). The median patient age was 60 years (range, 21–79 years) with 50% (8/16 patients) being female. The majority of patients (15/16 patients, 94%) had an Eastern Cooperative Oncology Group (ECOG) performance status of 0–1 and the predominant tumor types were mye-loma (6/16 patients, 38%) and diffuse large B-cell lymphoma (DLBCL; 4/16 patients, 25%). All subjects completed at least 1 cycle of therapy; one patient discontinued treatment due to dose-limiting toxicity (DLT) and one due to patient decision, whereas all others were withdrawn from the study following disease progression.

Dose Escalation Sixteen patients were treated in sequential cohorts at dose

levels of 25 mg/m 2 ( n = 3), 50 mg/m 2 ( n = 4), 100 mg/m 2 ( n = 4), 170 mg/m 2 ( n = 3), and 250 mg/m 2 ( n = 2; Table 1 ; the duration on study for each patient is presented in Fig. 1 ). The initial protocol for accelerated dose escalation was

Table 1. Patient demographics and baseline characteristics

Demographic parametersStudy population ( n = 16)

Age Median (range), years 60 (21–79) ≥65 year, n (%) 8 (50)

Sex, n (%) Male 8 (50) Female 8 (50)

ECOG Performance Status, n (%) 0 3 (19) 1 12 (75) 2 1 (6)

Disease status, n (%) Refractory 6 (38) Relapsed 10 (62)

TP53 mutational status, n (%) Wild-type 9 (56) Mutant 4 (25) Unknown 3 (19)

Tumor type, n (%) DLBCL 4 (25) Hodgkin lymphoma 2 (12) CLL – Richter transformation 1 (6) Multiple myeloma 6 (38) T-cell LPD 3 (19)

Median prior lines of therapy, n (range) 7 (1–14)

Dose level (mg/m 2 ), n (%) 25 3 (19) 50 4 (25) 100 4 (25) 170 3 (19) 250 2 (13)

NOTE: The clinical and pathologic features of 16 patients with ad-vanced hematologic malignancies who received CX-5461 therapy. Abbreviation: LPD, lymphoproliferative disorder.

changed to a standard 3+3 design during cohort 1, due to the observation of cutaneous adverse events in the fi rst patient treated at 25 mg/m 2 . Of the 16 subjects enrolled, the median treatment duration was 2 cycles, i.e., 6 weeks (range, 1–18 cycles). Early disease progression during cycle 1 resulted in the withdrawal of 2 patients from the study, both of whom were deemed ineligible for DLT assessment but were included in the pharmacokinetic and pharmacodynamic analysis. Fourteen patients were included in the main safety analysis for determination of the MTD. A DLT of palmar–plantar erythrodysesthesia (PPE) was observed in the fi rst patient treated at a dose level of 250 mg/m 2 , with a similar grade 2 adverse event noted in the second patient enrolled at this dose, although not fulfi lling DLT criteria. The MTD was determined by the safety committee as 170 mg/m 2 .

Safety Profi le All treatment-emergent adverse events (AE) occurring as

grade 3 are summarized in Table 2 , with investigator-assessed treatment-related AEs, reported as either possibly, probably, or






definitely related to CX-5461 treatment, indicated (all CX-5461 treatment-related AEs are shown in Supplementary Table S1; all additional treatment-emergent AEs are shown in Supple-mentary Table S2). A DLT of grade 3 PPE was observed in the first patient (PMC-16) enrolled into cohort 5 (250 mg/m2), with the same AE occurring as a grade 2 event in the second patient of this cohort (PMC-17). This toxicity was character-ized by pain, swelling, paresthesia, and/or erythema in the palms and/or soles of the feet (Supplementary Fig. S1A). Patient PMC-17 recommenced at a reduced dose of 170 mg/m2 and successfully received 17 more cycles at this level without any recurrence of symptoms.

Another significant but dose-independent toxicity noted was the development of grade 1–3 photosensitivity in 50% (8/16) of patients treated. This occurred within 48 hours of treatment and presented as a sunburn-like rash in sun-exposed areas (Supplementary Fig. S1B). After the observation of this AE in 2 patients in cohort 1 (25 mg/m2), subjects were subsequently asked at enrollment to adhere to strict photo-sensitivity precautions and were able to continue on therapy if sun protection was used for 72 hours following drug infu-sions. One patient (PMC-14) had a protocol-mandated dose reduction due to a grade 3 photosensitivity event and contin-ued on treatment for 3 more cycles without recurrence.

All cutaneous toxicities resolved without any sequelae. The grade 3 PPE was treated with corticosteroids and resolved in 3 weeks. No other significant drug-related toxicity was seen. Overall, CX-5461 was well tolerated, with the longest treat-ment durations extending up to 48 and 54 weeks in patients PMC-08 and PMC-17, respectively, and no significant hema-tologic toxicity was observed (Supplementary Table S1).

