first-in-human rna polymerase i transcription …...2019/05/15 · 1 first-in-human rna polymerase...

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First-in-Human RNA Polymerase I Transcription Inhibitor CX-5461 in Patients with

Advanced Hematological Cancers: Results of a Phase I Dose Escalation Study

#Amit Khot1, #Natalie Brajanovski2, ‡Donald P. Cameron3,4, Nadine Hein3, Kylee H.

Maclachlan1,2,4, Elaine Sanij2,4,5, ¥John Lim6, John Soong6, Emma Link4,7, Piers Blombery1,4,8,

Ella R. Thompson4,8, Andrew Fellowes2,8, Karen E. Sheppard2,4,9, Grant A. McArthur2,4,10,

§Richard B. Pearson2,4,9,11, §Ross D. Hannan2,3,4,9,11,12, §Gretchen Poortinga2,4,10*, §Simon J.

Harrison1,4*

1. Clinical Haematology, Peter MacCallum Cancer Centre and Royal Melbourne Hospital,

Melbourne, Victoria 3000, Australia.

2. Cancer Research Division, Peter MacCallum Cancer Centre, Melbourne, Victoria 3000,

Australia.

3. The ACRF Department of Cancer Biology and Therapeutics, The John Curtin School of

Medical Research, Australian National University, Acton 2601, Australia Capital

Territory, Australia.

4. Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville,

Victoria 3010, Australia.

5. Department of Pathology, University of Melbourne, Parkville, Victoria, 3010, Australia.

6. Senhwa Biosciences, Inc., 10F, No.225, Section 3, Pei-Hsin Road, Hsin-Tien District, New

Taipei City 23143, Taiwan, R.O.C.

7. Centre for Biostatistics and Clinical Trials, Peter MacCallum Cancer Centre, Melbourne,

Victoria 3000, Australia

8. Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, Victoria 3000,

Australia

9. Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville,


10. Department of Medicine, St. Vincent’s Hospital, University of Melbourne, Parkville,


11. Department of Biochemistry and Molecular Biology, Monash University, Clayton,

Victoria, 3800, Australia.

12. School of Biomedical Sciences, University of Queensland, Brisbane, Queensland, 4072,

Australia.

Cancer Research. on August 17, 2020. © 2019 American Association forcancerdiscovery.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on May 15, 2019; DOI: 10.1158/2159-8290.CD-18-1455

http://cancerdiscovery.aacrjournals.org/

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‡ Current address: Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm

171 65, Sweden.

¥ Current address: TP Therapeutics, Inc., 10628 Science Center Drive, Suite 225, San Diego,

CA 92121, U.S.A.

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

§ R.B. Pearson, R.D. Hannan, G. Poortinga and S.J. Harrison share senior authorship of this

article.

* Corresponding authors

Running Title: First-in-human study of CX-5461 in hematological cancers

Keywords (5): CX-5461, RNA Polymerase I, rDNA, hematological cancers, p53

Abbreviations:

RNA polymerase I (Pol I), ribosomal RNA (rRNA), rRNA genes (rDNA), Ataxia telangiectasia

mutated (ATM), Ataxia telangiectasia and Rad3 (ATR), Checkpoint kinases 1/2 (CHK1/2),

DNA damage response (DDR), double strand breaks (DSBs), nucleolar stress response (NSR),

acute myeloid leukemia (AML), multiple myeloma (MM), diffuse large B-cell lymphoma

(DLBCL), cutaneous T-cell lymphoma (CTCL), anaplastic large cell lymphoma (ALCL),

Eastern Cooperative Oncology Group (ECOG), dose-limiting toxicity (DLT), adverse event

(AE), maximum tolerated dose (MTD), palmar-plantar erythrodysesthesia (PPE), partial

response (PR), stable disease (SD), maximum plasma concentration (Cmax), terminal half-life

(t1/2), area under the curve (AUC), peripheral blood mononuclear cells (PBMCs), magnetic

activated cell sorting (MACS), fluorescent in-situ hybridization (FISH), 5’-external transcribed

spacer (5’ETS), G-quadruplex DNA (G4).

Financial Support:

This work was supported by the National Health and Medical Research Council (NHMRC) of

Australia Development grant (#1038852), Cancer Council Victoria (CCV) grants-in-aid




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(#1084545 and 1100892) and the Peter MacCallum Cancer Foundation. Senhwa provided

financial support with respect to drug supply and pharmacokinetic studies. Researchers were

funded by NHMRC Fellowships (G.A.M., R.B.P., R.D.H.) and the Snowdome Foundation

fellowship funding (AK).

Correspondence Footnote:

Gretchen Poortinga, Cancer Research Division, Peter MacCallum Cancer Centre, Melbourne,

Victoria, 3000, Australia.

E-mail: [email protected]

Simon Harrison, Clinical Haematology, Peter MacCallum Cancer Centre and Royal Melbourne

Hospital, Melbourne, Victoria, 3000, Australia.

