zemirciclib (AZD4573)

CDK9 inhibitors AZD4573 and atuveciclib reduced the IFN-γ induced PD-L1 expression in a dose-dependent manner. In conclusion, CDK9 inhibition could reduce the expression of PD-L1 in HCC cells. Using both CDK9 inhibitors and anti-PD-L1 antibodies is more effective than using either agent alone.

P2, N=52, Completed, AstraZeneca | Active, not recruiting --> Completed | Trial completion date: Dec 2024 --> Feb 2024

P1/2, N=40, Active, not recruiting, AstraZeneca | Trial completion date: Sep 2023 --> Dec 2024

P2, N=52, Active, not recruiting, AstraZeneca | N=81 --> 52 | Trial completion date: Aug 2024 --> Dec 2024

Current standard of care for pTCL revolves around the chemotherapy regimens CHOP/E and for CD30-postive pTCLs, brentuximab vedotin is approved...Using the MCL-1 inhibitor AZD5991, we have shown statistically significant benefit in survival when combined with CHOP in 2 MCL-1 dependent preclinical pTCL PDX models (Koch et al...These data suggested that treatment with AD4573 either as a monotherapy or in combination with CHOP, would be an effective therapeutic strategy in pTCL. AZD4573 monotherapy is currently being evaluated in a phase 2 study (NCT05140382) to assess the efficacy, safety, and PK in patients with relapsed/refractory pTCL.

We performed cell viability experiments on leukemia cell lines (MV4; 11, MOLM13, RS4; 11, THP1, SEM, KOPN8, NOMO1, HL60, ML2) with single, two and three drug combinations using a menin inhibitor (VTP50469), a DOT1L inhibitor (EPZ5676), and a CDK9 inhibitor (AZD4573). Based on our preclinical in vitro findings, the co-inhibition of menin-DOT1L or menin-CDK9 is a promising approach for the treatment of MLL-r leukemia. The varying responses of different MLL-r leukemia cell lines to drug combination treatment indicate that not all cases of MLL-r leukemia will respond uniformly to treatment combinations. Taken together, our data provide a strategy to determine optimal treatment combinations for personalized treatment of MLL-r leukemia.

AZD4573 is currently in a phase 2 study (NCT05140382) to assess the efficacy, safety, and PK of AZD4573 in patients with relapsed or refractory Peripheral T-cell lymphomas (pTCL). Our findings demonstrate that targeting CDK9 with AZD4573 can effectively induce apoptosis in pre-clinical BL models and could be an effective therapy for BL patients.

Compared with clinical single agent CDK9 inhibitor AZD4573, LCI139 is less toxic to normal human stromal cells (HS-5), whereas both LCI139 and AZD4573 have no significant cytotoxic effect on normal human foreskin fibroblast cells (Hs68). Finally, we show that LCI139 overcomes chronic ibrutinib resistance by decreasing viability in JeKO-1 IR and Mino IR cells with an IC50 of 79 nM and 89 nM, respectively. Therefore, the multitarget small molecule inhibitor LCI139 has superior potency against IR MCL cell lines and overcomes ibrutinib-resistance at nanomolar doses.

Our results support that the cooperation between MYC and XPO1 is associated with T-cell reductions characteristic of a Depleted or Cold TME, a key issue for CAR-T success. We applied the targeted therapies AZD4573 and Selinexor vs. cell line models to address this pathway, with both displaying anti-tumor effects as single agents.

IACS-010759, an inhibitor of complex I of the ETC which targets OxPhos, demonstrated synergy with AZD4573 in parental and ibr-resistant MCL cell lines. CDK9 inhibition with AZD4573 induced apoptosis, downregulated MYC and MCL1 and NFκB signaling, and overcame ibr resistance in preclinical MCL models. This was also noted in a sample obtained from a clinical trial. Prolonged CDK9 inhibition led to metabolic reprogramming towards OxPhos, which thus can serve as a therapeutic target in MCL.

