Dysregulation of PRMT5 in Chronic Lymphocytic Leukemia Supports Transformation to Diffuse Large B-Cell Lymphoma

Abstract

Background: Richter's transformation (RT) is the progression of chronic lymphocytic leukemia (CLL) to a high-grade lymphoma, most commonly resembling a diffuse large B-cell-like lymphoma, that has a bleak prognosis of 6-12 months. Many of the recurrent somatic alterations identified in RT are also associated with the CLL-phase of disease, suggesting additional unidentified genetic, epigenetic, or molecular events underlie transformation. Aberrant PRMT5 expression is known to promote and maintain oncogenic signaling in non-Hodgkin's lymphomas but its role in CLL and RT remains unclear. Methods: Tissue microarrays and western blots were used to evaluate PRMT5 expression in CLL/SLL and RT lymph nodes (LN). Differentially expressed genes between patient-matched RT LNs and CLL PBMCs were identified with scRNA-seq & scV(D)J-seq. ScRNA-seq was utilized to identify transcriptional changes between Eμ-PRMT5/TCL1 & Eμ-TCL1 mice. Tumor allografts were generated by administering 5 x 10^6 CD19+CD5+ spleen cells from Eμ-PRMT5/TCL1 mice intravenously to C57/BL6J mice. One week post-engraftment mice were enrolled in treatment groups (PRT382 10mg/kg, n=10; vehicle, n=7) with all treatments administered for 4 contiguous days per week by oral gavage. Results: Tissue microarray analysis on 70 CLL and 15 RT LN cases showed robust PRMT5 staining in RT LNs and minimal staining in CLL tissues. Immunoblot analysis revealed that CLL cells from patients that eventually undergo transformation displayed significant increase in PRMT5 expression months prior to transformation, whereas CLL patients maintained variable to minimal PRMT5 expression. ScRNA-seq & scV(D)J-seq on RT LN biopsies and CLL PBMCs from two patients revealed that PRMT5 expression was restricted to distinct nodal B cell subpopulations co-expressing MYC, BIRC5, CD83, and CD69. One PRMT5+ cluster displayed overlapping expression of proliferative markers (Mki67, TOP2A, PCNA, CALM2/3, and HMGB1/2) with enriched EIF4 signaling. Anti-apoptotic and immune modulating genes (BCL21A, IL4I1, TCL1A, and CCL3/4) were enriched in the second PRMT5+ nodal cluster. Interestingly, transcriptional diversity was enhanced in clonally related RT B cell populations with an increased proportion of cells expressing PRMT5 when compared to the CLL-phase of disease. To further evaluate the role of PRMT5 in CLL disease progression, we conducted histopathologic analysis on the spleen and lymph nodes of 6 month old animals from Eµ-PRMT5/TCL1 & Eµ-TCL1 mouse models, revealing a RT-like histology with loss of germinal centers and effacement of normal architecture exclusively in Eµ-PRMT5/TCL1 animals. Transcriptional changes underlying phenotypic differences between Eµ-PRMT5/TCL1 & Eµ-TCL1 mice were also assessed by scRNA-seq, where splenic B cells of the Eµ-PRMT5/TCL1 exhibited increased expression of oncogenic and immune regulating genes (Myc, Mki67, Egr1, Cxcr5, Ccr7, Il-10, Ctla4, and Pd-L1). Interestingly, the dominant B-cell clusters enriched in Eµ-PRMT5/TCL1 spleen and LNs showed significant transcriptional overlap with human RT PRMT5 expressing LN clusters (e.g., Myc, Cd83, and Cd69). With the observation that PRMT5 may play a significant role in CLL-to-RT evolution, we then aimed to evaluate the use of targeted PRMT5 inhibition against murine RT-like tumors in vivo. Using the selective SAM-competitive PRMT5 inhibitor, PRT382, in Eµ-PRMT5/TCL1 allografts, we observed PRT382 treatment delayed leukemic expansion, increased median survival (p<0.0001), and reduced spleen mass compared to vehicle treated mice. Conclusion: We show that PRMT5 is upregulated in the lymph nodes of CLL patients prior to and after transformation, suggesting a role for PRMT5 in CLL transformation to RT. Likewise, the Eμ-PRMT5/TCL1 mouse model develops a RT-like histology and shows significant overlap in the transcriptional profile to human RT tumors. And further, inhibition of PRMT5 with PRT382 provides significant improvement in median overall survival and slows disease progression in Eμ-PRMT5/TCL1 adoptive transfer models. Our preclinical data identifies PRMT5 as a high-risk marker in CLL and provides rationale for the clinical evaluation of PRMT5 inhibition in the treatment of high-risk CLL/RT.