Dual HDAC and EZH2 Inhibition Primes Mosunetuzumab for the Treatment of Germinal Center-Derived B-Cell Lymphoma

Chromatin-modifying genes, such as CREBBP and EZH2, are ubiquitously mutated in germinal center-derived B-cell (GCB) lymphomas, leading to impaired antigen presentation, immune escape, and malignancy progression. Belinostat (BEL), an HDAC inhibitor, and tazemetostat (TAZ), an EZH2 inhibitor, counteract the epigenetic derangements caused by CREBBP and EZH2 mutations. Mosunetuzumab (MOSUN) is an anti-CD20/CD3 bispecific antibody that relies on stimulated T cells and B:T-cell interaction for its activity. Though MOSUN exhibits an impressive overall response rate in follicular lymphoma, the progression-free survival significantly degrades over time. We hypothesize that dual HDAC and EZH2 inhibition primes the effects of MOSUN to improve its efficacy and deepen response in GCB lymphomas, while simultaneously targeting key drivers of lymphomagenesis.

Five GCB lymphoma cell lines with varying CREBBP and EZH2 mutations were treated on day 0 with nontoxic doses of BEL, TAZ, or dual BEL+TAZ, retreated on day 3, and collected on day 6. Samples were analyzed via flow cytometry to measure markers of immunogenicity. Monotherapy only induced marginal changes while combination BEL+TAZ caused a >1.7-, >1.9-, and >2.2-fold increase in MHC-I, MHC-II, and B2M, respectively, across all mutated cell lines compared to vehicle-treated controls (n=4 per cell line; all data presented herein have P values < 0.05 unless specified). Dual BEL+TAZ also led to a >1.4- and >1.7-fold increase in CD19 and CD20 expression as measured via flow cytometry (n=4 per cell line) and corroborated through Western blotting (n=3 per cell line) and immunofluorescence (n=3 in one cell line).

To assess the effect of epigenetic therapy on T cells, healthy donor CD3+ T cells were isolated, exposed to BEL, TAZ, or BEL+TAZ for 6 days, and analyzed via flow cytometry for cell surface markers of T-cell activity. Dual treatment increased CD8+ T cell populations and led to a 1.29-, 1.49-, and 1.37-fold increase in CD25, CD44, and CD69 expression, indicating T-cell activation (n=5). Intracellular markers of T-cell activity were similarly increased following BEL+TAZ: Ki-67, Granzyme A, and Granzyme B increased 1.74-, 2.11-, and 2.67-fold compared to controls (n=3). T cell-mediated IL-6 secretion increased 1.21-fold following BEL+TAZ treatment as measured in the culture medium by ELISA (n=2, P=0.08).

Primary samples from lymphoma patients enrolled in a clinical trial for BEL+TAZ (NCT05627245) were collected pre- and post-treatment and analyzed via flow cytometry. Dual BEL+TAZ treatment resulted in a 1.23- and 1.59-fold increase of MHC-I and CD20 expression on B cells, and a 1.3- and 1.21-fold increase in the CD8+ population and CD69 expression in T cells, indicating immune modulation in lymphoma patients (n=3). Peripheral blood mononuclear cells from a subject on the BEL+TAZ study were collected pre- and post-treatment and prepared for single-cell RNA sequencing. The samples were effectively sequenced with >320 million total reads each and >90% of reads mapped to the genome. Downstream bioinformatics analyses are underway to assess transcriptional changes across specific immune cell subtypes.

To evaluate BEL+TAZ priming on MOSUN activity, a B-cell lymphoma cell line, SU-DHL-4, and healthy T cells were exposed separately to BEL+TAZ or vehicle for 6 days before being co-cultured and exposed to MOSUN for 24 h. Co-cultures primed with BEL+TAZ before MOSUN exposure had 1.91-fold fewer viable B cells than those treated with MOSUN alone (n=3). Dual BEL+TAZ therapy also increased B:T-cell interactions as assessed by intercellular F-actin immunofluorescence and visualized through confocal microscopy (n=3). Lymphoma organoids were generated using SU-DHL-4 cells, pretreated with BEL+TAZ or vehicle for 6 days, co-cultured with BEL+TAZ-treated T cells, and exposed to MOSUN for 24 h (n=3). Organoids with BEL+ TAZ priming before MOSUN exposure exhibited a 1.63-fold decrease in viable B cells compared to unprimed organoids as measured via flow cytometry (n=3) and corroborated through immunofluorescence analyses (n=3).

In conclusion, dual BEL+TAZ treatment increases MHC/CD20 expression on malignant B cells and activates peripheral T cells, priming the tumor microenvironment for MOSUN therapy. Taken together, our findings support dual HDAC and EZH2 inhibition as a potential means to prime immunotherapy and improve outcomes in GCB lymphomas.