The androgen receptor (AR) is a well-established oncogenic driver of prostate cancer, and AR-targeting hormone therapy has proven an efficacious treatment option for patients with metastatic prostate cancer. Due to the importance of AR in prostate cancer disease progression, we leveraged small molecule microarray (SMM) screening technology to identify compounds that bind to transcriptional complexes of a common AR splice-variant, AR-V7, present along with AR in castration-resistant prostate cancer cell lysates. A subset of SMM hits were identified to modulate the AR Transcription Regulatory Network (TRN) in hormone-sensitive, but not castration-resistant prostate cancer models using a functional cell-based transcription signature approach. Several AR SMM hits directly engaged the AR ligand binding domain in a biophysical assay. One of these AR SMM hits altered AR nuclear translocation and antagonized steroid receptor co-activator recruitment to AR but did not antagonize the glucocorticoid receptor. Leveraging structural data for known AR ligands, we propose a computational model for binding of this molecule to the known AR ligand binding domain. Together, the data demonstrates that the AR SMM lysate screen successfully identified structurally novel small molecules that modulate the AR TRN and bind the AR ligand binding domain. Citation Format: Marek J Kobylarz, Michelle G Shum, Wayne L Glore, Ava Saffren, Shannon Carpenter, Mulini Pingili, Andrew Clay, Tamara D Hopkins, Hua Gao, Anna C Schinzel, Marius S Pop, Nikolaus D Obholzer, David B Freeman, Christopher J Dinsmore, Benjamin W Trotter, Charles Y Lin, Peter B Rahl. Small molecule microarray screening identifies novel androgen receptor ligands [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A057.

Abstract MYC is the most frequently amplified gene in human cancers and one of the most sought-after drug targets for cancer therapy. Its function as a transcription factor is essential for its oncogenic potential. However, development of small molecules that target oncogenic MYC function in cells has been intractable due to its lack of known ligand binding sites. To overcome these structural challenges, we leveraged Kronos Bio’s small molecule microarray (SMM) screening platform to identify small molecules that can bind to MYC transcriptional complexes in cell lysates from cancer cells with deregulated MYC function. Unbiased transcriptional signature-based profiling identified multiple SMM hits that modulated the MYC transcription regulatory network (TRN) in MYC-dependent cancer cell lines. One of the SMM hits mimicked signature changes due to MYC loss of function, which were also similar to signature changes effected by BET bromodomain inhibitors. Biochemical, biophysical, and structural analyses found that the SMM hit could directly engage with BET bromodomains in purified systems. Cell-based proximity labeling demonstrated binding to BET proteins in live cells, and comparative pharmacology approaches suggested that the BET protein engagement was likely driving the observed gene expression changes. These results demonstrate the utility of the SMM platform in identifying ligands that modulate transcription factor TRNs through binding to critical cofactors. Citation Format: Emily B Cohen, Zhihua Ma, Wayne L Glore, Oleg A Volkov, Tong Liang, Nikolaus D Obholzer, Andrew C Clay, Mulini Reddy Pingili, Yupeng Zheng, Jun Li, Shi Yun Wang, Tom Chen, David B Freeman, Hua Gao, Minyun Zhou, Priscilla Cheung, Tamara D Hopkins, Marius S Pop, Christopher J Dinsmore, B. Wesley Trotter, Charles Y Lin, Peter B Rahl. Small molecule microarray lysate screen identifies bromodomain ligands that target the MYC transcription regulatory network [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A059.

Transcriptional deregulation is a hallmark of many cancers and is exemplified by genomic amplifications of the MYC family of oncogenes, which occur in at least 20% of all solid tumors in adults. Targeting of transcriptional cofactors and the transcriptional cyclin-dependent kinase (CDK9) has emerged as a therapeutic strategy to interdict deregulated transcriptional activity including oncogenic MYC. Here, we report the structural optimization of a small molecule microarray hit, prioritizing maintenance of CDK9 selectivity while improving on-target potency and overall physicochemical and pharmacokinetic (PK) properties. This led to the discovery of the potent, selective, orally bioavailable CDK9 inhibitor 28 (KB-0742). Compound 28 exhibits in vivo antitumor activity in mouse xenograft models and a projected human PK profile anticipated to enable efficacious oral dosing. Notably, 28 is currently being investigated in a phase 1/2 dose escalation and expansion clinical trial in patients with relapsed or refractory solid tumors.

