PROJECT SUMMARY Epigenetic therapy aims to reprogram gene expression in cancer cells to achieve a therapeutic effect. To date, DNA methyltransferase (DNMT) inhibition (DNMTi) is the most effective form of epigenetic therapy, being particularly active in myeloid leukemias. Using a live cell assay to screen for drugs that achieve the same degree of epigenetic reprogramming as DNMTi, we discovered a new class of epigenetic drugs that activate silenced expression through inhibition of CDK9. Cyclin Dependent Kinases (CDKs) are of considerable clinical interest in cancer therapy and fall into two classes – cell cycle regulatory (e.g. CDK1,2,4,6 etc.) and transcriptional regulators (e.g. CDK7,9,12 etc.). Our new data place CDK9 at the heart of a node that regulates both gene silencing and activation. Targeting CDK9 has pleotropic effects on gene expression that appear ideal from an anti-tumor perspective: One observes simultaneous gene activation (of tumor suppressors), repression (of oncogenes), and induction of an interferon immune signature, which may be immunosensitizing for cancer therapy. This latter effect is of particular interest given the phenomenal recent development of immune based therapies, and our preliminary data suggest that CDK9 targeting may be a useful new approach to immunosensitization. Interestingly, CDK9 may not be the only CDK involved in immune regulation. Inhibition of CDK4/6 and of CDK5 have been shown to induce an IFN response and/or to be immunosensitizing in cancer therapy. Moreover, other transcriptional CDKs (e.g. CDK7) share phosphorylation targets with CDK9, raising the possibility that they also similarly affect gene silencing. Given that drugs that target CDK4 and/or 6 are in clinical use in breast cancer, and drugs targeting CDK7 and/or 9 are in clinical trials, it becomes important to know whether other CDKs are also immunosensitizing epigenetic regulators. Epigenetic effects may lead to different strategies for clinical development of these drugs (e.g. low doses rather than MTD, expectation of slow responses that take multiple cycles to occur, etc.) and for the design of combination strategies (combined epigenetic therapy, combinations with immune checkpoint inhibitors etc.). This project therefore aims to test the hypothesis that targeting CDKs leads to immunosensitizing epigenetic effects. We will confirm this hypothesis and test mechanisms and clinical/translational implications for cancer therapy in three aims: (i) Immunosensitization by CDK9 inhibition, in which we will study mechanisms and potential ways to enhance the effects; (ii) Epigenetic effects of CDKs, in which we will ask whether targeting CDK4,5,6 and 7 has similar epigenetic and immunosensitizing effects as targeting CDK9; and (iii) Preclinical studies and a clinical trial of combined CDK9 inhibition, DNMT inhibition and Immune checkpoint inhibition in AML and MDS, in which we will complete the necessary studies to bring CDK targeting as epigeneti...