Pharmacokinetic AnalysisMean plasma concentration–time profiles following the

first cycle of CX-5461 treatment and the resulting pharma-cokinetic parameters from these analyses are displayed in Supplementary Fig. S2 and Table 3, respectively. In sum-mary, following intravenous infusion, CX-5461 reached a maximum plasma concentration (Cmax) within 60 minutes of drug administration in all dose cohorts (Supplementary Fig. S2A). The terminal half-life (t1/2) ranged from 19.2 to 92.4 hours and showed a trend to increase with dose escala-tion, reaching the highest average of 83.3 hours in cohort 5 (250 mg/m2) with residual drug being detectable in patients in cohort 5 at day 15 postdose (Table 3). However, no residual drug was detectable at predose, cycle 2, day 1 (C2D1) in the 2 patients tested (PMC-15, PMC-17; data not shown), one each in cohorts 4 and 5, respectively, which is consistent with

Figure 1. Individual patient duration on study, graphed as a function of treatment duration (cycles completed). The plot legend depicts the colors used to denote best confirmed patient response: partial response (PR; green), stable disease (SD; blue), or progressive disease (PD; gray). One patient who achieved a radiologic and clinical response in an area of high-grade transformation of lymphoma following CX-5461 dosing is indicated (RR; purple). Red denotes the patient who had a DLT, and pink crosses signify patients who needed dose reductions (DR) due to the development of toxicities following the first cycle. Individual patient disease is notated with disease abbreviations as follows: ALCL, anaplastic large cell lymphoma; DLBCL, diffuse large B-cell lymphoma; MM, multiple myeloma; HL, Hodgkin lymphoma; CTCL, cutaneous T-cell lymphoma; T-PLL, T-cell prolymphocytic leukemia; CLL, chronic lym-phocytic leukemia. Presence of tumor TP53 or ATM gene mutation is indicated, as well as patients for whom no DNA sample was available for sequencing (N/A). Total patient number, n = 16; all patients are identified by patient ID number and have ceased treatment.

0 5 10 15 20

Treatment duration (cycles completed)

Pat

ient

ID

ATM

ATM

ALCL

DLBCL

DLBCL

MM

MM

MM

DLBCL

HL

T-PLL

MM

DLBCL

MM

MM

HL

CLL

CTCL

TP53

TP53

TP53

TP53

TP53

N/A

N/A

N/A

SD:PD:

PR:

RR:DLT:DR:

PMC-04

PMC-06

PMC-07

PMC-09

PMC-10

PMC-16

PMC-05

PMC-11

PMC-12

PMC-13

PMC-03

PMC-14

PMC-15

PMC-01

PMC-08

PMC-17






Table 2. Number and percentage of patients experiencing all AE types occurring as grade 3 events in >5% of the entire cohort over the full treatment period (AEs ordered by frequency of grade 3 events, alphabetical by term)

All patients ( n = 16) Cohort 1 ( n = 3) Cohort 2 ( n = 4) Cohort 3 ( n = 4) Cohort 4 ( n = 3) Cohort 5 ( n = 2)

All Grade 3 All Grade 3 All Grade 3 All Grade 3 All Grade 3 All Grade 3

Adverse event term N N % N N % N N % N N % N N % N N %Anemia 4 (3 a ) 2 13% 1 — — 2(2 a ) 2 50% 1(1 a ) — — — — — — — —

Abdominal pain 1 1 6% — — — 1 1 25% — — — — — — — — —

Atrial fi brillation 1 1 6% — — — 1 1 25% — — — — — — — — —

Blood bilirubin increased

1 1 6% 1 1 33% — — — — — — — — — — — —

Cardiac disorders 3 (1 a ) 1 6% — — — 2(1 a ) — — — — — — — — 1 1 50%

Creatinine increased 2 1 6% — — — 1 1 25% 1 — — — — — — — —

Diarrhea 2 1 6% — — — 1 1 25% 1 — — — — — — — —

Erythroderma 1 (1 a ) 1 6% — — — 1 1 25% — — — — — — — — —

Hypophosphatemia 1 1 6% — — — — — — 1 1 25% — — — — — —

Hypoxia 1 1 6% — — — 1 1 25% — — — — — — — — —

Infections and infestations

2 1 6% — — — 1 1 25% — — — 1 — — — — —

Investigations 3 1 6% — — — 1 — — 2 1 25% — — — — — —

Neutrophil count decreased

4 (4 a ) 1 6% 2 — — — — — 1 — — 1 1 33% — — —

PPE syndrome 2 (2 a ) 1 6% — — — — — — — — — — — — 2 1 50%

Photosensitivity 8 (8 a ) 1 6% 2 1 33% — — — 2 — — 2 — — 2 — —

Platelet count decreased

4 (4 a ) 1 6% — — — 2 — — 2 1 25% — — — — — —

Pulmonary edema 1 1 6% — — — 1 1 25% — — — — — — — — —

Renal and urinary disorders

2 1 6% — — — 1 — — 1 1 25% — — — — — —

Skin infection 1 1 6% — — — 1 1 25% — — — — — — — — —

Vasculitis 1 (1 a ) 1 6% 1 1 33% — — — — — — — — — — — —

a Indicates patients experiencing AEs possibly, probably, and defi nitely related to treatment over the full treatment period. See Supplementary Table S1 for complete list of treatment-related AEs with associated patient numbers and percentages.

the longest observed terminal half-life of 83.3 hours in cohort 5 ( Table 3 ). Pharmacokinetics were generally linear and dose-proportional in terms of both C max (Supplementary Fig. S2B) and AUC (Supplementary Fig. S2C) exposure parameters. Moreover, the appearance of multiple secondary peaks in plasma concentration–time profi les and a fl attened termi-nal slope resulting in a longer observed half-life, especially with increasing dose, suggested the presence of enterohepatic recirculation of the drug (Supplementary Fig. S2A; Table 3 ).

Effi cacy Analysis Of the 16 patients evaluated for effi cacy ( Fig. 1 ; Sup-

plementary Table S3), the best response was a confi rmed partial response (PR) sustained for more than 12 months. This patient (PMC-17), with anaplastic large cell lymphoma (ALCL), previously had very short durations of response to prior therapies including conventional chemotherapy, mAbs,

and an autologous stem-cell transplant. Another patient (PMC-05), with cutaneous T-cell lymphoma (CTCL) with large cell transformation, had clinical and radiologic evidence of response in the anatomic area of transformed disease. One patient (PMC-08) with heavily pretreated DLBCL had a period of sustained stable disease, receiving 16 cycles of treatment over 10 months, whereas another patient (PMC-15) with DLBCL achieved stable disease for 4 cycles. The best response noted in patients with myeloma was stable disease in 50% (3/6) of patients. Although all three patients with myeloma had actively progressing disease at study entry, their disease stabilization was maintained for 4–6 cycles.

