Hamaidi, Imène

Abstract BACKGROUND: Tumor-specific T cells are often characterized by reduced TCR signaling due to central and peripheral tolerance pathways that limit their responsiveness. These signals are further weakened by insufficient co-stimulation and active co-inhibitory pathways, collectively establishing a high threshold for activation. The integration of antigen recognition with co-stimulatory and inhibitory cues ultimately determines whether a T cell becomes activated or remains tolerant. Early TCR signaling must therefore be precisely regulated to prevent autoreactivity while still supporting protective immunity. Although phosphorylation and ubiquitination are well-established regulators of proximal TCR signaling, additional post-translational mechanisms remain less defined. SIRT2, a cytosolic NAD+-dependent deacetylase with emerging roles in immune regulation, has not been examined in the context of TCR signaling. METHODS: We evaluated proximal TCR signaling events in wild-type and SIRT2-deficient T cells using flow cytometry, immunoblotting, calcium flux assays, and RNA-sequencing. SIRT2-associated pathways were defined by mapping its interactome and acetylated substrates through mass spectrometry and immunoprecipitation. We screened LCK post-translational modifications by mass spectrometry and assessed SIRT2 enzymatic activity using an HPLC-based deacetylase assay. Conformational effects of LCK modification were examined using fluorescence-polarization binding assays and AlphaFold structural modeling. SIRT2 was deleted in human tumor infiltrating lymphocytes (TILs) via CRISPR/Cas9, and the impact of SIRT2 targeting was tested in lung cancer patient-derived xenograft models reconstituted with autologous TILs. RESULTS: SIRT2 deficiency amplified proximal TCR signaling, leading to elevated phosphorylation of early signaling mediators and increased calcium flux in both naïve and anergic T cells. Loss of SIRT2 also altered thymic selection dynamics and expanded TCR repertoire diversity. Mechanistically, SIRT2 interacted with and deacetylated LCK, the initiating kinase of proximal TCR signaling. Mass spectrometry identified lysine K228 in the LCK linker region as a SIRT2-regulated deacetylation site that governs LCK conformation and kinase activity. Functionally, SIRT2 inhibition in exhausted mouse and human TILs restored TCR signaling capacity and improved anti-tumor responses. CONCLUSION: Here we identify SIRT2-regulated deacetylation of LCK as a previously unrecognized mechanism that sets the strength and threshold of proximal TCR signaling. Accordingly, SIRT2 targeting reverses the exhausted phenotype of tumor-reactive T cells. Citation Format: Imene Hamaidi, Pingyan Cheng, Soo Young Jun, Min-Hsuan Wang, Min Zhang, Odesha Taylor, Luis Lopez-bailon, Ismail Can, Bin Fang, Anders Berglund, Bradford Perez, Ben Creelan, Andriy Marusyk, Virginia Shapiro, Haitao Ji, Jose R. Conejo-Garcia, Sungjune Kim. Sirt2 dictates TCR activation thresholds through post-translational control of LCK conformational state [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4250.

Abstract Background: While cancer immunotherapies have revolutionized treatment, their efficacy remains limited for most patients, highlighting tumors' capacity to evade immune detection. Epigenetic modifications, particularly DNA methylation, play a pivotal role in silencing immune-related pathways, thereby facilitating immune escape. By modulating gene expression, methylation disrupts immune activation, contributing to reduced tumor immunogenicity. This study examines the methylation landscape of co-stimulatory and immune checkpoint genes across various cancers to elucidate how these changes influence immune responses. Methods: Using The Cancer Genome Atlas (TCGA), we analyzed methylation and gene expression profiles across diverse cancer types. Data from 8,186 solid tumors and 745 adjacent normal tissues were processed via t-distributed stochastic neighbor embedding (t-SNE) with 247 probes targeting 20 immune-related genes. Principal component analysis (PCA) was applied to capture methylation variability across 8,931 tumor and normal samples. To explore the effects of methylation on immune gene expression, we analyzed data from 26 epithelial cancer cell lines treated with 5-azacitidine, a demethylating agent, using the GSE57342 dataset. Post-treatment changes in gene expression were assessed, and survival analyses from prior studies were used to explore clinical correlations. Results: We identified distinct methylation patterns in immune synapse genes, differentiating tumors from adjacent normal tissues. Co-stimulatory genes such as CD40 were hypermethylated in tumors, correlating with decreased expression, whereas immune checkpoint genes like PDL1 and HHLA2 were hypomethylated, correlating with increased expression. This inverse relationship between methylation and gene expression underscores the transcriptional regulatory role of DNA methylation. Treatment with 5-azacitidine reversed hypermethylation in co-stimulatory genes, including CD40, potentially enhancing immune recognition. Methylation levels of these genes were also associated with T cell infiltration into the tumor microenvironment and predicted better outcomes in melanoma patients, underscoring their clinical relevance. Conclusion: This study demonstrates that DNA methylation of immune synapse genes contributes to tumor immune evasion by modulating immune gene expression. Targeting these epigenetic alterations represents a promising strategy to restore tumor immunogenicity and improve immunotherapy outcomes, particularly when combined with immune checkpoint inhibitors. Reversing methylation-driven immune suppression could strengthen the immune response and enhance clinical outcomes for cancer patients. Citation Format: Imene Hamaidi, Anders Berglund, Matthew Mills, Ryan M. Putney, James J. Mulé, Sungjune Kim. DNA methylation of immune synapse genes drives tumor immune evasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4831.