Investigating Synergistic Effects of PRMT5 Inhibitors Paired with BH3 Mimetics in Double-Hit Lymphoma

Field: Medicine (Health Sciences)

Double Hit lymphoma (DHL) is an aggressive B cell malignancy and comprises 6% of diagnoses among lymphoma cases which is estimated to reach ~2000 cases annually in the US. It primarily affects adult males in their late fifties and sixties and poses unique clinical challenges as it lacks a definitive standard of care. DHL has a poor prognosis with an overall survival ranging from 22%-43% over the span of 1.4-5.0 years. An initial response to standard chemotherapy is commonly observed, but relapse is inevitable, and to date there is no cure. DHL has a poor prognosis due to the aggressive nature of the disease and older patient demographic, which can limit their options for intensive treatment regimens. The lack of effective treatment options and inevitable fatality of a DHL diagnosis emphasize the paramount need for novel therapeutic strategies for DHL. Our group, Baiocchi Lab, was the first to identify the overexpression of Protein Arginine Methyltransferase 5 (PRMT5) as a key driver of DHL pathogenesis. PRMT5 is an enzyme that catalyzes the methylation of arginine residues on histones and many key proteins for cell cycle progression and survival. This post-translational modification results in the silencing of tumor suppressor genes which contributes to cell transformation and cancer. In our work to explore the effects of PRMT5 inhibition and its potential to reprogram DHL cells, we have discovered that combining PRMT5 inhibition with Bcl-2 inhibition results in synergistic cell death. BCL-2 family proteins can be broadly categorized as pro- or anti-apoptotic and their relative abundances and binding interactions determine if a cell will live or die. The synergy observed between PRMT5 inhibition and Bcl-2 inhibition in other DHL cell lines made us curious about what other effects PRMT5 causes and what is the underlying reasoning for this synergy. We hypothesized that PRMT5 inhibition lowers the apoptotic threshold in DHL by modulating levels of BH3 family proteins to favor a pro-apoptotic state. Major experiments done to unfold our hypothesis were: MTS assay, which visualized our combined treatment results to see if synergy was present; Western blotting, to probe for what mechanisms were causing the synergy; and Immunofluorescence Microscopy, which allowed us to visualize changes in FOXO1 localization in DHL cell lines upon PRMT5 inhibition. Further investigation led us to believe that this upregulation in expression is because, in DHL, PRMT5 is inhibiting FOXO1 localization into the nucleus where it serves as a transcriptional factor for BH3-domain proteins. MTS assays showed that combined treatment induced synergy, especially in dosage concentrations high in PRT808 and low in Venetoclax. Western blotting revealed significant changes in abundance of pro-apoptosis proteins and BCL-2 family proteins with PRMT5 inhibition. IF confirmed upon PRMT5 inhibition via PRT808 treatment, FOXO1 is capable of localizing into the nucleus at greater quantities. In summary, we have successfully unveiled key characteristics of DHL and began the groundwork on the journey to create a specific, effective, and targeted therapy for DHL. Let alone amongst all DHL lines, within DHL cell lines that share a differentiation state, ABC or GBC, there are profound differences in their responses to treatments and phenotypes. This further emphasizes the importance of characterizing DHL to have a greater understanding of how we can target it effectively on a case-by-case basis.