Pharmacodynamic Analysis of CX-5461: On-Target Activity against rDNA Transcription

CX-5461 is a selective inhibitor of Pol I transcription of rDNA, functioning by occluding SL1, a complex crucial






Table 3. Pharmacokinetic parameters of CX-5461 following a single dose

Single CX-5461 dose, mg/m 2

PK parameter 25 ( n = 3) 50 ( n = 4) 100 ( n = 4) 170 ( n = 3) 250 ( n = 2) T max , median, (range), hr 1 (0.98–1) 0.75 (0.25–1.25) 0.5 (0.37–1) 0.52 (0.42–1) 1

C max , mean (SD), (ng/mL) 297 (46) 384 (174) 636 (235) 1,707 (455) 1,358 (526)

AUC 0–t , mean (SD), (hr • ng/mL) 2,057 (470) 3,646 (2,222) 10,146 (2,494) 16,792 (4,761) 27,147 (439)

AUC 0-∞ , mean (SD), (hr • ng/mL) 2,297 (516) 4,587 (2263) 12,152 (3,482) 17,943 (5,187) 28,612 (1006)

t 1/2 , mean (SD), hr a 23.2 (1.6) 39.8 (16.9) 58.4 (12.9) 45.5 (5.7) 83.3 (12.9)

NOTE: Pharmacokinetic (PK) parameters were reported as cohort mean (SD), except for T max , which was reported as cohort median (range). Abbreviations: C max , maximum concentration recorded; AUC 0–t , area under the curve from time 0 to last quantifi able concentration; AUC 0–∞ , area under the curve from time 0 extrapolated to infi nity; T max , time to reach C max . a Residual drug was detectable in patients in cohort 5 at day 15 postdose.

for the recruitment of transcription-competent Pol I to the rDNA promoter ( 20 ). To confi rm on-target drug activity, we developed a highly sensitive assay to measure Pol I–mediated transcription rates via FISH ( 16 ). The 5′-external transcribed spacer (5′ETS) of rRNA lies at the 5′ end of the 47S transcript and is rapidly processed following rRNA synthesis; therefore, its fl uorescent detection by FISH is used as a surrogate read-out of rDNA transcription rate, and the accuracy of the assay was previously validated by comparison with direct metabolic labeling of newly synthesized rDNA ( 10, 16 ). This allowed us to quantitate the abundance of 47S pre-rRNA levels in periph-eral blood mononuclear cells (PBMC; Fig. 2A and B ) and tumor tissue ( Fig. 3A–D ) in sequential samples during cycle 1.

To assess the pharmacodynamic effects of CX-5461 therapy, PBMCs were collected from all patients before treatment and then at 1, 4, 8, and 24 hours postinfusion of their fi rst cycle of treatment. Levels of rDNA transcription inhibition were analyzed as the median percentage change in 5′ETS signal intensity from baseline ( Fig. 2A and B ). A consistent and robust decrease in rDNA transcription rate was observed at 1 hour postinfusion in PBMCs ( Fig. 2A and B ). The average level of inhibition was 49% (range, 22.9%–69.9%), 51.1% (34.4%–64.4%), 19.6% (−72%–69.7%), 43.8% (37.1%–48%), and 38.6% (6.8%–70.5%) in cohorts 1–5, respectively ( Fig. 2B ). The excep-tion to this was in PBMC samples taken from patient PMC-11, where the baseline detected levels of 5′ETS signal intensity were unusually low. However, PBMC samples collected at later time-points displayed a rebound in rDNA rate, typically achieving levels similar to baseline by 24 hours post-treatment ( Fig. 2B ).

A comprehensive assessment of the quantitative dose–response relationship between CX-5461 plasma levels and Pol I–mediated transcription activity (5′ETS signal intensity) in PBMC samples across all dose cohorts is shown in Supplemen-tary Fig. S3. Blood sampling post-treatment revealed an inverse association, where the maximal inhibition of rDNA transcrip-tion observed at 1 hour postinfusion correlated with the initial peak in drug plasma concentration levels observed in each dose profi le (Supplementary Fig. S3). Moreover, falling plasma levels of CX-5461 during the linear phase of drug clearance was asso-ciated with a rebound in 5′ETS signal intensity in each patient, consistent with the utility of this assay for monitoring on-target

drug activity (Supplementary Fig. S3). We also noted that the extent of rDNA transcription inhibition did not correlate with increasing CX-5461 dose ( Fig. 2B ) despite dose-proportional pharmacokinetics (Supplementary Fig. S2B and S2C).

CX-5461 Treatment Inhibits rDNA Transcription in Patient Tumors

Paired tumor biopsy specimens were obtained from patients with accessible disease [11/16 patients; bone marrow trephine (BM), n = 4; lymph nodes, n = 5; liver, n = 1; skin lesions, n = 3]predose and 24 hours postinfusion for the determination of on-target activity in tumor cells following therapy ( Fig. 3A and B ). Inhibition of rDNA transcription was observed in the majority of tumor-infi ltrated specimens (10/13); however, the level of inhibition in rDNA transcription was variable (median range: 4%–68.5%) with no correlation to increasing dose ( Fig. 3B ). Tumor cells isolated from bone marrow by magnetic activated cell sorting (MACS; 4 patients) displayed a decrease in rDNA transcription rate in all patients ( Fig. 3C and D ). The degree of inhibition varied but was consist-ently inhibited at 4 hours and, again, did not correlate with CX-5461 dose ( Fig. 3C and D ). These data confi rm that 5′ETS signal intensity is a reproducible biomarker of on-target drug activity in patient tumor samples and is consistent with data in PBMC samples ( Fig. 2 ) showing that the amount of inhibition of rDNA transcription is independent of CX-5461 dose and also refl ecting the potential for a rebound in rDNA transcription rates by 24 hours post-treatment.

The best clinical response was detected in a 63-year-old patient (PMC-17) who had ALCL with hematologic and cutaneous involvement, where an objective PR was seen in both compartments after 6 months of CX-5461 treatment ( Fig. 4A ). Tumor tissue sampled from this patient for cor-relative studies also displayed a decrease in rDNA transcrip-tion rate at both the 4-hour (22.1%) and 24-hour (8.1%) time points post cycle 1 ( Fig. 4B ).

Clinical Response to CX-5461 Occurred in Patients with Both Wild-Type and Mutated TP53

CX-5461 inhibition of rDNA transcription has been dem-onstrated to mediate its therapeutic response by inducing a






Figure 2. The on-target effect of CX-5461 on rDNA transcription in normal peripheral blood mononuclear cells, as determined by RNA-FISH to the 5′ETS region of 47S pre-rRNA. A, In situ detection of 5′ETS 47S pre-rRNA transcript in PBMCs acquired from 5 representative patients from cohorts 1–5 treated with CX-5461. Samples were collected prior to treatment and at the 1, 4, 8, and 24 hour time points post CX-5461 infusion in cycle 1. Images were taken with a 60× objective, with a merged overlay of DAPI-stained nuclei (blue) and labeled Cy3 5′ETS probe (red) shown. Scale bars, 10 μm. (continued on following page)

Post CX-5461 treatment

Cohort 125 mg/m2

Cohort 250 mg/m2

Cohort 3100 mg/m2

Cohort 4170 mg/m2

Cohort 5250 mg/m2

A

PM

C-0

1P

MC

-06

PM

C-1

0P

MC

-13

PM

C-1

7

Baseline 1 hr 4 hr 8 hr 24 hr

DAPI/ETS

nucleolar stress response leading to activation of p53, but also via p53-independent mechanisms in a range of cancer cell types (10, 14, 15, 16, 22, 23). To further investigate the role of these pathways of therapeutic response to CX-5461, we performed targeted sequencing of 79 genes that were curated on the basis of their previously described role in the CX-5461 response mechanism (i.e., TP53, ATM, ATR, CHK2, MYC), their regulation of rDNA transcription (MYC, PI3K/AKT/mTOR signaling), and/or nucleolar function (NPM1, ribo-somal proteins) and including well-known components of DNA repair and DDR (BRCA1, BRCA2, RAD51; complete list in Supplementary Table S4) to determine their mutational status in tumors of enrolled patients as a potential biomarker of therapeutic response. DNA was extracted from available tumor samples (n = 13) and sequenced using hybridization-based next-generation sequencing (data summarized in Fig. 1; Supplementary Tables S3 and S5). Collectively, 5 patients in total were found to harbor mutations in TP53, with all but one being associated with an early progression of disease following treatment (Fig. 1). Notably, 1 patient with DLBCL that carried a TP53 mutation achieved the longest period of stable disease (PMC-08, 16 cycles), and another patient in whom the TP53 status was not available also achieved stable

disease (PMC-01; Fig. 1). Of the confirmed TP53 wild-type patients (n = 8), 1 achieved a prolonged PR (PMC-17, 18 cycles), 1 achieved a clinical and radiologic response in an area of transformed disease (PMC-05, 2 cycles), and 3 achieved periods of stable disease (all, 4 cycles; Fig. 1). The sequencing also identified 2 patients harboring mutations in ATM, one of the major known effectors of the CX-5461 p53-independent DDR-like response (14–16). Importantly, these patients were mutually exclusive from those who had tumors with TP53 mutations and were represented by a best confirmed response of stable disease (PMC-03) as well as rapid disease progres-sion (PMC-10; Fig. 1). Finally, patient tumor DNA was also assessed for copy-number variation (CNV) at the MYC and MDM2 loci; however, no CNVs were detected at either loci in any of the 13 patients (Supplementary Table S5).

To extend our interrogation of the role of p53 in the thera-peutic response to CX5461, we expanded our biomarker anal-ysis on the tumor sample from one TP53 wild-type patient, a 79-year-old patient (PMC-05) with CTCL who displayed a clinical and radiologic response in an area of high-grade transformation following 1 cycle of treatment (50 mg/m2, Fig. 4C–F). As seen by 18F-FDG-PET and digital photography, a reduction in tumor metabolic activity and corresponding






% C

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e in

5′E

TS

sig

nal i

nten

sity

from

bas

elin

e

B

Cohort 125 mg/m2

Cohort 5250 mg/m2

Cohort 4170 mg/m2

Cohort 250 mg/m2

Cohort 3100 mg/m2

PMC-01

PMC-03

PMC-04

PMC-05

PMC-06

PMC-07

PMC-08

PMC-09

PMC-10

PMC-11

PMC-12

PMC-13

PMC-14

PMC-15

PMC-16

PMC-17−100%

−50%

0%

50%

100%

150%

200%

1 hr 4 hr 8 hr 24 hr

Postinfusion

Peripheral blood mononuclear cells: cohorts 1–55′ETS signal intensity post cycle 1 of CX-5461 treatment