Additionally, in two independent endocrine- and palbociclib-resistance patient-derived xenografts, treatment with AZD4573 in combination with palbociclib and fulvestrant resulted in tumor regression. Tumor transcriptional profiling identified a set of transcriptional and cell cycle regulators differentially downregulated only in combination treated tumors. Together, these findings identify a clinically tractable combination strategy for overcoming resistance to endocrine therapy and CDK4/6 inhibitors in breast cancer and provide insight into the potential mechanism of drug efficacy in targeting treatment-resistant disease.

Even more, dual treatment of AZD 4573 with decitabine for 48 hours revealed additive to synergistic efficacy with combination indices (CI) between 0.6-1.0 in isobologram analyses. We provide a rationale for further evaluation of CDK-9 inhibition in acute myeloid leukemia, especially with Venetoclax resistance. Early clinical trials are ongoing.

Our results suggest that CDK7 and CDK9 targeted-therapies may be effective against TMZ-sensitive and resistant GBM.

Downstream inhibition of MAZ-NF1 chimeric transcript in an ALK- ALCL PDX using MEK-inhibitor AZD6244 produced a delay in tumor growth in-vivo...Finally, we trained a predictive model based on RNAseq data and viability upon drug exposure and found gene sets predicting response to specific compounds (i.e., ruxolitinib and belinostat)...1D) and showed close correspondence with patients' responses to various drugs, including chemotherapy (CHOP) as well as targeted agents (ALK-inhibitor crizotinib, JAK-inhibitor ruxolitinib, CD30-drug conjugate brentuximab-vedotin). In addition, we tested novel compounds emerging from the drug screenings including JAK/SYK inhibitor cerdulatinib and CDK9 inhibitor AZD4573: mice treated with the combination of the two drugs showed significantly better progression-free survival compared to single agents... We generated the largest TCL PDX bio-repository accounting for >90 models of different subtypes. PDXsmimicked donor lymphomas phenotypically and genotypically. We discovered novel driver fusions, oncogenic mutations, clonal evolution patterns, innovative therapeutics/combinations and response predictors.

AZD4573 plus acalabrutinib had a manageable safety profile with no new safety signals. The combination showedpromising clinical activity with durable responses in this heavily pretreated population, including pts with prior CAR-T. The study is ongoing and more mature data will be presented at the conference.

Preliminary results show encouraging clinical activity with AZD4573 monotherapy in pts with r/r PTCL, including 3 CRs and one complete metabolic response after initial PD. The PK and safety profiles of AZD4573 monotherapy in PTCL are consistent with the previous phase 1 study with no unexpected findings, and the study continues to expand in the PTCL population. Clinical trial, Phase II, relapsed/refractory, Peripheral T-cell lymphoma

Here we show that AZD4573, a selective inhibitor of CDK9, restricted growth of DLBCL cells...Thus, CDK9i induces reprogramming of the epigenetic landscape, and super-enhancer driven recovery of select oncogenes may contribute to resistance to CDK9i. PIM and PI3K represent potential targets to circumvent resistance to CDK9i in the heterogeneous landscape of DLBCL.

AZD4573 induced T-R conflicts and subsequent DNA damage. Accumulation of DNA damage caused caspase-3-dependent apoptotic cell death in sensitive breast cancer cells. However, less-sensitive breast cancer cells resolved T-R conflicts immediately and maintained genomic stability.

We thus examined the influence of CDK9 inhibition on the expression of PD-L1 in HCC and the potential of improving the efficacy of PD-1 blockade with the combination of CDK9 inhibitors. We first examined the influence of specific CDK9 inhibitors, AZD4573 and atuveciclib, on interferon-γ (IFN-γ) induced PD-L1 expression of human HCC cell lines HuH7 and Hep3B. We demonstrated that CDK9 inhibition could reduce the IFN-γ induced PD-L1 expression of HCC cells. Combination of CDK9 inhibitors and anti-PD-L1 antibodies was more effective than either therapy alone.

