Pediatric Acute Myeloid Leukemia associated S100A9 promotes T cell dysfunction

Abstract Despite immunotherapy revolutionizing cancer treatment, myeloid malignancies have remained largely refractory to T cell-based strategies, necessitating an in-depth analysis of the mechanisms inhibiting the therapeutic efficacy and raising the question of whether extrinsic factors in the tumor microenvironment could induce T cell dysfunction. To better understand the components limiting T cell function in myeloid settings, we performed phenotypic analyses and genome-wide DNA methylation profiling of T cells isolated from pediatric acute myeloid leukemia (AML) patients. We observed that AML-derived T cells contain a more differentiated methylation signature and are enriched for the more terminally differentiated TemRA population compared to acute lymphoblastic leukemia (ALL) patient-derived T cells. To identify putative mechanisms of T cell immune suppression in AML patients, we mined St. Jude's comprehensive multi-omic datasets for known immunosuppressive factors; S100A9 was among the most elevated inflammatory transcripts in AML blasts and was confirmed in the plasma protein using diagnostic samples (AML AUC, 0.770; T-ALL AUC, 0.729; B-ALL AUC, 0.561; AML < 13 years AUC, 0.538; AML ≥ 13 years AUC, 0.975). We have defined epigenetic regulation of T cell stemness disrupted by loss of DNA methylation genes known to drive myeloid malignancies (Kang et. al., Science, 2024; Prinzing et. al., STM, 2021; Ghoneim el. al., Cell, 2017). Thus, we applied the established exhaustion programs to interrogate the available patient T cell samples and found a significant decrease in cell stemness and multipotency in AML T cells compared to ALL T cells. Additionally, increased methylation was observed in genes associated with naïve T cells, cytotoxicity, and TLR activation in T cells. Decreased methylation was observed in genes related to T cell differentiation, changes in the mitochondria, and cellular metabolism. Analyzing the functional and transcriptional impact of S100A9 on healthy donor T cells revealed that both S100A9 protein and AML plasma induced in vitro T cell dysfunction, as demonstrated by the induction of a terminal effector phenotype, blunted cytotoxicity, and proliferation in response to TCR stimulation. S100A9 had a significant metabolic impact on treated T cells by Seahorse Metabolic Flux assay, as well as significant changes in mitochondrial membrane potential and increased total cellular ROS. These results suggest that S100A9 released from the AML patient's myeloid tumor drives inflammatory microenvironmental changes, which feed into T cell dysfunction on phenotypic, metabolic, RNA, and epigenetic levels, contributing to decreased T cell efficacy. These results provide mechanistic insight into the suppressive myeloid tumor microenvironment limiting T cell-based immunotherapy and highlight the need to better characterize the crosstalk between the innate and adaptive immune systems. Collectively, these data suggest that the decreased efficacy of T cell-based immunotherapies in pediatric AML is due to dysfunction in the patient-derived T cell, further exacerbated by the inflammatory myeloid microenvironment.