KB-0742 is an orally available, potent, and selective inhibitor of cyclin-dependent kinase 9 (CDK9). Sensitivity profiling across a panel of 800 adherent and suspension immortalized pan-cancer cell lines using the Broad Institute PRISM platform had previously demonstrated MYC amplification as being a driver of KB-0742 sensitivity. We sought to further determine the activity of KB-0742 in breast cancer cell lines, patient-derived cell and organoid cultures, and patient-derived xenograft (PDX) models. KB-0742 decreased the viability of cell lines derived from primary and metastatic breast cancers, including triple-negative breast cancers (TNBCs). In a panel of 17 breast cancer cell lines, including 2 human epidermal growth factor receptor 2-positive (HER2+), 7 estrogen-receptor positive (ER+), 1 HER2+/ER+ and 7 TNBCs, the effect of KB-0742 on viability was either cytostatic or cytotoxic over the duration of the culture period. KB-0742 was also tested in patient-derived cell lines derived from primary and metastatic tumors including TNBC, and in 2 TNBC MYC-high expressing patient-derived organoid cultures. KB-0742 demonstrated cytostatic and cytotoxic effects on cell growth and superior inhibitory effects on cell growth in the TNBC organoid cultures as compared with paclitaxel and gemcitabine. Since sensitivity to KB-0742 was observed in breast cancer cell lines and patient-derived models, including TNBCs, we evaluated efficacy and target engagement in several MYC-high expressing TNBC PDX animal models. Treatment with KB-0742 using an intermittent dosing schedule (3-days on/4-days off) was well tolerated and resulted in anti-tumor activity comparable to that observed with standard of care chemotherapeutic agents. Tumors collected at 2 and 8 hours post-final dose displayed a decrease in phosphorylated serine residue 2 (pSER2) in the C-terminal domain of RNA polymerase II (RNA pol-II) and decreased MYC protein levels consistent with CDK9 inhibition. These data demonstrate the efficacy of KB-0742 in preclinical models of breast cancer and supports clinical testing in TNBC patients. KB-0742 is currently being evaluated in a phase I dose-escalation trial in patients with relapsed or refractory solid tumors or non-Hodgkin lymphoma (NCT04718675). Citation Format: Douglas C. Saffran, Melinda A.L. Day, Nathalie Rioux, Tom Chen, Christina Lee, Suha Naffar-Abu Amara, David B. Freeman, Tressa Hood, Charles Y. Lin, Pavan Kumar, Jorge DiMartino. Preclinical activity of KB-0742, an oral, highly selective, CDK9 inhibitor, in cell lines and in MYC-high expressing, patient-derived models of multiple breast cancer subtypes [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-05.

Abstract Transcriptional deregulation is a hallmark of many cancers, including a subset that are “transcriptionally addicted” and depend on high levels of transcription for oncogenic program genes. Although these tumors are thought to be highly susceptible to targeting of the transcriptional apparatus, their molecular definition remains challenging. To better define molecular sensitivity to transcriptional inhibition, we profiled pan-cancer sensitivity to KB-0742 — a potent, selective, and orally bioavailable small molecule inhibitor of the transcription elongation cofactor CDK9. Multiplexed sensitivity profiling across ~1,000 adherent and suspension immortalized cell lines using the Broad PRISM platform revealed lineage and molecular determinants of sensitivity. Consistent with prior reports, MYC genomic amplification emerged as a key driver of CDK9 inhibitor sensitivity, and this was especially pronounced in non-small cell lung cancer. Sensitivity to CDK9 inhibition was further observed in MYC amplified/over-expressed ex vivo primary patient tumor cell cultures and patient-derived xenografts. Analysis of the temporal kinetics of CDK9 inhibition revealed a rapid collapse of oncogenic transcription programs comprised largely of short half-life transcripts including key oncogenes such as MYC and MCL1. In CDK9 sensitive ex vivo and in vivo models, suppression of oncogenic transcription for >8 hours was followed by apoptosis. In ex vivo models, CDK9 inhibitor sensitivity was observed for both treatment naïve and heavily pretreated patient samples. For in vivo models, CDK9 inhibition on an intermittent dosing schedule achieved sustained target coverage, as evidenced by both direct readouts of CDK9 activity and corresponding transcriptional response, and ultimately resulted in sustained tumor growth inhibition in multiple solid tumor types. These data suggest that MYC genomic amplification may serve as an important feature defining sensitivity to CDK9 inhibition in patients with advanced solid tumors. Citation Format: Melinda A. Day, Nikolaus D. Obholzer, Akanksha Pandey, Tom Chen, Tong Liang, Rosa A. Villagomez, Mulini Pingili, Jost V. Koren, David B. Freeman, Holly M. Nguyen, Jennifer L. Conor, Eva Corey, Matthew G. Reese, Andrew Boghossian, Brienne Engel, Melissa M. Ronan, Jennifer A. Roth, Joesph P. Vacca, Peter B. Rahl, Marius S. Pop, Benjamin W. Trotter, Charles Y. Lin, Jorge C. DiMartino, Pavan Kumar, Douglas C. Saffran. CDK9 inhibition is selective for transcriptionally addicted tumors harboring MYC genomic amplifications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1141.

