PROJECT SUMMARY The development of precision therapeutics – selective drugs that target oncogenic driver proteins – has revolutionized the treatment of cancer; but most of these drugs are plagued by the rapid emergence of resistance. Some drug resistance mechanisms can be readily deduced by genome sequencing (e.g., drug- resistant mutations in the oncogenic driver itself), but other drug-resistant states lack a clear genetic signature and therefore likely reflect proteomic alterations that reduce sensitivity to drug action. The CDK4/6-Cyclin D1- Retinoblastoma protein (Rb) axis is frequently dysregulated in specific cancers, including estrogen receptor- positive (ER+) breast cancers. Though CDK4/6 inhibitors, such as palbociclib and ribociclib, have emerged as compelling drugs to treat patients with ER+ breast cancer, resistance has already presented itself in clinical settings. Notably, these resistance mechanisms remain poorly understood and do not commonly involve mutations in CDKs or loss of the Rb, which is directly downstream of the CDK4/6-CyclinD1 complex and negatively regulates E2F transcription. The overall goal of this application is to utilize advanced chemoproteomic approaches to understand the molecular mechanisms of acquired resistance to cyclin dependent kinase inhibitors (CDKi’s) in ER+ breast cancer cells and provide novel therapeutic targets to overcome resistance to CDKi’s. Identifying protein targets in cancer based on their protein activity is an inevitable next step in advancing target cancer therapeutics. Our lab has established a suite of chemical probes that can collectively assess the reactivity and druggability of > 10,000 cysteine, lysine, and serine/threonine residues in > 5,000 human proteins. We have accordingly shown that we can use the activity-based protein profiling (ABPP) platform to discover novel druggable vulnerabilities in genetically defined cancers, including cysteines that show differential ligandability in KEAP1-mutant cancer cells with an activated NRF2 pathway. These findings point to the broader potential for ABPP as a method to search for differential ligandability and reactivity in specific disease states. In this respect, I hypothesize that the CDKi-resistant state harbors proteins with differential reactivity and/or ligandability that support loss of sensitivity to CDKi’s. To test this hypothesis, I propose two specific aims: 1) to identify and characterize proteins with differential reactivity and ligandability unique to the CDKi resistant state and 2) to characterize the role of MGLL and other proteins with differential reactivity and/or ligandability in acquired resistance to CDKi’s. I will examine CDKi resistant cell lines using the ABPP approach to understand the molecular underpinnings of CDKi resistance and to identify novel potential therapeutics, a crucial next step in combating drug resistance in the era of personalized medicine.