SUMMARY Acute myelogenous leukemia (AML) is organized as a cellular hierarchy initiated and maintained by a subset of self-renewing leukemia stem cells (LSC). We hypothesized that increased CD47 expression on human AML LSC contributes to pathogenesis by inhibiting their phagocytosis through the interaction of CD47 with an inhibitory receptor on phagocytes. We found that CD47 was more highly expressed on AML LSC than their normal counterparts, and that increased CD47 expression predicted worse overall survival in 3 independent cohorts of adult AML patients. Furthermore, blocking monoclonal antibodies directed against CD47 preferentially enabled phagocytosis of AML LSC and inhibited their engraftment in vivo. Finally, treatment of human AML LSC-engrafted mice with anti-CD47 antibody depleted AML and targeted AML LSC. In summary, increased CD47 expression is an independent poor prognostic factor that can be targeted on human AML stem cells with blocking monoclonal antibodies capable of enabling phagocytosis of LSC.

Multiparametric single-cell analysis is critical for understanding cellular heterogeneity. Despite recent technological advances in single-cell measurements, methods for analyzing high-dimensional single-cell data are often subjective, labor intensive and require prior knowledge of the biological system under investigation. To objectively uncover cellular heterogeneity from single-cell measurements, we present a novel computational approach, Spanning-tree Progression Analysis of Density-normalized Events (SPADE). We applied SPADE to cytometry data of mouse and human bone marrow. In both cases, SPADE organized cells in a hierarchy of related phenotypes that partially recapitulated well-described patterns of hematopoiesis. In addition, SPADE produced a map of intracellular signal activation across the landscape of human hematopoietic development. SPADE revealed a functionally distinct cell population, natural killer (NK) cells, without using any NK-specific parameters. SPADE is a versatile method that facilitates the analysis of cellular heterogeneity, the identification of cell types, and comparison of functional markers in response to perturbations.

Dysregulated Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling due to activation of tyrosine kinases is a common feature of myeloid malignancies. Here we report the first human disease-related mutations in the adaptor protein LNK, a negative regulator of JAK-STAT signaling, in 2 patients with JAK2 V617F-negative myeloproliferative neoplasms (MPNs). One patient exhibited a 5 base-pair deletion and missense mutation leading to a premature stop codon and loss of the pleckstrin homology (PH) and Src homology 2 (SH2) domains. A second patient had a missense mutation (E208Q) in the PH domain. BaF3-MPL cells transduced with these LNK mutants displayed augmented and sustained thrombopoietin-dependent growth and signaling. Primary samples from MPN patients bearing LNK mutations exhibited aberrant JAK-STAT activation, and cytokine-responsive CD34 ؉ early progenitors were abnormally abundant in both patients. These findings indicate that JAK-STAT activation due to loss of LNK negative feedback regulation is a novel mechanism of MPN pathogenesis. (Blood. 2010;116(6):988-992) IntroductionIn myeloproliferative neoplasms (MPNs), aberrant JAK-STAT signaling has primarily been attributed to activating mutations in tyrosine kinases. [1][2][3][4][5][6][7] However, approximately 40% to 50% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF) lack a defined genetic abnormality. Janus kinasesignal transducer and activator of transcription (JAK-STAT) activation can be demonstrated in some MPN patients lacking JAK2 or MPL mutations, [8][9][10] suggesting that other regulatory elements in this pathway are altered.One regulator of JAK-STAT signaling is LNK (SH2B3), a member of a family of adaptor proteins that share several structural motifs, including a proline-rich N-terminal dimerization domain, a pleckstrin homology (PH) domain, an Src homology 2 (SH2) domain, and a conserved C-terminal tyrosine residue (Fig 1A). 11 LNK binds to myeloproliferative leukemia virus oncogene (MPL) via its SH2 domain and colocalizes to the plasma membrane via its PH domain. 12,13 Upon cytokine stimulation with thrombopoietin (TPO), LNK binds strongly to JAK2 and inhibits downstream STAT activation, thereby providing critical negative feedback regulation. 12 LNK Ϫ/Ϫ mice exhibit an MPN phenotype, including an expanded hematopoietic stem cell compartment, megakaryocytic hyperplasia, splenomegaly, leukocytosis, and thrombocytosis. [14][15][16] These studies provide compelling evidence that loss of LNK activity is relevant to MPN pathobiology. Here we report the identification of LNK mutations in JAK2 V617F-negative MPNs that lead to dysregulated JAK-STAT signaling and cell proliferation. Methods Clinical samplesPatient samples were obtained with informed consent in accordance with the Declaration of Helsinki and approval by the Institutional Review Board of Stanford University School of Medicine. PCR and sequencingPolymerase chain reaction (PCR) and bidirectional sequencing were performed according ...

This paper addresses the process of career-interest formation as it relates to faculty careers in a diverse cohort of 38 recent biomedical sciences PhD graduates (including 23 women and 18 underrepresented minorities). The authors show that personal values and structural dynamics in the biomedical workforce play strong roles in shaping career interest.