SUMMARY We report a Jak2V617F knock-in mouse myeloproliferative neoplasm (MPN) model resembling human polycythemia vera (PV). The MPN is serially transplantable and we demonstrate that the hematopoietic stem cell (HSC) compartment has the unique capacity for disease initiation but does not have a significant selective competitive advantage over wild type HSCs. In contrast, myeloid progenitor populations are expanded and skewed towards the erythroid lineage, but cannot transplant the disease. Treatment with a JAK2 kinase inhibitor ameliorated the MPN phenotype, but did not eliminate the disease-initiating population. These findings provide insights into the consequences of JAK2 activation on HSC differentiation and function and have the potential to inform therapeutic approaches to JAK2V617F positive MPN. SIGNIFICANCE The JAK2V617F mutation is a promising candidate for molecularly targeted therapy in MPN. Early data from JAK2 inhibitor clinical trials have called into question the capacity of these compounds to alter the natural history of JAK2V617F mediated MPN. Determining the effect of JAK2 inhibitors on the disease-initiating population requires a model in which the JAK2V617F allele is expressed at physiological levels in hematopoietic stem and progenitor cells, as it is in humans. Our model demonstrates that JAK2V617F causes expansion of erythroid progenitors but that only the HSC compartment can initiate disease in a transplanted mouse. We further demonstrate that the HSC compartment, the definitive target for curative therapy of JAK2V617F mediated MPN, is resistant to treatment with a JAK2 inhibitor.

Haploinsufficiency for ribosomal protein genes has been implicated in the pathophysiology of Diamond-Blackfan anemia (DBA) and the 5q؊ syndrome, a subtype of myelodysplastic syndrome. The p53 pathway is activated by ribosome dysfunction, but the molecular basis for selective impairment of the erythroid lineage in disorders of ribosome function has not been determined. We found that p53 accumulates selectively in the erythroid lineage in primary human hematopoietic progenitor cells after expression of shRNAs targeting RPS14, the ribosomal protein gene deleted in the 5q؊ syndrome, or RPS19, the most commonly mutated gene in DBA. Induction of p53 led to lineagespecific accumulation of p21 and consequent cell cycle arrest in erythroid progenitor cells. Pharmacologic inhibition of p53 rescued the erythroid defect, whereas nutlin-3, a compound that activates p53 through inhibition of HDM2, selectively impaired erythropoiesis. In bone marrow biopsies from patients with DBA or del(5q) myelodysplastic syndrome, we found an accumulation of nuclear p53 staining in erythroid progenitor cells that was not present in control samples. Our findings indicate that the erythroid lineage has a low threshold for the induction of p53, providing a basis for the failure of erythropoiesis in the 5q؊ syndrome, DBA, and perhaps other bone marrow failure syndromes. (Blood. 2011;117(9):2567-2576) IntroductionHeterozygous deletions or mutations of ribosomal protein genes have been implicated in 2 human disorders, Diamond-Blackfan anemia (DBA) 1-3 and the 5qϪ syndrome, a subtype of myelodysplastic syndrome (MDS). 4,5 Both disorders are characterized by a severe macrocytic anemia. 2,6 In DBA, a congenital disorder, approximately 25% of patients have mutations in the RPS19 gene, 1 and mutations or deletions have been identified in at least 9 additional ribosomal protein genes. 3,7 In the 5qϪ syndrome, somatic deletion of one allele of chromosome 5q causes haploinsufficiency for the RPS14 gene. 5 A central outstanding question is how a defect in ribosomes, which are expressed in and are essential to all cells, causes a primarily erythroid phenotype.Multiple animal models have demonstrated the effects of ribosomal dysfunction on erythropoiesis and the role of p53, which is known to monitor ribosome function. 8 Morpholinos targeting RPS19 in zebrafish cause an accumulation of p53 and a block in erythropoiesis that is reversed in the absence of p53. 9 Mice with germline mutations in the RPS19 or RPS20 genes have hyperpigmented foot pads with p53 accumulation in the epidermis, a decreased hematocrit, and an increased erythrocyte mean cell volume. Crossing the RPS19 mutant mice with p53 null mice rescued the skin and hematopoietic phenotypes. 10 Finally, a conditional heterozygous deletion of the Cd74-Nid76 interval syntenic to human chromosome 5q, containing 6 genes, including RPS14, in mouse hematopoietic cells, causes a macrocytic anemia that is rescued by crossing to a p53 null background. 11Although p53 appears to play a critical role in these d...

Summary Over 80% of patients with the refractory anemia with ring sideroblasts subtype of myelodysplastic syndrome (MDS) have mutations in Splicing Factor 3B, Subunit 1 (SF3B1). We generated a conditional knock-in mouse model of the most common SF3B1 mutation, Sf3b1K700E. Sf3b1K700E mice develop macrocytic anemia due to a terminal erythroid maturation defect, erythroid dysplasia, and long-term hematopoietic stem cell (LT-HSC) expansion. Sf3b1K700E myeloid progenitors and SF3B1-mutant MDS patient samples demonstrate aberrant 3’ splice-site selection associated with increased nonsense-mediated decay. Tet2 loss cooperates with Sf3b1K700E to cause a more severe erythroid and LT-HSC phenotype. Furthermore, the spliceosome modulator, E7017, selectively kills SF3B1K700E-expressing cells. Thus, SF3B1K700E expression reflects the phenotype of the mutation in MDS and may be a therapeutic target in MDS.