Engrafted JAK2V617F Clone Hijacks Resident Non-Mutated Hematopoietic System to Drive Myeloproliferative Neoplasm
Authors: Bonal Dennis M , Chorzalska Anna Dorota , Oakes Alissa , Pardo Makayla R , Lee Seo-Ho , Clarke Michael Y , Petersen Max , Oh Hyunju , Brown Valeria , Treaba Diana Olguta , Reagan John L. , Olszewski Adam J. , Dooner Mark , Morgan John , Mills Joslyn , Bertone Paul , Dubielecka Patrycja M 
Published: December 2024

JAK2V617F is a major driver mutation in myeloproliferative neoplasms (MPN) and JAK2 inhibitors can alleviate the disease burden for patients with MPN. However, questions remain about the mechanistic role of JAK2V617F in driving the pleiotropy of phenotypes which range from low-variant allele frequency (VAF) clonal hematopoiesis (CHIP) to MPN to secondary AML; about the reason why highly specific JAK2 inhibitors fail to provide a curative potential; and about potential effects that the JAK2V617F clone might exert on both hematopoietic and non-hematopoietic components of the bone marrow. Models able to provide insights into these long-standing questions and inform optimized treatment strategies are critically needed.

Here we characterize a previously described traceable murine JAK2V617F MPN model (Bonal et al, 2023), with the disease driven by a low-VAF JAK2V617F clone and induced in unconditioned bone marrow transplantation (BMT) recipients. BMT recipients displayed profound BM microenvironmental alterations, with loss of osteo/mesenchymal clones and trabecular bone. Our model also revealed MPN-mimicking cells arising from the host hematopoietic system and contributing to the MPN-like phenotype.

Whole BMT was performed via a single tail vein injection of 5.0x106 donor cells into unconditioned C57BL/6 Ptprca (JAX#002014, CD45.1) recipient mice. Donor cells (JAK2V617F) were from a Poly I:C inducible MPN-like model created by crossing floxed-JAK2V617F (JAX#031658) with Mx1-Cre (JAX#003556) mice. All mice, including non-BMT wild type controls, were age- (10 weeks) and sex-matched. BMT of JAK2V67F clone resulted in a polycythemia vera-like phenotype (elevated hematocrit and white blood cell counts) with an average donor cell chimerism in peripheral blood of 2.74% (SD = 2.07). Eight months post-BMT, we performed bulk RNA-seq on whole BM sorted according to CD45 expression (CD45.1 recipient/control or CD45.2 donor). Gene set enrichment analysis of sorted recipient and donor fractions revealed significant upregulation of early erythroblast and downregulation of lymphoid pathways (B, T, NK cells) in donor-derived cells compared to controls. Surprisingly, recipient-derived cells showed upregulation in myeloid- (monocytes, eosinophils, neutrophils) and erythroblast-related gene sets, indicating a skewing of the non-JAK2V617F carrying recipient hematopoietic system towards an MPN-like phenotype.

Confirming our RNA-seq results, flow cytometry analyses of donor-derived (CD45.2) cells showed significantly higher levels of erythroid (CD71+/Ter119+), megakaryocyte (CD42d+/CD41+), myeloid (CD11b+), and granulocyte (Gr-1+/CD66a+) progenitors, with significantly lower T- (CD3+) and B-cell (B220+/CD19+) frequencies compared to controls. The most elevated cell type originating from donor cells was a megakaryocyte progenitor (CD42d+/CD41+). The erythroid progenitor output was similar between donor-derived and non-BMT wild type control populations, suggesting the small clone within recipients drove an equal output of erythroid cells compared to a healthy non-BMT animal. Intriguingly, recipient-derived (CD45.1) cells had significantly elevated monocytic (F4/80+/CD11b+) and erythroid (CD71+/Ter119+) progenitors, with nonsignificant trends towards higher myeloid and granulocyte progenitors and lower B cells, consistent with RNA-seq. We further observed an unexpected, significant increase in erythroid (CD71+/Ter119+) cells originating from the CD45.1 recipient population in the BMT mice, suggesting that the elevation in erythrocytes and the MPN phenotype partly results from the JAK2V617F clone on the CD45.1 recipient's bone marrow function. Our results suggest that the presence of the JAK2V617F clone, even in low frequencies, can profoundly impact the host hematopoietic system to contribute to the manifestation of an MPN phenotype.

Our model uncovers the impact of JAK2V617F donor cells on the host BM microenvironment and hematopoietic system, driving an MPN phenotype even with low donor cell chimerism. The observation that non-JAK2V617F carrying hematopoietic cells participate in MPN progression suggests that the MPN clone not only impacts hematopoiesis-supporting stroma, but may influence non-mutated cells, and may change our understanding and therapeutic approaches to human MPN, especially in patients with low VAFs.