In this heavily pretreated population including CAR-T failures, the combination of AZD4573 and acalabrutinib resulted in high response rates (ORR 63.6%, CR rate 36.4%). No DLTs were detected, the combination was well tolerated with a manageable safety profile, and no new safety signals were identified. The study is currently enrolling in the Phase 2a expansion phase and updated data will be presented at the meeting.

Introduction Brexucabtagene autoleucel (BA) cellular therapy is the most recent milestone for treating patients with mantle cell lymphoma (MCL) and the only CAR-T cell therapy approved by the FDA for MCL...Indeed, targeting CDK9 with AZD4573 induced impressive anti-MCL activity in vitro and overcomes the dual resistance in vivo in patient-derived xenograft models. Targeting HSP90 with two independent inhibitors PU-H71 or 17-AAG was also potent in MCL preclinical models...Conclusion Our data demonstrated that an unexpected but novel axis led by HSP90-MYC-CDK9 drives the dual resistance to BTKi and CAR T therapies. Our study sheds light on the underlying mechanism of CAR-T resistance in addition to BTKi resistance in MCL and provides compelling preclinical evidence for therapeutic targeting of the HSP90-MYC-CDK9 axis to overcome the dual resistance in patients with MCL.

AZD4573 had manageable safety and a PK profile suitable for QW dosing in a broad range of hematologic malignancies including lymphoma and leukemia. The reduction in pSer2 and MCL-1 supports the RP2D of 12 mg for lymphoma and 9 mg for leukemia. A combination study of AZD4573 and acalabrutinib (NCT04630756) is ongoing to further examine the DLBCL response signal seen in this study.

We demonstrated the in vitro and in vivo activity of CDK9 inhibition on multiple HCC cell lines. Our data support further clinical development of CDK9 inhibitors as a treatment for HCC.

To demonstrate the utility of a selective CDK9 inhibitor for treatment of hematologic patients with an unmet medical need such as high-grade B cell lymphoma (HGBL) and chronic lymphocytic leukemia who relapse or are refractory to ibrutinib and venetoclax (R/R CLL)...Differential expression (DE) analysis from VIP152, atuveciclib and KB-0742 treatment of 2 lymphoma cell lines by RNA seq is compared to DE of samples from 7 VIP152 treated HGBL patients...With high ATP, VIP152, AZD4573 and alvocidib maintain potency in low nM range, while fadraciclib and KB-0742 have 760 nM-1.7 µM IC50s...Selective transcriptional downregulation is observed with 30mg IV weekly. Samples from patients with DLBCL or CLL are sensitive to VIP152 and updated PK, PD and ctDNA dynamics from the ongoing phase 1 trials (NCT02635672, NCT04978779) will be presented.

10058-F4 reduces c-Myc protein levels and suppresses HepG2 cell proliferation, possibly by upregulating cyclin-dependent kinase (CDK) inhibitors, p21WAF1, and reducing intracellular alpha-fetal protein (AFP) levels. The combination of AZD4573 and 10058-F4 has a synergistic anti-araC-resistant AML activity, providing a solid database for the aforementioned scientific hypothesis.

Background: Resistance to AraC (cytarabine) is a major obstacle for effective treatment and limits survival rates of children with acute myeloid leukemia (AML). Our results demonstrate promising antileukemic activity of the combination of AZD4573 and Venetoclax against AraC-resistant AML cells. Future studies will determine the in vivo efficacy of this promising combination therapy against AraC-resistant xenograft NSGS mouse models.

Several compounds targeting BCL-2 signaling, which are under clinical investigation, were tested (BCL-2: venetoclax, BCL-2/Xl: AZD4320, MCL-1: AZD5991 and CDK9: AZD4573)...Priming with a hypomethylating agent (HMA, decitabine) resulted in additive (CI close to 1) to synergistic (CI 0.25 – 0.7) proapoptotic effects in isobologram analysis and led to a release of drug resistance in primary resistant cell lines...attenuation thereof after forcely expressing the dominant-negative splicing variant). To summarize, we show that inhibition of BCL-2 signaling is a promising approach to target acute leukemia – as a monoagent as well as in combination with HMA: Further, we provide a path for exploration of ASPP2k as a predictive tool as well as a therapeutic sensitizer of pro-apoptotic drugs, which will be addressed in future studies.