E-mail: [email protected]

Conflict of Interest

J. Soong is Chief Medical Officer at Senhwa Biosciences, Inc. J. Lim has stock ownership in

Senhwa Biosciences, Inc. R.D. Hannan is a Chief Scientific Advisor to Pimera, Inc. No

potential conflicts of interest were disclosed by the other authors.

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 first-in-class selective rDNA transcription inhibitor, in a first-in-

human, phase I dose escalation study in advanced hematological cancers. Administration of

CX-5461 intravenously once every 3 weeks to 5 cohorts determined a maximum tolerated dose

of 170 mg/m2, with a predictable pharmacokinetic profile. 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 lymphoma attained a prolonged partial

response and 5 patients with myeloma and diffuse large B-cell lymphoma achieved stable




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disease as best response. CX-5461 is safe at doses associated with clinical benefit and

dermatologic AEs are manageable.

Statement of Significance: CX-5461 is a first-in-class selective inhibitor of rDNA

transcription. This first-in-human study establishes feasibility of targeting this process,

demonstrating single-agent anti-tumor activity against advanced hematological cancers with

predictable pharmacokinetics and a safety profile allowing prolonged dosing. Consistent with

preclinical data, anti-tumor activity was observed in TP53 wildtype and mutant malignancies.

Introduction

Despite significant progress in the treatment of hematological malignancies with chemotherapy,

monoclonal antibodies and cellular therapies over the last 40 years, with corresponding

improvements in survival outcomes, there remain many patients who are not cured with existing

therapies, necessitating the investigation of agents with novel modes of action (1-4).

The availability of functional ribosomes is a fundamental requirement for growth and

proliferation in mammalian 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 over 100 years (5, 6). RNA polymerase I (Pol I) transcription of

the ribosomal RNA (rRNA) genes (rDNA) is rate-limiting for ribosome biogenesis, a high-

energy consumption process that is significantly elevated in rapidly dividing tumor cells (7, 8).

As rDNA transcription underpins this process, it requires precise regulation; Pol I-mediated

transcription is tightly controlled by oncogenes such as MYC, RAS and PI3K, which when

activated in cancer cells, contribute to hyper-activation of rDNA transcription (9-13).

Furthermore, perturbation of rDNA transcription 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 signalling 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 non-specific targeting of rDNA




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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 inhibitor 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 SL-1 with the rDNA promoter,

exhibiting greater than 200-fold selectivity relative to the inhibition 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 non-

canonical p53-independent nucleolar-localized DNA damage response (DDR) that requires

activation of the Ataxia telangiectasia mutated (ATM) and Ataxia telangiectasia and Rad3

related (ATR) kinase signaling pathways (10, 14, 15). Importantly, as a single agent, CX-5461

shows a robust survival benefit in murine models of a range of hematological cancers including

MYC-driven B-cell lymphoma (10, 22), acute myeloid leukemia (AML) (16) and multiple

myeloma (MM) (17, 23), leading to rapid tumor cell clearance and/or disease reduction with

minimal toxicity.

On the basis of this encouraging preclinical data, we initiated a first-in-human dose escalation

study of CX-5461 in patients with relapsed and refractory hematological malignancies

(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 pharmacodynamic profile of CX-5461, preliminary anti-tumor activity

and to investigate the impact of TP53 mutational status as well as mutations in other potential

CX-5461 response factors including ATM/ATR pathway members, as predictive biomarkers

of efficacy. Dose escalations were planned in 7 cohorts (from 25 to 450 mg/m2), in an

accelerated design, with change to a 3+3 design based on predefined toxicity criteria. We report

here on the findings of this first-in-human, first-in-class study.

Results

Patient Demographics and Disease Characteristics

Between July 27, 2013, and May 4, 2016, 17 patients with advanced hematological

malignancies were recruited, of whom 16 received CX-5461 at the Peter MacCallum Cancer




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Centre, 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 enrolment and were representative of a heavily pre-treated 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 myeloma (6/16 patients, 38%) and diffuse large B-cell

lymphoma (DLBCL) (4/16 patients, 24%). 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 while 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/m2 (n = 3), 50 mg/m2

(n = 4), 100 mg/m2 (n = 4), 170 mg/m2 (n = 3) and 250 mg/m2 (n = 2) (Table 1; the duration on

study for each patient is presented in Figure 1). The initial protocol for accelerated dose

escalation was changed to a standard 3+3 design during cohort 1, due to the observation of

cutaneous adverse events in the first patient treated at 25 mg/m2. 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 PK and PD analysis.

Fourteen patients were included in the main safety analysis for determination of the maximum

tolerated dose (MTD). A DLT of palmar-plantar erythrodysesthesia (PPE) was observed in the

first patient treated at a dose level of 250 mg/m2, with a similar grade 2 adverse event noted in

the second patient enrolled at this dose, though not fulfilling DLT criteria. The MTD was

determined by the safety committee as 170 mg/m2.

