CDK9 Blockade Exploits Context-dependent Transcriptional Changes to Improve Activity and Limit Toxicity of Mithramycin for Ewing Sarcoma.

There is a need to develop novel approaches to improve the balance between efficacy and toxicity for transcription factor-targeted therapies. In this study, we exploit context-dependent differences in RNA polymerase II processivity as an approach to improve the activity and limit the toxicity of the EWS-FLI1-targeted small molecule, mithramycin, for Ewing sarcoma. The clinical activity of mithramycin for Ewing sarcoma is limited by off-target liver toxicity that restricts the serum concentration to levels insufficient to inhibit EWS-FLI1. In this study, we perform an siRNA screen of the druggable genome followed by a matrix drug screen to identify mithramycin potentiators and a synergistic "class" effect with cyclin-dependent kinase 9 (CDK9) inhibitors. These CDK9 inhibitors enhanced the mithramycin-mediated suppression of the EWS-FLI1 transcriptional program leading to a shift in the IC50 and striking regressions of Ewing sarcoma xenografts. To determine whether these compounds may also be liver protective, we performed a qPCR screen of all known liver toxicity genes in HepG2 cells to identify mithramycin-driven transcriptional changes that contribute to the liver toxicity. Mithramycin induces expression of the BTG2 gene in HepG2 but not Ewing sarcoma cells, which leads to a liver-specific accumulation of reactive oxygen species (ROS). siRNA silencing of BTG2 rescues the induction of ROS and the cytotoxicity of mithramycin in these cells. Furthermore, CDK9 inhibition blocked the induction of BTG2 to limit cytotoxicity in HepG2, but not Ewing sarcoma cells. These studies provide the basis for a synergistic and less toxic EWS-FLI1-targeted combination therapy for Ewing sarcoma.

PIM kinase inhibitor AZD1208 for treatment of MYC-driven prostate cancer.

BACKGROUND: PIM1 kinase is coexpressed with c-MYC in human prostate cancers (PCs) and dramatically enhances c-MYC-induced tumorigenicity. Here we examine the effects of a novel oral PIM inhibitor, AZD1208, on prostate tumorigenesis and recurrence. METHODS: A mouse c-MYC/Pim1-transduced tissue recombination PC model, Myc-CaP allografts, and human PC xenografts were treated with AZD1208 (n = 5-11 per group). Androgen-sensitive and castrate-resistant prostate cancer (CRPC) models were studied as well as the effects of hypoxia and radiation. RNA sequencing was used to analyze drug-induced gene expression changes. Results were analyzed with χ(2) test. Student's t test and nonparametric Mann-Whitney rank sum U Test. All statistical tests were two-sided. RESULTS: AZD1208 inhibited tumorigenesis in tissue recombinants, Myc-CaP, and human PC xenograft models. PIM inhibition decreased c-MYC/Pim1 graft growth by 54.3 ± 39% (P < .001), decreased cellular proliferation by 46 ± 14% (P = .016), and increased apoptosis by 326 ± 170% (P = .039). AZD1208 suppressed multiple protumorigenic pathways, including the MYC gene program. However, it also downregulated the p53 pathway. Hypoxia and radiation induced PIM1 in prostate cancer cells, and AZD1208 functioned as a radiation sensitizer. Recurrent tumors postcastration responded transiently to either AZD1208 or radiation treatment, and combination treatment resulted in more sustained inhibition of tumor growth. Cell lines established from recurrent, AZD1208-resistant tumors again revealed downregulation of the p53 pathway. Irradiated AZD1208-treated tumors robustly upregulated p53, providing a possible mechanistic explanation for the effectiveness of combination therapy. Finally, an AZD1208-resistant gene signature was found to be associated with biochemical recurrence in PC patients. CONCLUSIONS: PIM inhibition is a potential treatment for MYC-driven prostate cancers including CRPC, and its effectiveness may be enhanced by activators of the p53 pathway, such as radiation.