Figure 2. (Continued) B, Quantitative analysis of rDNA transcription inhibition in PBMCs from all 16 patients following CX-5461 dosing (as in A, at indicated times in cycle 1), expressed as a median percentage change in FISH signal intensity from baseline. Spot intensity was measured using a pipeline solution developed in Definiens Tissue Studio 3.6.

clinical improvement was observed in the focal aggressive scalp lesion (Fig. 4C). As observed with other patient tumor samples assayed (Figs. 3 and 4B), this tumor response was associated with a decrease in rDNA transcription rates when compared with baseline (8.7%, Fig. 4D). Given this patient’s wild-type TP53 tumor status, we assayed total p53 protein levels by IHC in a punch biopsy that directly sampled the cutaneous lesion pretreatment and 24 hours post-treatment and observed elevated p53 expression in the CX-5461–treated sample when compared with baseline (Fig. 4E). Furthermore, Western blot analysis of tumor samples showed that along with stabilization of p53 protein levels, a corresponding increase in the p53-target protein p21 after 24-hour CX-5461 exposure was observed (Fig. 4F). These data demonstrate that in a setting where the tumor was TP53 wild-type and the patient had a clinical response, inhibition of rDNA transcrip-tion was associated with activation of p53.

DISCUSSIONHere we report the results of a first-in-human study

assessing the tolerability, safety, and anticancer activity of the small-molecule RNA polymerase I inhibitor CX-5461 in patients with advanced hematologic malignancies. We

have determined an MTD of 170 mg/m2 when the drug is administered by intravenous infusion once every 3 weeks. A DLT of grade 3 PPE was observed at a dose of 250 mg/m2. An additional AE of photosensitivity was noted in 50% of the patients treated, independent of dose level, and this was manageable with avoidance of sun exposure for 72 hours after drug dosing. Although these cutaneous AEs were not anticipated from the preclinical data, they resolved without any sequelae. No other significant hematologic or other AEs were noted. Moreover, the patients in the study were heav-ily pretreated, with a median of 7 prior lines of therapy and with 10 patients having prior high-dose therapy followed by autologous or allogeneic hematopoietic progenitor cell trans-plant. Despite this, one patient with ALCL had a prolonged partial response for more than 12 months, and 5 patients with either multiple myeloma or DLBCL achieved a period of sta-ble disease. Interestingly, a patient with CTCL demonstrated clinical benefit and radiologic response in a site of trans-formed disease, suggesting that T-cell lymphoma may be a tumor type which warrants further specific investigation.

Here, analysis of the on-target effect of CX-5461 in humans has been demonstrated by a decrease in rDNA tran-scription rates in both PBMCs and tumor tissue by RNA-FISH, establishing the utility of this assay for monitoring






A B

SD: Stable disease RR: Radiological response

Best overall response

PMC-01: MM25 mg/m2

PMC-07: CLL50 mg/m2

PMC-10: T-PLL100 mg/m2

PMC-13: MM170 mg/m2

Baseline4 hr postCX-5461

24 hr postCX-5461

C

−100%

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PMC-13170 mg/m2

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DAPI/ETS

PMC-04: DLBCLLymph node

25 mg/m2

PMC-07: CLLliver

50 mg/m2

PMC-09: MMbone marrow

trephine100 mg/m2

PMC-08: DLBCLbone marrow

trephine50 mg/m2

PMC-11: DLBCLlymph node100 mg/m2

PMC-15: DLBCLlymph node170 mg/m2

DAPI/ETS

Baseline24 hr postCX-5461

Best overall response

PD: Progressive disease SD: Stable disease

PD: Progressive disease PR: Partial response

Post CX-5461

50 mg/m2

100 mg/m2

25 mg/m2

170 mg/m2

250 mg/m2

PMC-03: BM

PMC-04: Lymph node

PMC-05: Skin lesion

PMC-05: Lymph node

PMC-07: Liver

PMC-07: BM

PMC-08: BM

PMC-09: BM

PMC-10: Skin lesion

PMC-11: Lymph node

PMC-12: Lym

ph node

PMC-15: Lym

ph node

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lesio

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−80%

−60%

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on-target drug activity. Maximum inhibition occurs 1–4 hours postdose and correlates with peak drug levels, with a return to baseline levels by 24 hours postdose. This raises the possibility that more frequent dosing (i.e., once-weekly administration) may improve the efficacy profile of this agent, and therefore studies designed to investigate this dosing schedule in the same population are planned to commence in the near future. Furthermore, a phase I dose-escalation study with day 1 and day 8 dosing of CX-5461 in a 4-week cycle in patients with advanced solid cancers is also currently ongoing (Canadian Cancer Trials Group; ClinicalTrials.gov identifier: NCT02719977; ref. 24). Interestingly, the inhibi-tion of Pol I–mediated transcription occurred independently of dose level, providing a strong rationale for trialing more frequent dosing strategies at or below the MTD determined in this study. The pharmacokinetic profile of CX-5461 was generally predictable, linear, and dose-proportional, with a mean plasma half-life of 45.5 hours at the MTD. Drug was detectable in the plasma for up to 2 weeks following infusion in the highest dose cohort. The suggestions of enterohepatic recirculation of the drug and the possibility of drug accumu-lation with repeated doses have been taken into account in the protocol design, incorporating more frequent albeit lower dosing strategies.

There is now extensive preclinical evidence for improved efficacy using combinations of CX-5461 with other agents in clinical use or trials, including everolimus (mTOR inhibitor) and AZD7762 (CHK1/2 inhibitor) in B-cell lymphoma (15, 22), VE-822 (ATR inhibitor) in acute lymphoblastic leukemia (14), and carfilzomib (proteasome inhibitor) and panobi-nostat (pan-HDAC inhibitor) in myeloma (23), as well as PIM kinase inhibitors in prostate cancer (25). These are diseases which have previously been most effectively treated by com-bination drug therapy, and the demonstration of on-target effects at low doses of CX-5461 is encouraging for the pos-sibility of clinical synergy with low toxicity in combination therapies.

