Progression of aggressive prostate cancers (PCa) with androgen receptor splice variants or neuroendrocrine features is currently untreatable in the clinic. Therefore novel therapies are urgently required. We conducted RNA-seq using tumors from a unique murine transplant mouse model which spontaneously progresses to metastatic disease. Differential gene expression analysis revealed a significant increase of topoisomerase IIα, Top2a (Top2a) in metastatic tumors. Interrogation of human data revealed that increased Top2a expression in primary tumors selected patients with more aggressive disease. Further, significant positive correlation was observed between Top2a and the histone methyltransferase, Ezh2. Combination of the Top2 poison etoposide with the Ezh2 inhibitor GSK126 or DZNep significantly increased cell death in vitro in murine and human prostate cancer cell lines. Additionally, combination therapy extended time to progression and increased therapeutic efficacy in vivo. Overall, our studies demonstrate that patients screened for Top2a and Ezh2 expression would exhibit significant response to a combinational treatment involving low dose etoposide combined with Ezh2 inhibition. In addition, our data suggests that this combination therapeutic strategy is beneficial against aggressive PCa, and provides strong rationale for continued clinical development.

Background: Approximately 40% of B-cell lymphomas are characterized by the presence of recurrent chromosomal translocation affecting the Myc/8q24 locus. Further, similar breakpoints may also occur as secondary events giving rise to “Double Hit” (DH) lymphomas. DH lymphomas frequently involve Myc with concurrent, Bcl-2 or Bcl6 translocations. These Myc associated B cell lymphomas are linked with poor survival, with a median overall survival of only 0.2-1.5 years, and novel therapeutic strategies are urgently required. EZH2 is the catalytic subunit of the polycomb repressive complex 2 (PRC2) and is shown to correlate with disease progression in many cancer types. EZH2 is involved in gene repression via tri-methylation of histone H3 lysine 27 (H3K27me3) and its expression is demonstrated to be increased by Myc, highlighting a potential therapeutic target towards Myc driven B cell lymphomas. In this study we determined the antitumor activity of a specific EZH2 inhibitor, GSK-126. Taking advantage of the well characterized Eμ-myc B cell lymphoma mouse model, we screened tumors with defined genetic alterations in apoptotic pathways to determine GSK-126 antitumor activity. Methods: The anti-tumor effects of GSK126 or ectopic expression of an EZH2 mutant deficient for histone methyltransferase activity (ΔSET-EZH2) was assessed towards Eµ-myc B cell lymphomas, and compound mutant B cell lymphomas (Eµ-myc/Bcl2, Eµ-myc/p53-/-, Eµ-myc/p19-/-, and Eµ-myc/apaf1-/-) by uptake of propidium iodide. Further apoptotic assessment was performed using annexin, cell cycle analysis, mitochondria damage and caspase activity by flow cytometry. GSK-126 inhibition of EZH2 activity was determined by H3K27me3 expression by immunoblot. In vivo anti-tumor activity was determined by transplanting Eµ-myc lymphomas and Eµ-myc compound mutants expressing control of ΔSET-EZH2 plasmids to wild type mice. Overall survival was determined by Kaplan-Meier analysis. Results: Preliminary data demonstrates that GSK-126 treatment of Eµ-myc lymphomas results in rapid apoptotic cell death in a dose dependent manner. GSK-126 mediated apoptosis is independent of p53, though requires mitochondria damage. Further experiments described above are currently being performed. Conclusion: Our current data demonstrate that EZH2 inhibition induces apoptosis in Eµ-myc B cell lymphomas independent of p53, but requires mitochondrial damage. Overall, this study will lead to a more comprehension of the apoptotic mechanisms underlying GSK-126 therapeutic effect and will provide translational tools to predict the therapeutic response in DH lymphomas overexpressing Bcl2 or Bcl6. Citation Format: Elena Lasorsa, Kevin Schaarschuch, Zafardjan Dalimov, Leigh Ellis. Determining the apoptotic pathways required for the EZH2 inhibitor, GSK-126, in a mouse model of B cell lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5527. doi:10.1158/1538-7445.AM2014-5527