Mantle cell lymphoma (MCL) is an aggressive form of NHL where frequent relapse following standard therapies remains a serious concern, even for promising new treatments such as combinations of a BTK inhibitor with the selective Bcl2 inhibitor venetoclax. Combination of AZD4573 with acalabrutinib resulted in greater anti-tumor activity than either monotherapy. Altogether, these data suggest that AZD4573, alone or in combination with acalabrutinib, could be an effective therapy for patients with r/r MCL.

However, 10–50% of newly diagnosed patients with AML may not respond to venetoclax and HMA or LDAC, and 3–15% patients may not respond to venetoclax with intensive or non-intensive chemotherapy.1–6 In addition, up to 40% of responding patients may relapse with low rates of response of 20% to salvage therapy and poor overall survival of 2 months after relapse.7 Clinical and biological factors associated with primary and acquired resistance to venetoclax include secondary AML, monocytic differentiation, complex cytogenetics, mutations in TP53, BAX, dependence on other anti- apoptotic proteins, altered metabolism of nicotinamide, fatty acids, and oxidative phosphortylation.3,8–14 Several novel inhibitors of BCL-2 are currently being tested in clinic, including BGB 11417, APG-2575, LP-108 and others...There is strong pre-clinical rationale for targeting MCL-1 alone as well as in conjunction with BCL-2 inhibition in AML.15 Recently several selective and highly potent MCL-1 inhibitors have entered pre-clinical and clinical development including S63845, AZD5991, AMG397, and others. Questions remain regarding the therapeutic window of these inhibitors given the important physiologic role of MCL-1 in vital organs and early reports of cardiac adverse events from the AMG176 phase 1 trial.15,16 Multiple pre-clinical studies have expectedly shown synergism between BCL- 2 and MCL-1 inhibition making it a promising path for clinical development of these agents.17,18 Multifactorial challenges in design of specific MCL-1 inhibitors also led to interest in compounds which downregulate MCL-1 expression. Cyclin dependent kinase (CDK) inhibitors including alvocidib, dinaciclib, voruciclib are in various stages of evaluation. Although addition of alvocidib to intensive chemotherapy improved response rates but failed to improve event-free or overall survival.19 Novel CDK inhibitors are currently in early phase trials including AZD4573, CYC065, TG02-101, and others. Inhibition of Nedd8 activating enzyme has complex repercussions for the intrinsic apoptotic pathway with eventual increase in Noxa leading to MCL-1 neutralization.20 Pevonedistat has shown promising early results in AML and myelodysplastic syndrome and is being investigated multiple clinical trials for solid tumors as well. BCL-xL Inhibition Another anti-apoptotic protein BCL-xL had been long recognized as a potential therapeutic target in AML, in particular AML from preceding MPN and AML recurrent post venetoclax failure, but toxicity of earlier inhibitors precluded clinical development.21–23 Recently, AZD0466, a dual BCL-2/xL inhibitor with a favorable therapeutic index and robust activity has been developed and is undergoing pre-clinical development and planned for phase iin hematological malignancies.24 Targeting the Extrinsic Apoptosis Pathway Inhibitor of apoptosis protein (IAP) inhibition: X-linked IAP (XIAP), cellular IAP (cIAP) and survivin have been of long- standing interest in AML. Prior clinical trials with XIAP inhibitor AEG35156, cIAP targeting agent birinapant, and survivin targeting agent LY2181308 have not succeeded in clinc.16,25 ASTX660 is a dual antagonist of XIAP and cIAP which is currently being investigated in phase 1/2 trials in solid tumors and in combination with HMA in relapsed or refractory AML.26,27 TRAIL Agonism Agonists of the TNF-related apoptosis-inducing ligand (TRAIL) receptors have been tested in AML with low response rates.28,29 Previous agents have had limited success in part due to suboptimal clustering of TRAIL receptors.30 Novel antibodies against TRAILR1 and TRAILR2 including an IgM molecule IGM-8444, a tetravalent compound INBRX-109, and HLX56 are currently in phase 1 trials and preclinical data suggests potential synergy with venetoclax.31 FLIP Inhibition FLICE-like inhibitor protein (FLIP or CFLAR) is a key regulator of the death-inducing signaling complex (DISC) involved in the extrinsic apoptotic pathway...This can be augmented by inhibiting p53 degradation via MDM2, which is often upregulated in AML.34 Idasanutlin in combination with venetoclax showed anti- Figure 1 leukemic activity in the dose finding stage in R/R AML.35 Several other inhibitors of MDM2 and dual MDM2/X inhibitors are currently in various stages of pre-clinical and clinical development including HDM-201, KRT-232, BI-9078282, and others.34 Conclusions Opportunities to target the apoptosis machinery in AML has considerably evolved in the last decade. While venetoclax heralded a paradigm shift for patients, we are now faced with challenges in patients who relapse or remain refractory. We have novel clinical stage compounds to methodologically target different facets of the apoptotic pathway and optimize novel combinations with the goal to improve the cure rates in AML patients.

