Somatic chromosomal deletions in cancer are thought to indicate the location of tumor suppressor genes, whereby complete loss of gene function occurs through biallelic deletion, point mutation, or epigenetic silencing, thus fulfilling Knudson's two-hit hypothesis.1 In many recurrent deletions, however, such biallelic inactivation has not been found. One prominent example is the 5q- syndrome, a subtype of myelodysplastic syndrome (MDS) characterized by a defect in erythroid differentiation.2 Here, we describe an RNA interference (RNAi)-based approach to discovery of the 5q- disease gene. We find that partial loss of function of the ribosomal protein RPS14 phenocopies the disease in normal hematopoietic progenitor cells, and moreover that forced expression of RPS14 rescues the disease phenotype in patient-derived bone marrow cells. In addition, we identified a block in the processing of pre-rRNA in RPS14 deficient cells that is highly analogous to the functional defect in Diamond Blackfan Anemia, linking the molecular pathophysiology of the 5q- syndrome to a congenital bone marrow failure syndrome. These results indicate that the 5q- syndrome is caused by a defect in ribosomal protein function, and suggests that RNAi screening is an effective strategy for identifying causal haploinsufficiency disease genes.

Lineage specification is a critical issue in developmental and regenerative biology. We hypothesized that microRNAs (miRNAs) are important participants in those processes and used the poorly understood regulation of megakaryocyte-erythrocyte progenitors (MEPs) in hematopoiesis as a model system. We report here that miR-150 modulates lineage fate in MEPs. Using a novel methodology capable of profiling miRNA expression in small numbers of primary cells, we identify miR-150 as preferentially expressed in the megakaryocytic lineage. Through gain- and loss-of-function experiments, we demonstrate that miR-150 drives MEP differentiation toward megakaryocytes at the expense of erythroid cells in vitro and in vivo. Moreover, we identify the transcription factor MYB as a critical target of miR-150 in this regulation. These experiments show that miR-150 regulates MEP fate, and thus establish a role for miRNAs in lineage specification of mammalian multipotent cells.

Key Points • A gene expression profile consistent with activated JAK2 signaling is seen in all MPN patients, including in patients with CALR mutations. • Transcriptional profiling discriminates subsets of MPNs based on JAK2V617F allele burden and on the presence of CALR and TET2 mutations. Genomic studies have identified somatic alterations in the majority of myeloproliferative neoplasms (MPN) patients, including JAK2 mutations in the majority of MPN patients and CALR mutations in JAK2-negative MPN patients. However, the role of JAK-STAT pathway activation in different MPNs, and in patients without JAK2 mutations, has not been definitively delineated. We used expression profiling, single nucleotide polymorphism arrays, and mutational profiling to investigate a well-characterized cohort of MPN patients. MPN patients with homozygous JAK2V617F mutations were characterized by a distinctive transcriptional profile. Notably, a transcriptional signature consistent with activated JAK2 signaling is seen in all MPN patients regardless of clinical phenotype or mutational status. In addition, the activated JAK2 signature was present in patients with somatic CALR mutations. Conversely, we identified a gene expression signature of CALR mutations; this signature was significantly enriched in JAK2-mutant MPN patients consistent with a shared mechanism of transformation by JAK2 and CALR mutations. We also identified a transcriptional signature of TET2 mutations in MPN patent samples. Our data indicate that MPN patients, regardless of diagnosis or JAK2 mutational status, are characterized by a distinct gene expression signature with upregulation of JAK-STAT target genes, demonstrating the central importance of the JAK-STAT pathway in MPN pathogenesis. (Blood. 2014;123(22):e123-e133) Introduction The Philadelphia-negative myeloproliferative neoplasms (MPN) are hematopoietic disorders characterized by clonal expansion of mature myeloid elements. These include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Genomic analysis of the MPNs has shown frequent mutational events in the JAK-STAT signaling pathway including JAK2V617F mutations in 90% to 95% of patients with PV, and in 50% to 60% of patients with ET and PMF. 1-5 In addition, somatic mutations in the thrombopoietin receptor MPL in a subset of patients with JAK2V617F-negative ET and PMF, JAK2 exon 12 mutations 6,7 in JAK2V617F-negative PV, 8,9 and LNK mutations in JAK2V617F-negative MPN 10-12 have been identified. These genetic data indicate that mutations that lead to constitutive JAK-STAT signaling are a common genetic event in the different MPNs. The identification of mutations in the JAK-STAT pathway in the majority of MPN patients led to the development of JAK inhibitors, and approval by the US Food and Drug Administration of the JAK1/2 inhibitor ruxolitinib for the treatment of myelofibrosis (MF). 13,14 Treatment with ruxolitinib and other JAK kinase inhibitors leads to substantive clinical benefit including marked reduction in spl...