Myelofibrosis Is Initiated and Sustained By Rare Multipotent Stem Cells

Abstract Background: Myelofibrosis (MF) is a chronic myeloproliferative neoplasm (MPN) characterized by marked bone marrow fibrosis, extramedullary hematopoiesis and significant risk for leukemic transformation. Most patients carry recurrent MPN driver mutations in JAK2, CALR and MPL and many carry additional mutations in epigenetic regulators, splicing and signaling pathways. MF pathophysiology remains poorly understood, particularly with respect to the cellular identity of the MF clone(s) bearing mutations that initiate and maintain the disease. Therefore, here we tracked somatic mutations from nine MF patients in sorted hematopoietic cell populations from serial time-points as well as patient-derived xenografts (PDXs), to identify clinically relevant MF cell populations for future targeted therapies. Methods: Genomic DNA was isolated from the peripheral blood mononuclear cells (PBMCs) of nine chronic-phase MF patients (5 JAK2+ and 4 CALR+) and sequenced using the 54-gene TruSight Myeloid targeted panel. PBMCs from the same patients at serial time-points (if available) were sorted into hematopoietic stem and progenitor cell (HSPC) and mature cell populations. In addition, PDXs were generated following injection of CD34+ peripheral blood cells into sub-lethally irradiated NSG and/or NSG-SGM3 mice. Human myeloid (CD45+33+) and B (CD45+19+) cells were isolated from PDXs after 8-16 weeks. Digital droplet PCR was used to interrogate known oncogenic variants, identified from the targeted sequencing, in all sorted primary and PDX cell fractions. Results: We reveal that the vast majority of genetic lesions used to interrogate the hierarchy were acquired at the level of immunophenotypically defined hematopoietic stem/multipotent progenitor cells (HSC/MPP) (CD45+34+38-RA-, termed MF-HSC). MF-HSC variants were generally shared with most HSPC, differentiated myeloid (CD33+) and lymphoid (B and/or T cell) populations sorted from primary patient samples. In our cohort, MPN driver mutations were invariably acquired in MF-HSC and consistently maintained over longitudinal clinical follow-up (median of 17 months) in both MF-HSCs and differentiated myeloid cells (CD33+). Interestingly however, in 2 cases (1 CALR+ and 1 JAK2+ MF), ASXL1 mutations in MF-HSCs were observed at the first time-point but restricted to the CD33+ compartment at a later time-point. Further, in one patient, SF3B1 mutation was restricted to the CD33+ fraction at the first time-point and was completely absent at the second time-point. Thus, certain mutations, like ASXL1, may be required for MF initiation but not necessarily disease maintenance; or alternatively, independent MF-HSC clones may outcompete others over time. Additionally, some acquired downstream mutations may be transient in nature and not clinically relevant. Together these data suggest that MF-HSCs are essential for MF clonal maintenance. To complement our genetic interrogation of the hematopoietic hierarchy in MF, we demonstrate that CD34+ HSPCs generate multi-lineage grafts in xenotransplanted mice bearing mutations identified in MF-HSCs. CALR+ samples invariably demonstrate multi-lineage potential and clonal dynamics in PDXs suggest that CALR mutations are likely the first genetic lesion acquired in multipotent MF-HSCs. JAK2+ samples generated more variable data, but by combining the genetic data of both PDXs and lymphoid cells sorted from primary patient samples, we show that JAK2+ MF-HSCs may also be multipotent. Taken together, these data suggest that MF-HSCs are multipotent, functionally relevant MF-initiating cells capable of propagating downstream HSPC populations and the CD33+ MF-disease clone that harbors all interrogated mutations. Thus, rare MF-HSCs are both disease-initiating and disease-maintaining cells. Conclusions: Our approach combining high-resolution cell sorting and xenograft assays with genomic interrogation demonstrates that MF is initiated and maintained by rare multipotent MF-HSCs. Our study underscores the complex evolutionary events that occur in the stem cell compartment during disease progression and identifies MF-HSCs as the relevant cellular target for future therapeutic intervention. Disclosures Gupta: Novartis: Consultancy, Honoraria, Research Funding; Incyte: Research Funding.

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