More effective treatment options for elderly acute myeloid leukemia (AML) patients are needed as only 25–50% of patients respond to standard-of-care therapies, response duration is typically short, and disease progression is inevitable even with some novel therapies and ongoing clinical trials. Anti-apoptotic BCL-2 family inhibitors, such as venetoclax, are promising therapies for AML. Nonetheless, resistance is emerging. We demonstrate that venetoclax combined with cyclin-dependent kinase (CDK) inhibitor alvocidib is potently synergistic in venetoclax-sensitive and -resistant AML models in vitro, ex vivo and in vivo. Alvocidib decreased MCL-1, and/or increased pro-apoptotic proteins such as BIM or NOXA, often synergistically with venetoclax. Over-expression of BCL-XL diminished synergy, while knock-down of BIM almost entirely abrogated synergy, demonstrating that the synergistic interaction between alvocidib and venetoclax is primarily dependent on intrinsic apoptosis. CDK9 inhibition predominantly mediated venetoclax sensitization, while CDK4/6 inhibition with palbociclib did not potentiate venetoclax activity. Combined, venetoclax and alvocidib modulate the balance of BCL-2 family proteins through complementary, yet variable mechanisms favoring apoptosis, highlighting this combination as a promising therapy for AML or high-risk MDS with the capacity to overcome intrinsic apoptosis mechanisms of resistance. These results support clinical testing of combined venetoclax and alvocidib for the treatment of AML and advanced MDS.

Alvocidib is a potent inhibitor of cyclin-dependent kinase-9 (CDK9) and induces apoptosis in cancer cells by reducing the expression of short-lived, anti-apoptotic proteins such as MCL-1. Alvocidib, as a part of a sequential combination regimen with cytarabine and mitoxantrone (ACM), is currently in a Phase II clinical trial in relapsed/refractory acute myeloid leukemia (AML). Patients with AML that have a high dependence on MCL-1 are considered more likely to benefit from the alvocidib-containing regimen. MCL-1 has emerged as a key protein in drug resistance of multiple solid tumor types including breast, prostate and lung cancers. The use of alvocidib in clinical settings beyond the ACM regimen is somewhat limited by the current intravenous route of administration. An orally administered form of alvocidib would allow prolonged repression of MCL-1 through chronic dosing and scheduling. Alvocidib itself is highly permeable in CACO-2 monolayers and is soluble at acidic pHs but solubility is strikingly reduced at neutral or basic conditions, which could hamper the development of an oral formulation. We hypothesized that a phosphate prodrug of alvocidib would improve solubility under neutral or basic conditions and enable the efficient systemic delivery of alvocidib via oral administration. We synthesized a phosphate prodrug of alvocidib, TP-1287, in three steps from the parent compound. The solubility of TP-1287, was determined at various pH levels. It was found to be highly soluble under acidic, neutral, and basic conditions (1.5 mg/mL at pH 2.2; 1.8 mg/mL at pH 4.5; 9.5 mg/mL at pH 6.8 and 9.3 mg/mL at pH 8.7) compared to alvocidib (4.4 mg/mL at pH 2.2; 1.3 mg/mL at pH 4.5; 0.02 mg/mL at pH 6.8 and 0.02 mg/mL at pH 8.7). Pharmacokinetic studies were conducted in mice in which TP-1287 was efficiently converted to the parent alvocidib (Cmax = 1922.7 ng/ml, t1/2 = 4.4 hr) with high oral bioavailability (%F = 182.3, compared to intravenous alvocidib). Efficacy and pharmacodynamic studies (measuring MCL-1 expression levels), were evaluated in tumor xenograft models. TP-1287 demonstrated significant anti-tumor efficacy in the MV4-11 AML mouse