CD47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells.

Acute myeloid 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 three 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.

Rapid development of exhaustion and down-regulation of eomesodermin limit the antitumor activity of adoptively transferred murine natural killer cells.

Natural killer (NK) cells are potent anti-viral and antitumor "first responders" endowed with natural cytotoxicity and cytokine production capabilities. To date, attempts to translate these promising biologic functions through the adoptive transfer of NK cells for the treatment of cancer have been of limited benefit. Here we trace the fate of adoptively transferred murine NK cells and make the surprising observation that NK cells traffic to tumor sites yet fail to control tumor growth or improve survival. This dysfunction is related to a rapid down-regulation of activating receptor expression and loss of important effector functions. Loss of interferon (IFN)γ production occurs early after transfer, whereas loss of cytotoxicity progresses with homeostatic proliferation and tumor exposure. The dysfunctional phenotype is accompanied by down-regulation of the transcription factors Eomesodermin and T-bet, and can be partially reversed by the forced overexpression of Eomesodermin. These results provide the first demonstration of NK-cell exhaustion and suggest that the NK-cell first-response capability is intrinsically limited. Further, novel approaches may be required to circumvent the described dysfunctional phenotype.

Single-cell phospho-specific flow cytometric analysis demonstrates biochemical and functional heterogeneity in human hematopoietic stem and progenitor compartments.

The low frequency of hematopoietic stem and progenitor cells (HSPCs) in human BM has precluded analysis of the direct biochemical effects elicited by cytokines in these populations, and their functional consequences. Here, single-cell phospho-specific flow cytometry was used to define the signaling networks active in 5 previously defined human HSPC subsets. This analysis revealed that the currently defined HSC compartment is composed of biochemically distinct subsets with the ability to respond rapidly and directly in vitro to a broader array of cytokines than previously appreciated, including G-CSF. The G-CSF response was physiologically relevant-driving cell-cycle entry and increased proliferation in a subset of single cells within the HSC compartment. The heterogeneity in the single-cell signaling and proliferation responses prompted subfractionation of the adult BM HSC compartment by expression of CD114 (G-CSF receptor). Xenotransplantation assays revealed that HSC activity is significantly enriched in the CD114(neg/lo) compartment, and almost completely absent in the CD114(pos) subfraction. The single-cell analyses used here can be adapted for further refinement of HSPC surface immunophenotypes, and for examining the direct regulatory effects of other factors on the homeostasis of stem and progenitor populations in normal or diseased states.

Novel mutations in the inhibitory adaptor protein LNK drive JAK-STAT signaling in patients with myeloproliferative neoplasms.

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.

From pipettes to policy: Reflections on a decade working to expand opportunity and equity in science.

Science policy focuses on the allocation of resources within the scientific enterprise and the downstream impacts of these investments. Here, I describe my journey from being a curious kid, to becoming a signaling biologist, to my current role as a science policy professional focusing on the areas of biomedical research training, workforce diversity, and promoting basic research. I provide insights on skills important in this career track-collaboration, diplomacy, adaptability, and resilience. Finally, I share the vision that animates my work-"science by all, science for all"-and encourage you with the career advice my mother gave: "never self-eliminate."

The funders' perspective: Lessons learned from the National Institutes of Health Diversity Program Consortium evaluation.

Advancing diversity in the biomedical research workforce is critical to the ability of the National Institutes of Health (NIH) to achieve its mission. The NIH Diversity Program Consortium is a unique, 10-year program that builds upon longstanding training and research capacity-building activities to promote workforce diversity. It was designed to rigorously evaluate approaches to enhancing diversity in the biomedical research workforce at the student, faculty, and institutional level. In this chapter we describe (a) the program's origins, (b) the consortium-wide evaluation, including plans, measures, challenges, and solutions, and (c) how lessons learned from this program are being leveraged to strengthen NIH research-training and capacity-building activities and evaluation efforts.

Decoupling of the minority PhD talent pool and assistant professor hiring in medical school basic science departments in the US.

