Dual HDAC and EZH2 inhibition modulates RFX5 activity to increase the immunogenicity of germinal center-derived B-cell lymphoma Free

Chromatin-modifying genes, such as CREBBP and EZH2, are ubiquitously mutated in germinal center-derived B-cell (GCB) lymphomas, leading to impaired antigen presentation, a “cold” tumor environment, and malignancy progression. Belinostat (BEL), an HDAC inhibitor, and tazemetostat (TAZ), an EZH2 inhibitor, counteract the epigenetic derangements caused by CREBBP and EZH2 mutations, and together increase immune activity and antigen presentation targets to create a “hot” tumor environment. We hypothesize that dual HDAC and EZH2 inhibition increases antigen presentation pathways and T cell-directed cytotoxicity to deepen response rates in GCB lymphoma.

We previously reported that combination BEL+TAZ treatment increases numerous cell surface markers of immunogenicity, including MHC-I, MHC-II, and beta-2-microglobulin on B-cell lymphoma cell lines harboring various CREBBP/EZH2 mutations. Furthermore, BEL+TAZ increases markers of activation in healthy T cells compared to BEL/TAZ monotherapy. To evaluate epigenetic priming on T-cell directed cell kill, a luciferase+ B-cell lymphoma cell line, SU-DHL-4, and partially HLA-matched T cells were exposed separately to vehicle, BEL, TAZ, or BEL+TAZ for six days before being co-cultured for 24 h. Co-cultures primed with BEL+TAZ had fewer viable B cells compared to controls via luminescent assays (n=3, all data herein have P values < 0.05). B cell viability was protected with pretreatment of an MHC-I blocking antibody, showing that BEL+TAZ-induced cell kill is dependent on MHC-I.

Combo therapy also increased B:T-cell interactions as visualized by co-culturing BEL+TAZ-treated SU-DHL-4 cells with GFP+ F-actin Jurkat T cells and performing live cell imaging (n=2). Lymphoma spheroids were generated using Karpas-422 cells, pretreated with vehicle, BEL, TAZ, or BEL+TAZ for six days, and co-cultured with T cells for 24 h. Spheroids with BEL+TAZ priming exhibited increased T-cell infiltration through confocal microscopy (n=3).

To assess epigenetic therapy on peripheral immune cells in vivo, primary blood samples from lymphoma patients enrolled in a clinical trial for BEL+TAZ (NCT05627245) were collected pre- and post-treatment and analyzed using single-cell RNA-seq. Combo BEL+TAZ induced various antigen processing and presentation pathways in CD4+ and CD8+ T cells through gene ontology over-representation analyses. Furthermore, CD8+ T cells exhibited increased calcium-mediated signaling and T-cell proliferation signals, revealing an activated state following treatment.

To identify the transcriptional targets of BEL+TAZ therapy in malignant cells, SU-DHL-4 cells were treated and analyzed using bulk RNA- and ATAC-seq (n=4). The expression/enrichment of many antigen presentation genes and pathways all increased following treatment. Transcription factor analyses revealed that RFX5, a component of the regulatory factor X complex that regulates MHC expression, was significantly enriched following dual treatment, suggesting its involvement in BEL+TAZ-induced immunogenicity. Dual BEL+TAZ therapy increased HOMER ATAC-seq enrichment of RFX5, further highlighting its regulation by BEL+TAZ treatment.

Chromatin immunoprecipitation (ChIP) PCR for RFX5 was performed on vehicle- and BEL+TAZ-treated SU-DHL-4 cells using primers targeting the promoters of multiple MHC genes. Combo therapy led to increased RFX5 binding at the HLA-DQA1 and HLA-DRA promoters compared to controls (n=2). Dual BEL+TAZ also increased the protein expression of RFX5 and its binding partner, CIITA, measured via Western blots (n=3, four cell lines). RFX5 expression was knocked down (KD) in SU-DHL-4 cells using siRNAs. RFX5 WT and KD cells were treated with vehicle, BEL, TAZ, or BEL+TAZ for six days and co-cultured with T cells for 24 h. Decreased cell viability was observed in the RFX5 WT cells but not the KDs following treatment (n=3), revealing the critical role of RFX5 expression in BEL+TAZ-mediated cell kill. Lastly, RFX5 knockout (KO) murine A20 lymphoma cells were generated using CRISPR-Cas9 gene editing. An experiment is currently underway evaluating tumor growth and overall survival in vehicle-, BEL-, TAZ-, and BEL+TAZ-treated BALB/c mice xenografted with either RFX5 WT or KO murine A20 cells.

In conclusion, dual HDAC and EZH2 inhibition modulates RFX5 activity to increase antigen presentation and T cell-directed targeting, proposing a novel epigenetic pathway by which to induce an immune-activated tumor environment in GCB lymphomas.