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Purpose: Cyclin-dependent kinase 9 (CDK9) is a transcriptional regulator and potential therapeutic target for many cancers. Multiple nonselective CDK9 inhibitors have progressed clinically but were limited by a narrow therapeutic window. This work describes a novel, potent, and highly selective CDK9 inhibitor, AZD4573. Experimental Design: The antitumor activity of AZD4573 was determined across broad cancer cell line panels in vitro as well as cell line-and patient-derived xenograft models in vivo. Multiple approaches, including integrated transcriptomic and proteomic analyses, loss-of-function pathway interrogation, and pharmacologic comparisons, were employed to further understand the major mechanism driving AZD4573 activity and to establish an exposure/ effect relationship. Results: AZD4573 is a highly selective and potent CDK9 inhibitor. It demonstrated rapid induction of apoptosis and subsequent cell death broadly across hematologic cancer models in vitro, and MCL-1 depletion in a dose-and time-dependent manner was identified as a major mechanism through which AZD4573 induces cell death in tumor cells. This pharmacodynamic (PD) response was also observed in vivo, which led to regressions in both subcutaneous tumor xenografts and disseminated models at tolerated doses both as monotherapy or in combination with venetoclax. This understanding of the mechanism, exposure, and antitumor activity of AZD4573 facilitated development of a robust pharmacokinetic/PD/efficacy model used to inform the clinical trial design. Conclusions: Selective targeting of CDK9 enables the indirect inhibition of MCL-1, providing a therapeutic option for MCL-1dependent diseases. Accordingly, AZD4573 is currently being evaluated in a phase I clinical trial for patients with hematologic malignancies (clinicaltrials.gov identifier: NCT03263637). See related commentary by Alcon et al., p. 761

The bromodomain and extraterminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Pharmacological targeting of BRD4 bromodomains by small molecule inhibitors has proven to be an effective means to disrupt aberrant transcriptional programs critical for tumor growth and/or survival. Herein, we report AZD5153, a potent, selective, and orally available BET/BRD4 bromodomain inhibitor possessing a bivalent binding mode. Unlike previously described monovalent inhibitors, AZD5153 ligates two bromodomains in BRD4 simultaneously. The enhanced avidity afforded through bivalent binding translates into increased cellular and antitumor activity in preclinical hematologic tumor models. In vivo administration of AZD5153 led to tumor stasis or regression in multiple xenograft models of acute myeloid leukemia, multiple myeloma, and diffuse large B-cell lymphoma. The relationship between AZD5153 exposure and efficacy suggests that prolonged BRD4 target coverage is a primary efficacy driver. AZD5153 treatment markedly affects transcriptional programs of MYC, E2F, and mTOR. Of note, mTOR pathway modulation is associated with cell line sensitivity to AZD5153. Transcriptional modulation of MYC and HEXIM1 was confirmed in AZD5153-treated human whole blood, thus supporting their use as clinical pharmacodynamic biomarkers. This study establishes AZD5153 as a highly potent, orally available BET/BRD4 inhibitor and provides a rationale for clinical development in hematologic malignancies. Mol Cancer Ther; 15(11); 2563-74. ©2016 AACR.

Proteins of the bromodomain and extraterminal (BET) family, in particular bromodomain-containing protein 4 (BRD4), are of great interest as biological targets. BET proteins contain two separate bromodomains, and existing inhibitors bind to them monovalently. Here we describe the discovery and characterization of probe compound biBET, capable of engaging both bromodomains simultaneously in a bivalent, in cis binding mode. The evidence provided here was obtained in a variety of biophysical and cellular experiments. The bivalent binding results in very high cellular potency for BRD4 binding and pharmacological responses such as disruption of BRD4-mediator complex subunit 1 foci with an EC of 100 pM. These compounds will be of considerable utility as BET/BRD4 chemical probes. This work illustrates a novel concept in ligand design-simultaneous targeting of two separate domains with a drug-like small molecule-providing precedent for a potentially more effective paradigm for developing ligands for other multi-domain proteins.

BH3 mimetics like Venetoclax target pro-survival Bcl-2 family proteins and are important therapeutics in the treatment of hematological malignancies. We demonstrate endogenous Bfl-1 expression can render preclinical lymphoma tumor models insensitive to Mcl-1 and Bcl-2-inhibitors. However, suppression of Bfl-1 alone was insufficient to fully induce apoptosis in Bfl-1-expressing lymphomas, highlighting the need for targeting additional pro-survival proteins in this context. Importantly, we demonstrated that CDK9 inhibitors rapidly downregulate both Bfl-1 and Mcl-1, inducing apoptosis in BH3 mimetic resistant lymphoma cell lines in vitro and driving in vivo tumor regressions in DLBCL PDX models expressing Bfl-1. This data underscores the need to clinically develop CDK9 inhibitors, like AZD4573, for the treatment of lymphomas using Bfl-1 as a selection biomarker.