Safety Profile

All treatment-emergent adverse events (AEs) 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

Supplementary 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 characterised by pain, swelling, paraesthesia,




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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 hr of treatment and

presented as a sunburn-like rash in sun-exposed areas (Supplementary Fig. S1B). Following the

observation of this AE in 2 patients in cohort 1 (25 mg/m2), subjects were subsequently asked

at enrolment to adhere to strict photosensitivity precautions and were able to continue on

therapy if sun protection was used for 72 hr following drug infusions. One patient (PMC-14)

had a protocol-mandated dose reduction due to a grade 3 photosensitivity event and continued

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 treatment durations extending up to 48

and 54 weeks in patients PMC-08 and PMC-17, respectively and no significant hematological

toxicity was observed (Supplementary Table S1).

Pharmacokinetic Analysis

Mean plasma concentration-time profiles following the first cycle of CX-5461 treatment and

the resulting pharmacokinetic parameters from these analyses are displayed in Supplementary

Figure 2 and Table 3, respectively. In summary, 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

hr and showed a trend to increase with dose escalation, reaching the highest average of 83.3 hr

in cohort 5 (250 mg/m2) with residual drug being detectable in patients in cohort 5 at day 15

post-dose (Table 3). However, no residual drug was detectable at pre-dose, 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 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 Cmax (Supplementary Fig. S2B) and area under the curve (AUC) (Supplementary Fig. S2C)

exposure parameters. Moreover, the appearance of multiple secondary peaks in plasma

concentration-time profiles and a flattened terminal slope resulting in a longer observed half-




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life, especially with increasing dose, suggests the presence of enterohepatic recirculation of the

drug (Supplementary Fig. S2A, Table 3).

Efficacy Analysis

Of the 16 patients evaluated for efficacy (Fig. 1, Supplementary Table S3), the best response

was a confirmed partial response (PR) sustained for over 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, monoclonal antibodies 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 pre-treated diffuse

large B-cell lymphoma (DLBCL) had a period of sustained stable disease, receiving 16 cycles

of treatment over 10 months, while 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. While all three myeloma patients 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 the

transcription initiation selectivity factor 1 (SL-1), a complex crucial for the recruitment of

transcription-competent Pol I to the rDNA promoter (20). To confirm on-target drug activity,

we developed a highly sensitive assay to measure Pol I-mediated transcription rates via

fluorescent in-situ hybridization (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 fluorescent detection by FISH is used as a surrogate readout of rDNA transcription

rate and the accuracy of the assay was previously validated by comparison to direct metabolic

labelling of newly synthesised rDNA (10, 16). This allowed us to quantitate the abundance of

47S pre-rRNA levels in peripheral blood mononuclear cells (PBMCs) (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 hr post-infusion of their first cycle of

treatment. Levels of rDNA transcription inhibition were analysed as the median percentage




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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 hr post-infusion in PBMCs (Fig. 2A and

B). The average level of inhibition was 49.0% (range, 22.9-69.9%), 51.1% (34.4-64.4%), 19.6%

(-72.0-69.7%), 43.8% (37.1-48.0%) and 38.6% (6.8-70.5%) in cohorts 1-5, respectively (Fig.

2B). The exception 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 hr 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 Supplementary Figure S3. Blood sampling post-

treatment revealed an inverse association, where the maximal inhibition in rDNA transcription

observed at 1 hr post-infusion correlated with the initial peak in drug plasma concentration

levels observed in each dose profile (Supplementary Fig. S3). Moreover, falling plasma levels

of CX-5461 during the linear phase of drug clearance was associated 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 C).

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) pre-dose and 24 hr post-infusion for 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-infiltrated specimens (10/13); however, the level of inhibition in rDNA

transcription was variable (median range: 4.0-68.5%) 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 consistently inhibited at 4 hr and again, did not correlate

with CX-5461 dose (Fig. 3C and D). These data confirm 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




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transcription is independent of CX-5461 dose and also reflecting the potential for a rebound in

rDNA transcription rates by 24 hr post-treatment.

The best clinical response was detected in a 63-year-old patient (PMC-17) who had ALCL with

hematological and cutaneous involvement, where an objective PR was seen in both

compartments following 6 months of CX-5461 treatment (Fig 4A). Tumor tissue sampled from

this patient for correlative studies also displayed a decrease in rDNA transcription rate at both

the 4 hr (22.1%) and 24 hr (8.1%) time points post cycle 1 (Fig. 4B).

Clinical Response to CX-5461 Occurred in Patients with Both Wildtype and Mutated

TP53

CX-5461 inhibition of rDNA transcription has been demonstrated to mediate its therapeutic

response by inducing a 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 based on their previously described role in

the CX-5461 response mechanism (i.e. p53, ATM, ATR, CHK2, MYC), their regulation of

rDNA transcription (MYC, PI3K/AKT/mTOR signalling) and/or nucleolar function (NPM1,

ribosomal 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 hybridisation-based next

generation sequencing (data summarized in Fig. 1, Supplementary Table S3 and S5).

