Faculty Bibliography

The classical model of hematopoiesis has long held that hematopoietic stem cells (HSCs) sit at the apex of a developmental hierarchy in which HSCs undergo long-term self-renewal while giving rise to cells of all the blood lineages. In this model, self-renewing HSCs progressively lose the capacity for self-renewal as they transit into short-term self-renewing and multipotent progenitor states, with the first major lineage commitment occurring in multipotent progenitors, thus giving rise to progenitors that initiate the myeloid and lymphoid branches of hematopoiesis. Subsequently, within the myeloid lineage, bipotent megakaryocyte-erythrocyte and granulocyte-macrophage progenitors give rise to unipotent progenitors that ultimately give rise to all mature progeny. However, over the past several years, this developmental scheme has been challenged, with the origin of megakaryocyte precursors being one of the most debated subjects. Recent studies have suggested that megakaryocytes can be generated from multiple pathways and that some differentiation pathways do not require transit through a requisite multipotent or bipotent megakaryocyte-erythrocyte progenitor stage. Indeed, some investigators have argued that HSCs contain a subset of cells with biased megakaryocyte potential, with megakaryocytes directly arising from HSCs under steady-state and stress conditions. In this review, we discuss the evidence supporting these nonclassical megakaryocytic differentiation pathways and consider their relative strengths and weaknesses as well as the technical limitations and potential pitfalls in interpreting these studies. Ultimately, such pitfalls will need to be overcome to provide a comprehensive and definitive understanding of megakaryopoiesis.

Histiocytic neoplasms are clonal, hematopoietic disorders characterized by an accumulation of abnormal, monocyte-derived dendritic cells or macrophages in Langerhans Cell (LCH) and non-Langerhans (non-LCH) histiocytoses, respectively. The discovery of BRAFV600E mutations in ~50% of these patients provided the first molecular therapeutic target in histiocytosis. However, recurrent driving mutations in the majority of BRAFV600E-wildtype, non-LCH patients are unknown, and recurrent cooperating mutations in non-MAP kinase pathways are undefined for the histiocytic neoplasms. Through combined whole exome and transcriptome sequencing, we identified recurrent kinase fusions involving BRAF, ALK, and NTRK1, as well as recurrent, activating MAP2K1 and ARAF mutations in BRAFV600E-wildtype, non-LCH patients. In addition to MAP kinase pathway lesions, recurrently altered genes involving diverse cellular pathways were identified. Treatment of MAP2K1- and ARAF-mutated, non-LCH patients using MEK and RAF inhibitors, respectively, resulted in clinical efficacy demonstrating the importance of detecting and targeting diverse kinase alterations in these disorders.

Somatic PTPN11 mutations cause juvenile myelomonocytic leukemia (JMML). Germline PTPN11 defects cause Noonan syndrome (NS), and specific inherited mutations cause NS/JMML. Here, we report that hematopoietic cells differentiated from human induced pluripotent stem cells (hiPSCs) harboring NS/JMML-causing PTPN11 mutations recapitulated JMML features. hiPSC-derived NS/JMML myeloid cells exhibited increased signaling through STAT5 and upregulation of miR-223 and miR-15a. Similarly, miR-223 and miR-15a were upregulated in 11/19 JMML bone marrow mononuclear cells harboring PTPN11 mutations, but not those without PTPN11 defects. Reducing miR-223's function in NS/JMML hiPSCs normalized myelogenesis. MicroRNA target gene expression levels were reduced in hiPSC-derived myeloid cells as well as in JMML cells with PTPN11 mutations. Thus, studying an inherited human cancer syndrome with hiPSCs illuminated early oncogenesis prior to the accumulation of secondary genomic alterations, enabling us to discover microRNA dysregulation, establishing a genotype-phenotype association for JMML and providing therapeutic targets.

We adapted UV CLIP (cross-linking immunoprecipitation) to accurately locate tens of thousands of m(6)A residues in mammalian mRNA with single-nucleotide resolution. More than 70% of these residues are present in the 3'-most (last) exons, with a very sharp rise (sixfold) within 150-400 nucleotides of the start of the last exon. Two-thirds of last exon m(6)A and >40% of all m(6)A in mRNA are present in 3' untranslated regions (UTRs); contrary to earlier suggestions, there is no preference for location of m(6)A sites around stop codons. Moreover, m(6)A is significantly higher in noncoding last exons than in next-to-last exons harboring stop codons. We found that m(6)A density peaks early in the 3' UTR and that, among transcripts with alternative polyA (APA) usage in both the brain and the liver, brain transcripts preferentially use distal polyA sites, as reported, and also show higher proximal m(6)A density in the last exons. Furthermore, when we reduced m6A methylation by knocking down components of the methylase complex and then examined 661 transcripts with proximal m6A peaks in last exons, we identified a set of 111 transcripts with altered (approximately two-thirds increased proximal) APA use. Taken together, these observations suggest a role of m(6)A modification in regulating proximal alternative polyA choice.