In this study, although 5 of 8 patients who were wild-type and 1 of 5 patients who were mutant for TP53 showed beneficial clinical responses and 4 patients with TP53 muta-tions showed early progression, the overall small number of patients allows only speculation as to the degree by which TP53 mutation status can be used as a predictor of CX-5461 efficacy. It has also been reported that CX-5461 therapeu-tic activity requires mechanisms which are independent of p53, including activation of an ATM/ATR-dependent DDR

(14–16) and an NSR-driven downregulation of MYC (17). Furthermore, in addition to inhibiting rDNA transcription, a recent study reported that CX-5461 induces replication-dependent DNA damage through stabilization of G-quad-ruplex (G4) DNA structures (26). The breadth of activity demonstrated in these preclinical studies, all of which can be mediated through targeting of rDNA, illustrates that the extent of CX-5461 therapeutic efficacy may depend on cancer cell type and the presence or absence of key molecular pathways, which could serve as predictors of response. For example, elevated MYC is predicted to sensitize cancers to Pol I inhibition (10, 13) and was shown to confer sensitivity to CX-5461 in prostate cancer models (25), whereas homolo-gous recombination–deficient cancer cells lacking p53 display an enhanced response to CX-5461 (26). These studies suggest therapeutic potential for CX-5461 in a broad range of tumor types, and importantly a phase I trial evaluating CX-5461 in advanced solid tumors is ongoing (24). The patient tumor sequencing performed here also revealed 2 patients harbor-ing ATM mutations that did not co-occur with TP53 muta-tions, with 1 of these patients achieving stable disease (Fig. 1; Supplementary Tables S3 and S5), which may suggest that one of these key CX-5461 response pathways must be intact for drug efficacy. Moreover, although future studies will examine the extent to which these mechanisms of action and their downstream responses contribute to the therapeutic efficacy of CX-5461, the data in this study demonstrate that CX-5461 shows on-target rDNA transcription inhibition in parallel with drug plasma levels, and this on-target activity in tumor samples correlates with activation of p53 in a patient in whom a clinical response was demonstrable.

The observation of PPE and photosensitivity as the only significant toxicities in our study has important implications for the ongoing development of the drug. Both were noted within 48 hours of drug dosing, which provides a timeframe for maximum risk of the adverse event. Precautions requiring strict sun protection are essential, including sunscreens that block UVA, as one patient experienced photosensitivity after sitting behind glass, which absorbs up to 97% of UVB. Impor-tantly, adherence to these measures for 72 hours after drug dosing prevented recurrence of these events in all patients and allowed continuing treatment for prolonged periods. Similar toxicities have also been seen with drugs such as the BRAF inhibitor vemurafenib, and it has been possible to con-tinue their use with appropriate supportive care and without dose reduction (27), as noted in our study.

Figure 3. CX-5461 displays on-target rDNA transcription inhibition in paired tumor biopsy specimens and MACS isolated tumor cells. Needle-core biopsies of tumor tissue were collected from patients with accessible tumors (n = 11) representing cohorts 1–5, pretreatment and 24 hours post CX-5461 administration (A and B). Samples were formalin-fixed and paraffin-embedded (FFPE) before being evaluated by RNA-FISH to assess on-target Pol I–mediated transcription inhibition. A, In situ detection of 5′ETS 47S pre-rRNA transcript in 6 representative patient tumors (tumor tissue as indi-cated). Images were taken at 60× magnification and a merged overlay of DAPI-stained nuclei (blue) and labeled Cy3 5′ETS probe (red) is shown in each case. Scale bars, 10 μm. B, Quantitative analysis of rDNA transcription inhibition in tumor biopsy specimens [tumor tissue as indicated; bone marrow tre-phine (BM)], expressed as a median percentage change in FISH signal intensity from baseline in patients achieving either stable disease, partial response, radiologic response, or progressive disease. In those patients with bone marrow infiltration, aspirate samples were also collected (n = 4) and MACS-sorted with negative selection for malignant cells prior to analysis (C and D). C, In situ detection of 5′ETS pre-rRNA transcript in MACS-sorted tumor cells acquired from 4 patients treated with CX-5461. Representative images were taken at 60× magnification and a merged overlay of DAPI-stained nuclei (blue) and labeled Cy3 5′ETS probe (red) is shown in each case. Scale bars, 10 μm. D, As in B, bar graphs displaying the median percentage change in FISH signal intensity from baseline, as detected in individual patients prior to treatment and at 4 hours (yellow) and 24 hours (green) post CX-5461 infusion. Spot intensity was measured using a pipeline solution developed in Definiens Tissue Studio 3.6.






Tot

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2.0 × 107

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1.0 × 107

5.0 × 106

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24 hr post CX-5461Baseline

4 hr post CX-5461

Baseline11 months post

cycle 1 (250 mg/m2)

PMC-17: ALCLCutaneous lesion, right knee/thigh

Total p53

p21

β-actin

Scalp lesionTP53 wild-type

Base 24 hr






escalations were planned in 7 cohorts (25–450 mg/m2), initially in an accelerated design, with change to a 3+3 dose-escalation schema based on the predefined toxicity criteria and DLTs of CX-5461. Patients remained on trial until disease progression, significant toxic-ity, or a clinical observation satisfying another withdrawal criterion was evident. The primary objective of the study was to define the safety and tolerability of CX-5461, by determining the DLTs and the MTD. The secondary objectives were to assess the pharmacokinetic and pharmacodynamic profile of CX-5461, investigate any prelimi-nary clinical effects on tumor response, and to identify predictive biomarkers of efficacy. The secondary endpoints were assessment of grade 3+ adverse events, overall response, and determination of the pharmacokinetic profile of CX-5461.