Collectively, 5 patients in total were found to harbour mutations in TP53, with all but one being

associated with an early progression to 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) while another patient in whom the TP53 status was not available also achieved

stable disease (PMC-01) (Fig. 1). Of the confirmed TP53 wildtype patients (n = 8), 1 achieved

a prolonged partial response (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 harbouring mutations in ATM,

one of the major known effectors of the CX-5461 p53-independent DDR-like response (14, 15,

16). Importantly, these patients were mutually exclusive from those that had tumors with TP53

mutations and were represented by a best confirmed response of stable disease (PMC-03) as




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well as rapid disease progression (PMC-10) (Fig. 1). Lastly, 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 therapeutic response to CX5461, we

expanded our biomarker analysis on the tumor sample from one TP53 wildtype 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 clinical improvement was observed in the focal aggressive scalp lesion (Fig. 4C).

As observed with other patient tumor samples assayed (Fig. 3 and 4B), this tumor response was

associated with a decrease in rDNA transcription rates when compared to baseline (8.7%, Fig.

4D). Given this patient’s wildtype TP53 tumor status, we assayed total p53 protein levels by

immunohistochemistry (IHC) in a punch biopsy that directly sampled the cutaneous lesion pre-

and 24 hr post-treatment and observed elevated p53 expression in the CX-5461-treated sample

when compared to 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 following 24 hr CX-5461 exposure was observed (Fig. 4F). These data

demonstrate that in a setting where the tumor was TP53 wildtype and the patient had a clinical

response, inhibition of rDNA transcription was associated with activation of p53.

Discussion

Here 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 hematological 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 adverse event 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 hr after drug dosing. While these cutaneous adverse events

were not anticipated from the preclinical data, they resolved without any sequelae. No other

significant hematological or other adverse events were noted. Moreover, the patients in the

study were heavily pre-treated, 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 transplant. Despite this, one patient with ALCL had a prolonged partial response for over




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12 months, and 5 patients with either MM or DLBCL achieved a period of stable disease.

Interestingly, a patient with CTCL demonstrated clinical benefit and radiologic response in a

site of transformed 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 transcription rates in both PBMCs and tumor tissue by RNA-FISH,

establishing the utility of this assay for monitoring on-target drug activity. Maximum inhibition

occurs 1-4 hr post-dose and correlates with peak drug levels, with a return to baseline levels by

24 hr post-dose. 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) (24). Interestingly, the

inhibition 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 PK profile of CX-5461 was generally predictable, linear and dose-proportional,

with a mean plasma half-life of 45.5 hr at the MTD. Drug was detectable in the plasma for up

to 2 weeks following infusion in the highest dose cohort. The suggestion of enterohepatic

recirculation of the drug and the possibility of drug accumulation 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), also carfilzomib (proteasome inhibitor) and panobinostat (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 combination drug

therapy and the demonstration of on-target effects at low doses of CX-5461 is encouraging for

the possibility of clinical synergy with low toxicity in combination therapies.




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In this study, while 5/8 patients who were wildtype and 1/5 patients who were mutant for TP53

showed beneficial clinical responses and 4 patients with TP53 mutations showed early

progression, the overall small number of patients only allows 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 therapeutic activity requires mechanisms which are independent of p53,

including activation of an ATM/ATR-dependent DDR (14-16) and a NSR-driven down-

regulation 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-quadruplex (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 sensitise cancers to Pol I inhibition (10, 13) and was shown to

confer sensitivity to CX-5461 in prostate cancer models (25), while homologous 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 harbouring ATM mutations that did not co-

occur with TP53 mutations, with 1 of these patients achieving stable disease (Fig. 1;

Supplementary Table S3 and S5), which may suggest that one of these key CX-5461 response

pathways must be intact for drug efficacy. Moreover, while 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 hr

of drug dosing, which provides a timeframe for maximum risk of the adverse event. Precautions

requiring strict sun protection are essential, including sunscreens which block UVA as one

patient experienced photosensitivity after sitting behind glass, which absorbs up to 97% of

UVB. Importantly, adherence to these measures for 72 hr after drug dosing prevented

recurrence of these events in all patients and allowed continuing treatment for prolonged




14

periods. Similar toxicities have also been seen with drugs such as the BRAF inhibitor

vemurafenib and it has been possible to continue their use with appropriate supportive care and

without dose reduction (16, 17), as noted in our study.

In summary, in this first-in-human, first-in-class study we have determined a MTD for CX-

5461 in patients with advanced hematological cancers and demonstrated tolerability 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 during future development of this agent. This data provides 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 unexplored targeted therapeutic approach.

Methods

Patient Selection

Patients were eligible for participation in the study, if they had any measurable, relapsed or

refractory advanced hematological malignancy, without any standard therapeutic options

available, aged ≥ 18 years, with adequate organ and bone marrow function, an Eastern

Cooperative Oncology Group (ECOG) performance status of 0-2 at screening and life

expectancy ≥ 3 months. Adequate organ and bone marrow function are defined by the

following: organ, creatinine clearance greater than 50 ml/min, 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 x 109/L without the use of

GCSF for 7 days prior to Day 1 and platelets ≥ 100 x 109/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 (CNS) 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 fungal infection. All subjects provided

written informed consent prior to trial enrolment. The trial protocol was approved by the

Institutional Review Board and the trial was conducted in accordance with the Good Clinical




15

Practice (GCP) guidelines and the ethical principles outlined in the Declaration of Helsinki and

the International Conference on Harmonisation.

