A selective CDK12/13 non-covalent inhibitor with potent anti-breast cancer activity Victor Quereda1, Simon Bayle1, Francesca Vena1, Andrii Monastyrskyi2, William R. Roush2 and Derek Duckett1 Dec 4-8, 2018 1Moffitt Cancer Center and Research Institute, Tampa, FL. 2The Scripps Research Institute, Jupiter, FL.

Abstract Selective silencing or inhibition of CDK12/13 activity downregulates Inhibition of CDK12/13 blocks tumor progression in PDX models of the expression of certain DNA damage response TNBC and provokes enhanced efficacy over single agent treatment -dependent (CDKs)12 and 13 are Ser/Thr protein and members of the CDK family of when dosed in combination with selected DNA damaging agents . CDKs are regulated by and certain members of this family have been characterized as key regulators of the cell cycle. Other members - CDK7, CDK8, CDK9, CDK12 and CDK13 - have been A B CRISPR v2 Cdk12 Cdk13 12+13 A B described as transcription-regulating kinases. CDK12 and CDK13 phosphorylate the C-terminal domain Comp. A 30nM Comp. A 90nM A BCM-4013 PDX C Cdk12 Vehicle Comp. A Cisplatin Combo 1.5 231 Control 6hr Time (hr) 0 4 8 24 0 4 8 24 (CTD) of rpb1 (the largest subunit of RNA polymerase II), and interact with RNA processing factors, playing 231 Comp. A 30nM 6hr 1000 Cdk13 pSer2 CTD 60 pivotal roles controlling transcription initiation, elongation, and co-transcriptional RNA processes. 231 Comp. A 90nM 6hr Vehicle ** pSer2 CTD pSer5 CTD 800 Comp. A Cisplatin * 40 ** Surprisingly, CDK12 and CDK13 have been shown to control a subset of genes. Within this subset, CDK12 100 1.0 *** pSer7 CTD 3 600 Combo Ki67 *** CTD * 20 acts as the limiting factor for the transcription of genes important in the response to DNA damage and ** *** CTD mm 400 ATM ** *** 50 *** *** *** *** 0 CDK13 controls the expression of genes involved in growth signaling pathways. Notably, CDK12 and 0.5 *** ** *** ATM 200 cells Positive % V A Ci Co *** Rad51 *** *** CDK13 have been shown to be promising therapeutic targets for several cancer types. We have generated a *** *** Rad51 0 ** *** % proliferation % 10 *** 0 γH2AX 0 20 40 60 novel, selective, and orally bioavailable small molecule inhibitor of CDK12 and CDK13. Our lead molecule γH2AX Day Relative mRNA expression mRNA Relative 8 CRISPR v2 Cdk12 Cdk13 12+13 0.0 downregulates key DNA damage repair proteins including ATM, ATR, Brca1 and Rad51 leading to induction cParp cParp 6 B γH2AX * 4 * of apoptotic cell death in all triple negative breast cancer (TNBC) cell lines tested. We have confirmed the GAPDH GAPDH 100 2 0 selectivity of our compound by kinase profiling and by comparing the expression profile by RNA-seq of (A) Clonogenic growth of MDA-MB-231 cells infected with CRISPR/Cas9 80 cells Positive % (A) qPCR analysis of indicated genes, 6 hr. after administration of MDA- V A Ci Co cells treated with compound or infected with CRISPR sgRNA targeting CDK12 and/or CDK13. Importantly, tumors 60

lentivirus containing sgRNA to CDK12, CDK13 or control. ***p < 0.001 3 Vehicle 30 MB-231 cells with the indicated concentrations of Comp. A or vehicle. ** our compound acts in synergy with the chemotherapeutic agents Cisplatin, Irinotecan and Olaparib. Finally, in 40 Comp. A by t-test. Cisplatin 20 *p<0.05, **p<0.01, ***p<0.001 by t-test. 20 preclinical efficacy studies, our lead compound blocked tumor progression in two patient derived xenograft (B) Immunoblot analysis of total protein derived form MDA-MB-231 cells Combo C3A (B) Immunoblot and analysis of protein expression level after treatment of 1000mm *** infected with CRISPR/Cas9 lentivirus containing sgRNAs to CDK12, 0 10 *** (PDX) models of TNBC and demonstrated tumor regression and enhanced efficacy over single treatment MDA-MB-231 cells with Comp. A at the selected time points. less Percentwith mice 0 20 40 60 CDK13 or control (v2). Day 0 when dosed in combination with selected chemotherapeutic agents. cells Positive % V A Ci Co

D BCM-3887 PDX F Vehicle Comp. A Irinotecan Combo 100 ** 1000 * Vehicle 80 CDK12/13 inhibition synergizes with chemotherapeutics agents 800 Comp. A 60 Irinotecan * Ki67 40 3 600 Combo Selective inhibition of CDK12/13 kills TNBC Cells 20

promoting TNBC cell death through downregulation of DDR proteins mm 400 0

% Positive cells Positive % V A Ir Co 200 ** 4 0 *** 0 10 20 30 40 50 3 *** A B C A B C F Day 2 Kinase Kd (nM) Cisplatin Olaparib Irinotecan Hoechst Brca1 γH2AX Merge E γH2AX ** CDC2L5 4.9 1:1 Cispl:CompA CI<0.3 1:1 Olap:CompA CI<0.1 1:1 Irino:CompA CI<0.2 1 120 4:1 Cispl:CompA CI<0.3 120 4:1 Olap:CompA CI<0.5 120 4:1 Irino:CompA CI<0.1 100 CDK12 98 100 1:4 Cispl:CompA CI<0.2 100 1:4 Olap:CompA CI<0.1 100 1:4 Irino:CompA CI<0.1 0 Vehicle cells Positive % CDK4-cyclinD1 >10000 80 80 80 80 V A Ir Co 60 24 hr. 60 60 60