Cyclin-dependent kinase 9 (Cdk9) is a serine/threonine kinase that regulates elongation of transcription through phosphorylation of RNA polymerase II at serine 2 (pSer2-RNAPII). Mcl1, an anti-apoptotic protein that has been linked to increased survival and chemotherapy resistance in various cancers, can be indirectly modulated through transient inhibition of Cdk9 due to it having a short-lived transcript and being a labile protein. Transient inhibition of Cdk9, therefore, represents a potential therapeutic opportunity in tumors dependent on Mcl1 for survival, including various hematological malignancies. AZ5576 is a potent, highly selective, and orally bioavailable inhibitor of Cdk9 that inhibits Cdk9 enzyme activity with an IC50 <5nM and decreases phosphorlyation of Ser2-RNAPII in cells with an IC50 of 96nM. In the MLL-fusion containing acute myeloid leukemia (AML) line, MV411, short-term treatment with AZ5576 led to a rapid dose-dependent decrease in pSer2-RNAPII with concomitant loss of Mcl1 mRNA and protein, resulting in the cleavage and activation of caspase 3 and loss of cell viability. However, protein expression of other Bcl2 family members (e.g. Bcl2, BclxL, Bim) remained unchanged. Further, AZ5576 induced rapid caspase activation and loss of viability in a diverse set of hematological cancer cell lines, including those from acute myeloid leukemia (16/20), multiple myeloma (10/19), and diffuse large B-cell lymphoma (8/13). In vivo efficacy with intermittent dosing of AZ5576 was also demonstrated in multiple xenograft models from each hematological tumor type. Finally, AZ5576 was still able to induce loss of viability in various multiple myeloma lines cultured in the presence of cytokines or bone marrow stromal cells that represent potential protective effects of the tumor microenvironment. Together, these results highlight the therapeutic potential for selective CDK9 inhibition in the treatment of hematological malignancies. Citation Format: Justin Cidado, Minhui Shen, Michael Grondine, Scott Boiko, Haiyun Wang, Alexandra Borodovsky, Anne Marie Mazzola, Alan Wu, Deborah Lawson, Douglas Ferguson, Beirong Gao, Andy Cui, Celina D’Cruz, Lisa Drew. AZ5576, a novel potent and selective CDK9 inhibitor, induces rapid cell death and achieves efficacy in multiple preclinical hematological models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3572.

Cyclin-dependent kinase 9 (CDK9) regulates elongation of transcription through phosphorylation of RNA polymerase II (pSer2-RNAPII), and its short-term inhibition downregulates genes with short-lived transcripts and labile proteins. We developed a novel and selective CDK9 inhibitor, AZD4573, with nanomolar potency and physicochemical properties suitable for IV administration and short exposure. Initial transcriptomic and proteomic analyses were performed on MCF7 breast cancer cells treated with AZD4573 for 4h or 8h to determine which genes were most rapidly modulated by CDK9 inhibition. Of the 1,290 genetically-linked, cancer-associated genes analyzed on these platforms, MCL1 ranked amongst the top as most robustly and sustainably suppressed at both the mRNA and protein level with AZD4573 treatment. MCL1 is an anti-apoptotic member of the BCL2 gene family and is frequently amplified and/or over-expressed in various cancers, conferring a survival advantage to the tumor cell. Mcl1 has also been notoriously difficult to drug with small molecules, although inhibitors are now in early clinical development. Transient inhibition of CDK9 with AZD4573, however, provides a means to indirectly target Mcl1 and induce apoptosis in Mcl1-addicted tumors. In human cancer cell line panel screens, AZD4573 demonstrated the ability to induce rapid caspase activation (6h) and loss of viability (24h) across a diverse set of hematological cancers (median caspase EC50=30nM, GI50=11nM) but with minimal effect on solid tumors (median EC50 & GI50 >30μM). The activity of AZD4573 in the cell line panels displayed a strong correlation with that of the Mcl1 inhibitor, AZD5991 (r2 = 0.837), suggesting transient CDK9 inhibition induces cell death through an Mcl1-dependent mechanism. Furthermore, knockdown of BAK and/or BAX in sensitive cell lines rescued the cells from AZD4573-mediated caspase induction and cell death, underscoring the mechanistic involvement of the intrinsic apoptosis pathway. In the AML cell line, MV411, AZD4573 led to a rapid dose- and time-dependent decrease in pSer2-RNAPII with concomitant loss of Mcl1 mRNA and protein, resulting in caspase induction and loss of cell viability. In contrast, protein levels of the other anti-apoptotic proteins, Bcl2 and BclxL, remained unchanged out to 6h post-AZD4573 treatment of MV411 cells. With intermittent dosing of AZD4573, in vivo efficacy was observed in multiple hematological tumor xenograft models. AZD4573 also exhibited the ability to drive deeper and more durable regressions in combination with multiple targeted agents. Based on our findings, AZD4573 could be an effective treatment option, either as single agent or in combination, for patients with hematological malignancies. Thus, we have initiated a first-time-in-human study with AZD4573 in October 2017 (NCT03263637). Citation Format: Justin Cidado, Theresa Proia, Scott Boiko, Maryann San Martin, Steven Criscione, Douglas Ferguson, Wenlin Shao, Lisa Drew. AZD4573, a novel CDK9 inhibitor, rapidly induces cell death in hematological tumor models through depletion of Mcl1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 310.