Abstract Tumorigenesis is driven by the accumulation of adverse genetic changes resulting in dysregulated transcription promoting altered gene expression and the cancer cell state. Thus, tumors can develop dependencies on the transcriptional regulators that promote the reprogrammed gene expression landscape. One such regulator is cyclin-dependent kinase 9 (CDK9), which regulates transcriptional elongation and promotes activation of transcription factors. We developed a potent, selective, and orally bioavailable CDK9 inhibitor, KB-0742. KB-0742 is highly selective for CDK9 as compared to other CDKs and has shown minimal off-target effects in kinase and receptor panel screens. Here we present the nonclinical pharmacologic characterization of KB-0742. The pharmacokinetics (PK) of KB-0742 in rats and dogs showed rapid absorption and high bioavailability with a %F of ≥84 after oral administration in rats and >100 in dogs. KB-0742 exhibited low turnover in human microsome and hepatocyte preparations and is projected to have low clearance and a high volume of distribution in humans based on allometric scaling from nonclinical species. These properties may facilitate achievement of sustained target engagement in patients using intermittent dosing which may mitigate the toxicity of CDK9 inhibition. To test this hypothesis, mice bearing MV4-11 (acute myeloid leukemia) xenografts were treated with KB-0742 at either 60 mg/kg for 3-days on/4-days off or continuous dosing at 25 mg/kg. Intermittent dosing showed similar tumor growth inhibition (81%) as seen with continuous dosing (74%). Target engagement was assessed in the tumors by measuring the inhibition of RNA polymerase II (pSER2). KB-0742 treatment resulted in an over 50% decrease in pSER2 compared with vehicle-treated controls. To support the phase I clinical trial, 2 pharmacodynamic assays were developed to measure drug target engagement in peripheral blood mononuclear cells (PBMCs). The first used RNA-expression profiling to measure alterations in gene expression with treatment, and the second used MSD protein analysis to measure changes in pSER2 levels. Ex vivo experiments using PBMCs from 3 healthy donors showed an over 80% reduction in pSER2 levels at 4 hours post-exposure to 1 µM of KB-0742. Similar results were observed using the RNA-expression profiling assay, in which decreased gene expression was observed in key genes at 1 µM exposures. KB-0742’s high specificity, oral bioavailability, and favorable PK properties distinguish it from other available CDK9 inhibitors. We are currently testing the hypothesis in the clinic with a phase I trial (NCT04718675) that the ability to dose intermittently will allow for favorable anti-tumor activity while minimizing adverse effects. Citation Format: Melinda A. L. Day, Douglas C. Saffran, Nathalie Rioux, Tom Chen, Christina Lee, David B. Freeman, Crystal MacKenzie, Joseph P. Vacca, Peter B. Rahl, Benjamin Wesley Trotter, Charles Y. Lin, Pavan Kumar, Jorge DiMartino. Preclinical pharmacokinetics and pharmacodynamics of KB-0742, a selective, oral CDK9 inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P228.

Castration-resistant prostate cancers (CRPCs) lose sensitivity to androgen-deprivation therapies but frequently remain dependent on oncogenic transcription driven by the androgen receptor (AR) and its splice variants. To discover modulators of AR-variant activity, we used a lysate-based small-molecule microarray assay and identified KI-ARv-03 as an AR-variant complex binder that reduces AR-driven transcription and proliferation in prostate cancer cells. We deduced KI-ARv-03 to be a potent, selective inhibitor of CDK9, an important cofactor for AR, MYC, and other oncogenic transcription factors. Further optimization resulted in KB-0742, an orally bioavailable, selective CDK9 inhibitor with potent anti-tumor activity in CRPC models. In 22Rv1 cells, KB-0742 rapidly downregulates nascent transcription, preferentially depleting short half-life transcripts and AR-driven oncogenic programs. In vivo, oral administration of KB-0742 significantly reduced tumor growth in CRPC, supporting CDK9 inhibition as a promising therapeutic strategy to target AR dependence in CRPC.

