EZH1/2 Inhibition Improves the Anti-Tumor Efficacy of CAR and TCR T-Cell Based Therapies Against Multiple Liquid and Solid Tumors
Authors: Nason Siena , Yang Ziqi , Ghilardi Guido , Paruzzo Luca , Carturan Alberto , Fardella Eugenio , Guruprasad Puneeth , Padmanabhan Anushka Anant , Blanchard Tatiana , Linette Gerald , Carreno Beatriz M. , Susanibar-Adaniya Sandra , Garfall Alfred L. , Ruella Marco , Porazzi Patrizia 
Published: December 2024

Background: Chimeric antigen receptor (CAR) T cells have shown unprecedented success in refractory or relapsed B cell malignancies, leading to six FDA-approved products targeting CD19 or BCMA. However, many patients still experience treatment failure following CAR T-cell therapy. Additionally, CART immunotherapies have so far failed against solid tumors because T cells cannot reach, fully activate, and function within the suppressive tumor microenvironment. Epigenetic modulation presents a promising strategy to overcome immunotherapy resistance by simultaneously targeting cancer cells, T cells, and the tumor microenvironment.

Abnormal activity and mutations of the epigenetic modulators EZH2 and the homolog EZH1 are critical determinants of the transcriptome of various cancers and associate with disease progression, therapy resistance, and poor prognosis. By catalyzing tri-methylation of histone 3 on Lysine 27 (H3K27me3), EZH1 and EZH2 regulate critical cancer pathways such as cell cycle checkpoints, interferon response, and DNA damage. EZH1 and EZH2 are druggable targets, and we here hypothesized that combining EZH1/2 inhibitors with CAR T-cell therapy could enhance immunotherapy efficacy across multiple malignancies.

Previously, we showed that the FDA-approved EZH2 inhibitor tazemetostat significantly enhanced anti-CD19 CAR T-cell (CART19) therapy in large B-cell lymphoma (LBCL) models (Porazzi P., Blood, 2023, 142 (Supplement 1); 1018). Here, we explored whether EZH2 inhibition could enhance CART efficacy against other targets and cancers, as well as other adoptive T-cell therapies. Finally, we also investigated the potential benefit of inhibiting both EZH1 and EZH2 to further boost the anti-tumor effect of CART cells.

Methods and Results: We first explored the synergy between tazemetostat (taz), and novel CAR T-cell products designed for B-cell lymphoma (anti-CD22 and anti-CD79b CART). We combined anti-CD22 CART and anti-CD79b CART with tazemetostat in LBCL models (e.g. OCI-Ly18, SU-DHL-4). We observed that CART22 and CART79b co-cultured with tazemetostat showed enhanced cytotoxicity (72 hs, CART22-taz vs -DMSO p&lt;0.001; CART79b-taz vs -DMSO p&lt;0.01).

We then investigated whether EZH2 inhibition could augment CAR T-cell-mediated cytotoxicity in multiple myeloma (MM) and acute myeloid leukemia (AML) models. Pretreating an array of MM (e.g. RPMI-8226) and AML (e.g. THP-1, KG-1) cell lines with tazemetostat before exposure to anti-BCMA (CARTBCMA) or anti-CD33 CART (CART33) significantly increased killing efficacy at multiple E:T ratio and even with ineffective single-agent CART doses (72 hs; CARTBCMA-taz vs -DMSO p&lt;0.01; CART33-taz vs -DMSO p&lt;0.001).

Recognizing the potential impact beyond hematological cancers, we investigated EZH2 inhibition in solid tumors overexpressing EZH2, such as HER2+ prostatic adenocarcinoma and ovarian cancer. Pretreatment of cancer cells (PC3, SKOV-3) with tazemetostat significantly potentiated the long-term (120 hs) cytotoxic activity of HER2-specific CAR T cells. Given tazemetostat's approval for advanced sarcomas, we extended our study to Ewing Sarcoma using the A673 cell line. In this setting, combining tazemetostat with TCR T cells targeting the LOXHD1 epitope (Deng, Q. et al., Cell Rep. 39, 110971) significantly enhanced long-term T-cell-mediated killing of sarcoma cells (T-cell-taz vs -DMSO P&lt;0.05).

Finally, we speculated that targeting EZH1 in combination with EZH2 would enhance the synergy of the combination with CART because of the potential compensatory mechanism triggered by EZH1 and its expression in non-proliferating cells. To this goal, we employed the dual EZH1/EZH2 inhibitor valemetostat. In the context of clinically relevant models of LBCL (OCI-Ly18, SU-DHL-4, Toledo and Karpass 422 cell lines) and MM (RPMI cell line), the combination of valemetostat and CART19 or CARTBCMA substantially enhanced CART killing (72 hs) at multiple E:T ratios.

Conclusions: In summary, this study demonstrates that EZH2 inhibition enhances CART efficacy in multiple models of solid and liquid cancers, suggesting its potential clinical relevance. Furthermore, we show that the novel EZH2/1 inhibitor valemetostat lead to an increased improvement of tumor killing as compared to EZH2i alone. Mechanistically, we demonstrate that EZH1/EZH2 modulation can unlock the full potential of adoptive T-cell immunotherapy.