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The super enhancer complex (SEC) is a group of transcription regulatory proteins that coordinate the expression of genetic programs which determine cell identity and drive disease states, such as cancer. In acute myeloid leukemia (AML), SECs have been shown to turn on transcriptional programs that drive tumorigenesis and disease progression. The SEC is replete with potential therapeutic targets that have been the focus of many drug development efforts; including cyclin-dependent kinases (CDK), bromodomain proteins (BRD), histone deacetylases (HDAC), and histone methyltransferases (HMT). SEC-regulated transcription begins as CDK9/cyclin T1 is recruited from an inhibitory complex by BRD4 and brought to the transcriptional start site of genes. CDK9 phosphorylates RNA polymerase II, releasing it from the SEC and leading to transcriptional elongation and gene expression. Considering the close association of CDK9 and BRD4, we hypothesized that the combination of CDK9 and BRD4 inhibitors would have synergistic effects, particularly in AML, a disease largely driven by SEC function. Alvocidib is a potent CDK9 inhibitor with validated clinical activity in AML from multiple Phase II studies in over 400 patients. Additionally, BRD4 inhibitors have demonstrated early promise in clinical studies with a focus on AML. We found that CDK9 inhibitors combined with bromodomain inhibitors produced a synergistic effect by inhibiting the SEC more effectively than either of these compounds alone. For example, cell viability studies with various combinations resulted in an increase in potency. This was observed with alvocidib combined with JQ-1 (BRD4 inhibitor) in MV4-11 AML cells. Furthermore, the combination of alvocidib with JQ-1 completely abrogated SEC function, as measured by c-myc expression through RT-qPCR. Similar results were achieved with other combinations of CDK9 and BRD4 inhibitors. The alvocidib and JQ-1 combination was also evaluated in an MV4-11 mouse xenograft model. As single agents, alvocidib (2.5 mg/kg) exhibited a 44% tumor growth inhibition and JQ-1 (25 mg/kg) a 1% growth inhibition. When these two doses were combined there was 100% tumor growth inhibition. These data, primarily focused on alvocidib and JQ-1, suggest a strong rational for combining CDK9 and BRD4 inhibitors as a treatment strategy for AML. Furthermore, these findings could be more broadly applied to additional therapeutic targets in the SEC, such as DOT1L and HDACs. These strategies yield synergistic effects at inhibiting SEC function and are highly active in tumor growth studies of AML in vivo. Clinical studies utilizing these combination strategies are the next steps to further explore this approach. Citation Format: Brigham L. Bahr, Kyle S. Maughan, Katherine K. Soh, Jeremiah J. Bearss, Wontak Kim, Peter Peterson, Clifford Whatcott, Adam Siddiqui-Jain, Steve L. Warner, David J. Bearss. Combination strategies to target super enhancer transcriptional activity by CDK9 and BRD4 inhibition in acute myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2698. doi:10.1158/1538-7445.AM2015-2698

Anemia of chronic disease (ACD) is an inflammatory cytokine driven disease characterized by hypoferremia despite adequate iron stores. This is largely due to hepcidin, a master regulator of iron homeostasis, which blocks enterocytes from absorbing iron and preventing iron release from macrophages by binding to ferroportin. It is known that bone morphogenetic proteins (BMP) up-regulate hepcidin by activating the SMAD signaling pathway through the activin-like kinase receptor 2 (ALK2). Therefore, ALK2 has emerged as a potential therapeutic target to modulate hepcidin levels and treat ACD. We have developed a novel series of small molecule ALK2 inhibitors with promising activity in preclinical models of ACD. Using well-established cell-based and animal models of hepcidin signaling and anemia, we optimized and validated the activity of the most promising preclinical lead candidates. These compounds demonstrate significant activity in downregulating hepcidin expression in BMP-induced cell culture studies at concentrations of 100 nM or lower. Importantly, this hepcidin lowering activity was observed at concentrations that exhibited no cytotoxicity suggesting the compounds have a clean selectivity profile. The compounds also demonstrated remarkable activity in animal models of anemia, including an acute model induced by the administration of turpentine oil and a more chronic model induced by tumor formation and growth. Treatment with the lead candidates completely reversed the induction of hepcidin expression in these models and also decreased the symptoms of anemia as measured by serum iron and red blood cell levels. From these data, we have nominated a candidate to advance into IND-enabling studies that has favorable drug-like properties. We anticipate a clinical development strategy that focuses on anemia of cancer with subsequent expansion into anemia associated more broadly with other inflammatory and chronic diseases Disclosures Kim: Tolero Pharmaceuticals: Employment. Maughan:Tolero Pharmaceuticals: Employment. Soh:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Bahr:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Warner:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties.