Neutrophil extracellular traps drive myeloproliferation and immune dysfunction in chronic myelomonocytic leukemia

Abstract Background: Chronic myelomonocytic leukemia (CMML) is an aggressive hematologic malignancy characterized by myelodysplastic and myeloproliferative features, with high risk of transformation to acute myeloid leukemia. CMML is associated with systemic inflammation, clonal monocytosis, and susceptibility to life-threatening cytokine release in response to stressors. We hypothesized that clonal neutrophils contribute to chronic inflammation, excess myeloproliferation and decreased T cell immunosurveillance in CMML via dysregulated neutrophil extracellular trap (NET) formation (NETosis), and use a novel inhibitor of NETosis, neonatal NET-inhibitory factor (nNIF), to therapeutically target NET formation in CMML. Methods: Neutrophils (PMNs) were isolated via magnetic negative selection from the peripheral blood of healthy donors (HD) and CMML patients. NET formation by PMNs +/- nNIF was assessed in vitro under both basal and stimulated (phorbol myristate acetate; PMA) conditions and analyzed by myeloperoxidase (MPO)-DNA ELISA and confocal microscopy. Colony-forming assays with primary CD34+ hematopoietic stem and progenitor cells (HSPCs) from HD and CMML patients were performed in semisolid media supplemented with primary patient plasma or cell-free NET assay supernatant (NET-S) +/- nNIF. PMNs from CMML and HD samples were further analyzed for distinct transcriptional profiles using bulk RNA-seq. T-cell differentiation and exhaustion markers were assessed in primary T-cells from HD and CMML patients by flow cytometry. Results: We found that CMML patient-derived PMNs show elevated spontaneous NET formation compared to HD PMNs. CMML NET formation did not increase significantly upon PMA stimulation, indicating that CMML PMNs are at maximal basal NET induction. Preincubation of CMML PMNs with nNIF significantly inhibited both baseline and PMA-stimulated NET formation. Further, plasma from CMML patients induced NETosis in HD PMNs, an effect completely reversed by the nNIF preincubation. To assess the ability of NET components to drive myeloproliferation, we performed colony-forming assays using HD and CMML-derived HSPCs cultured with HD or CMML plasma or NET-S. HD HSPCs cultured with CMML plasma or CMML NET-S showed significantly increased colony number and size compared to controls. Preincubation of CMML PMNs with nNIF prior to NET-S collection abrogated this pro-proliferative effect and reduced HD colony formation. Similarly, CMML HSPCs cultured with CMML NET-S demonstrated increased colony formation that was reversed by preincubation of CMML PMNs with nNIF, confirming that soluble factors released during CMML NETosis promote myeloproliferation. These findings were recapitulated in an in vivo CMML mouse model. We next investigated CMML-associated immune dysfunction. Flow cytometric analysis of peripheral blood T-cells from HD and CMML patients revealed significantly increased expression of exhaustion markers PD-1 and TIGIT on CD4+ T cells and PD-1, TIGIT, and KLRG1 on CD8+ T cells in CMML patients versus HD. Bulk RNA-seq of CMML versus HD PMNs showed distinct gene expression profiles, with CMML PMNs demonstrating upregulated expression of multiple NETosis-related genes, including neutrophil elastase (ELANE), MPO, neutrophil collagenase (MMP8), and lactoferrin (LTF), and downregulation of gene terms associated with T-cell activation and co-stimulation. These transcriptomic data suggest that chronic NET activation in CMML may impair T-cell priming and immunosurveillance. Conclusion: Our data suggest dysregulated NETosis as a mechanism linking inflammation, myeloproliferation, and immune dysfunction in CMML. CMML PMNs are primed for constitutive NETosis, and NET-derived factors promote both healthy and malignant HSPC proliferation while impairing T-cell function. Inhibition of NETosis using nNIF suppresses augmented NETosis at baseline and under stimulated conditions in CMML PMNs and hinders the proliferative capacity of CMML HSPCs. These findings provide a compelling rationale for targeting NETs as a novel therapeutic strategy in CMML.