Arginine methylation on MSI2 drives resistance to PRMT1/5 inhibitors in lymphoma, controls translation and dictates cell fate

Abstract Dysregulation of protein arginine methyltransferases (PRMTs) is implicated in many cancers. MTAP-deleted tumors are selectively sensitive to next-generation PRMT5 inhibitors now in clinical trials, including for lymphomas. However, mechanisms of resistance and the role of arginine methylation remain poorly understood. A genome-wide CRISPR screen identified the RNA-binding protein MSI2 as the top gene conferring resistance to PRMT5 inhibition in B-cell lymphoma (Erazo et al., Nat Commun, 2022). MSI2 is essential for stem cell self-renewal, and its overexpression is associated with poor outcomes in hematologic malignancies (Kharas et al., Nat Med, 2010; Park et al., JCI, 2015; Palacios et al., Leukemia, 2021). We hypothesized MSI2 function may be post-translationally regulated by PRMTs. Mass spectrometry of immunoprecipitated MSI2 revealed five arginine methylation sites (R197, R199, R201, R228, R261). In vitro methylation assays showed that PRMT1 preferentially methylates R197/199/201, while PRMT5 targets R261. Mutation of all five sites to lysine (5K mutant) abolished MSI2 methylation. Novel methyl-specific antibodies against R197/201 and R261 were validated using overexpression, genetic knockout, and pharmacologic inhibition models. Elevated MSI2 R197/201 methylation was observed in 12/16 lymphoma lines, and R261 in 15/16. Sensitivity to PRMT5 inhibitors significantly correlated with R261 methylation (p=0.0079). Dual PRMT1/5 inhibition synergistically reduced viability in Z138, OCILY19, and SUDHL4 cells (p<0.000001). MSI2 overexpression conferred resistance to PRMT1 inhibitors and partially rescued viability during dual inhibition (from 12% to 30%, p=0.0012; and from 30% to 50% in PRMT knockout cells, p=0.003). In contrast, the MSI2-5K mutant failed to rescue (p=0.003, p=0.0007), indicating arginine methylation is required for MSI2's function in drug resistance. We next explored the functional role of MSI2 methylation in mRNA metabolism. HyperTRIBE profiling showed that WT and MSI2-5K bound nearly identical RNA targets (94% 3′UTR, 78% gene overlap), including MYC and BCL2, with no significant differences in binding by RIP-qPCR. However, AHA labeling revealed WT MSI2 enhanced nascent c-MYC protein by 50%, while MSI2-5K reduced it by 20% relative to control. c-MYC 3′UTR luciferase assays showed a 2-fold increase with WT MSI2 and ~50% decrease with MSI2-5K, confirming that methylation is dispensable for RNA binding but required for translational enhancement of MYC. MSI2-5K also acted as a dominant negative, suppressing MYC translation. To study MSI2 arginine methylation in vivo, we generated knock-in mice with lysine substitutions at the arginine methylation sites (MSI2-Arg-mut) using sequential CRISPR targeting. MSI2-Arg-mut mice exhibited decreased peripheral blood counts, smaller spleens and Peyer's patches, and reduced bone marrow cellularity. While HSPC frequencies were preserved, total HSCs, MPP2, MPP4, and ST-HSCs were significantly reduced. In competitive transplants, MSI2-Arg-mut marrow showed impaired multilineage engraftment by 16 weeks (p=0.00005), partially phenocopying MSI2 loss (Park et al., JEM 2014; Andrés-Aguayo et al., Blood, 2011). Proteomic profiling of HSPCs revealed 430 upregulated and 999 downregulated proteins (log2FC >|0.5|, p<0.05). Downregulated proteins overlapped significantly with MSI2-HyperTRIBE targets (20%) and were enriched for MYC, mTOR, and E2F signaling pathways. Upregulated proteins overlapped 40% with previously defined MSI2 targets. MSI2 protein levels were unchanged, indicating reduced functional activity due to loss of methylation. Surprisingly, MSI2-Arg-mut mice had enhanced germinal center (GC) formation following sheep red blood cell challenge (2-fold increase, p=0.042), with reduced BCL2 and MSI2 protein levels in B cells by IHC and flow cytometry. These data suggest cell context-dependent effects of methylation on MSI2 abundance. In summary, MSI2 is a dual substrate of PRMT1 and PRMT5, and arginine methylation is critical for its role in promoting translation of targets such as MYC and conferring resistance to PRMT inhibitors. The MSI2-Arg-mut mouse model uncovers a translational mechanism for PRMT function, where loss of methylation reduces HSC fitness, alters the proteome, and increases GC formation. These findings position MSI2 methylation as a central regulatory node in hematopoiesis, B-cell activation, and lymphomagenesis.