BACKGROUND: BRAF V600E is the genetic lesion underlying hairy-cell leukemia. We assessed the safety and activity of the oral BRAF inhibitor vemurafenib in patients with hairy-cell leukemia that had relapsed after treatment with a purine analogue or who had disease that was refractory to purine analogues. METHODS: We conducted two phase 2, single-group, multicenter studies of vemurafenib (at a dose of 960 mg twice daily)--one in Italy and one in the United States. The therapy was administered for a median of 16 weeks in the Italian study and 18 weeks in the U.S. study. Primary end points were the complete response rate (in the Italian trial) and the overall response rate (in the U.S. trial). Enrollment was completed (28 patients) in the Italian trial in April 2013 and is still open (26 of 36 planned patients) in the U.S. trial. RESULTS: The overall response rates were 96% (25 of 26 patients who could be evaluated) after a median of 8 weeks in the Italian study and 100% (24 of 24) after a median of 12 weeks in the U.S. study. The rates of complete response were 35% (9 of 26 patients) and 42% (10 of 24) in the two trials, respectively. In the Italian trial, after a median follow-up of 23 months, the median relapse-free survival was 19 months among patients with a complete response and 6 months among those with a partial response; the median treatment-free survival was 25 months and 18 months, respectively. In the U.S. trial, at 1 year, the progression-free survival rate was 73% and the overall survival rate was 91%. Drug-related adverse events were usually of grade 1 or 2, and the events most frequently leading to dose reductions were rash and arthralgia or arthritis. Secondary cutaneous tumors (treated with simple excision) developed in 7 of 50 patients. The frequent persistence of phosphorylated ERK-positive leukemic cells in bone marrow at the end of treatment suggests bypass reactivation of MEK and ERK as a resistance mechanism. CONCLUSIONS: A short oral course of vemurafenib was highly effective in patients with relapsed or refractory hairy-cell leukemia. (Funded by the Associazione Italiana per la Ricerca sul Cancro and others; EudraCT number, 2011-005487-13; ClinicalTrials.gov number NCT01711632.).

Telomere shortening is common in bone marrow failure syndromes such as dyskeratosis congenita (DC), aplastic anemia (AA) and myelodysplastic syndromes (MDS). However, improved knowledge of the lineage-specific consequences of telomere erosion and restoration of telomere length in hematopoietic progenitors is required to advance therapeutic approaches. We have employed a reversible murine model of telomerase deficiency to compare the dependence of erythroid and myeloid lineage differentiation on telomerase activity. Fifth generation Tert-/- (G5 Tert-/-) mice with shortened telomeres have significant anemia, decreased erythroblasts and reduced hematopoietic stem cell (HSC) populations associated with neutrophilia and increased myelopoiesis. Intracellular multiparameter analysis by mass cytometry showed significantly reduced cell proliferation and increased sensitivity to activation of DNA damage checkpoints in erythroid progenitors and in erythroid-biased CD150hi HSC, but not in myeloid progenitors. Strikingly, Cre-inducible reactivation of telomerase activity restored hematopoietic stem and progenitor cell (HSPC) proliferation, normalized the DNA damage response, and improved red cell production and hemoglobin levels. These data establish a direct link between the loss of TERT activity, telomere shortening and defective erythropoiesis and suggest that novel strategies to restore telomerase function may have an important role in the treatment of the resulting anemia.

We synthesized "mesoscale" nanoparticles, approximately 400 nm in diameter, which unexpectedly localized selectively in renal proximal tubules and up to 7 times more efficiently in the kidney than other organs. Although nanoparticles typically localize in the liver and spleen, modulating their size and opsonization potential allowed for stable targeting of the kidneys through a new proposed uptake mechanism. Applying this kidney targeting strategy, we anticipate use in the treatment of renal disease and the study of renal physiology.