Abstract Castration resistant prostate cancers (CRPCs) lose sensitivity to hormone therapy, but remain dependent on oncogenic transcription programs driven by the androgen receptor (AR) and other oncogenic transcription factors such as MYC. Using small molecule microarrays (SMMs), we screened HEK293 cellular lysates for compounds binding to exogenously expressed ARv7, a mutant splice form of AR that drives castration resistance. Although transcription factors like ARv7 and MYC are considered classically undruggable, SMMs are able to identify small molecule interactors of druggable co-factors and other proteins in complex with the target protein – in this case ARv7. SMM hits were triaged for the ability to selectively inhibit an AR dependent transcriptional reporter, and also for their ability to reduce proliferation in AR dependent tumor cells. From this screen, we identified KI-ARv3, a potent and selective inhibitor of CDK9. CDK9 is a cyclin-dependent kinase (CDK) that functions primarily as a general co-factor in RNA Polymerase II (RNA Pol II) transcription elongation. CDK9 is a well-characterized and important cofactor for AR, MYC, and other oncogenic transcription factors. In prostate cancer, CDK9 has been shown to modulate and be required for AR-specific gene expression. More broadly, transcriptional CDK inhibitors including those selective for CDK9 have shown strong potential as therapeutic agents owing to their ability to selectively downregulate oncogenic transcription programs and target tumors addicted to transcription factors such as AR or MYC. However, as CDK9 also plays a global role in transcription, it is unclear whether there exists a sufficient therapeutic index for clinical benefit. Prior clinical investigation of transcriptional CDK inhibitors has also been confounded by off-target interactions with other kinases and especially other CDKs that also play important roles in transcription and the cell cycle. We found that KI-ARv3 demonstrated excellent selectivity for CDK9 versus other CDKs and kinases, and further optimization of KI-ARv3 resulted in KB-00130742, an oral bioavailable CDK9 inhibitor with a biochemical IC50 of 15nM against CDK9 and greater than 50-fold selectivity for all profiled CDKs and greater than 100-fold selectivity against cell cycle CDKs. Both KI-ARv3 and KB-00130742 exhibited potent anti-tumor activity in CRPC models, as well as other models known to be dependent on MYC-driven transcription. In 22Rv1 CRPC cells, KB-00130742 rapidly downregulated nascent transcription, and preferentially depleted short half-life transcripts and AR driven oncogenic programs. In vivo, oral administration of KB-00130742 was well-tolerated and significantly reduced tumor growth in models of CRPC and leukemia. Overall these data support CDK9 inhibition using KB-00130742 as a therapeutic strategy to target AR dependence in CRPC and oncogenic transcription in other tumor types. Citation Format: André Richters, David Freeman, Christina Lee, Florian Kabinger, Shelby Doyle, Becky Leifer, Peter Mikochik, Sajjeev Jagannathan, Jost Vrabic Koren, Kristen Karlin, Calla M. Olson, Christopher Wilfong, Charles Y. Lin, Doug Saffran, Joseph Vacca, Norbert Bischofberger, Marius Pop, Angela N. Koehler. Targeting oncogenic transcription in prostate cancer with a novel, oral bioavailable, and ultra-selective CDK9 inhibitor [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1771.

The transcription factor Max is a basic-helix-loop-helix leucine zipper (bHLHLZ) protein that forms homodimers or interacts with other bHLHLZ proteins, including Myc and Mxd proteins. Among this dynamic network of interactions, the Myc/Max heterodimer has crucial roles in regulating normal cellular processes, but its transcriptional activity is deregulated in a majority of human cancers. Despite this significance, the arsenal of high-quality chemical probes to interrogate these proteins remains limited. We used small molecule microarrays to identify compounds that bind Max in a mechanistically unbiased manner. We discovered the asymmetric polycyclic lactam, KI-MS2-008, which stabilizes the Max homodimer while reducing Myc protein and Myc-regulated transcript levels. KI-MS2-008 also decreases viable cancer cell growth in a Myc-dependent manner and suppresses tumor growth in vivo. This approach demonstrates the feasibility of modulating Max with small molecules and supports altering Max dimerization as an alternative approach to targeting Myc.