Inhibition of PRMT5 with JNJ-64619178 sensitizes B-cell non-Hodgkin lymphoma cells to both intrinsic and extrinsic apoptosis Free

Introduction: Protein arginine methyltransferase 5 (PRMT5), a type II arginine methyltransferase, is overexpressed in several aggressive B cell malignancies and facilitates cancer cell growth. JNJ-64619178 (JNJ-9178) is a PRMT5-selective small molecule inhibitor that has been investigated in a clinical trial in patients with advanced solid tumors, B-cell non-Hodgkin lymphoma (NHL) and lower risk myelodysplastic syndromes [NCT03573310]. However, clinical activity was limited despite robust target engagement. Here, we investigated the effects of JNJ-9178 on both the intrinsic and extrinsic apoptosis pathways and identified rational combination partners for JNJ-9178 to achieve improved activity in B-cell NHL.

Methods: Dynamic BH3 profiling (DBP) was employed to quantify the net pro-apoptotic signaling triggered by ex vivo drug treatments. Other standard techniques include gene knockdown via shRNA, Western blotting, PI/Annexin V and FACS analysis. Various cell lines, including DLBCL (TMD8, Ri-1, OCI-Ly1, OCI-Ly1R, SUDHL4), double-hit lymphoma patient-derived xenograft (DW19), MCL (Mino, Jeko-1), and BL (Raji, BL-70) were utilized to investigate the in vitro anti-cancer properties of JNJ-9178, BH3 mimetics (venetoclax [Ven, BCL-2i], S63845 [S63, MCL-1i], A1331852 [A133, BCL-xLi]) and TRAIL analogs (rhTRAIL [recombinant human TRAIL], Conatumumab [DR5 agonist], and Mapatumumab [DR4 agonist]). Drug synergism was calculated using SynergyFinder. A luciferase-labeled OCI-Ly1 cell line-derived xenograft (CDX) mouse model was employed for in vivo drug evaluation.

Results: BH3 profiling in a panel of B-cell NHL cell lines identified 7 partially BCL-2-dependent cell lines (DW19, TMD8, Ri-1, SUDHL4, OCI-Ly1, Jeko-1 and Mino). These cell lines were not sensitive to Ven (except for OCI-Ly1 and Ri-1) due to co-dependence on other anti-apoptotic proteins. DBP demonstrated that JNJ-9178 exposure increased overall mitochondrial apoptotic priming and further increased BCL-2 dependence in all 7 cell lines. The combination of JNJ-9178 and Ven synergistically induced apoptosis in all 7 cell lines.

We also identified 3 less BCL-2-dependent cell lines, Raji (BCL-2 sufficient), BL-70 (BCL-2 deficient) and OCI-Ly1R (with Ven-acquired resistance generated from the OCI-Ly1 cell line). All 3 cell lines were primarily MCL-1 dependent and sensitive to S63 but resistant to Ven. DBP in these cell lines demonstrated that JNJ-9178 increased overall mitochondrial apoptotic priming and MCL-1 dependence while having negligible impact on BCL-2 dependence. Consistently, the cells became more sensitive to S63-induced apoptosis in the presence of JNJ-9178 while remaining resistant to Ven. Together, these data suggest that JNJ-9178 increases overall mitochondrial apoptotic priming without switching BCL-2-less dependent cell lines to become BCL-2-dependent. In a CDX mouse model, we found the combination of JNJ-9178 with Ven significantly delayed tumor growth and prolonged survival compared to single drug treatment.

Examining death receptor expression on the cell membrane via flow cytometry, we found that JNJ-9178 led to increased membrane expression of the TRAIL receptors DR4 and DR5 in B-cell NHL cell lines including DLBCL, MCL and BL, and sensitized these cells to rhTRAIL-induced apoptosis. Utilizing targeted knockdown and the agonist antibodies Conatumumab and Mapatumumab, we found the synergism between JNJ-9178 and rhTRAIL required activation of both DR4 and DR5 in most cell lines. Mechanistically, the increased expression of membrane DR4 and DR5 by JNJ-9178 could be due to a cellular response to global splicing alterations as we found that splicing modulators targeting different components of the splicing machinery other than SmB, SmD1 and SmD3 also led to increased membrane DR4 and DR5 expression and sensitized tumor cells to rhTRAIL-induced apoptosis. We tested the combination of JNJ-9178 with conatumumab in the CDX mouse model and found this combination significantly delayed tumor growth and prolonged survival compared to single drug treatment.

Conclusions: We identified PRMT5 as an important regulator of both intrinsic and extrinsic apoptosis. Our data suggest that DBP has the potential to optimize the selection of BH3 mimetics to combine with JNJ-9178 to maximize the activity of this drug across certain B-cell NHL subtypes. Additionally, JNJ-9178 sensitizes B-cell NHL cell lines to TRAIL-induced cancer cell-selective extrinsic apoptosis.