Session: 631. Myeloproliferative Syndromes and Chronic Myeloid Leukemia: Basic and Translational: Poster III
Hematology Disease Topics & Pathways:
MPN, Chronic Myeloid Malignancies, Diseases, Myeloid Malignancies
Methods Transformation assays were carried out using Ba/F3 cells transduced with MPL R514_P518delinsK (Ba/F3MPLRPK) or MPL W515L (Ba/F3MPLW515L), and Ba/F3 cells engineered to express MPL (Ba/F3MPL) that were transduced with JAK2 H608Y or JAK2 V617F. Additionally, hTERT-transduced immortalized human stromal cells (hTERT stromal cells), shown by single-cell RNA sequencing to have a CXCL12-abundant reticular cell-like genetic profile in terms of FOXC1, EBF3, LEPR, PDGFRB, SCF, and CXCL12 expression, were also transduced with JAK2 H608Y or wild-type JAK2. We quantitatively analyzed the levels of 12 cytokines (IL-6, FLT3L, GM-CSF, IL-3, IL-34, IL-11, SCF, LIF, CXCL12, IL-15, M-CSF, and IL-7) in the culture supernatant using LEGENDplex™ bead-based multiplex assays. RNA sequencing analysis was performed on hTERT stromal cells transduced with wild-type JAK2 (hTERT stromal cellsJAK2WT) or JAK2 H608Y (hTERT stromal cellsJAK2HY). The gene expression levels were calculated, and analysis of the differentially expressed genes was conducted using the R package edgeR.
Results Ba/F3MPLRPK cells demonstrated a cytokine-independent growth capability at a level comparable to Ba/F3MPLW515L, confirming that MPL R514_P518delinsK, a novel somatic mutation, could be a driver mutation for ET. Ba/F3MPL cells expressing JAK2 H608Y exhibited IL-3-independent proliferation, albeit less efficiently than cells with the JAK2 V617F mutation. hTERT stromal cellsJAK2HY showed a remarkable increase in GM-CSF production and a slight decrease in CXCL12. Expression quantification analysis comparing hTERT stromal cellsJAK2HY and hTERT stromal cellsJAK2WT detected a total of 3,409 significantly differentially expressed genes. The gene expression levels of IL-1 cytokine superfamily members, including IL36B, IL1A, and IL1B, in addition to CSF2 (GMCSF) and CSF3 (GCSF), were significantly higher in hTERT stromal cellsJAK2HY than in hTERT stromal cellsJAK2WT. Additionally, hTERT stromal cellsJAK2HY showed a significant decrease in the expression of genes encoding collagen, such as COL3A1 and COL1A1, and a significant increase in matrix metalloproteinases, such as MMP1 and MMP3, compared with hTERT stromal cellsJAK2WT. These data indicate that the JAK2 H608Y mutation induces an inflammatory environment in the bone marrow niche and endorses the remodeling of stromal cells. These changes may promote the acquisition of a novel MPL driver mutation in hematopoietic stem cells (HSCs) carrying this germline JAK2 mutation, potentially contributing to the onset and pathology of MPNs.
Conclusion Our findings provide the first evidence that germline JAK2 mutations not only influence the proliferation and differentiation trajectories of HSCs, but also remodel the bone marrow niche through an increase in the secretion of inflammatory cytokines, such as the IL-1 cytokine superfamily and GM-CSF.
Disclosures: Sugimoto: Novartis Pharmaceutical: Honoraria; Incyte Biosciences Japan G.K.: Research Funding; Toyo Kohan K.K.: Research Funding; MSD K.K.: Research Funding; Pharmaessentia Japan K.K.: Honoraria. Nagaharu: The Naito Science and Engineering Foundation: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding. Tawara: Novo Nordisk Pharma Ltd.: Honoraria; Novartis Japan: Honoraria; Ono: Honoraria; AstraZeneca: Honoraria; Alexion: Honoraria; Asahi Kasei: Honoraria; Astellas: Honoraria; Chugai: Honoraria, Research Funding; Kyowa Kirin: Honoraria, Research Funding; Sumitomo Pharma: Research Funding; Bristol Myers Squibb: Honoraria; Daiichi Sankyo: Honoraria; Eisai: Honoraria; Janssen: Honoraria; Meiji Seika: Honoraria; MSD: Honoraria; AbbVie: Honoraria; Otsuka: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria; Takeda: Honoraria. Ohishi: Pharmaessentia Japan K.K.: Research Funding.