NRAS mutation drives excessive netosis and differentiation syndrome in acute promyelocytic leukemia Free

Acute promyelocytic leukemia (APL) is highly curable with all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). However, differentiation syndrome (DS) remains a life-threatening complication in ~25% of patients. The pathogenesis of DS is linked to an extreme inflammatory response during APL induction therapy, including cytokine surge and neutrophil differentiation. Neutrophil extracellular traps (NETs) can mediate tissue damage and thrombosis when overproduced, suggesting a potential role in DS. NRAS mutations, present in almost 10% of APL, have been observed to correlate with early clinical complications, including early death and DS. In this study, we aim to uncover the mechanism by which NRAS mutations induce NETosis and lead to DS.

Methods We integrated transcriptomic analysis of primary APL samples with functional studies in NB4 cells engineered to express mutant NRAS (NRAS^mut, G12D), wild-type NRAS overexpression (NRAS^oe), or empty vector. Cells were treated with ATRA to induce differentiation, assessed by CD11b expression via flow cytometry. ATO-induced apoptosis was evaluated by Annexin V assay. NET formation was quantified by measuring cfDNA, MPO-DNA, and NE-DNA via Picogreen and ELISA, and visualized by immunofluorescence for MPO/NE. Inhibitors targeting CXCR1/2 (Reparixin), NE(Sivelestat), MEK (Trametinib), PAD4(GSK484), and JAK1/2 (Ruxolitinib) were applied to evaluate their inhibitory effects on NETosis and differentiation. HUVECs were exposed to NET-containing supernatants to assess endothelial activation via ICAM-1, E-selectin, and VCAM-1 expression.

Results NRAS^mut APL samples showed enriched expression of ATRA-induced differentiation signatures. GSEA confirmed significantly upregulated IL-8–CXCR1/2 signaling in NRAS^mut versus NRAS^oe and control patients. In vitro, NRAS^mut NB4 cells demonstrated significantly increased early differentiation at 24 hours (P<0.001 vs. control; P=0.0009 vs. NRAS^oe), but no difference at 48 hours. ATO-induced apoptosis was reduced in NRAS^mut cells compared to others (P=0.0011 vs. control, P=0.0250 vs NRAS^oe). Upon ATRA stimulation, NRAS^mut cells released dramatically more NETs than NRAS^oe (measured by cfDNA, MPO-DNA, and NE-DNA, P＜0.0001, P=0.0079, and P=0.0099) or controls (P＜0.0001, P=0.0005, and P=0.0004), with significantly elevated IL-8 in supernatants (P＜0.0001 vs. NRAS^oe, P=0.0007 vs.controls). Immunofluorescence also visually confirmed more extensive NET structures in NRAS^mut cells. To sum up, while ATRA alone induced an appropriate amount of NETs in control cells, NRAS mutation remarkably amplified this effect.

Reparixin selectively inhibited NETosis in NRAS^mut cells, showing significant reductions when combined with ATRA (P < 0.0001), and even when administered at a high dose prior to ATRA stimulation. In contrast, Sivelestat significantly suppressed NET release in all NB4 cell lines after ATRA administration (vs. control, NRAS^oe, and NRAS^mut; all P<0.0001) and more importantly did not impair the differentiation process by ATRA. Trametinib inhibited differentiation but had limited effect on repressing NETosis. GSK484 and Ruxolitinib modestly reduced NET formation at higher concentrations. NET-rich supernatants from NRAS^mut cells induced pronounced endothelial activation: HUVECs showed elevated ICAM-1 and E-selectin expression compared to other groups.

Conclusion NRAS mutations in APL synergized with ATRA to accelerate leukemic differentiation and promote excessive NETosis through IL-8–CXCR1/2 signaling pathway. NETs mediated endothelial injury, which may underlie DS-related hemorrhage complications. Targeting this pathway with reparixin or sivelestat attenuates NET formation without compromising cell differentiation, offering a promising therapeutic strategy to mitigate DS-induced tissue damage while preserving anti-leukemic efficacy. These findings provide mechanistic insights into NRAS-driven inflammation in APL and support NETs-targeted therapy for prophylaxis of DS.