xenograft model and produced as much as a 61.7% inhibition of the pharmacodynamic biomarker MCL-1 in xenografted tumors, demonstrating a wide, 75-fold therapeutic dosing window. In addition, TP-1287 strongly inhibited tumor growth, achieving 109.1% tumor growth inhibition (%TGI) at the 7.5 mg/kg dose level. TP-1287 is highly soluble over a broader pH range than alvocidib and is efficiently metabolized to the parent compound in vivo, following oral administration. Tumor xenograft models and pharmacodynamic studies indicate that oral delivery of TP-1287 is efficacious in mice. Based on these results, we anticipate moving TP-1287, as an orally delivered CDK9 inhibitor, into a forthcoming clinical trial directed towards solid tumors vulnerable to the suppression of MCL-1. Citation Format: Wontak Kim, Hillary Haws, Peter Peterson, Clifford J. Whatcott, Steven Weitman, Steven L. Warner, David J. Bearss, Adam Siddiqui-Jain. TP-1287, an oral prodrug of the cyclin-dependent kinase-9 inhibitor alvocidib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5133. doi:10.1158/1538-7445.AM2017-5133

In individuals with chronic inflammatory diseases, such as cancer or rheumatoid arthritis, constitutive signaling through ALK2, a member of the bone morphogenetic protein (TGFβ/BMP) receptor family, leads to debilitating anemia, commonly referred to as anemia of chronic disease (ACD). Activation of ALK2, like other members of the BMP receptor family, leads to the phosphorylation and activation of SMAD family transcription factors via signal transduction and subsequent activation of gene expression. Activation of ALK2 in the liver induces the SMAD-driven transcription of the peptide hormone hepcidin which, by promoting the degradation of the iron transporter ferroportin, leads to reduced serum iron levels and subsequent functional anemia. Lowering constitutively elevated hepcidin levels by inhibiting ALK2 kinase activity is a potentially viable therapeutic strategy for ACD. Current therapeutic approaches for ACD rely on transfusions, intravenous iron and the use of erythropoietin-based therapies, none of which address the underlying pathological deficit of functionally low iron levels. TP-0184 is a small-molecule, selective inhibitor of ALK2 kinase activity (IC50 = 5 nM). TP-0184 has demonstrated profound preclinical activity in three mouse efficacy models for ACD. In model 1, TP-0184 reversed hepcidin induction in mice treated with turpentine oil. In model 2, TP-0184 abrogated reductions in hemoglobin and total red blood cell counts induced by intraperitoneal injection with heat-inactivated Brucella abortus. In model 3, TP-0184 reversed elevated hepcidin levels in TC-1 tumor bearing mice. Plasma and liver pharmacokinetics in mice revealed that TP-0184 has a high volume of distribution (Vd = 30.8) and accumulates at high concentrations in the liver (Cmax of 292 mM following a single oral dose of at 20 mg/kg). In rat multi-dose tolerability studies, TP-0184 caused no adverse effects when dosed at 200 mg/kg for 7 days, far exceeding the dose levels required to produce efficacy (25 mg/kg). These data suggest that favorable distribution to the liver may play a significant role in the preclinical efficacy of TP-0184 and provide evidence of a significant therapeutic window. Collectively these studies support the clinical evaluation of TP-0184 as an alternative treatment for ACD. Disclosures Peterson: Tolero Pharmaceuticals: Employment. Kim:Tolero Pharmaceuticals: Employment. Haws:Tolero Pharmaceuticals: Employment. Whatcott:Tolero Pharmaceuticals: Employment. Siddiqui-Jain:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Warner:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties.