Pharmacokinetic Sampling and AnalysisSerial heparinized blood samples were collected from a peripheral

vein on the contralateral side of the body to the site of injection. These samples were acquired prior to dosing; at 15 and 30 minutes during infusion; immediately upon completion of infusion; and then at 15 minutes, 30 minutes, and 1, 2, 4, 6, and 8 hours after dose admin-istration. Subsequently, all patients returned to the clinic on days 2, 3, and 4, for additional blood sample collections at 24, 48, and 72 hours after day 1 treatment. After collection, samples were shipped frozen to CPR Pharma Services Pty Ltd where analysis of all phar-macokinetic parameters was performed. Parameters calculated and reported on the basis of actual sampling times included: maximum observed plasma concentration (Cmax), time of maximum observed plasma concentration (Tmax), effective half-life (t1/2), and area under the plasma concentration–time curve. The pharmacokinetic popu-lation consisted of patients who received at least one intravenous dose of CX-5461 and who had evaluable pharmacokinetic data from plasma.

Correlative Sampling and Pharmacodynamic AnalysisTo confirm on-target drug activity and identify predictive biomark-

ers of therapeutic response, all patients were asked to provide periph-eral blood samples at baseline and then at 1, 4, 8, and 24 hours post day 1 infusion. In those patients who had biopsy-accessible disease, tumor tissue specimens (from bone marrow, lymph nodes, liver, skin lesions) were obtained prior to treatment and 24 hours post cycle 1. When available, aspirate samples from the bone marrow were also harvested by MACS and the tumor cells collected by negative selection (antibodies used for MACS sorting listed in Supplementary Table S6). Pol I transcription levels were measured indirectly in these samples via FISH targeting the 5′ETS region of 47S pre-ribosomal RNA (RNA-FISH). Detailed methods describing cell isolation and preparation as well as RNA-FISH and immunoblotting (antibodies used for Western blot analysis listed in Supplementary Table S7) are provided in the Supplementary Methods. A custom targeted hybridization-based next-generation sequencing panel was used to identify sequence

In summary, in this first-in-human, first-in-class study, we have determined an MTD for CX-5461 in patients with advanced hematologic cancers and demonstrated tolerabil-ity with extended periods of dosing. Clinical responses have also been noted, which have been sustained and beneficial in some cases. Importantly, cutaneous adverse events have been observed, which will need to be actively managed dur-ing future development of this agent. These data provide a basis for further studies in appropriate tumor groups to explore more frequent dosing and combination strategies. Taken together, this study demonstrates for the first time that Pol I–mediated transcription of rDNA can be selectively and safely targeted in humans and validates a previously unex-plored targeted therapeutic approach.

METHODSPatient Selection

Patients were eligible for participation in the study if they had any measurable, relapsed, or refractory advanced hematologic malig-nancy, without any standard therapeutic options available, aged ≥18 years, with adequate organ and bone marrow function, an ECOG performance status of 0–2 at screening, and life expectancy ≥3 months. Adequate organ and bone marrow function were defined by the following: organ, creatinine clearance greater than 50 mL/min-ute, a total bilirubin ≤ 2 times the upper limit of normal and hepatic transaminases ≤ 2 times the upper limit of normal; bone marrow, hemoglobin (Hb) ≥ 9 g/dL, absolute neutrophil count ≥ 1.0 × 109/L without the use of granulocyte colony-stimulating factor for 7 days prior to day 1 and platelets ≥ 1 × 1011/L (except in cases of bone marrow infiltration, where lower thresholds were allowed). Patients with other malignancies requiring concurrent anticancer therapy or known active central nervous system disease were excluded from the study. Other key exclusion criteria included patients with a QT interval greater than 450 msec or significant bacterial, viral, or fun-gal infection. All subjects provided written informed consent prior to trial enrolment. The trial protocol was approved by the Insti-tutional Review Board and the trial was conducted in accordance with the Good Clinical Practice guidelines and the ethical principles outlined in the Declaration of Helsinki and the International Con-ference on Harmonisation.

Study Design and ObjectivesThis was a single-center, first-in-human, phase I, open-label, dose-

escalation study designed to establish the safety, pharmacokinetic, and pharmacodynamic characteristics of CX-5461 in patients with advanced hematologic malignancies. CX-5461 was administered as a 1-hour intravenous infusion on day 1 of each 21-day cycle. Dose

