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Background: Chronic Lymphocytic Leukemia (CLL) is a genetically heterogeneous disease characterized by clonal expansion of B-lymphocytes that induce secondary immune suppression. CLL is now treated with inhibitors of Bruton tyrosine kinase (BTK) and BCL2. Virtually all patients respond to therapy, however resistance to these therapies has been described justifying the need for novel CLL therapies. Broad inhibition of cyclin dependent kinases (CDK) and associated alternative target enzymes with agents such as flavopiridol or dinaciclib have demonstrated significant clinical activity in CLL but are hindered by a relatively narrow therapeutic window. VIP152 is a highly specific inhibitor of CDK9 - considered the most important CDK kinase member for CLL clinical activity. VIP152 has favorable pharmacokinetic properties and has demonstrated durable, preliminary single-agent clinical activity in double-hit diffuse large B-cell lymphoma. Herein, we report the efficacy of VIP152 preclinically in CLL. Methods: On-target activity of VIP152 was measured using a KinomeScan from DiscoveryRx at 100nM and 1000nM. Kinase profiling for VIP152 was performed on 6 kinases in a 10-dose assay at ReactionBio. Cell-based viability and proliferation assays (MTS, Annexin-V (AV), and propidium iodine (PI)) were performed in primary CLL cells and the CLL cell lines, HG3 and MEC1. Transcriptional activity after VIP152 exposure was measured via qPCR and limiting-cell RNA sequencing (lcRNAseq). Proteomic and immunoblot studies were performed to measure perturbations in CDK9 binding partners and on-target activity of VIP152. A genome-wide CRISPR/CAS9 knockout screen was performed to identify any synthetically lethal targets and pathways. Results: The KinomeScan identified CDK9 as the kinase with maximal inhibition upon VIP152 treatment and no other CDKs were identified at 100nM. Kinase profiling revealed the IC 50 of VIP152 was lowest for CDK9/Cyclin T1 and CDK9/Cyclin T2 with close similarity to dinaciclib and greater than 1 log superiority over KB-0742. Co-immunoprecipitation and proteomics experiments have identified a CDK9 specific mechanism of action relating to perturbations of CDK9 binding partners. Specifically, we showed that CDK9 nuclear immunoprecipitation resulted in decreased co-immunoprecipitation of 7SK RNA components (HEXIM1 & MEPCE) as well as decreased RNA Polymerase II (RNAP2). The decrease in RNAP2 CoIP was further seen via proteomics. A 2-hour exposure of VIP152 against HG3 and MEC1 demonstrated growth inhibition, with an IC 50 of 0.9814µM and 1.092µM respectively. Continuous exposure of the compound for 24 hours resulted in a statistically significant drop in relative viability of 30% across a 10-fold dose range (0.1µM to 1.0µM) as measured by AV/PI. Primary CLL cells (n=10) responded with similar dosing strategies with a 54% reduction in viability at 1µM; moreover, stromal cell co-culture experiments demonstrated VIP152's ability to induce cell death and overcome stromal protection with short exposure. Induction of apoptosis was observed with pro-caspase-3 and PARP cleavage on immunoblot. qPCR and immunoblot studies demonstrated a time dependence of phosphorylated serine 2 (pS2) RNAP2 decreases alongside diminishment of MYC and MCL1 mRNA and protein. pS2 was shown to decrease as early as 2 hours after VIP152 treatment with similar decreases in MCL1 and MYC at both the protein and mRNA levels. We identified several pathways which are disrupted following treatment via lcRNAseq, including TNFR1 and TNFR2 signaling as well as upregulation of autophagy signals. Finally, CRISPR screen identified several potentially synergistically lethal targets, including transcriptional co-activators, DNA binding proteins, and cell proliferation pathways. Validation of these is ongoing as is an in vivo study of VIP152 in a CLL mouse model. Conclusions: Our data demonstrate VIP152 to be a highly selective and potent CDK9 inhibitor that disrupts the CDK9 nuclear complex and mediates significant preclinical activity against CLL cell lines and primary CLL cells. VIP152 also demonstrates predictable and new pharmacodynamic markers to assess target engagement. Collectively, these data support the recently initiated CLL clinical trial (NCT04978779). Disclosures Johnson: Vincerx: Current Employment; Janssen: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Frigault: Vincerx Pharma Inc: Current Employment; AstraZeneca: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months, Patents & Royalties. Greer: Gilead: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Vincerx Pharma Inc: Current Employment. Hamdy: Vincerx Pharma Inc: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Acerta Pharma Inc: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Ended employment in the past 24 months, Patents & Royalties. Izumi: Acerta Pharma Inc: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Ended employment in the past 24 months, Patents & Royalties; Vincerx Pharma Inc: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Hwang: Vincerx Pharma Inc: Current Employment, Current equity holder in publicly-traded company. Blachly: KITE: Consultancy, Honoraria; INNATE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria. Byrd: Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria; Newave: Membership on an entity's Board of Directors or advisory committees.