Faculty diversity is a longstanding challenge in the US. However, we lack a quantitative and systemic understanding of how the career transitions into assistant professor positions of PhD scientists from underrepresented minority (URM) and well-represented (WR) racial/ethnic backgrounds compare. Between 1980 and 2013, the number of PhD graduates from URM backgrounds increased by a factor of 9.3, compared with a 2.6-fold increase in the number of PhD graduates from WR groups. However, the number of scientists from URM backgrounds hired as assistant professors in medical school basic science departments was not related to the number of potential candidates (R2=0.12, p>0.07), whereas there was a strong correlation between these two numbers for scientists from WR backgrounds (R2=0.48, p<0.0001). We built and validated a conceptual system dynamics model based on these data that explained 79% of the variance in the hiring of assistant professors and posited no hiring discrimination. Simulations show that, given current transition rates of scientists from URM backgrounds to faculty positions, faculty diversity would not increase significantly through the year 2080 even in the context of an exponential growth in the population of PhD graduates from URM backgrounds, or significant increases in the number of faculty positions. Instead, the simulations showed that diversity increased as more postdoctoral candidates from URM backgrounds transitioned onto the market and were hired.

Career Development among American Biomedical Postdocs.

Recent biomedical workforce policy efforts have centered on enhancing career preparation for trainees, and increasing diversity in the research workforce. Postdoctoral scientists, or postdocs, are among those most directly impacted by such initiatives, yet their career development remains understudied. This study reports results from a 2012 national survey of 1002 American biomedical postdocs. On average, postdocs reported increased knowledge about career options but lower clarity about their career goals relative to PhD entry. The majority of postdocs were offered structured career development at their postdoctoral institutions, but less than one-third received this from their graduate departments. Postdocs from all social backgrounds reported significant declines in interest in faculty careers at research-intensive universities and increased interest in nonresearch careers; however, there were differences in the magnitude and period of training during which these changes occurred across gender and race/ethnicity. Group differences in interest in faculty careers were explained by career interest differences formed during graduate school but not by differences in research productivity, research self-efficacy, or advisor relationships. These findings point to the need for enhanced career development earlier in the training process, and interventions sensitive to distinctive patterns of interest development across social identity groups.

An Assessment of Hospital-Based Palliative Care in Maryland: Infrastructure, Barriers, and Opportunities.

CONTEXT: Maryland recently passed legislation mandating that hospitals with more than 50 beds have palliative care (PC) programs. Although the state's health agency can play a key role in ensuring successful implementation of this measure, there is little actionable information from which it can guide resource allocation for enhancing PC delivery statewide. OBJECTIVES: To assess the PC infrastructure at Maryland's 46 community-based nonspecialty hospitals and to describe providers' perspectives on barriers to PC and supports that could enhance PC delivery. METHODS: Data on PC programs were collected using two mechanisms. First, a survey was sent to all 46 community-based hospital chief executive officers by the Maryland Cancer Collaborative. The Maryland Health Care Commission provided supplementary survey and semistructured interview data. RESULTS: Twenty-eight hospitals (60.9%) provided information on their PC services. Eighty-nine percent of these hospitals reported the presence of a structured PC program. The profile of services provided by PC programs was largely conserved across hospital geography and size. The most common barriers reported to PC delivery were lack of knowledge among patients and/or families and lack of physician buy-in; most hospitals reported that networks and/or conferences to promote best practice sharing in PC would be useful supports. CONCLUSION: Systematic collection of state-level PC infrastructure data can be used to guide state health agencies' understanding of extant resources and challenges, using those data to determine resource allocation to promote the timely receipt of PC for patients and families.

Biomedical Science Ph.D. Career Interest Patterns by Race/Ethnicity and Gender.

Increasing biomedical workforce diversity remains a persistent challenge. Recent reports have shown that biomedical sciences (BMS) graduate students become less interested in faculty careers as training progresses; however, it is unclear whether or how the career preferences of women and underrepresented minority (URM) scientists change in manners distinct from their better-represented peers. We report results from a survey of 1500 recent American BMS Ph.D. graduates (including 276 URMs) that examined career preferences over the course of their graduate training experiences. On average, scientists from all social backgrounds showed significantly decreased interest in faculty careers at research universities, and significantly increased interest in non-research careers at Ph.D. completion relative to entry. However, group differences emerged in overall levels of interest (at Ph.D. entry and completion), and the magnitude of change in interest in these careers. Multiple logistic regression showed that when controlling for career pathway interest at Ph.D. entry, first-author publication rate, faculty support, research self-efficacy, and graduate training experiences, differences in career pathway interest between social identity groups persisted. All groups were less likely than men from well-represented (WR) racial/ethnic backgrounds to report high interest in faculty careers at research-intensive universities (URM men: OR 0.60, 95% CI: 0.36-0.98, p = 0.04; WR women: OR: 0.64, 95% CI: 0.47-0.89, p = 0.008; URM women: OR: 0.46, 95% CI: 0.30-0.71, p<0.001), and URM women were more likely than all other groups to report high interest in non-research careers (OR: 1.93, 95% CI: 1.28-2.90, p = 0.002). The persistence of disparities in the career interests of Ph.D. recipients suggests that a supply-side (or "pipeline") framing of biomedical workforce diversity challenges may limit the effectiveness of efforts to attract and retain the best and most diverse workforce. We propose incorporation of an ecological perspective of career development when considering strategies to enhance the biomedical workforce and professoriate through diversity.

