Abstract PO-040: Mechanisms of action of immune checkpoint blockade therapy in EZH2/Bcl2-mutant B-cell lymphomas
Authors: Sarapulov Alexey V , Inna Serganova , Bucktrout Ryan , Isshiki Yusuke , Santella Anthony , Rodriguez Marc-Anthony , Chakraborty Sanjukta , Brunschvig Solene , Vardhana Santhosha , Tripodo Claudio , Melnick Ari , Beguelin Wendy , Zappasodi Roberta 
Published: June 2024

Abstract
Background Diffuse large B cell lymphoma (DLBCL) is the most frequent subtype of non-Hodgkin lymphomas (B-NHLs). B-NHLs respond poorly to currently available immune checkpoint blockade (ICB) therapies, such as anti-PD-1 or anti-CTLA-4 + anti-PD-1 combination and the reason for this is unknown. New genetic classifications of DLBCL have been recently proposed, providing rationale for studying ICB resistance mechanisms according to these novel molecular DLBCL subtypes.
Methods Genetic aberration involving Bcl2 and EZH2 are commonly found in follicular lymphoma (FL) and DLBCL in patients. Here, we used mouse models of EZH2/Bcl2-mutant (EZB) lymphoma, constitutively expressing Bcl-2 and EZH2Y641F mutation in germinal center B (GCB) cells (Cg1-Cre; Rosa26LSL.BCL2.IRES.GFP; EZH2Y641F), including a genetically engineered mouse model (GEMM) which spontaneously develops diseases resembling FL and wild type mice transplanted with a GEMM-derived EZB cell line resembling DLBCL lymphoma. Mice were treated with ICB therapy alone or in combination with the EZH2 inhibitor, tazemetostat (TAZ). Immune and lymphoma cells from spleens and lymph nodes were analyzed by flow cytometry and immunofluorescence staining of tissue sections. These parameters were correlated with tumor progression by bioluminescence imaging, MRI, and overall survival.
Results We found that TAZ alone or in combination with anti-CTLA-4 moderately decreased tumor burden but did not significantly improve survival of EZB GEMM mice. This was associated with increased frequencies of CXCR5+ CD8+ T cells in spleen and lymph nodes. We then evaluated the effect of dual anti-CTLA-4 + anti-PD-1 ICB therapy to target the additional immune checkpoint, PD-1. In EZB GEMM mice, at 150 days post-treatment, anti-CTLA-4 + anti-PD-1 therapy with or without TAZ extended survival, compared to single agents (71.4% vs 42.6%). Of note, tumor burden irrespective of treatment positively correlated with frequencies of CXCR5+Bcl6+CD4+ T follicular helper cells and MHC-I/II+CD86+ normal B cells, suggesting ongoing immune reaction. Interestingly, mice receiving TAZ + ICB had lower tumor burden and highest percentages of NK cells. In the more aggressive transplantable EZB-DLBCL model, anti-CTLA-4 + anti-PD-1 combination led to complete tumor regressions, extending survival, and inducing anti-tumor immunological memory. Combination-treated tumors had greater proportions of CXCR5+GzmB+CD8+ T cells and greater CXCR5 expression levels on conventional T cells compared to monotherapy or control tumors. Anti-PD-1 or ICB combination increased the proportion of Ly6c+ lymphoma cells, suggesting diversion to plasmablast phenotype.
Conclusions Our results indicate that CXCR5+ T-cell:B cells interactions may be implicated in EZH2-driven lymphomas, with different outcomes depending on the disease stage (FL vs. DLBCL). Ongoing work is focused on the mechanistic role of increasing access of cytolytic immune cells to lymphoma niches via CXCR5 and promoting lymphoma cell reprogramming in the responses of EZB lymphoma to ICB therapy.
Citation Format: Alexey V Sarapulov, Serganova Inna, Ryan Bucktrout, Yusuke Isshiki, Anthony Santella, Marc-Anthony Rodriguez, Sanjukta Chakraborty, Solene Brunschvig, Santhosha Vardhana, Claudio Tripodo, Ari Melnick, Wendy Beguelin, Roberta Zappasodi. Mechanisms of action of immune checkpoint blockade therapy in EZH2/Bcl2-mutant B-cell lymphomas [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-040.