Targeting epigenetic regulation and post-translational modification with 5-Aza-2’ deoxycytidine and SUMO E1 inhibition Augments T Cell Receptor Therapy

Cellular immunotherapy using modified T cells offers new avenues for cancer treatment. T cell receptor (TCR) engineering of CD8 T cells enables these cells to recognize tumor associated antigens (TAA) and tumor specific neo-antigens. Improving TCR T cell therapy through increased potency and in vivo persistence will be critical for clinical success. We evaluated a novel drug combination to enhance TCR therapy in mouse models for acute myeloid leukemia (AML) and multiple myeloma (MM). Combining TCR therapy with the SUMO E1 inhibitor TAK981 and the DNA methylation inhibitor 5-Aza-2’ deoxycytidine resulted in strong anti-tumor activity in a persistent manner against two in vivo tumor models of established AML and MM. We uncovered that the drug combination caused strong T cell proliferation, increased cytokine signaling in T cells, improved persistence of T cells, and reduced differentiation towards exhausted phenotype. Simultaneously the drug combination enhanced immunogenicity of the tumor by increasing HLA and costimulation and surprisingly reducing inhibitory ligand expression. Combining T cell therapy with TAK981 and 5-Aza-2’ deoxycytidine may be an important step towards improved clinical outcome. This study utilizes in vivo tumor models in NSG mice to investigate the efficacy of TAK981 and/or 5-Aza-2’ treatment on OCI-AML3 and U266 tumors. Luciferase-tdTomato tagged cells enable real-time monitoring of tumor growth and T cell dynamics. Ex vivo analysis involves the isolation of bone marrow and tumors for comprehensive molecular profiling, including flow cytometry and single-cell RNA sequencing using the 10x Genomics Chromium X platform. Samples were collected from both bone marrow and tumor tissues. For each mouse, bone marrow from both paws was combined to ensure representative sampling. The bone marrow samples underwent negative MACS enrichment to isolate human cells, using anti-human CD45 (APC) antibodies and APC-beads. This process specifically targeted human cells within the mouse bone marrow population. To further purify the enriched human cell population, positive selection was performed using anti-human CD45 antibodies, sorting for cells expressing this marker. This ensured a high level of purity in the final cell population. Subsequently, the purified human cells were labeled with unique hashtags, allowing for individual sample identification and tracking throughout downstream analyses. Following cell isolation and labeling, a single run of 10x Genomics 3’ v3.1 chemistry was conducted. This involved the preparation of single-cell gene expression libraries using the 10x Genomics Chromium X platform, following the manufacturer's instructions.