SUMMARY We used an in vivo short hairpin RNA (shRNA) screening approach to identify genes that are essential for MLL-AF9 acute myeloid leukemia (AML). We found that Integrin Beta 3 (Itgb3) is essential for murine leukemia cells in vivo, and for human leukemia cells in xenotransplantation studies. In leukemia cells, Itgb3 knockdown impaired homing, downregulated LSC transcriptional programs, and induced differentiation via the intracellular kinase, Syk. In contrast, loss of Itgb3 in normal HSPCs did not affect engraftment, reconstitution, or differentiation. Finally, we confirmed that Itgb3 is dispensable for normal hematopoiesis and required for leukemogenesis using an Itgb3 knockout mouse model. Our results establish the significance of the Itgb3 signaling pathway as a potential therapeutic target in AML.
Key Points• IFNa targets Jak2V617F MPN stem cells.Interferon-a (IFNa) is an effective treatment of patients with myeloproliferative neoplasms (MPNs). In addition to inducing hematological responses in most MPN patients, IFNa reduces the JAK2V617F allelic burden and can render the JAK2V617F mutant clone undetectable in some patients. The precise mechanism underlying these responses is incompletely understood and whether the molecular responses that are seen occur due to the effects of IFNa on JAK2V617F mutant stem cells is debated. Using a murine model of Jak2V617F MPN, we investigated the effects of IFNa on Jak2V617F MPN-propagating stem cells in vivo. We report that IFNa treatment induces hematological responses in the model and causes depletion of Jak2V617F MPN-propagating cells over time, impairing disease transplantation. We demonstrate that IFNa treatment induces cell cycle activation of Jak2V617F mutant long-term hematopoietic stem cells and promotes a predetermined erythroid-lineage differentiation program. These findings provide insights into the differential effects of IFNa on Jak2V617F mutant and normal hematopoiesis and suggest that IFNa achieves molecular remissions in MPN patients through its effects on MPN stem cells. Furthermore, these results support combinatorial therapeutic approaches in MPN by concurrently depleting dormant JAK2V617F MPNpropagating stem cells with IFNa and targeting the proliferating downstream progeny with JAK2 inhibitors or cytotoxic chemotherapy. (Blood. 2013;121(18):3692-3702)
Large chromosomal deletions are among the most common molecular abnormalities in cancer, yet the identification of relevant genes has proven difficult. The 5q؊ syndrome, a subtype of myelodysplastic syndrome (MDS), is a chromosomal deletion syndrome characterized by anemia and thrombocytosis. Although we have previously shown that hemizygous loss of RPS14 recapitulates the failed erythroid differentiation seen in 5q؊ syndrome, it does not affect thrombocytosis. Here we show that a microRNA located in the common deletion region of 5q؊ syndrome, miR-145, affects megakaryocyte and erythroid differentiation. We find that miR-145 functions through repression of Fli-1, a megakaryocyte and erythroid regulatory transcription factor. Patients with del(5q) MDS have decreased expression of miR-145 and increased expression of Fli-1. Overexpression of miR-145 or inhibition of Fli-1 decreases the production of megakaryocytic cells relative to erythroid cells, whereas inhibition of miR-145 or overexpression of Fli-1 has a reciprocal effect. Moreover, combined loss of miR-145 and RPS14 cooperates to alter erythroid-megakaryocytic differentiation in a manner similar to the 5q؊ syndrome. Taken together, these findings demonstrate that coordinate deletion of a miRNA and a protein-coding gene contributes to the phenotype of a human malignancy, the 5q؊ syndrome. IntroductionThe 5qϪ syndrome is a subtype of myelodysplastic syndrome (MDS) characterized by a macrocytic anemia, a normal or elevated platelet count, and hypolobated micromegakaryocytes. 1 Patients have heterozygous deletions of chromosome 5q, and no genetic lesions have been identified on the intact allele that would cause homozygous inactivation of a gene, raising the hypothesis that haploinsufficiency for one or more genes is sufficient to cause the clinical phenotype of the 5qϪ syndrome. A common deleted region (CDR) for the 5qϪ syndrome has been mapped that spans approximately 1.5 megabases, encompassing 40 protein-coding genes. 2 In a screen of each of the genes within this CDR, we found that decreased expression of RPS14, encoding a member of the 40S ribosomal subunit, causes the erythroid phenotype of the 5qϪ syndrome. 3 Similarly, inherited mutations that inactivate one allele of other ribosomal proteins cause Diamond-Blackfan anemia (DBA), another disease in which patients have a severe macrocytic anemia. 4 Whereas macrocytic anemia is a defining feature of the 5qϪ syndrome and DBA, both of which are associated with haploinsufficiency for ribosomal genes, thrombocytosis is characteristic of the 5qϪ syndrome but not DBA. 5 We hypothesized that additional genes must contribute to the molecular basis of the 5qϪ syndrome given that RPS14 deficiency probably does not explain the full clinical phenotype of the 5qϪ syndrome, that deletions are universally large, and that no mutations have been found that inactivate RPS14 or any other single gene within the 5qϪ syndrome CDR.In our genetic screen of the 5qϪ syndrome CDR, we evaluated all protein coding genes in the CDR fo...
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