Study Design and Objectives

This was a single centre, first-in-human, phase I, open-label, dose escalation study designed to

establish the safety, pharmacokinetic (PK) and pharmacodynamic (PD) characteristics of CX-

5461 in patients with advanced hematological malignancies. CX-5461 was administered as a 1

hr intravenous infusion on day 1 of each 21-day cycle. Dose 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 dose-limiting toxicities of CX-5461.

Patients remained on trial until disease progression, significant toxicity 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 PK and PD profile of CX-5461, investigate any

preliminary clinical effects on tumor response, and to identify predictive biomarkers of efficacy.

The secondary endpoints were assessment of grade 3+ adverse events, overall response (OR)

and determination of the pharmacokinetic profile of CX-5461.

Pharmacokinetic Sampling and Analysis

Serial 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; 15 and 30

minutes during infusion; immediately upon completion of infusion; and then at 15 minutes, 30

minutes, 1 hr, 2 hr, 4 hr, 6 hr and 8 hr following dose administration. Subsequently, all patients

returned to the clinic on Days 2, 3, and 4, for additional blood sample collections at 24, 48 and

72 hr following Day 1 treatment. Following collection, samples were shipped frozen to CPR

Pharma Services Pty Ltd, Thebarton, Australia where analysis of all PK parameters was

performed. Parameters calculated and reported based on 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 (AUC). The pharmacokinetic population consisted of patients who received at least 1

intravenous dose of CX-5461 and who had evaluable pharmacokinetic data from plasma.

Correlative Sampling and Pharmacodynamic Analysis




16

To confirm on-target drug activity and identify predictive biomarkers of therapeutic response,

all patients were asked to provide peripheral blood samples at baseline and then at 1, 4, 8 and

24 hr post day 1 infusion. In those patients that had biopsy-accessible disease, tumor tissue

specimens (from bone marrow, lymph nodes, liver, skin lesions) were obtained prior to

treatment and 24 hr 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 fluorescent in situ hybridization 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 hybridisation-based next generation sequencing panel was used to identify

sequence variants in 79 genes following extraction of DNA from available tumor samples (see

Supplementary Methods for extended methods). All correlative samples in this study were

collected, de-identified and processed according to a protocol specified standard operating

procedure (see Supplementary Methods for details).

Statistical Analysis

All statistical analyses were performed in SAS Analytics Software (version 9.3; SAS Institute,

Inc.). Demographics, baseline characteristics, pharmacokinetic parameters and clinical

laboratory evaluations were summarized with descriptive statistics. For analysis of

pharmacodynamic response during drug treatment, levels of Pol I transcription inhibition were

analysed by calculating the median percentage change in FISH signal intensity from each

patient’s baseline measurement.

Disclosure of Potential Conflicts of Interest

J. Soong is Chief Medical Officer at Senhwa Biosciences, Inc. John Lim has stock ownership

in Senhwa Biosciences, Inc. R.D. Hannan is a Chief Scientific Advisor to Pimera, Inc. No

potential conflicts of interest were disclosed by the other authors.

Authors’ Contributions

Conception and design: A. Khot, G.A. McArthur, R.B. Pearson, R.D. Hannan, G. Poortinga,

S.J. Harrison




17

Development of methodology: A. Khot, N. Brajanovski, D.P. Cameron, N. Hein, E. Sanij, E.

Link, P. Blombery, E.R. Thompson, G.A. McArthur, R.B. Pearson, R.D. Hannan, G. Poortinga,

S.J. Harrison

Acquisition of data (provided animals, acquired and managed patients, provided

facilities, etc.): A. Khot, N. Brajanovski, D.P. Cameron, J. Lim, P. Blombery, E.R. Thompson,

A. Fellowes

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational

analysis): A. Khot, N. Brajanovski, D.P. Cameron, K.H. Maclachlan, E. Link, P. Blombery,

E.R. Thompson, A. Fellowes, R.B. Pearson, R.D. Hannan, G. Poortinga, S.J. Harrison

Writing, review, and/or revision of the manuscript: N. Brajanovski, A. Khot, K.H.

Maclachlan, E. Sanij, J. Soong, P. Blombery, E.R. Thompson, A. Fellowes, K.E. Sheppard,

R.B. Pearson, R.D. Hannan, G. Poortinga, S.J. Harrison

Administrative, technical, or material support (i.e., reporting or organizing data,

constructing databases): E. Link,

Study supervision: R.B. Pearson, R.D. Hannan, G. Poortinga, S.J. Harrison

Acknowledgments

We 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.

Grant Support

This work was supported by the National Health and Medical Research Council (NHMRC) of

Australia project grants (#1038852), Cancer Council Victoria (CCV) grants-in-aid (#1084545

and 1100892) and the Peter MacCallum Cancer Foundation. Senhwa provided financial support

with respect to drug supply and pharmacokinetic studies. Researchers were funded by NHMRC

Fellowships (G.A.M., R.B.P., R.D.H.) and the Snowdome Foundation fellowship funding

(AK).