The proteasome inhibitor bortezomib is widely used in the treatment of patients with multiple myeloma (MM). The expression levels of many proteins increase as a result of bortezomib treatment, including the pro-apoptotic protein NOXA. NOXA functions to sequester the anti-apoptotic BCL-2 family member, MCL-1. High levels of MCL-1 and/or low levels of NOXA have been implicated in bortezomib resistance and negative patient outcomes, including short duration of treatment response. The BCL-2-specific BH3 mimetic venetoclax (ABT-199) has also been explored in multiple hematological malignancies, including the treatment of MM. Venetoclax induces apoptosis in a BCL-2 specific manner by directly inhibiting BCL-2 function. However, intrinsic resistance to venetoclax treatment observed in MM patient samples has been attributed to a low BCL-2-to-MCL-1 gene expression ratio, suggesting a central role for MCL-1 in cell survival in this context as well. Increased MCL-1 expression is a known resistance mechanism to venetoclax treatment in a variety of cell types including chronic lymphocytic leukemia and lymphomas. Considering the central role of MCL-1 to treatment efficacy in MM, we investigated the ability of an MCL-1-lowering agent, namely the CDK9 inhibitor alvocidib, to potentiate the activity of venetoclax in MM. Alvocidib suppresses MCL-1 expression via CDK9-mediated regulation of RNA polymerase II. Alvocidib has achieved robust improvements in the clinical response rates of high-risk, newly diagnosed acute myeloid leukemia (AML) patients as part of the time-sequential ACM regimen (alvocidib + cytarabine + mitoxantrone). We therefore hypothesized that alvocidib would potentiate the activity of venetoclax in MM through an MCL-1-dependent mechanism. In this report, we demonstrate that alvocidib inhibits the protein expression of MCL-1 in MM cells in a time-dependent fashion, up to 96 hours. In cell viability assays, the addition of up to 100 nM venetoclax resulted in a 2.8-fold reduction in the IC50 of alvocidib in the cultured OPM-2 cell line. Conversely, the potentiation of venetoclax activity with the addition of alvocidib resulted in a more than 500-fold decrease in IC50 in the relatively venetoclax-resistant OPM-2 cells. Additional studies are currently underway to investigate the efficacy of alvocidib and venetoclax in the context of bortezomib resistance where low NOXA may contribute to enhanced cell survival via MCL-1. Taken together, our data suggest that the combination of alvocidib with venetoclax may constitute a novel therapeutic regimen in the treatment of MM. Further, it suggests that CDK9-mediated targeting of MCL-1 may offer a clinical route to addressing intrinsic resistance in MM patients. Citation Format: Mark Livingston, Wontak Kim, Hillary Haws, Peter Peterson, Clifford J. Whatcott, Adam Siddiqui-Jain, Steven Weitman, David J. Bearss, Steven L. Warner. Alvocidib potentiates the activity of venetoclax in preclinical models of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1106. doi:10.1158/1538-7445.AM2017-1106

Introduction Venetoclax (ABT-199) is an approved BCL-2 inhibitor for the treatment of patients with chronic lymphocytic leukemia (CLL). Multiple clinical trials are underway to explore its efficacy in additional indications. While venetoclax demonstrated high remission rates in combination with azacitidine in early stage clinical trials, the question of durability of responses and primary and acquired resistance remain, especially given the modest activity and rapid development of resistance as a single agent. One reported mechanism of intrinsic resistance is high expression of other BCL-2 family proteins, including MCL-1. We and others have demonstrated that the CDK9 inhibitor, alvocidib, can mediate transcriptional repression of anti-apoptotic MCL-1. It has also been shown that alvocidib can increase pro-apoptotic BIM, a dual activator and sensitizer BH3-only protein that can directly induce apoptosis and simultaneously inactivate anti-apoptotic BCL-2 family proteins such as MCL-1 and BCL-2, thus having the same effect on mitochondria-associated apoptosis as MCL-1 down-regulation, with the potential to directly induce apoptosis. An alvocidib-containing cytotoxic chemotherapy regimen demonstrated favorable remission rates in high-risk AML patients over standard therapy in a randomized Phase 2 trial indicating its potential role and safety in AML. We hypothesized that alvocidib and venetoclax would synergize against AML cells by shifting the overall balance of pro- and anti-apoptotic BCL-2 proteins in favor of apoptosis and thus represent a novel active treatment regimen in AML. Aims This study seeks to examine the efficacy of a treatment regimen containing alvocidib and venetoclax in multiple preclinical studies, including in vivo models of AML. Methods Cell viability assays interrogating alvocidib and venetoclax activity in cell lines were performed using CellTiter-Glo according to manufacturer's protocol. mRNA/miRNA expression changes were assessed using standard RT-PCR technique. Protein expression changes were assessed using standard western immunoblotting technique. To assess the efficacy of an alvocidib and venetoclax