Figure 4. Antitumor activity observed with CX-5461 treatment. PMC-17, diagnosed with ALCL, had a partial response in cutaneous disease and remained on trial for the longest duration (18 cycles; A and B). A, Clinical photographs of the right lateral thigh/knee, taken pretreatment and 11 months after commencing CX-5461 therapy (at 250 mg/m2 in cycle 1). B, Box-and-whisker plots showing the total levels of FISH signal intensity detected per nucleus (arbitrary units) in paired skin punch biopsies taken from the cutaneous lesions of PMC-17, pretreatment (red), and at the 4-hour (yellow) and 24-hour (green) time points post CX-5461 infusion. The horizontal line within the box indicates the median, the “+” within the box denotes the mean, and whiskers indicate the 10th and 90th percentile (data points outside this range are not depicted). Representative images were taken at 60× magnification, and a merged overlay is shown in each case. Scale bars, 10 μm. PMC-05, diagnosed with CTCL, exhibited a radiologic response in a scalp lesion with high-grade transformation following one cycle of CX-5461 treatment (50 mg/m2; C–F). C, Digital photographs (left) and 18F- FDG-PET scans (right) of the left scalp, demonstrating a clinical benefit and a visible reduction in tumor metabolic activity at 7 and 22 days post–first infusion, respectively. D, Box-and-whisker plots as in B displaying the total levels of FISH signal intensity detected per nucleus (arbitrary units) in paraffin-embedded skin punch biopsies taken from the cutaneous scalp lesion of PMC-05, pretreatment (red) and 24 hours (green) post CX-5461 treatment. Representative images were taken at 60× magnification, and a merged overlay of DAPI-stained nuclei (blue) and labeled Cy3 5′ETS probe (red) is shown in each case (bottom). Scale bars, 10 μm. E, IHC staining (20×) showing robust nuclear accumulation of p53, 24 hours post CX-5461 therapy. F, Immunoblot analyses of total p53 and p21 protein levels in tumor tissue lysates extracted from the scalp lesion of PMC-05 (TP53 wild-type tumor status), prior to and 24 hours post CX-5461 infusion. Equal amounts of protein from each sample were probed and β-actin was used as a loading control.






variants in 79 genes after extraction of DNA from available tumor samples (see Supplementary Methods for extended methods). All correlative samples in this study were collected, deidentified, and processed according to a protocol-specified standard operating pro-cedure (see Supplementary Methods for details).

Statistical AnalysisAll statistical analyses were performed in SAS Analytics Software

(version 9.3; SAS Institute, Inc.). Demographics, baseline character-istics, pharmacokinetic parameters, and clinical laboratory evalu-ations were summarized with descriptive statistics. For analysis of pharmacodynamic response during drug treatment, levels of Pol I transcription inhibition were analyzed by calculating the median percentage change in FISH signal intensity from each patient’s base-line measurement.

Disclosure of Potential Conflicts of InterestA. Khot is a consultant/advisory board member for Celgene, Jans-

sen, and Amgen. N. Hein reports receiving a commercial research grant from Pimera, Inc. J. Lim is a vice president, clinical & regula-tory affairs at Senhwa Biosciences, Inc. and has ownership interest (including stock, patents, etc.) in the same. J. Soong has ownership interest (including stock, patents, etc.) in Senhwa Biosciences Inc. G.A. McArthur is a consultant/advisory board member for Pimera, Inc. R.B. Pearson is a consultant/advisory board member for Pimera, Inc. R.D. Hannan is an executive group manager for the Centre for Health and Medical Research at ACT Health Directorate and a lab head at Peter MacCallum Cancer Centre, reports receiving a com-mercial research grant from Pimera Inc., and is a consultant/advisory board member for the National Breast Cancer Fund. No potential conflicts of interest were disclosed by the other authors.

Authors’ ContributionsConception and design: A. Khot, J. Lim, E. Link, G.A. McArthur, R.B. Pearson, R.D. Hannan, G. Poortinga, S.J. HarrisonDevelopment of methodology: A. Khot, N. Brajanovski, D.P. Cameron, N. Hein, E. Sanij, J. Lim, E. Link, P. Blombery, G.A. McArthur, R.D. Hannan, G. Poortinga, S.J. HarrisonAcquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A. Khot, N. Brajanovski, D.P. Cam-eron, J. Lim, P. Blombery, E.R. Thompson, A. Fellowes, S.J. HarrisonAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A. Khot, N. Brajanovski, D.P. Cameron, E. Sanij, J. Lim, E. Link, P. Blombery, G.A. McArthur, R.B. Pearson, R.D. Hannan, G. Poortinga, S.J. HarrisonWriting, review, and/or revision of the manuscript: A. Khot, N. Brajanovski, K.H. Maclachlan, E. Sanij, J. Lim, J. Soong, E. Link, P. Blombery, A. Fellowes, K.E. Sheppard, G.A. McArthur, R.B. Pearson, R.D. Hannan, G. Poortinga, S.J. HarrisonAdministrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J. Soong, E. Link, R.D. Hannan, S.J. HarrisonStudy supervision: G.A. McArthur, G. Poortinga, S.J. Harrison

AcknowledgmentsWe thank the patients and their families for their participation

in the study. We also thank the Peter MacCallum Cancer Centre Research Nursing Team. We thank Dr. Megan Bywater and Dr. Stephen Lade for advising on assays used in this study. This work was supported by the National Health and Medical Research Coun-cil (NHMRC) of Australia Development grant (#1038852), Cancer Council Victoria (CCV) grants-in-aid (#1084545 and #1100892) and the Peter MacCallum Cancer Foundation. Senhwa Biosciences pro-vided financial support with respect to drug supply and pharma-cokinetic studies. Researchers were funded by NHMRC Fellowships

(to G.A. McArthur, R.B. Pearson, R.D. Hannan) and the Snowdome Foundation fellowship funding (to A. Khot).

Received December 12, 2018; revised April 11, 2019; accepted May 10, 2019; published first May 15, 2019.

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2019;9:1036-1049. Published OnlineFirst May 15, 2019.Cancer Discov Amit Khot, Natalie Brajanovski, Donald P. Cameron, et al. Results of a Phase I Dose-Escalation StudyCX-5461 in Patients with Advanced Hematologic Cancers: First-in-Human RNA Polymerase I Transcription Inhibitor

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http://cancerdiscovery.aacrjournals.org/content/suppl/2019/05/15/2159-8290.CD-18-1455.DC1

http://cancerdiscovery.aacrjournals.org/content/9/8/1036.full#ref-list-1

http://cancerdiscovery.aacrjournals.org/content/9/8/1036.full#related-urls

http://cancerdiscovery.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerdiscovery.aacrjournals.org/content/9/8/1036


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