Abstract 2952: Targeting CDK9 reactivates epigenetically silenced genes in cancer

In cancer, the epigenomeis aberrantly reprogrammed leading to a wide range of heritable changes in gene expression, such as silencing of tumor suppressor genes (TSG). Altered epigeneticmarks in cancer involve DNA methylation and histone post-translational modifications, and these come about as a result of agingand acquisitionof genetic and epigeneticchanges in readers/writers/editors of the epigenome. Given the reversible nature of epigeneticmodifications, one goal of epigenetic therapyof cancer is to induce TSG reactivation, leading to cancer cell differentiation and cancer cell death. To identify novel targets that can reactivate epigeneticallysilenced genes, we developed a phenotypic-based system, YB5. YB5 is a colon cancer cell line generated by stably transfecting SW48 cells with a vector containing GFP driven by a methylated and silenced CMV promoter. GFP re-expression can be achieved by known epigeneticdrugs that lead to demethylation or induce active chromatin marks in the CMV promoter. We screened a natural compound library for GFP activation in YB5 and identified a novel drug classthat shares an aminothiazolecore structure, and has epigeneticeffects that are equivalent to DNA methyltransferaseinhibitor (DNMTi). Target deconvolution identified CDK9 as the target of these drugs, which reactivate gene expression without affecting DNA methylation. It is well established that CDK9, the catalytic subunit of p-TEFb, is a transcriptional activator. CDK9 in complex with its regulatory subunit, Cyclin T1or T2, is recruited by multiple mechanisms to promote RNAPII promoter-proximal pause release by phosphorylating negative elongation factors( DSIFand NELF). In addition, phosphorylation of the C-terminal domain (CTD) of RNAPII on Serine-2 allows recruitment of RNA processing factors, which work on the nascent RNA as it emerges from RNAPII. Our new data show that long-term CDK9 inhibition can reactivate epigeneticallysilenced genes with minimal downregulation effects, effects which are the opposite of the canonical role of CDK9-mediated transcriptional elongation. Mechanistically, we showed that CDK9 inhibition dephosphorylates the SWI/SNFprotein SMARCA4and represses HP1α expression, both of which contribute to gene reactivation. Based on gene activation, we developed the highly selective and potent CDK9 inhibitor MC180295 (IC50 =5nM) that has broad anti-cancer activity in-vitro and is effective in in-vivo cancer models. Additionally, CDK9 inhibition sensitizes with the immune checkpointinhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapyof cancer. This is the first study that links CDK9 to maintaining gene silencing at epigeneticallyrepressed loci in mammals. Excitingly, this is also the first example of kinase inhibitors as potential drugs that reverse epigeneticsilencing. Citation Format: Hanghang Zhang, Somnath Pandey, Meghan Travers, Jittasak Khowsathit, George Morton, Hongxing Sum, Carlos A. Barrero, Carmen Merali, Yasuyuki Okamoto, Takahiro Sato, Judit Garriga, Natarajan V. Bhanu, Johayra Simithy, Bela Patel, Jozef Madzo, Noel Raynal, Benjamin A. Garcia, Marlene A. Jacobson, Salim Merali, Yi Zhang, Wayne Childers, Magid Abou-Gharbia, John Karanicolas, Stephen B. Baylin, Cynthia A. Zahnow, Jaroslav Jelinek, Xavier Grana, Jean-Pierre J. Issa. Targeting CDK9 reactivates epigeneticallysilenced genes in cancer [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 2952.