combination on tumor growth in an in vivo model, the OCI-AML3 xenograft mouse model and ex vivo studies with AML patient samples were performed. Results Herein we demonstrate that alvocidib inhibits both mRNA and protein expression of MCL-1 in a time and concentration-dependent fashion in 3 out of 4 AML cell lines analyzed, while in cells where alvocidib did not reduce MCL-1 protein levels (i.e. MOLM-13) a dose-dependent decrease in miR17-92, and concomitant increase in BIM protein was observed after 24 hours of alvocidib treatment. The alvocidib-venetoclax combination resulted in very strong synergistic reductions of cell viability (with combination indices [CI] of 0.4 to 0.7), both in venetoclax-sensitive and resistant cells. The venetoclax-sensitive lines, MV4-11 and MOLM-13, exhibited 5- to 10-fold reduction of venetoclax EC50 values in the low nM range when combined with only 80 nM alvocidib. Importantly, venetoclax-resistant lines, OCI-AML3 and THP-1, exhibited at least 20-fold reduction of venetoclax EC50 values from near 1 µM to 10-50 nM, when combined with 80 nM alvocidib.In the venetoclax-resistant OCI-AML3 xenograft model, single agent alvocidib and venetoclax achieved tumor growth inhibition (TGI) of 9.7 and 31.5%, respectively, while the combination achieved 87.9% TGI at the same dose levels of individual drugs. Conclusions Taken together, our data suggest that the combination of alvocidib with venetoclax is highly synergistic in vitro and in vivo, in both venetoclax-sensitive and -resistant AML across a heterogeneous genomic background. The particularly high level of synergy achieved in venetoclax-resistant cell lines highlights the central importance of both BCL-2 and MCL-1-mediated cell survival in AML. Importantly, the addition of alvocidib to venetoclax treatment reduced IC50s to clinically achievable concentrations. Therefore, we conclude that an alvocidib/venetoclax combination may be a novel approach for the treatment of AML and warrants further pre-clinical and clinical validation. Disclosures Whatcott: Tolero Pharmaceuticals: Employment. Kim:Tolero Pharmaceuticals: Employment. Haws:Tolero Pharmaceuticals: Employment. Mesa:Celgene: Research Funding; Galena: Consultancy; Promedior: Research Funding; Ariad: Consultancy; Novartis: Consultancy; CTI: Research Funding; Incyte: Research Funding; Gilead: Research Funding. Peterson:Tolero Pharmaceuticals: Employment. Siddiqui-Jain:Tolero Pharmaceuticals: Employment. Weitman:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Warner:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties.

Of the more than 240,000 estimated new cases of breast cancer in 2016 (US), triple-negative breast cancer (TNBC) was expected to comprise roughly 12%. So named because of their lack of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression, TNBCs pose a unique therapeutic challenge, with only chemotherapeutic options currently available. New therapeutic options are desperately needed for patients with TNBC. Recent reports have identified a cellular addiction of TNBCs to PIM1, suggesting that the addiction is mediated by PIM regulation of Myc and BH3 proteins MCL-1 or BCL-2. The PIM family of serine/threonine kinases are highly conserved and the link between PIM and Myc function has been well studied. The second-generation PIM inhibitor, TP-3654, has been shown to suppress Myc expression in vitro. Additionally, CDK9 inhibitors are also known to suppress Myc expression. CDK9 inhibition mediates specific reductions in transcription of short-lived mRNAs such as Myc and MCL-1. The CDK9/cyclin T complex is a critical component of the P-TEFb complex, promoting productive RNA elongation through phosphorylation of serine 2 of the heptapeptide repeats of the C-terminal domain of RNA Polymerase II (RNAPII-CTD). We are developing a potent CDK9 inhibitor, alvocidib, and an oral prodrug form of alvocidib named TP-1287, and are investigating the potential clinical utility of CDK9 inhibition in acute myeloid leukemia (AML). We hypothesized that PIM and CDK9 inhibition would be an active combination in models of TNBC due to the added effect of targeting Myc through two independent mechanisms. In the TNBC cell line, MDA-MB-231, TP-3654 reduced relative Myc protein expression by 74%, while alvocidib reduced expression by 71%. The combination of TP-3654 and alvocidib reduced detectable expression 100%, as measured by standard immunoblotting, at concentrations of 100 nM with a 3-hour treatment. To test this hypothesis in vivo, TP-3654 and TP-1287 were tested in the MDA-MB-231 xenograft. Single-agent TP-3654 (150 mg/kg) reduced tumor growth (%TGI) 40.7%, while TP-1287 (3.75 mg/kg) reduced tumor growth 11.6%. The combined-treatment regimen reduced tumor growth by 58.3% at day 18 of treatment. These results support a rationale for further clinical investigation of PIM and CDK9 inhibitors for the suppression of Myc in patients with TNBC. Citation Format: Hillary Haws, Wontak Kim, Adam Siddiqui-Jain, David J. Bearss, Steven L. Warner, Clifford J. Whatcott. Targeting Myc in triple-negative breast cancer models through the dual inhibition of PIM kinases and CDK9 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B178.