18

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13. Poortinga G, Quinn LM, Hannan RD. Targeting RNA polymerase I to treat MYC-driven cancer. Oncogene. 2015;34:403–12.




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14. Negi SS, Brown P. rRNA synthesis inhibitor, CX-5461, activates ATM/ATR pathway in acute lymphoblastic leukemia, arrests cells in G2 phase and induces apoptosis. Oncotarget. 2015;6:34846-58.

15. Quin J, Chan KT, Devlin JR, Cameron DP, Diesch J, Cullinane C, et al. Inhibition of RNA polymerase I transcription initiation by CX-5461 activates non-canonical ATM/ATR signaling. Oncotarget. 2016;7:49800–18.

16. Hein N, Cameron DP, Hannan KM, Nguyen N-YN, Fong CY, Sornkom J, et al. Inhibition of Pol I transcription treats murine and human AML by targeting the leukemia-initiating cell population. Blood. 2017;129:2882–95.

17. Lee HC, Wang H, Baladandayuthapani V, Lin H, He J, Jones RJ, et al. RNA Polymerase I Inhibition with CX-5461 as a Novel Therapeutic Strategy to Target MYC in Multiple Myeloma. Br J Haematol. 2017;177:80–94.

18. Burger K, Mühl B, Harasim T, Rohrmoser M, Malamoussi A, Orban M, et al. Chemotherapeutic drugs inhibit ribosome biogenesis at various levels. Journal of Biological Chemistry. 2010;285:12416–25.

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20. Drygin D, Lin A, Bliesath J, Ho CB, O'Brien SE, Proffitt C, et al. Targeting RNA polymerase I with an oral small molecule CX-5461 inhibits ribosomal RNA synthesis and solid tumor growth. Cancer research. 2010 ed. 2011;71:1418–30.

21. Haddach M, Schwaebe MK, Michaux J, Nagasawa J, O'Brien SE, Whitten JP, et al. Discovery of CX-5461, the First Direct and Selective Inhibitor of RNA Polymerase I, for Cancer Therapeutics. ACS Med Chem Lett. 2012;3:602–6.

22. Devlin JR, Hannan KM, Hein N, Cullinane C, Kusnadi E, Ng PY, et al. Combination Therapy Targeting Ribosome Biogenesis and mRNA Translation Synergistically Extends Survival in MYC-Driven Lymphoma. Cancer Discov. 2016;6:59–70.

23. Maclachlan KH, Cuddihy A, Hein N, Cullinane C, Harrison SJ, Hannan R, et al. Novel Combination Therapies with the RNA Polymerase I Inhibitor CX-5461 Significantly Improve Efficacy in Multiple Myeloma. Blood. New York: Saunders; 2017;130:1805.

24. Hilton J, Cescon DW, Bedard P, Ritter H, Tu D, Soong J, et al. 44OCCTG IND.231: A phase 1 trial evaluating CX-5461 in patients with advanced solid tumors. Annals of Oncology. 2018;29.

25. Rebello RJ, Kusnadi E, Cameron DP, Pearson HB, Lesmana A, Devlin JR, et al. The Dual Inhibition of RNA Pol I Transcription and PIM Kinase as a New Therapeutic




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21

Table Legends:

Table 1. Patient demographics and baseline characteristics: The clinical and pathologic

features of 16 patients with advanced hematological malignancies who received CX-5461

therapy.

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 (adverse events ordered

by frequency of grade 3 events, alphabetical by term)

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




22

Figure Legends

Figure 1. Individual patient duration on study, graphed as a function of treatment

duration (cycles completed). The plot legend depicts the colours 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 dose-limiting toxicity (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 abbreviation as follows: ALCL, anaplastic large cell

lymphoma; DLBCL, diffuse large B-cell lymphoma; MM, multiple myeloma; HL, Hodgkin’s

lymphoma; CTCL, cutaneous T-cell lymphoma; T-PLL, T-cell prolymphocytic leukemia; CLL,

chronic lymphocytic 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.

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 peripheral blood mononuclear

cells (PBMCs) acquired from 5 representative patients from cohorts 1-5 treated with CX-5461.

Samples were collected prior to treatment and at the 1 hr, 4 hr, 8 hr and 24 hr time points post

CX-5461 infusion in cycle 1. Images were taken with a 60x objective, with a merged overlay

of DAPI stained nuclei (blue) and labelled Cy3 5’ETS probe (red) shown (scale bar = 10µm).

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.

