ST2 promotes leukemia transformation by reshaping the immune landscape in Acute Myeloid Leukemia Free

Acute myeloid leukemia (AML) is an aggressive hematological malignancy with a 5-year survival rate of 30%, largely due to treatment failure and frequent relapse after therapy. A major factor in disease progression and persistence is the ability of leukemia cells to evade immune surveillance. We previously applied a state-transition modeling framework to conceptualize AML development as transcriptomic trajectories within a leukemia state-space. This leukemia state-space was constructed using time-series bulk RNA-seq data from a conditional Cbfb::MYH11 (CM) knock-in murine model of AML, which recapitulates the inv(16)(p13.1q22) chromosomal inversion found in 8–10% of AML patients. State-transition analysis identified IL1RL1 (encoding ST2) as a leukemia-promoting gene upregulated early and progressively increased during the transition from health to AML. Notably, increased ST2 expression is found in AML patients particularly in the inv(16) and t(8;21) subtypes in the TCGA, BEAT-AML, and TARGET-AML datasets (inv(16): fold change 10.9 vs. healthy controls, p < 0.001; t(8;21): fold change 16.6, p < 0.001) and high ST2 level is associated with poor prognosis (TCGA, n = 139, p < 0.001).

ST2, the receptor for interleukin-33 (IL-33), regulates inflammatory responses, including Th2 immune response and mast cell activation. Although dysregulation of the IL-33/ST2 axis has been implicated in inflammatory diseases and a variety of solid tumors, its contribution to immune evasion and leukemic transformation in AML remains largely unexplored. We propose that ST2 fosters an immunosuppressive bone marrow microenvironment to support AML development. We investigated the contribution and underlying mechanism of ST2 using CM AML as a model. Flow cytometry revealed early and progressive ST2 expression in peripheral blood during disease development. Both flow cytometry and qPCR showed upregulation of ST2 in hematopoietic stem and progenitor cells (HSPCs) as early as 2 weeks after induction of CM fusion oncogene. Mechanistically, CM expression upregulates the transcription factor GATA2, which occupies and transactivates ST2 promoter based on our chromatin immunoprecipitation (ChIP) and reporter assays. To test the contribution of ST2 to leukemogenesis, we generated conditional ST2 knockout, CM knock-in mice (CM-ST2-KO mice). CM-ST2-KO mice show delayed leukemia onset, reduced circulating cKit+ blasts, and significantly extended survival (median survival 192 days; p < 0.0001) compared to CM mice (103 days). To further decipher the leukemia-promoting mechanism of ST2, we performed scRNA-seq on bone marrow cells from pre-leukemic CM, CM-ST2-KO, and WT mice. First, cell type annotation by SingleR revealed that the leukemia cells are enriched in stem cells, erythrocytes, and mast cells. Cell-cell communication analysis via CellChat showed increased crosstalk between leukemic-enriched cell types and other immune cells (T cells, myeloid cells) in CM vs. WT mice. Compared to CM mice, CM-ST2-KO mice have decreased crosstalk between leukemia cells and immune cells. This analysis identified PD-1/PD-L1 and ALOX5 as candidates of ST2-regulated immunosuppressive pathways. We confirmed that Cd274 (encoding PD-L1) and Alox5 were upregulated in CM bone marrow cells and reversed in CM-ST2-KO. Consistent with CM expression, ST2-overexpression resulted in increased Cd274 and Alox5 expression upon IL-33 stimulation. Increased PD-L1 protein level was found in HPSCs and CD11b+ myeloid populations, which were skewed toward immunosuppressive subsets [i.e, M2 macrophages, PD-L1^high Ly6C+ myeloid-derived suppressor cells (MDSCs)] with reduced PD-L1^low Ly6G+ MDSCs in CM mice. Concurrent with upregulation of PD-L1 and Alox5, we observed increased expression of T cell checkpoint markers, including PD-1 and TIGIT on CD8+ T cells and CD160 on CD4+ T cells during CM-induced leukemia progression. These immunosuppressive phenotypes were all reversed in CM-ST2-KO mice.

In summary, this study identified ST2 as a key regulator of leukemia progression and immune suppression in AML. Our data indicate that ST2 promotes leukemic transformation and reshapes the bone marrow immune landscape by upregulating ALOX5 and PD-L1, expanding immunosuppressive myeloid populations, and enhancing T cell exhaustion. Genetic knockout of ST2 reverses these immunosuppressive phenotypes and prolongs survival, supporting the therapeutic potential of targeting ST2 to improve immune-based therapies in AML.