JAK2-V617F Expression Results in a Cell-Intrinsic Expansion of Megakaryocyte Progenitors with Evidence of Myc Pathway Activation in Both Mature and Precursor Megakaryocytes By Single Cell RNA Sequencing

Myeloproliferative neoplasms (MPNs) are characterized by recurrent driver mutations in JAK2, CALR and MPL, with JAK2 being the most frequently enriched in patients with polycythemia vera. There is an expansion of often dysmorphic megakaryocytes, which occurs with disease progression to myelofibrosis. This is of potential biological and clinical significance, since megakaryocytes are considered an important component of the stem cell niche producing key niche factors, such as TGF-β and CXCL4. However, the mechanisms mediating megakaryocyte expansion and the impact of mutant JAK2 on megakaryocyte gene expression are not fully understood.

To address these issues, we established bone marrow chimeras by transplanting congenic wildtype and inducible Jak2^V617F cells into irradiated recipients; the wildtype cells included a GFP transgene to distinguish wildtype from Jak2^V617F cells. Specifically, a 5:1 ratio of wildtype to Jak2^V617F cells was used and tamoxifen was given 4 weeks after transplantation to induce UBC-ERT2-Cre, and thereby activate the Jak2^V617F allele, and mice were analyzed 12 weeks later. Whereas the contribution of Jak2^V617F cells to hematopoietic stem cells and myeloid progenitors increased modestly (~2-fold) from input, a marked (~5-fold) expansion of Jak2^V617F megakaryocytic-erythroid progenitors (MEPs) was observed, suggesting that Jak2^V617F induces a selective cell-intrinsic expansion of MEPs. Single cell sequencing of sorted Kit+ lineage- Sca+ cells from these mice was performed (n = 4) using the 10X Genomics platform: this experimental approach allowed us to compare MEP transcriptomes under a shared altered (bone marrow microenvironment. We identified 148 differentially expressed genes that were enriched for cell cycling genes. Enriched Hallmark gene expression signatures included Myc and E2F targets and MTORC1 signaling.

To address the impact of Jak2^V617F on megakaryocyte gene expression, we performed scRNA sequencing of enriched megakaryocyte populations from wildtype or Jak2^V617F mice (n = 3, each). In brief, wildtype or Jak2^V617F mice (8-10 weeks old) were treated with polyinosinic:polycytidylic acid to induce Jak2^V617F expression. Six weeks later, bone marrow was harvested and megakaryocytes enriched by bovine serum albumin gradient followed by CD61-positive magnetic bead selection. Single cell RNA sequencing was then performed using the 10X Genomics platform. Megakaryocytes were identified by common protein identifiers (Pf4, Vwf, Cd34, Gp1ba and Mpl) as well as unbiased cell identifiers from the literature. In total, 1,228 differentially expressed genes were identified in Jak2^V617F megakaryocytes. The top most enriched gene expression signature was Myc targets (FDR = 0). Consistent with this, we identified increased Myc mRNA expression in Jak2^V617F megakaryocytes (mean 0.67 vs 0.28, adjusted p-value = 3.77E-16.). We also observed significant enrichment for TNF-a/NF-kB, p53 and JAK/STAT signaling pathways. Megakaryocytes are a major source of important stem cell niche factors, including TGF-β, CXCL4, IL6, IL12, CCL3 and others). We observed increase in the number TGF- β transcripts (adjusted p-value = 2.14E-7) in megakaryocytes, which colocalized with mature megakaryocytes (expressing high Gp1ba/Gp1bb/Vwf) and was in contrast to higher Myc expression in the megakaryocyte precursors (expressing high Kit/Cd34.)

Altogether, these data show that Jak2^V617F expression results in a marked cell-intrinsic expansion of MEPs and the production of atypical megakaryocytes marked by increased Myc expression and expression of selected stem cell niche genes.

Oh:Novartis: Consultancy; Kartos Therapeutics: Consultancy; CTI BioPharma: Consultancy; Celgene/Bristol Myers Squibb: Consultancy; Disc Medicine: Consultancy; Blueprint Medicines: Consultancy; PharmaEssentia: Consultancy; Constellation: Consultancy; Geron: Consultancy; AbbVie: Consultancy; Sierra Oncology: Consultancy; Incyte: Consultancy.