Figure 3. CX-5461 displays on-target rDNA transcription inhibition in paired tumor

biopsy specimens and MACS isolated tumour cells. Needle core biopsies of tumour tissue

were collected from patients with accessible tumors (n = 11) representing cohorts 1-5, pre-

treatment and 24 hr post CX-5461 administration (A, B). Samples were formalin-fixed and

paraffin-embedded 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 tumours (tumor tissue as indicated). Images were taken at 60x




23

magnification and a merged overlay of DAPI stained nuclei (blue) and labelled Cy3 5’ETS

probe (red) is shown in each case (scale bar = 10µm). B, Quantitative analysis of rDNA

transcription inhibition in tumour biopsy specimens (tumor tissue as indicated; bone marrow

trephine (BM)), expressed as a median percentage change in FISH signal intensity from

baseline in patients achieving either stable disease (SD), partial response (PR), radiologic

response (RR) or progressive disease (PD). 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, D). C, In situ detection of 5’ETS pre-rRNA transcript in

MACS sorted tumour cells acquired from 4 patients treated with CX-5461. Representative

images were taken at 60x magnification and a merged overlay of DAPI stained nuclei (blue)

and labelled Cy3 5’ETS probe (red) is shown in each case (scale bar = 10µm). 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 hr (yellow) and 24 hr (green) post CX-

5461 infusion. Spot intensity was measured using a pipeline solution developed in Definiens

Tissue Studio® 3.6. Patient disease abbreviated as in Figure 1: DLBCL, diffuse large B-cell

lymphoma; CLL, chronic lymphocytic leukemia; MM, multiple myeloma; T-PLL, T-cell

prolymphocytic leukemia.

Figure 4. Antitumor Activity observed with CX-5461 Treatment. PMC-17, diagnosed with

anaplastic large cell lymphoma (ALCL), had a partial response in cutaneous disease and

remained on trial for the longest duration (18 cycles) (A, B). A, Clinical photographs of the

right lateral thigh/knee, taken pre-treatment 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, pre-treatment (red) and at the 4 hr (yellow) and 24 hr (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 60x

magnification and a merged overlay is shown in each case (scale bar = 10µm). PMC-05,

diagnosed with cutaneous T-cell lymphoma (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 (right) and 18F- FDG-PET scans (left) 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




24

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, pre-treatment (red) and 24 hr

(green) post CX-5461 treatment. Representative images were taken at 60x magnification and a

merged overlay of DAPI stained nuclei (blue) and labelled Cy3 5’ETS probe (red) is shown in

each case (lower panel) (scale bar = 10µm). E, Immunohistochemistry staining (20x) showing

robust nuclear accumulation of p53, 24 hr post CX-5461 therapy. F, Immunoblot analyses of

total p53 and p21 protein levels in tumour tissue lysates extracted from the scalp lesion of PMC-

05 (TP53 wildtype tumor status), prior to and 24 hr post CX-5461 infusion. Equal amounts of

protein from each sample were probed and ß-actin was used as a loading control.




Table 1. Patient demographics and baseline characteristics: The clinical and pathologic features of 16 patients with advanced hematological malignancies who received CX-5461 therapy.

Demographic Parameter Study 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 (%) Wildtype 9 (56) Mutant 4 (25) Unknown 3 (19)

Tumour Type, n (%) DLBCL 4 (25) Hodgkin’s Lymphoma 2 (12) CLL – Richter’s Transformation 1 (6) Multiple Myeloma T-cell LPD

6 (38) 3 (19)

Median Prior Lines of Therapy, n (range) 7 (1-14)

Dose Level (mg/m2), n (%) 25 3 (19) 50 4 (25) 100 4 (25) 170 3 (19) 250 2 (13)

Abbreviations: DLBCL, Diffuse Large B-Cell Lymphoma; CLL, Chronic Lymphocytic Leukemia; LPD, Lymphoproliferative Disorder; ECOG, Eastern Cooperative Oncology Group




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 (adverse events ordered by frequency of grade 3 events, alphabetical by term)

Adverse event 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

N N % N N % N N % N N % N N % N N %

Anemia 4 (3*) 2 13% 1 . . 2(2*) 2 50% 1(1*) . . . . . . . . Abdominal Pain 1 1 6% . . . 1 1 25% . . . . . . . . . Atrial Fibrillation 1 1 6% . . . 1 1 25% . . . . . . . . . Blood Bilirubin Increased 1 1 6% 1 1 33% . . . . . . . . . . . . Cardiac Disorders 3(1*) 1 6% . . . 2(1*) . . . . . . . . 1 1 50% Creatinine Increased 2 1 6% . . . 1 1 25% 1 . . . . . . . . Diarrhea 2 1 6% . . . 1 1 25% 1 . . . . . . . . Erythroderma 1(1*) 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*) 1 6% 2 . . . . . 1 . . 1 1 33% . . . Palmar-Plantar Erythrodysesthesia Syndrome 2(2*) 1 6% . . . . . . . . . . . . 2 1 50%

Photosensitivity 8(8*) 1 6% 2 1 33% . . . 2 . . 2 . . 2 . . Platelet Count Decreased 4(4*) 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*) 1 6% 1 1 33% . . . . . . . . . . . . * indicates patients experiencing AEs possibly, probably and definitely 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.

