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Primary myelofibrosis (PMF) is a clonal hematologic malignancy characterized by BM fibrosis, extramedullary hematopoiesis, circulating CD34+ cells, splenomegaly, and a propensity to evolve to acute myeloid leukemia. Moreover, the spleen and BM of patients harbor atypical, clustered megakaryocytes, which contribute to the disease by secreting profibrotic cytokines. Here, we have revealed that megakaryocytes in PMF show impaired maturation that is associated with reduced GATA1 protein. In investigating the cause of GATA1 downregulation, our gene-expression study revealed the presence of the RPS14-deficient gene signature, which is associated with defective ribosomal protein function and linked to the erythroid lineage in 5q deletion myelodysplastic syndrome. Surprisingly, reduced GATA1 expression and impaired differentiation were limited to megakaryocytes, consistent with a proproliferative effect of a GATA1 deficiency on this lineage. Importantly, expression of GATA1 effectively rescued maturation of PMF megakaryocytes. Together, these results suggest that ribosomal deficiency contributes to impaired megakaryopoiesis in myeloproliferative neoplasms.

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Primary myelofibrosis (PMF) is a clonal hematologic malignancy resulting from the transformation of a pluripotent hematopoietic progenitor cell leading to increased hematopoiesis and overproduction of abnormal blood cells. PMF is characterized by bone marrow (BM) fibrosis, extramedullary hematopoiesis, increased numbers of circulating CD34+ cells, splenomegaly, and a propensity to evolve to AML. Patients also display anemia and thrombocytopenia and harbor abnormal, immature megakaryocytes (Mks) in their BM and spleen. Numerous mutations are associated with PMF including JAK2, CALR, MPL, TET2, CBL, ASXL1, and IDH. The presence of abnormal Mks in the BM of PMF patients is an important characteristic of the disease, as these cells are believed to contribute to the fibrosis.Here, we performed an analysis of Mk differentiation from individuals with PMF compared to healthy individuals as a way to better understand the mechanism leading to their defects. CD34+ cells obtained from PMF and healthy donors were cultivated in presence of thrombopoietin to induce Mks differentiation. Under these conditions, we observed that the proliferative capacity of CD34+ cells from PMF patients was increased as compared to normal CD34+ cells. At the same time, the percentage of mature Mks obtained was reduced. Semi-solid culture conditions confirmed the proliferative advantage of the PMF CD34+ cells. To investigate the mechanism responsible for this phenotype, we performed genome wide expression analysis of sorted Mks from patients versus controls. Nine PMF patients with different mutations (5 JAK2V617F, 1 MPLW515L, 2 CALR and 1 triple negative) and 4 healthy donors were included in the study. Gene Set Enrichment Analysis (GSEA) analysis revealed that the top scoring pathways included the RPS14 signature, ribosome biogenesis, rRNA metabolic process, and rRNA processing. We first studied the RPS14 geneset, which consists of a group of genes that were up-regulated in CD34+ cells after knock-down of RPS14, a component of the small ribosome subunit. Using shRNA against RPS14, we discovered that its knock down leads to a significant reduction in the development of mature Mks from normal CD34+ cells. The microarray data also revealed changes in the expression of several myeloid genes. CEBPa and GFI-1 were over-expressed in PMF Mks, whereas GFI-1b and PF4, which are associated with Mk differentiation, were down-regulated. Surprisingly the expression of GATA1, a master regulator of megakaryopoiesis, was not changed. However, consistent with previous report, we discovered that GATA1 protein level was reduced in PMF Mks. GATA1 deficient Mks are impaired in their maturation, so we reasoned that the reduction in GATA1 contributes to the defects in PMF. Indeed, we found that overexpression of GATA1 in PMF CD34+ cells rescued Mk differentiation. To understand how GATA1 protein but not mRNA, is reduced, we knocked down RPS14 in the JAK2 mutant SET2 megakaryoblastic cell line. As predicted, we observed a reduction in GATA1 protein, but not mRNA. By ribosomal profiling, we saw that the shRPS14 led to an abnormal ribosomal profile with a reduction of the 40s peak. Finally, by RT-PCR analysis of the ribosomal and polysomal fractions, we found that GATA1 mRNA was less abundant in the polysomal fraction in cells expressing the shRPS14. This finding is consistent with recent data showing that alterations in ribosomal genes cause a decrease in GATA1 protein in Diamond-Blackfan Anemia. Although we identified the RPS14 gene signature, we did not observe changes in RPS14 expression or in any of the ribosomal subunits. Since our GSEA analysis also identified deficiencies in three ribosomal biogenesis pathways, we looked for genes present in all three signatures. NOP58, a ribonucleoprotein required for ribosome biogenesis, was significantly reduced in all samples of PMF Mks. To link the reduction in NOP58 expression with PMF associated mutations, we expressed MPLW515L or the CALR type II mutant in hematopoietic progenitor cells and detected a reduction in NOP58 expression. Our findings suggest that the activation of JAK/STAT signaling leads to a reduction in NOP58, which contributes to defects in ribosome assembly, decreased GATA1 expression and subsequent impairment in Mk differentiation. This work reveals, for the first time, that defective ribosomes contribute to the pathogenesis of PMF and suggests new avenues for therapeutic intervention. Disclosures Stein: Incyte Corporation: Honoraria, Speakers Bureau; Sanofi Oncology: Honoraria.