Project Summary/Abstract Chronic lymphocytic leukemia (CLL) is the most prevalent adult leukemia in Western countries. Despite recent treatment advances, CLL remains incurable. In the era of emerging targeted therapies, disease progression and relapse increasingly are key challenges faced by CLL patients. Therefore, there is an ongoing need for the identification of targetable vulnerabilities in the context of therapeutic resistance in CLL. Our studies, as well as recent data from others, have identified aberrant splicing as a molecular characteristic of CLL. Our preliminary data also suggests that the proprietary small-molecule spliceosome modulator sudemycin D6 (SD6; Wildflower Biopharma, Inc.), which has recently progressed to GLP IND-enabling studies, effectively inhibits CLL cell growth and induces apoptosis both in vitro and especially in vivo. We also showed that SD6 can overcome pro-survival and pro-growth signals and markedly enhance the activity of the clinically approved CLL therapies ibrutinib, idelalisib and venetoclax to induce apoptosis in primary CLL cells co-cultured with bone marrow stromal cells and T-cell-derived cytokines. Collectively, our preliminary results provide a strong rationale for future clinical development of spliceosome modulators and potential combination therapies for CLL. The main objective of this application is to acquire high quality pre-clinical data that supports the first-in-human clinical trial of the novel splicing modulator SD6 for CLL. Our central hypothesis is that SD6 is able to effectively kill CLL cells with selectivity relative to normal B and other hematopoietic cells and to lead to disease regression in vivo, as a novel potentially curative drug for CLL especially when used in combination with other agents. We further hypothesize that SD6 has significant synergies with FDA-approved CLL drugs at the molecular level and may overcome drug resistance of the current clinical CLL drugs when used in combination. To test our hypotheses, we propose the following Specific Aims: 1) Identify and validate novel therapeutic vulnerabilities to spliceosome modulation in CLL; 2) Determine the synergistic interactions of SD6 with ibrutinib (BTK inhibitor), idelalisib (PI3K inhibitor), and venetoclax (BCL2 inhibitor) in primary human CLL samples, as well as CLL cell lines harboring drug resistance mutations; 3) Evaluate the in vivo therapeutic efficacy of SD6 as a single agent or in combination with ibrutinib, idelalisib, and venetoclax using a syngeneic CLL mouse model. Our long-term goal is to pursue commercial development of spliceosome modulators for the treatment of CLL and other cancers with splicing-related susceptibilities.