What do I want to be with my PhD? The roles of personal values and structural dynamics in shaping the career interests of recent biomedical science PhD graduates.

Interest in faculty careers decreases as graduate training progresses; however, the process underlying career-interest formation remains poorly defined. To better understand this process and whether/how it differs across social identity (i.e., race/ethnicity, gender), we conducted focus groups with 38 biomedical scientists who received PhDs between 2006 and 2011, including 23 women and 18 individuals from underrepresented minority (URM) backgrounds. Objective performance and quality of advisor relationships were not significantly different between scientists with high versus low interest in faculty careers. Career interests were fluid and formed in environments that generally lacked structured career development. Vicarious learning shaped similar outcome expectations about academic careers for all scientists; however, women and URMs recounted additional, distinct experiences and expectations. Scientists pursuing faculty careers described personal values, which differed by social identity, as their primary driver. For scientists with low interest in faculty careers, a combination of values, shared across social identity, and structural dynamics of the biomedical workforce (e.g., job market, grant funding, postdoc pay, etc.) played determinative roles. These findings illuminate the complexity of career choice and suggest attracting the best, most diverse academic workforce requires institutional leaders and policy makers go beyond developing individual skill, attending to individuals' values and promoting institutional and systemic reforms.

Decoupling of tumor-initiating activity from stable immunophenotype in HoxA9-Meis1-driven AML.

Increasing evidence suggests tumors are maintained by cancer stem cells; however, their nature remains controversial. In a HoxA9-Meis1 (H9M) model of acute myeloid leukemia (AML), we found that tumor-initiating activity existed in three, immunophenotypically distinct compartments, corresponding to disparate lineages on the normal hematopoietic hierarchy--stem/progenitor cells (Lin(-)kit(+)) and committed progenitors of the myeloid (Gr1(+)kit(+)) and lymphoid lineages (Lym(+)kit(+)). These distinct tumor-initiating cells (TICs) clonally recapitulated the immunophenotypic spectrum of the original tumor in vivo (including cells with a less-differentiated immunophenotype) and shared signaling networks, such that in vivo pharmacologic targeting of conserved TIC survival pathways (DNA methyltransferase and MEK phosphorylation) significantly increased survival. Collectively, H9M AML is organized as an atypical hierarchy that defies the strict lineage marker boundaries and unidirectional differentiation of normal hematopoiesis. Moreover, this suggests that in certain malignancies tumor-initiation activity (or "cancer stemness") can represent a cellular state that exists independently of distinct immunophenotypic definition.

Extracting a cellular hierarchy from high-dimensional cytometry data with SPADE.

The ability to analyze multiple single-cell parameters is critical for understanding cellular heterogeneity. Despite recent advances in measurement technology, methods for analyzing high-dimensional single-cell data are often subjective, labor intensive and require prior knowledge of the biological system. To objectively uncover cellular heterogeneity from single-cell measurements, we present a versatile computational approach, spanning-tree progression analysis of density-normalized events (SPADE). We applied SPADE to flow cytometry data of mouse bone marrow and to mass cytometry data of human bone marrow. In both cases, SPADE organized cells in a hierarchy of related phenotypes that partially recapitulated well-described patterns of hematopoiesis. We demonstrate that SPADE is robust to measurement noise and to the choice of cellular markers. SPADE facilitates the analysis of cellular heterogeneity, the identification of cell types and comparison of functional markers in response to perturbations.

Dendritic cells cross-dressed with peptide MHC class I complexes prime CD8+ T cells.

The activation of naive CD8+ T cells has been attributed to two mechanisms: cross-priming and direct priming. Cross-priming and direct priming differ in the source of Ag and in the cell that presents the Ag to the responding CD8+ T cells. In cross-priming, exogenous Ag is acquired by professional APCs, such as dendritic cells (DC), which process the Ag into peptides that are subsequently presented. In direct priming, the APCs, which may or may not be DC, synthesize and process the Ag and present it themselves to CD8+ T cells. In this study, we demonstrate that naive CD8+ T cells are activated by a third mechanism, called cross-dressing. In cross-dressing, DC directly acquire MHC class I-peptide complexes from dead, but not live, donor cells by a cell contact-mediated mechanism, and present the intact complexes to naive CD8+ T cells. Such DC are cross-dressed because they are wearing peptide-MHC complexes generated by other cells. CD8+ T cells activated by cross-dressing are restricted to the MHC class I genotype of the donor cells and are specific for peptides generated by the donor cells. In vivo studies demonstrate that optimal priming of CD8+ T cells requires both cross-priming and cross-dressing. Thus, cross-dressing may be an important mechanism by which DC prime naive CD8+ T cells and may explain how CD8+ T cells are primed to Ags that are inefficiently cross-presented.

