Project Summary Cancer relapse and the emergence of chemotherapy resistance are driven by epigenetic and transcriptional profiles that repress tumor suppressor genes (TSGs). Small molecules that alter the epigenome to stimulate re- expression of TSGs can re-sensitize tumors to chemotherapy; however, current drugs that target epigenetic processes fail due to off-target effects (toxicity) or due to compensation from other epigenetic players (resistance). Combination therapies target multiple nodes in an epigenetic network to reduce toxicity while avoiding resistance, but exploring vast combinatorial space to identify such therapies is prohibitive using current technologies. The overarching goal of this proposal is to unravel the interconnected networks that govern TSG expression and identify small molecules that induce re-expression of TSGs using novel combinatorial platforms. The approaches and technologies proposed may be applied to many cancers and TSGs; the expression of the TSG CDKN2A in acute lymphoblastic leukemia (ALL) will be the focus of this work, as 20% of ALL cases result in relapse, leading to grim prognoses, and CDKN2A is frequently repressed in relapsed ALL. To identify small molecule combinations that may induce re-expression of CDKN2A in relapsed ALL, a proven combinatorial drug screening platform developed in the Blainey Lab will be employed to screen the Broad Institute Drug Repurposing Library for compounds that synergize with FDA-approved chemotherapies and epigenetic modifiers to induce CDKN2A:GFP expression in an ALL cell line (Aim 1). In parallel, the protein networks that govern CDKN2A expression in ALL will be identified by using high-throughput, barcoded, single-cell RNA sequencing (Seq-Well) to screen a combinatorial CRISPR-Cas9 knockout library for CDKN2A expression (Aim 2). Finally, to look for chemogenetic perturbations that cause re-expression of any TSG in ALL, a novel platform for high-throughput RNA-sequencing of combinatorial chemogenetic libraries will be developed and used to sequence the transcriptional outputs of all combinations of known epigenome-modifying drugs with the knockdown of each annotated epigenomic regulator expressed in leukemia cells, including any identified in Aims 1 and 2. Validated combinations of small molecules and genes from Aims 1-3 will constitute a new collection of lead compounds and gene targets for the development of therapies that may extend the life-span and health-span of patients with relapsed ALL. The proposed research will be conducted in the Blainey Lab at the Broad Institute of MIT and Harvard, world- leaders in the development of technologies that drive medical discovery. The results of this work will be presented at national and international conferences and published in peer-reviewed journals as appropriate; all data will be made available through public databases.