AZD5991 is a highly selective BH3-mimetic that demonstrates high in vitro potency in MCL1-dependent cell lines with an IC50 <1 nM.7 We have demonstrated that direct MCL1 inhibition with AZD5991 disrupts survival of neoplastic B-cells in lymph-node mimicking conditions, induces mitochondrial dysfunction and cooperates with BCL2/X inhibitors in vitro and in vivo.8 A phase 1 clinical trial of AZD5991 alone or in combination with venetoclax in hematologic malignancies is ongoing (NCT03218683)...AMG-176 demonstrated synergy with venetoclax in AML models; however, it also caused cytopenias.9 AMG-176 was shown to induce apoptosis of CLL cells independent of prognostic markers and overcame the protective effect of stromal microenvironment.10 However, suppressive effects on hematopoiesis will likely become the dose- limiting factor in clinical trials of MCL1-targeting agents. The effect of MCL1 inhibition on cord blood-derived CD34+ cells was studied using a small-molecule inhibitor, S63845,11 resulting in almost complete depletion of human hematopoietic stem and progenitor cells, while mature blood cells survived normally...We have shown that SYK inhibition with entospletinib leads to downmodulation of MCL1 protein in CLL both in vitro and in the clinic.12,13 Treatment of CLL cells with entospletinib, but not other BCR-signaling inhibitors, led to a disruption of BAFF-BCR cross-talk and downmodulation of MCL1 mRNA and protein, thus implicating SYK in transduction of multiple pro-survival signals emanating from the tumor microenvironment.13 Entospletinib has shown promising clinical activity in CLL.12,14 It is currently being developed in myeloid malignancies.15 A novel dual SYK/BTK inhibitor luxeptinib also was shown to downregulate MCL1 and other pro-survival BCL2 proteins in CLL.16 Transcriptional cyclin-dependent kinases (CDK7/9) regulate activity of RNA polymerase, thereby controlling production of mRNA. Downmodulation of MCL1 has been considered a key mechanistic event accounting for the pro-apoptotic activity of CDK inhibitors in neoplastic B-cells.17,18 Pan-CDK inhibitors (flavopiridol, dinaciclib) demonstrate clinical efficacy in lymphoid malignancies. We and others reported that preclinical efficacy of AZ5576, a selective inhibitor of CDK9, and its clinical congener AZD4573 in lymphoid tumors depended on rapid downmodulation of MCL1, BFL1, and MYC.19–21 CDK9 inhibition can increase efficacy of BH3-mimetics, but safety of this combination will need to be explored.22,23 CDK9 inhibitors in development, in addition to AZD4573, include VIP152, CYC065, and voruciclib...Navitoclax is a small-molecule inhibitor of BCL2, BCLX, and BCL2L2; however, its use in CLL has been plagued by thrombocytopenia.24 AZD4320 is an alternative agent that co-targets BCL2/X...ABBV-155, an antibody-drug conjugate targeting BCLX, where BCLX is being used as a payload to avoid “off-tumor” effects, demonstrated promise in solid tumor models.26 Finally, as many microenvironment-driven pro-survival pathways converge on NFB, leading to induction of chemokines, cell cycle regulators, and BCL2 family proteins themselves, this transcription factor continues to be an attractive target in CLL...When combined with ibrutinib in murine CLL models, VAY-736 produced prolonged survival compared with either therapy alone.28 Alternative targets for therapeutic antibodies include ROR1 (cirmtuzumab), CD19 (tafasitamab), and others.29 As resistance to novel agents is becoming an unmet need in CLL, exploration of novel targets is of paramount importance...Multiple CDK inhibitors are currently being explored and have anti-tumor effects not restricted to MCL1 inhibition. CAR T cells and bi-specific antibodies have exceptional efficacy in lymphoid malignancies and thus are of high relevance in CLL.

