Mantle cell lymphoma (MCL) is an incurable B-cell non-Hodgkin's lymphoma. MCL patients who relapse on targeted therapies have a particularly poor prognosis. Protein arginine methyltransferase 5 (PRMT5), an enzyme that drives symmetric dimethylation of arginine (SDMA) residues, is overexpressed in MCL and promotes MCL growth and survival. Thus, PRMT5 has emerged as an attractive therapeutic target in MCL. We developed a small molecule PRMT5 inhibitor (PRT-382) that exhibits significant anti-MCL activity in cell lines (50-100 nM) and in vivo preclinical MCL murine models (10 mg/kg, daily, four days on; 3 days off). Despite the anti-tumor activity of PRMT5 inhibition, we have observed some animals develop drug resistance leading to rapid progression of MCL despite continued treatment with PRT-382. While treatment with PRT-382 significantly prolonged the survival of our MCL patient derived xenograft (PDX) model (83D vs. 48D, p-value: 0.0045), one mouse developed a rapid and much greater expansion of MCL cells both in the peripheral blood and in multiple organs when it reached early removal criteria at day 90. Despite the significantly increased tumor burden in this mouse while on PRMT5 inhibitory therapy, SDMA reduction in MCL tumor cells was still achieved in comparison to the vehicle control treated mice. MCL tumor cells from this PRMT5 inhibitor refractory mouse and PRMT5 inhibitor naïve mice were re-engrafted and treated with PRT-382. Compared to mice engrafted with treatment naïve cells, the mice engrafted with PRMT5 inhibitor refractory cells had decreased survival (p-value: 0.004). Thus, in vivo resistance was retained allowing generation of additional samples for mechanistic studies. In addition to in vivo models of resistance, some MCL cell lines (Maver, Mino, UPN1, and Jeko) show primary resistance to PRMT5 inhibition based on high IC50s of PRT-382 (mean: 800; range: 300-2000 nM) compared to the sensitivity to this drug in other MCL cell lines (mean: 80; range: 50-100 nM). Prolonged culture of four PRT-382 sensitive MCL lines (CCMCL, Rec-1, SP53, and Z-138) with drug escalation produced cell lines with acquired PRT-382 resistance with IC50s 3 to 5 times (200-500 nM) that of parental lines. We found this PRMT5 inhibitor resistance persisted after prolonged culture (30d) in the absence of drug. As observed in the MCL PDX PRMT5 inhibitor refractory mice, SDMA reduction was still achieved at the original PRT-382 doses. Utilizing single cell RNA sequencing (scRNA-seq) and bulk RNA-sequencing, these multiple in vivo and in vitro PRMT5 inhibitor resistant samples were evaluated in comparison with their parental/untreated counterparts to identify compensatory pro-survival pathways amplified with PRMT5 inhibitor resistance. ScRNA-seq analysis on MCL PDX samples demonstrated a strong shift in global gene expression depending on treatment duration and condition. Upregulation of Insulin Receptor, PI3K, MAPK, and MTOR signaling were identified using Ingenuity Pathway Analysis (p-value threshold of 0.001 and z-score of 1.1) based on differentially expressed genes with PRMT5 inhibitor treatment in the resistant cell lines and PDX in comparison to their sensitive or parental counterparts. In vitro drug combination analysis has demonstrated synergy inhibiting PRMT5 (PRT-382) in combination with PI3K/MTORC1 and 2 (Omipalisib), MTORC1 (Temsirolimus), and EIF1A (Silvestrol) in MCL cell lines with primary (Mino) and acquired (Rec-1, SP53, and Z-138) resistance to PRMT5 inhibition. We are currently mechanistically validating these pathways in our in vitro models and invivo preclinical MCL models.

Acquired Resistance to PRMT5 Inhibition in Mantle Cell Lymphoma Is Associated with Compensatory Activation of Multiple Signaling Pathways