Mutant megakaryocytes orchestrate marrow immune remodeling via inflammatory and LINE‑1–Driven mechanisms in MPNs

Abstract Introduction Megakaryocytes (MKs), traditionally recognized as the precursors of platelets, have emerged as key regulators of the hematopoietic stem cell (HSC) function. Recent studies suggest that MKs also contribute to pathogen surveillance and immune response. How diseased MKs alter their immune modulatory function to impact HSC behavior in normal and neoplastic hematopoiesis remains poorly understood. In this study, we investigated the immunomodulatory functions of MKs and their impact on the marrow microenvironment of myeloproliferative neoplasms (MPNs), a group of clonal stem cell disorders marked by dysregulated megakaryopoiesis and neoplastic HSC expansion. Methods Murine models expressing human JAK2V617F specifically in MKs (Pf4-cre+FF1+) were generated. Bone marrow biopsies from MPN patients and age-matched orthopedic surgery controls were analyzed by immunohistochemistry (IHC). All studies were conducted under approved animal and human research protocols. Results Previously, we showed that JAK2V617F mutant MKs drive a myeloproliferative syndrome characterized by HSC expansion and a progressive decline of wild-type HSC function over two years. Here, using this aging MPN murine model, we found that mutant MKs enhanced uptake and MHC I presentation of exogenous peptide antigens and activated CD8⁺ T cells in an antigen-specific manner. They also exhibited an inflammatory secretory profile, with elevated PF4, TGFβ, and IL-1β in aged Pf4-cre⁺FF1⁺ mice versus controls, cytokines known to promote immune cell recruitment, differentiation, and suppression. These activities shape a pro-inflammatory niche that may favor mutant clonal expansion and drive MPN progression. Single-cell RNA-sequencing (scRNAseq) on unfractionated marrow cells from young (4mo old) and aged (1yr old) Pf4-cre⁺FF1⁺ and control mice revealed that aging amplifies JAK2V617F mutant MK–driven immune modeling. In aged mutants, HSCs and MKs upregulated proliferation and inflammatory pathways, neutrophils and T cells were activated, but CD8⁺ T cell cytotoxicity was impaired, and monocytes/macrophages shifted toward an immune-suppressive phenotype, indicating widespread immune remodeling. RT-qPCR of bone marrow MKs from aged Pf4-cre⁺FF1⁺ mice revealed significantly increased transcripts across the 5′UTR, ORF1, ORF2, and 3′UTR of active LINE-1 retrotransposon elements—a change absent in young mice and other marrow cell types. Lamivudine treatment reduced LINE-1 RNA in mutant MKs, implicating retrotransposition. Given LINE-1's role in aging and innate immunity, its upregulation in aged mutant MKs may drive innate immune activation and inflammatory marrow remodeling, consistent with elevated cGas and STING expression detected in these cells. We validated these findings in marrow biopsies from MPN patients (n=13), using hip replacement surgery samples as controls (n=5). Using IHC for CD41 and ORF1p, we found no ORF1p in MKs from age-matched controls but detected it in 12/13 MPN patients (average age 71 yr), with 40–100% of MKs positive. While largely MK-restricted, ORF1p expression was also detected in non-MK marrow cells in two advanced cases, suggesting LINE-1 activation is a hallmark of MPN MKs and may extend to other marrow cells in progressive disease. Conclusions These findings identify megakaryocytes as active immune modulators within the marrow niche. In MPNs, JAK2V617F mutation and aging synergistically enhance MK-mediated inflammatory and immunoregulatory functions. The discovery of elevated LINE-1 activity in aged mutant MKs links retrotransposon biology to innate immune activation and chronic inflammation, providing a novel mechanism by which diseased MKs reshape the marrow environment to favor mutant clonal expansion and potentially accelerat MPN progression.