CDK6 5100 tumors 40 40 40 15 3 Vehicle ** CDK7 >10000 40 20 20 20 Comp. A *** CDK9 >10000 0 0 0 Cisplatin Irinotecan 10

-8 -6 -4 -2 20 % DMSO control DMSO% % DMSO control DMSO% -8 -6 -4 -2 Combo *** GSK3A 1200 control DMSO% -8 -6 -4 -2 50µM C3A

1000mm 5 GSK3B 810 [M] Cisplatin [M] Olaparib [M] Irinotecan 0 24 hr. 0 20 40 60 ** 120 less Percentwith mice Day 0 D 100 cells Positive % V A Ir Co Comp. A Comp. A 120 80 D DNA DAMAGE/ E 100 90nM Comp. A 60 REPLICATION ERRORS Control Cisplatin 50µM Comp. A 90nM Combo Control Tumor volume measurements of BCM-4013 PDX (A) or BCM-3887 PDX (D) treated with the indicated drug; Comp. A (p.o. 20mg/kg daily 5days/week), 80 24 hr. 60 40 Time (hr) 0.5 0.5 6 24 48 0.5 6 24 48 0.5 6 24 48 48 Cisplatin (IP 6mg/kg once a week, 2 consecutive treatments), Irinotecan (IP 50mg/kg single dose), combination, or vehicle (n ≥ 8 per arm). 40 H2AX 20 pSer2 CTD *p < 0.05, **p < 0.01, ***p<0.001 by t-test. (B and E) Kaplan-Meyer curves for experiment described in (A or D) presenting percentage of animals with 20 Control DMSO% Combo 3 0 0 tumors smaller than 1000mm at the indicated day. (E and F) Representative immunohistochemistry images of selected proteins in mice treated with pSer2 CTD/CTD ratio CTD/CTD pSer2 24 hr. % DMSO control DMSO% pSer5 CTD -10 -8 -6 -4 -9 -8 -7 -6 -5 -4 HR the indicated drug or vehicle at the end of the study. Scale bar represent 40µm. Quantification of immunohistochemistry images is plotted on the right. [M] [M] CTD *p < 0.05, **p < 0.01, ***p<0.001 by t-test. ATM 50 (A) Kinase tree representing inhibition of comp. A at 10µM, over CtIP ATM G EXO1 RPA % + γH2AX Legend: ATR PARP 40 % + γH2AX pan-nuclear 450 kinases were tested. MRN BRCA1 Inh = 40-60% Rad51 % + γH2AX/Brca1 Inh = 60-80% (B) Compound binding selectivity values (Kd) for kinases with 30 Inh = 80-90% RAD51 RAD52 γH2AX 20 Inh = 90-100% measurable binding affinity (DiscoverX). BRCA2 Conclusions (C) In-Cell Western Blot of MDA-MB-231 cells treated with of cells % 10 End Synthesis cParp increasing concentrations of comp. A for 4 hr. and stained with processing repair 0 Cispl (µM) 0 50 0 50 0 50 0 50 GAPDH E MDA-MB-231 F pSer2 CTD antibody (green) and total CTD (red). Top panel Comp. A (nM) 0 0 90 90 0 0 90 90 • We have developed a potent, selective and orally bioavailable, inhibitor targeting CDK12 and CDK13. MDA-MB-436 represents pSer2 and total Ser2 merged image. Bottom panel – HS578T 6 hr. 24 hr. 150 120 MDA-MB-468 concentration response curve. • Our CDK12/CDK13 inhibitor downregulates DNA damage repair proteins. 100 (D) In vitro ADP-glo® kinase assay. Effect of increasing the (A-C) Effect on cell proliferation after treatment (72 hr.) of MDA-MB-231 cells with the indicated drugs alone or in combination with Comp. A. Average of 100 80 concentration of Comp. A on the phosphorylation of RNApol II Chou-Talalay synergy index (CI) values are indicated. (D) Schematic representation of Homologous Recombination (HR) repair process after a double strand • Comp. A replicates the effect of a CRISPR targeting to CDK12 and CDK13, consistent with on target action. 60 break has been generated in the DNA. The main proteins involved are indicated and proteins downregulated in our system are in red. (E) Immunoblot analysis 50 CTD repeat by Cdk12. 40 of indicated proteins derived from MDA-MB-231 cells after treatment with Cisplatin, Comp. A or the combination. Representative images (F) and quantification 20 *** *** (E) Anti-proliferative activity of Comp. A in the indicated cell lines

% proliferation % • CDK12/CDK13 inhibition acts in synergy with various DNA damaging chemotherapeutic agents. % DMSO control DMSO% 0 0 as measured by Celltiter-Glo® for 72 hr. (G) of MDA-MB-231 cells treated with Cisplatin, Comp. A or the combination for the indicated times. +γH2AX depict cells with more than ten γH2AX foci/cell. -10 -8 -6 -4 0 10 30 90 +γH2AX pan-nuclear are cells in which the intensity of the signal surpass a threshold selected for each experiment based on qualitative observations. [M] Comp. A [nM] Comp. A (F) Clonogenic growth of MDA-MB-231 cells in the presence of • Comp. A (20mg/kg, PO) significantly decrease tumor growth in vivo and greatly synergizes with chemotherapeutic agents +γH2AX/+Brca1 are cells with more than ten γH2AX and more than ten Brca1 foci detected in the cell. Scale bar represents 10µm. comp. A or vehicle. ***p<0.001 by t-test. resulting in fulminant in two PDX orthotopic TNBC models, resistant to current treatment options. This presentation is the intellectual property of WRR/ TSRI and DD/MCC. Contact [email protected] for reprint and/or distribution.