Palbociclib could reverse afatinib, docetaxel, & radiation resistance...CDK7/8/9/12/13 are involved in RNA polymerase II function with RNA elongation pause/release, transcription & onco-histones control, and SWI/SNF superfamily with epigenetic modifications. HNSCC cell lines(SCC4, SCC9, SCC15, SCC25, FaDu, KB, Cal27, SAS, and TW2.6, betel-nuts related) were tested in (1)in vitro sensitivity to CDK inhibitors; (2)synergistic effect of AZD4573(CDK9 inhibitor) with other therapies by MTT assay, colony formation, flow cytometry, & western blotting; (3) NGS studies to elucidate molecular mechanisms. Palbociclib response correlated to CCND1 gain & CDKN2A deletion; but FaDu had poor response with both and TW2.6 had good response without both... CCND1 gain & CDKN2A deletion could not fully predict CDK4/6 inhibitor response. TW2.6 is responsive to CDK4/6 inhibitors(palbociclib>ribociclib>abemaciclib). FAT1 loss, CCND3 amplification, PI3K/AKT/mTOR derangements, & FGFR amplification might imply CDK4/6 inhibitor resistance.

BH3 mimetics like Venetoclax target pro-survival Bcl-2 family proteins and are important therapeutics in the treatment of hematological malignancies. 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.

In the present study, we sought to determine sensitivities of a panel of follicular cell-derived thyroid cancer cell lines to the CDK7 inhibitor, THZ1 and CDK9 inhibitor, AZD4573...qRT-PCR did not reveal reduced mRNA levels in TPC1 cells with THZ1 treatment while, Bortezomib (proteasome inhibitor) co-treatment with THZ1 rescued RNAPII protein...qRT-PCR of both cell lines treated THZ-1 showed stable BRAF gene expression; further mechanistic studies are ongoing. In summary, human thyroid cancer cell lines are sensitive to inhibition of CDK7 and CDK9 likely through several mechanisms not all directly attributable to RNAPII inhibition.

In the present study, we sought to determine sensitivities of a panel of follicular cell-derived thyroid cancer cell lines to the CDK7 inhibitor, THZ1 and CDK9 inhibitor, AZD4573...qRT-PCR did not reveal reduced mRNA levels in TPC1 cells with THZ1 treatment while, Bortezomib (proteasome inhibitor) co-treatment with THZ1 rescued RNAPII protein...qRT-PCR of both cell lines treated THZ-1 showed stable BRAF gene expression; further mechanistic studies are ongoing. In summary, human thyroid cancer cell lines are sensitive to inhibition of CDK7 and CDK9 likely through several mechanisms not all directly attributable to RNAPII inhibition.