The positive-transcription elongation factor (P-TEFb) complex is critical in stimulating the transcription of non-abortive transcripts by RNA polymerase II. The CDK9/cyclin-T heterodimer comprises the P-TEFb complex and promotes transcription elongation through phosphorylation of serine 2 of the heptapeptide repeats of the c-terminal domain of RNA Polymerase II (RNAPII-CTD). Alvocidib, a potent CDK9 inhibitor, has been shown to suppress expression of MCL-1 and Myc mRNAs, and clinical pharmacodynamic data suggest that alvocidib's activity is primarily mediated through the suppression of MCL-1 mRNA and protein expression. In addition to MCL-1 suppression, we hypothesized that CDK9 inhibition affects other RNA Polymerase II target RNAs, such as microRNAs. Recently, it was reported that inhibition of bromodomain and extraterminal domain (BET) protein by JQ1 suppresses miR17-92 expression. miR17-92 negatively regulates expression of the pro-apoptotic BH3-only protein, BIM, leading to suppression of BIM expression, thereby decreasing the cells' ability to induce apoptosis. It was hypothesized that CDK9 kinase activity is key to miR17-92 expression and suppression of BIM expression and that targeting CDK9 would lead to a decrease in miR17-92, and increases in BIM. MV4-11, OCI-AML3, MOLM13, and THP1 AML cell lines were used to determine the effects of alvocidib treatment on microRNAs. RT-qPCR was utilized to determine microRNA expression levels of miR17-92 and mRNA levels of BIM and other markers. Protein changes were determined using standard gel electrophoresis and immunoblotting technique. CellTiter-Glo and Caspase-Glo were used for all cell viability and apoptosis assays interrogating alvocidib. With CDK9 inhibition mediated via alvocidib, a dose- and time-dependent decrease in miR17-92 expression in MV4-11, OCI-AML3, MOLM13, and THP1 cells was observed. A decrease was observed even 3 hours post-treatment, persisting for up to 24 hours. MicroRNA suppression following treatment ranged in magnitude, with a maximal effect of between 3.3 to 4.2-fold suppression, depending on the cell line and timepoint. This suppression coincided with an increase in BIM mRNA and protein expression in the MV4-11, OCI-AML3, and MOLM13 cells. However, BIM protein increase was not observed in THP1 cells. Maximal increase in BIM mRNA levels reached 5.4-fold in the MV4-11 cell line. Our data suggest that CDK9 inhibition suppresses RNA Polymerase II-mediated expression of miR17-92, which in turn leads to increased expression of BIM. Combined with MCL-1 reduction, increased BIM protein expression mediated by alvocidib leads to enhanced apoptosis. Taken together, the data provide additional understanding of CDK9 as a potential therapeutic target, and are consistent with the hypothesis that CDK9 activity is necessary for miR17-92 expression. Citation Format: Hillary Haws, Hubert F. Arokium, James L. Bogenberger, Adam Siddiqui-Jain, David J. Bearss, Raoul Tibes, Steven L. Warner, Clifford J. Whatcott. Alvocidib-mediated inhibition of CDK9 upregulates BIM via suppression of miR17-92 [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 883.