Cyclin-dependent kinase 9 (CDK9) regulates transcription elongation through phosphorylation of RNA polymerase II at serine 2 (pSer2-RNAPII), and its short-term inhibition downregulates genes with short-lived transcripts and labile proteins, thus providing a mechanism to inhibit key survival proteins like Mcl1 and Myc. We developed a selective CDK9 inhibitor, AZD4573, that exhibits anticancer activity across a diverse set of hematologic cell lines and is being evaluated in patients with hematologic malignancies. Through efforts to identify combinations with AZD4573, we observed that combined treatment with acalabrutinib, our Bruton tyrosine kinase (BTK) inhibitor, led to more robust and durable responses in preclinical ABC-DLBCL models. We demonstrate BTK inhibition with acalabrutinib upregulates the proapoptotic BH3-only protein Bim in BTKi sensitive ABC-DLBCL cell lines, priming cells for apoptosis induction and further sensitizing them to CDK9 inhibition with AZD4573. Specifically, acalabrutinib upregulated proapoptotic BH3-only proteins Bim and Bmf (>3-fold, 24h) in two BTK-dependent ABC-DLBCL cell lines, OCILy10 and TMD8 but not in a BTK-independent GCB-DLBCL cell line Karpas-422. In each model, treatment with 100nM AZD4573 resulted in rapid induction of cleaved caspase-3 (CC3), and subsequent combination with acalabrutinib accelerated induction of CC3 and increased the magnitude of cell death in the BTK-dependent cell lines. Apoptotic priming by acalabrutinib was validated in vitro in OCILy10 cells by siRNA-mediated knockdown of BH3-only proteins followed by a phenotypic comparison of combination dosing schedules. Both inhibitors dosed concurrently (unprimed) resulted in a minimal increase in the percentage of apoptotic cells compared to treatment with AZD4573 alone (49% of population). In contrast, when dosed sequentially with a pretreatment of acalabrutinib (primed) before the addition of AZD4573, nearly all cells were apoptotic (>90%). Consistent with the in vitro results, the combination of AZD4573 with acalabrutinib resulted in rapid and complete tumor growth inhibition in both BTK-dependent ABC-DLBCL xenograft models. In the OCILy10 model, the combination led to complete tumor regressions and 100% of animals treated in the combination arm (8/8) demonstrated a 60-day delay to regrowth off treatment compared to single-agent AZD4573. Similarly in TMD8, the combination resulted in complete regressions in 100% of treated animals with a highly durable response (17/19 animals tumor-free at 100+ days) whereas the combination yielded no additional benefit in Karpas-422. Acalabrutinib was recently approved for relapsed/refractory mantle cell lymphoma and is being evaluated in patients with additional subtypes of lymphomas. Based on our findings, the combination of acalabrutinib with AZD4573 could be an effective treatment option for patients with ABC-DLBCL. Citation Format: Scott Boiko, Theresa Proia, Maryann San Martin, Justin Cidado, Wenlin Shao, Lisa Drew. A mechanistic rationale for combining acalabrutinib with CDK9 inhibitor, AZD4573, in ABC-DLBCL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 306.