Srsf2P95H and Nf1 deficiency synergistically activate JAK-STAT signaling and exacerbate age-related clonal hematopoiesis in mice Free

Clonal hematopoiesis (CH) is an age-related condition characterized by clonal expansion of hematopoietic stem cells (HSCs) bearing somatic mutations in CH-defined genes. Different mutation combinations contribute concurrently or mutually exclusively to CH progression and disease subtypes. SRSF2 is among the most frequently mutated splicing factors, with the P95 hotspot driving aberrant splicing and predicting poor prognosis in hematologic malignancies. While SRSF2^P95H recapitulates myelodysplastic syndromes (MDS) in mice and has been studied with co-mutations in IDH1/2, ASXL1, RUNX1, TET2 and JAK2, its interplay with RAS signaling remains unclear. NF1, a negative RAS regulator and tumor suppressor, when deleted in hematopoietic cells, causes progressive myeloproliferative neoplasms in mice.

To assess the combinatorial impacts of SRSF2 and NF1, we retrospectively analyzed patients from our hospital over five years. Patients harboring both SRSF2^P95H and NF1 mutations (SN) were compared to those with SRSF2^P95H instead of NF1 (S), or NF1 without SRSF2^P95H (N). SN patients were more frequently diagnosed with myeloid malignancies (96.2%), spanning AML, MDS/MPN, MPN, MDS, with one T-ALL case. Elevated WBC, neutrophil, and monocyte counts also suggested enhanced myeloid skewing in SN individuals.

We modeled this in vivo by generating mice with hematopoietic-specific Srsf2^P95H mutation and/or Nf1 deletion: wildtype (WT), Srsf2^P95H alone (S), Nf1 homozygous deletion (NN), and the double mutant (SNN). Compared to S and NN mice, SNN exhibited progressive neutrophilia and lymphopenia in peripheral blood (PB). PB smears showed increased hyposegmented neutrophils. Bone marrow (BM) flow cytometry revealed expansions of LKS⁺, HSC, MPP1, CMP, PreGM, Mac-1⁺ cells and Mac-1⁺ Ly6G⁺ neutrophils, partially at the expense of CLP proportion and B-lineage maturation. In BM transplantation (BMT) assays, SNN recipients exhibited marked HSPCs reduction despite lower donor chimerism; Mac-1⁺ cells maintained advantageous expansion. Gene set enrichment analysis (GSEA) revealed upregulation of RNA splicing, apoptosis, and notably, JAK-STAT pathways (STAT3, STAT5, and phosphorylation). Western blotting confirmed STAT3/5 hyperphosphorylation. Differential gene expression analysis showed increased Il6ra, Jak2, Ccnd1, Ccnd2 and other JAK-STAT components. Splicing analysis confirmed known Ezh2 poison exon inclusion, revealed aberrant skipping of Jak2 exons 13 and 14, and identified a mis-splicing landscape of JAK-STAT genes in SNN HSCs. We hypothesized that under RAS hyperactivation, Srsf2^P95H amplifies JAK-STAT signaling via mis-splicing of its key components.

To validate this, we treated BMT mice with DMSO, ruxolitinib (RUX) or CTX-712 (CTX). RUX is a classic selective JAK inhibitor; CTX targets CDC2-like kinases to suppress SRSF2 phosphorylation. Developed by Seishi Ogawa's team in 2017, CTX is currently in global phase I/II trials. Unexpectedly, RUX significantly restored donor chimerism and HSC frequency with reductions in Mac-1⁺ cells and neutrophils to levels seen in CTX-treated SNN or DMSO-treated WT. Colony formation assays confirmed improved HSPC output and reduced CFU-GM bias. GSEA of RUX-treated SNN HSCs showed downregulation of IL6-JAK-STAT3 signaling and upregulation of DNA repair.

Since CH prevalence rises with age and SRSF2 mutations occur more frequently in the elderly, we analyzed the correlation between co-mutation and aging. SN patients were diagnosed at an older age than SRSF2-WT ones. In mice, SNN HSCs showed significant downregulation of focal adhesion, a hallmark of HSC senescence. Whole-exome sequencing revealed that aged SN/SNN mice accumulated more somatic mutations, consistent with human CH. Interestingly, we discovered a novel Jak2 exon 13 missense mutation, validated by Sanger sequencing.

Taken together, we demonstrate that under Nf1 deficiency, Srsf2^P95H impairs HSC function and exacerbates myeloid-biased hematopoiesis by augmenting JAK-STAT signaling, which can be rescued by JAK inhibition. Our findings elucidate the synergistic effects of Srsf2^P95H and Nf1 loss in CH progression, uncovering an unanticipated JAK-STAT-dependent mechanism mediated by Srsf2^P95H. Furthermore, we highlight a conserved, age-related pathophysiological trajectory of CH between humans and mice, providing a preclinical model to dissect molecular interactions in age-related CH and to support therapeutic development in hematologic malignancies.