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Mantle Cell Lymphoma (MCL) is an incurable B-cell Non-Hodgkin's Lymphoma, which is characterized by neoplastic proliferation of B Lymphocytes. If treated aggressively, the median survival of an MCL patient is about 8 to 12 years. However, the majority of MCL cases eventually relapse with a median survival of only 3 to 8 months. For this reason, novel therapeutic strategies in the refractory setting of MCL are needed. Our lab group introduced Protein Arginine Methyltransferase 5 (PRMT5) as a key driver for MCL lymphomagenesis. PRMT5 shares its function with a family of nine Protein Arginine Methyltransferases (PRMTs) to catalyze the formation of arginine methylation on histone tails. PRMT5, as a type II PRMT, generates symmetric dimethylarginine (SDMA) residues. PRMT5's upregulation has been associated with the growth and survival of MCL. For this reason, our lab, in collaboration with Prelude Therapeutics, has developed the drug PRT-382, a potent and selective inhibitor of PRMT5. We have found that PRMT5 inhibitor therapy prolongs survival and decreases the overall disease burden in MCL murine models and cell lines. However, despite that anti-tumor efficacy, some MCL cell lines have shown primary resistance to PRMT5 inhibition. We have also observed some mice progress over the course of this therapy, which is indicative of acquired PRMT5 inhibitor resistance. For this reason, we sought 1) to evaluate the upfront susceptibility of 9 MCL cell lines to the PRMT5 inhibitor; 2) to generate acquired resistant counterparts to those cell lines sensitive to the PRMT5 inhibitor; 3) to utilize next-generation sequencing technologies to compare these resistant cell lines and murine samples to their sensitive/untreated counterparts. We hypothesized that comparing the multiple PRMT5 inhibitor resistant cell lines and murine samples with their parental/untreated counterparts will highlight pro-survival pathways amplified with PRMT5 inhibitor resistance. To investigate this hypothesis, the half-maximal inhibitory concentrations (IC50) of nine MCL cell lines were determined by measuring viability using Annexin V/PI staining and flow cytometry following 9 days of PRMT5 inhibitor titration. The five cell lines with IC50s of approximately 100 nM or less (Z-138, CCMCL, Rec-1, SP53, and Granta-519) were defined as having primary sensitivity to PRMT5 inhibition. Conversely, cell lines with an approximately three to ten-fold higher IC50 (Maver-1, Jeko, Mino, and UPN1) were defined as having primary resistance to PRMT5 inhibition. Four of the five primary sensitive cell lines (Z-138, CCMCL, Rec-1, and SP53) were utilized to model acquired resistance to PRMT5 inhibition by employing drug escalation protocols to the cell lines in continuous culture. Resistance of the acquired resistant cell lines was reassessed by IC50s for PRT 382 on day 9 measured using Annexin V/PI staining and flow cytometry. In this way, these MCL cell lines were generated with acquired PRMT5 inhibitor resistance (IC50s 3 to 5 times that of the original). To verify sustained resistance, acquired resistant cell lines were cultured for one month in the absence of the inhibitor and rechallenged at their new IC50 three times. DNA and RNA of these PRMT5 inhibitor resistant cell lines and murine samples and their parental/untreated counterparts were submitted for next-generation sequencing technologies including bulk RNA and whole-exome sequencing. Ingenuity Pathway Analysis (IPA) was utilized to prioritize candidate genes and pathways upregulated with disease progression. Sequencing analysis on these PRMT5 inhibitor acquired and primary resistant cell lines and murine samples have highlighted several pathways including MAPK and PI3K signaling which are amplified with PRMT5 inhibitor resistance. These pathways are currently being subjected to further evaluation as possible targets for combination therapy with PRMT5 inhibition. In addition, prioritized targets within these pathways will be further validated via evaluation of mRNA and protein expression levels using quantitative reverse transcription PCR (RT-qPCR) and western blot, respectively. Controlled expression of candidate genes will be performed via shRNA knockdown in resistant cell lines to assess for changes in the IC5O for PRT 382. This proposal is novel in its investigation of the evolution and development of PRMT5 inhibitor resistance as it seeks to characterize the genetic, epigenetic, and cell signaling mechanisms that evolve with this phenotype. In this way, candidate genes and compensatory signaling pathways that confer resistance and sensitivity to PRMT5 inhibitor therapy will be highlighted. Such analysis will provide valid targets for combinatorial therapies that will be further evaluated in vivo using multiple preclinical murine models.