Tumor-specific CD4+ T cells are activated by "cross-dressed" dendritic cells presenting peptide-MHC class II complexes acquired from cell-based cancer vaccines.

Tumor cells that constitutively express MHC class I molecules and are genetically modified to express MHC class II (MHC II) and costimulatory molecules are immunogenic and have therapeutic efficacy against established primary and metastatic cancers in syngeneic mice and activate tumor-specific human CD4+ T lymphocytes. Previous studies have indicated that these MHC II vaccines enhance immunity by directly activating tumor-specific CD4+ T cells during the immunization process. Because dendritic cells (DCs) are considered to be the most efficient APCs, we have now examined the role of DCs in CD4+ T cell activation by the MHC II vaccines. Surprisingly, we find that DCs are essential for MHC II vaccine immunogenicity; however, they mediate their effect through "cross-dressing." Cross-dressing, or peptide-MHC (pMHC) transfer, involves the generation of pMHC complexes within the vaccine cells, and their subsequent transfer to DCs, which then present the intact, unprocessed complexes to CD4+ T lymphocytes. The net result is that DCs are the functional APCs; however, the immunogenic pMHC complexes are generated by the tumor cells. Because MHC II vaccine cells do not express the MHC II accessory molecules invariant chain and DM, they are likely to load additional tumor Ag epitopes onto MHC II molecules and therefore activate a different repertoire of T cells than DCs. These data further the concept that transfer of cellular material to DCs is important in Ag presentation, and they have direct implications for the design of cancer vaccines.

Hereditary prostate cancer in Finland: fine-mapping validates 3p26 as a major predisposition locus.

In a recent genome-wide linkage (GWL) analysis of Finnish families at high risk for prostate cancer, we found two novel putative susceptibility loci at 3p25-p26 and 11q14. Here, we report the fine-mapping of these two critical regions at high resolution with 39 microsatellite markers in 16 families, including multiplex families that were not used in the GWL scan. The maximum multipoint HLOD was 3.39 at 3p26 and 1.42 at 11q14. The highest LOD scores were seen around markers D3S1270 and D3S4559 (alpha=0.89), covering approximately two megabases. The two known genes in this region CHL1 (cell adhesion molecule with homology to L1CAM) and CNTN6 (contactin 6) were screened for exonic mutations in the families showing the strongest linkage, but no disease-segregating sequence variants were observed. The recombination map pointed to a region proximal to the area of best linkage, suggesting that more genes may need to be investigated as candidates. These results provide strong evidence for the existence of a prostate cancer susceptibility gene at 3p26 in Finnish prostate cancer families. This locus has not been strongly linked with hereditary prostate cancer in other populations. However, the mildly positive 3p LOD scores in a recent GWL analysis of patients from the United States suggest that the locus may also be important in other populations.

Genome-wide scan for linkage in finnish hereditary prostate cancer (HPC) families identifies novel susceptibility loci at 11q14 and 3p25-26.

BACKGROUND: In order to identify predisposition loci to hereditary prostate cancer (HPC), we performed a genome-wide linkage analysis using samples from a genetically homogeneous population, with 13 Finnish multiplex prostate cancer families. METHODS: Altogether 87 DNA samples were genotyped from 13 families. Logarithm-of-odds (LOD) scores were calculated for all autosomes using FASTLINK and GENEHUNTER designating all unaffected men and all women as unknown. RESULTS: The highest LOD scores in the affected-only analyses were found at 11q14, where the two-point LOD score was 2.97 (theta = 0.0 at D11S901), GENEHUNTER heterogeneity LOD (HLOD) of 3.36, and a non-parametric-linkage (NPL) score of 2.67 (P = 0.008). A second positive site was at 3p25-26, with a two-point LOD score of 2.57 (theta = 0.01 at D3S1297), HLOD of 2.15, and NPL score of 2.27 (P = 0.02). CONCLUSIONS: The results suggest two HPC regions in the Finnish population, which have not been reported previously and warrant further study.