Short-term and long-term sting activation elicit divergent cellular responses in distinct hematopoietic populations in chronic myelomonocytic leukemia

Abstract Activation of the stimulator of interferon genes (STING) modulates cellular responses in a cell-context-dependent manner. Intrinsic IFN-stimulated gene (ISG) expression enhances cellular responses to STING activation. We previously demonstrated that chronic myelomonocytic leukemia (CMML) is characterized by cell-intrinsic ISG upregulation. While STING-activation can mediate hematopoietic stem-cell (HSC) self-renewal or exhaustion and CD14+-monocyte depletion, the distinct effects of acute, short-term and long-term STING activation in distinct clonal hematopoietic populations remains unclear. Therefore, we sought to evaluate the effects of STING activation dynamics in normal and CMML HSC and monocyte populations. To do so we first treated BM mononuclear cells (MNCs) from patients (pts) with CMML (n=11) or healthy donors (HDs, n=6) in co-culture with mesenchymal stromal cells with a STING agonist (dazostinag). Given that monocytic lineage is characterized by high expression of cysteine-cysteine chemokine receptor 2 (CCR2), MNCs were also treated with TAK-500, an IgG1 anti-CCR2 antibody linked to a STING agonist payload, to evaluate the specific effects of STING activation in monocyte populations. Dazostinag or TAK-500 depleted total and CCR2+ MNCs from CMML pts but not from HDs Accordingly, STING activation with dazostinag or TAK-500 induced a dose-dependent depletion of monocytic cell lines (MOLM13, THP1) through IRF3 phosphorylation and caspase 3 cleavage. To evaluate if acute STING activation impairs CMML's HSPCs repopulation potential we performed colony-formation studies in BM CD34+ cells from CMML pts (n=8) and observed that TAK-500 and dazostinag impaired myeloid, but not erythroid, colony-formation. We previously reported that Vav-KDM6B/Tet2f/f/Vav-Cre (KDM6B/Tet2)mice recapitulate features of CMML. Single-cell RNA-sequencing (scRNA-seq) of MNCs from these mice and control (Vav-cre) mice confirmed myeloid bias and expansion of CCR2+ monocytes that upregulate IFN response genes and inflammatory signals in KDM6B/Tet2 mice. BM CCR2+ monocyte expansion in KDM6B/Tet2 mice was confirmed by flow cytometry. Therefore, to evaluate the effects of acute STING activation in vivo, we treated KDM6B/Tet2 mice with a single dose of TAK-500. TAK-500 enhanced CCR2+ BM cell apoptosis and reduced LSK colony formation capacity. In vivo depletion of MOLM-13 and human CD45+ cells was also observed after acute TAK-500 treatment in xenograft models and in a CMML patient-derived xenograft model, respectively. To dissect the effects of short-term STING activation, we then treated KDM6B/Tet2 mice with vehicle or TAK-500 weekly for 3 consecutive weeks. Therapy was associated with peripheral blood monocyte depletion, BM CCR2+ cell depletion, and apoptosis. TAK-500 also induced LSK expansion and depletion of granulomonocytic, common-myeloid, and megakaryocytic/erythroid progenitors. LSK expansion with reduced colony-forming capacity and CD14+ monocyte differentiation was sustained in colony-formation assays. Bulk RNA-sequencing of sorted LSKs demonstrated downregulation of genes involved in RNA metabolism and translation, G1/S transition and neutrophil degranulation, suggesting reduced cell activity and differentiation. scRNA-seq of BM MNCs after 3 weeks of therapy confirmed depletion of CCR2+ cells, myelomonocytic precursors and monocyte populations with TAK-500 but not with vehicle. STING activation induced type I IFN gene expression (e.g Irf7) in monocytes and splenic extramedullary hematopoiesis. Analysis of cell-cell communication networks revealed increased number of interactions involved in cell migration, inflammation and immune interactions between monocyte and other cell types. To compare the effects of short vs long-term STING activation, we treated control and KDM6B/Tet2 mice with TAK-500 over a period of 14 weeks. In contrast to short-term STING activation, long-term TAK-500 exposure was not associated with CCR2+ cell depletion, changes in LSK or progenitor populations or transformation in KDM6B/Tet2 mice. scRNA-seq of MNCs revealed decreased expression of inflammatory genes on monocyte and progenitor populations compared to short-term exposure which suggests cell-adaptive responses to overcome inflammatory and cell death programs associated with acute STING activation.Together, these data highlight the complex effects of STING activation dynamics in distinct hematopoietic populations and their role in CMML pathophysiology.