Summary (30 lines, broad, long-term) With the advent of more effective therapies in CLL, there is a greater priority on identifying better treatment of Richter's Syndrome (RS), as this has become one of the most important unmet medical needs to address for CLL patients. The main goals of this Project are to utilize functional precision medicine techniques to better understand biological vulnerabilities in Richter's cells and to identify novel pharmacologic interventions to increase apoptotic signaling in these cells. Our priority is on the direct study of viable samples from RS patients obtained in collaboration with the Biospecimens core. We seek to identify the most promising combinations to subsequently explore in early phase clinical trials for patients with RS, funded by other mechanisms. The job of precision medicine is to match the right patient to the right drug. A strategy of genomic sequencing has yielded several successes, such as treating EGFR mutant lung cancer with EGFR inhibitors; however, to date RS has not yielded to the identification of targetable genetic abnormalities, and this will be the focus of Project 1. In Project 3, we propose an alternative approach, which we call functional precision medicine. Rather than rely on static -omic data, we propose to perturb cellular function in ways that allow us to identify active drugs. Central to this strategy is BH3 profiling, a technique in which we expose mitochondria from living cells to synthetic BH3 peptides and measure mitochondrial permeabilization. From this assay we can learn which anti- apoptotic proteins the cell relies on for survival, whether BCL-2, BCL-XL, MCL-1, some combination thereof, or none of these proteins. This information has direct translational application, as clinical BH3 mimetic inhibitors of all three listed proteins now exist. In fact, BH3 profiling was used to direct therapy of the BCL-2 inhibitor venetoclax to CLL and AML, two indications for which venetoclax has now received FDA approval. We propose to use BH3 profiling to identify which BH3 mimetic(s) would be most active in RS. BH3 profiling can also provide a summary measure of how close a cell is to the threshold of apoptosis. When coupled with a brief preceding drug exposure, dynamic BH3 profiling (DBP) can identify drugs from any class that induce apoptotic signaling in cancer cells, moving them closer to the threshold of apoptosis. We have previously demonstrated in several liquid and solid tumor contexts that this strategy accurately identifies drugs with in vivo activity for individual tumors, and can predict clinical response in patients. We propose to use DBP to identify active drugs, after which we will explore their use in combination with each other and with the appropriate BH3 mimetics identified above. As we identify drug vulnerabilities for the panel of RS samples to be studied in this Project, we will compare them with clinical, genomic, transcriptomic, and proteomic annotations prepa...