Mechanistic Insights and Therapeutic Potential of a PRMT5 Inhibitor Combined with Venetoclax in B Cell Malignancies

Introduction: PRMT5, a type II arginine methyltransferase, is overexpressed in several aggressive B cell malignancies and facilitates cancer cell growth. Small molecule inhibitors targeting PRMT5 show promising pre-clinical activity, particularly in mantle cell lymphoma (MCL) and diffuse large B cell lymphoma (DLBCL). There have been a number of clinical trials evaluating PRMT5 inhibitors in patients with advanced solid tumors or non-Hodgkin lymphoma (NHL), revealing manageable dose-dependent toxicity and initial signs of anti-tumor activity in various tumor types [NCT06137144, NCT05528055, and NCT03573310]. The BCL-2 inhibitor venetoclax demonstrates promising monotherapy activity in NHL. Here, we explore the mechanisms underlying the therapeutic potential of PRMT5i-1 (P1), a SAM and ATP competitive PRMT5 inhibitor, in combination with venetoclax in B cell malignancies.

Methods: Dynamic BH3 profiling (DBP) was employed to quantify the net pro-apoptotic signaling triggered by ex vivo drug treatments. Other standard techniques include RNA sequencing, gene knockdown via shRNA, immunoprecipitation (IP), Western blotting, PI/Annexin V and CellTiter-Glo. DLBCL (TMD8, RI-1, OCI-Ly1, SUDHL4), double-hit lymphoma (DHL) patient-derived xenograft (PDX) cell line (DW19), MCL (Mino, Jeko-1), and Burkitt lymphoma (Raji) cell lines were utilized to investigate the in vitro anti-cancer properties of P1 and BH3 mimetics (venetoclax [BCL2i], S63845 [S63, MCL-1i], A1331852 [A133, BCL-xLi], Selleckchem). Drug synergism was calculated using SynergyFinder, and a OCI-Ly1 cell line-derived xenograft (CDX) mouse model was employed for in vivo drug evaluation.

Results: By utilizing a synergy score of 10 as a threshold, combining P1 with venetoclax synergistically increased apoptosis in all 8 cell lines tested with the exception of Raji cells. Interestingly, Raji cells were resistant to P1, venetoclax, and A133, but sensitive to S63. In contrast, the combination of P1 with S63 or A133 each led to synergistic apoptosis in only 4 of the cell lines tested (S63: DW19, TMD8, OCI-Ly1, Mino; A133: DW19, OCI-Ly1, Jeko, Mino). Therefore, venetoclax appears to be a more effective partner for P1 compared to S63 and A133. Knockdown of PRMT5 also increased the sensitivity of cells to venetoclax. In a CDX mouse model, combining P1 with venetoclax significantly delayed tumor growth and prolonged mouse survival compared to single-drug treatment.

Through DBP analysis, P1 was found to enhance the overall mitochondrial apoptotic priming of DLBCL and MCL cell lines (>15% increase in cytochrome c loss). Notably, Raji cells exhibited lower basal levels of apoptosis priming, and P1 had minimal impact on apoptotic priming in this particular cell line.

To elucidate the mechanism behind the synergy between P1 and venetoclax, we performed RNA sequencing analysis on P1 treated OCI-Ly1 and SUDHL4 cell lines and found a significant (Q value< 0.001) enrichment of genes in several pathways, including alternative splicing and apoptosis. However, the protein expression of BCL-2 family members had minimal alterations upon P1 treatment. Subsequently, through IP we observed an enhanced interaction between BCL-2 and BIM following P1 treatment, potentially contributing to the heightened sensitivity to venetoclax.

To determine if the combination of P1 and venetoclax activates cell death other than mitochondrial apoptosis, BAX and BAK double knockdown (DKD) cell lines were created and tested for their sensitivities to the two drugs. DKD cells were partially protected from venetoclax and P1 induced cell apoptosis. Furthermore, the pan-caspase inhibitor Z-VAD-FMK rescued the drug combination-induced cell death, indicating the involvement of caspases. The caspase 8 inhibitor Z-IETD-FMK also rescued drug-induced cell death, but not as much as Z-VAD-FMK, suggesting potential activation of the extrinsic apoptosis pathway by the combined use of P1 and venetoclax.

Conclusions: Our study suggests that the combination of a PRMT5 inhibitor P1 with venetoclax potently induces both intrinsic and extrinsic apoptotic cell death and may serve as a potential therapeutic strategy to explore further for DLBCL and MCL.