Crispino, John D.

Abstract 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.

Abstract Purpose: Myelofibrosis is characterized by bone marrow fibrosis, atypical megakaryocytes, splenomegaly, constitutional symptoms, thrombotic and hemorrhagic complications, and a risk of evolution to acute leukemia. The JAK kinase inhibitor ruxolitinib provides therapeutic benefit, but the effects are limited. The purpose of this study was to determine whether targeting AURKA, which has been shown to increase maturation of atypical megakaryocytes, has potential benefit for patients with myelofibrosis. Patients and Methods: Twenty-four patients with myelofibrosis were enrolled in a phase I study at three centers. The objective of the study was to evaluate the safety and preliminary efficacy of alisertib. Correlative studies involved assessment of the effect of alisertib on the megakaryocyte lineage, allele burden, and fibrosis. Results: In addition to being well tolerated, alisertib reduced splenomegaly and symptom burden in 29% and 32% of patients, respectively, despite not consistently reducing the degree of inflammatory cytokines. Moreover, alisertib normalized megakaryocytes and reduced fibrosis in 5 of 7 patients for whom sequential marrows were available. Alisertib also decreased the mutant allele burden in a subset of patients. Conclusions: Given the limitations of ruxolitinib, novel therapies are needed for myelofibrosis. In this study, alisertib provided clinical benefit and exhibited the expected on-target effect on the megakaryocyte lineage, resulting in normalization of these cells and reduced fibrosis in the majority of patients for which sequential marrows were available. Thus, AURKA inhibition should be further developed as a therapeutic option in myelofibrosis. See related commentary by Piszczatowski and Steidl, p. 4868

Key PointsDNA methylation changes during the development of DS-AMKL occur in sequential waves of opposing losses and gains of methylation. Each wave of DNA methylation abnormalities targets specific gene networks that contribute to distinct biological features of the disease.

We used high resolution mate‐pair sequencing (HRMPS) in 15 patients with primary myelofibrosis (PMF): eight with normal karyotype and seven with PMF‐characteristic cytogenetic abnormalities, including der(6)t(1;6)(q21–23;p21.3) (n = 4), der(7)t(1;7)(q10;p10) (n = 2), del(20)(q11.2q13.3) (n = 3), and complex karyotype (n = 1). We describe seven novel deletions/translocations in five patients (including two with normal karyotype) whose breakpoints were PCR‐validated and involved MACROD2, CACNA2D4, TET2, SGMS2, LRBA, SH3D19, INTS3, FOP (CHTOP), SCLT1, and PHF17. Deletions with breakpoints involving MACROD2 (lysine deacetylase; 20p12.1) were recurrent and found in two of the 15 study patients. A novel fusion transcript was found in one of the study patients (INTS3‐CHTOP), and also in an additional non‐study patient with PMF. In two patients with der(6)t(1;6)(q21–23;p21.3), we were able to map the precise translocation breakpoints, which involved KCNN3 and GUSBP2 in one case and HYDIN2 in another. This study demonstrates the utility of HRMPS in uncovering submicroscopic deletions/translocations/fusions, and precise mapping of breakpoints in those with overt cytogenetic abnormalities. The overall results confirm the genetic heterogeneity of PMF, given the low frequency of recurrent specific abnormalities, identified by this screening strategy. Currently, we are pursuing the pathogenetic relevance of some of the aforementioned findings. Am. J. Hematol. 88:741–746, 2013. © 2013 Wiley Periodicals, Inc.