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

Single CX-5461 dose, mg/m2

PK Parameter 25 (n = 3) 50 (n = 4) 100 (n = 4) 170 (n = 3) 250 (n = 2)

Tmax, median, (Range), hr

1.00 (0.98-1.00)

0.75 (0.25 -1.25)

0.50 (0.37-1.00)

0.52 (0.42-1.00) 1.00

Cmax, mean (SD), (ng/mL)

297 (46)

384 (174)

636 (235)

1,707 (455)

1,358 (526)

AUC0–t, mean (SD), (hr*ng/mL)

2,057 (470)

3,646 (2,222)

10,146 (2,494)

16,792 (4,761)

27,147 (439)

AUC0-∞, mean (SD), (hr*ng/mL)

2,297 (516)

4,587 (2263)

12,152 (3,482)

17,943 (5,187)

28,612 (1006)

t1/2, mean (SD), hr* 23.2 (1.6)

39.8 (16.9)

58.4 (12.9)

45.5 (5.7)

83.3 (12.9)

NOTE: PK parameters were reported as cohort mean (SD), except for Tmax, which was reported as cohort median (range). *Residual drug was detectable in patients in cohort 5 at day 15 post dose. Abbreviations: Cmax, Maximum concentration recorded; AUC0–t, Area Under the Curve from time 0 to last quantifiable concentration; AUC0–∞, Area Under the Curve from time 0 extrapolated to infinity; SD, standard deviation; Tmax, time to reach Cmax




0 5 10 15 20

Treatment Duration (Cycles Completed)

Patie

nt ID

A.

Figure. 1

ATM

ATM

ALCL

DLBCL

DLBCL

MM

MM

MM

DLBCLHL

T-PLLMM

DLBCL

MM

MM

HLCLL

CTCL

TP53

TP53

TP53

TP53

TP53

N/A

N/A

N/A

SD:PD:

PR:

RR:DLT:DR:

PMC-04PMC-06PMC-07PMC-09PMC-10PMC-16PMC-05PMC-11PMC-12PMC-13PMC-03PMC-14PMC-15PMC-01PMC-08PMC-17




Figure. 2

% C

hang

e in

5’E

TS S

igna

l Int

ensi

ty F

rom

Bas

elin

ePost CX-5461 Treatment

Cohort 125 mg/m²

Cohort 250 mg/m²

Cohort 3100 mg/m²

Cohort 4170 mg/m²

Cohort 5250 mg/m²

A.

B.

Cohort 125 mg/m²

Cohort 5250 mg/m²

Cohort 4170 mg/m²

Cohort 250 mg/m²

Cohort 3100 mg/m²

PMC-

01PM

C-06

PMC-

10PM

C-13

PMC-

17Baseline 1 hr 4 hr 8 hr 24 hr

DAPI / ETS

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

Post Infusion

Peripheral Blood Mononuclear Cells: Cohort 1-55’ETS Signal Intensity Post Cycle 1 of CX-5461 Treatment




A.

Figure. 3

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%

-50%

0%

50%

150%

200%

24 hr 4 hr

SD PD PD PD

PMC-13170 mg/m²

PMC-10100 mg/m²

PMC-0750 mg/m²

PMC-0125 mg/m²

% C

hang

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5’E

TS S

igna

l Int

ensi

ty

From

Bas

elin

e

D.

��PE T�mo�r Tiss�e Sections� Co�orts 1-55’ETS Signal Intensity 24 hr Post CX-5461 Treatment

% C

hang

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5’E

TS S

igna

l Int

ensi

ty

From

Bas

elin

e

MACS Sorted Bone Marrow - Malignant Cells5’ETS Signal Intensity Post CX-5461 Treatment

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


100 mg/m²


170 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/m²100 mg/m²

25 mg/m²

170 mg/m²250 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: Lymph Node

PMC-15: Lymph Node

PMC-17: Skin Lesion

-80%

-60%

-40%

-20%

0%

20%

40%

60%

80%

100%

120%

SD

PD

RR

RR

PD PDSD

PD

PD

PD

PD

SD

PR

Dose Level




Tota

l rR

NA

-FIS

H S

igna

l / N

ucle

us (a

.u)

Baseline

24 hr (+)

0.0

DAPI / ETS

C. D.

E. F.

Digital Image ��F-FDG-PET Scan

PMC-05: CTCLCutaneous Lesion - Left Scalp

Baseline 24 hr Post CX-54617 Days 22 Days

Bas

elin

ePo

st C

ycle

1(5

0 m

g/m²)

p53IHC

(20x)

A.

Baseline4 hr (+

)

24 hr (+)

B.

0.0

Tota

l rR

NA

-FIS

H S

igna

l / N

ucle

us (a

.u)

DAPI / ETS

Baseline 24 hr Post CX-5461

Figure. 4

2.0 x 10�

1.5 x 10�

1.0 x 10�

1.5 x 10�

1.0 x 10�

5.0 x10�

5.0 x10�

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

ß-Actin

Scalp LesionTP53 Wildtype

Base 24 hr




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 Hematological Cancers: First-in-Human RNA Polymerase I Transcription Inhibitor

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