PROJECT SUMMARY Deregulation of the epigenetic reader protein Bromodomain and PHD finger-containing transcription factor (BPTF) has recently been shown to play a role in various cancers, yet remains an understudied protein. A significant limitation is the lack of potent chemical probes to study its function. One mechanism for regulating cancer progression is through association of BPTF with the oncogene c-MYC and genetic knockdown studies have revealed that BPTF is vital for overexpressing cells, but not c-MYC-driven apoptosis. Preliminary studies by the Pomerantz lab with a first-in-class BPTF inhibitor, AU1, suggest that BPTF bromodomain inhibition alone may not be sufficient for inhibiting BPTF in the context of cancer cell proliferation, but the compound has been useful in demonstrating synergy with existing chemotherapeutics both in vitro and in vivo. At this juncture, the development of a higher affinity tool compound with improved physiochemical properties is necessary in order to further cellular studies of BPTF in cancers. We envision this tool compound as a Proteolysis Targeting Chimera (PROTAC), consisting of an inhibitor for the target protein, a linker, and an E3 ligase. This proposed heterobifunctional molecule would allow for comparison of BPTF inhibition versus BPTF degradation in K562 cells, a chronic myelogenous leukemia cell line with BPTF growth dependency. Takeda Pharmaceutical and the Structural Genomics Consortium recently reported a potent inhibitor of CECR2 (IC50=30 nM), another bromodomain-containing protein involved in chromatin remodeling, but a synthesis of the molecule was not reported. Interestingly, TP-238 also had off-target binding to BPTF (IC50= 350 nM). Motivated by the potency of BPTF to TP-238 and the significant insights gained from PROTAC constructs targeting other bromodomains, we hypothesize that a TP-238-based PROTAC would be an ideal tool compound with which to further inform our understanding of BPTF inhibition and degradation in cancer cells while evaluating selectivity. In Aim 1, my recently reported synthesis of TP-238 will be modified to allow for linker and E3 ligand attachment. A focused library of TP238-based PROTACs with varying linker composition and length will be synthesized and subsequently analyzed using in-house biophysical assays including, NMR, Surface Plasma Resonance (SPR) and AlphaScreen. In Aim 2, the ability of each PROTAC to degrade BPTF will be analyzed using gel-based assays in a concentration and time-dependent manner. In Aim 3, the efficacy of our bifunctional construct of TP- 238 will be compared to the monovalent, bromodomain inhibitor to verify that BPTF proteosomal degradation is beneficial. Investigation of BPTF target genes c-MYC, ERK, BCL2 and BCL2L with ATAQ-seq will follow and shape our mechanistic understanding of BPTF function in K562 cells. A key preliminary result for this work was the first BPTF small molecule cocrystal structure we published with TP-238 bound. T...