7. Project Summary/Abstract The overarching goal of this NGC Center is to leverage expertise in small molecule drug discovery, target identification, and protein and IDR biochemistry to identify and advance small molecules that target transcriptional fusion oncoproteins by 1) targeting the transcriptional machinery that is co-opted by fusion proteins to drive the oncogenic transcriptional programs, and 2) Directly impair IDR function with small molecules. Transcriptional fusion proteins induce oncogenic gene expression programs that drive cancer development and progression. These proteins act as neomorphic transcription factors by recruiting and re-wiring transcriptional regulatory complexes. Rational drug design to directly target transcriptional fusion proteins remains a major challenge because these proteins lack enzymatic activity and do not have obvious pockets amenable for small molecule binding. In addition, the mechanisms by which fusion oncoproteins co-opt transcriptional regulatory complexes are incompletely understood. As such, the development of new therapies to target transcriptional fusion proteins has lagged efforts to develop targeted therapies for mutant oncoproteins involved in cellular signaling. Our team has discovered that the Mediator transcriptional co-activator complex is co-opted to control the oncogenic gene expression programs induced by transcriptional fusion proteins. We have further identified the CDK8 kinase module as a druggable entry point by which to impair Mediator function through a novel trapping mechanism. Our team will probe the biochemical and transcriptional mechanisms through which CDK8/19 inhibitors alter Mediator function and develop CDK8/19 inhibitors that maximize Mediator impairment and study these small molecules in advanced preclinical models of Ewing sarcoma and rhabdomyosarcoma. We further identified that vaccinia-related kinase 1 (VRK1) is a kinase that becomes synthetically lethal following CDK8/19 impairment, and our team will develop novel specific VRK1 inhibitors and evaluate their efficacy in single agent and combination strategies. A central theme has recently emerged that many fusion proteins consist of a DNA binding domain linked to an intrinsically disordered region (IDR). The IDR mediates self-association or phase separation that has been shown to be essential for the oncogenic nature of these fusion proteins. Our team has developed an innovative biochemical method of identifying the key protein regions within IDRs that mediate self-association. We will map the key self-association residues in three fusion protein IDRs and identify small molecules that impair the ability of these regions to drive phase separation.