ALBERT

Description: Exosomes are extracellular vesicles that function in cell-cell communication by trafficking protein and RNA species to bystander cells. While exosomes are produced by all cell types, those released by cancer cells have come to the forefront of investigation for their potential to modulate the tumorigenic niche. We recently reported that exosomes released from acute myelogenous leukemia (AML) cells impact the phenotype and function of stromal and hematopoietic stem and progenitor cells (HSPC) found in the bone marrow (Huan et al. Cancer Res. 2013). As part of these studies, we observed a decrease in clonogenic potential of murine HSPCs exposed to exosomes isolated from the Molm-14 AML cell line in vitro and in xenograft transplantation studies. To determine if this observation was more widely applicable, we next exposed murine c-kit-selected HSPCs to exosomes isolated from both primary patient samples and the HL-60 AML cell line in vitro. Strikingly, exposure to primary AML patient and HL-60 exosomes produced a significant reduction in colony formation; on average, only 7.3% as many colonies formed in exposed conditions compared to controls. We also previously showed that exosomes traffic a complex mixture of protein and RNA to bystander cells. A recent report demonstrated increased DNA damage in mammary epithelial cells due to elevated reactive oxygen species (ROS) following exposure to exosomes derived from multiple breast cancer cell lines (Dutta et al. PLOS One 2014). A similar mechanism has been shown to restrict the replicative capacity of human HSPCs. Here, we hypothesized that exosome transfer might elicit a DNA damage response in murine HSPCs, contributing to the decreased ability of exposed cells to form colonies. When we performed 48 hours of in vitro HL-60 exosome exposure of c-kit enriched progenitor cells, we found a statistically significant upregulation of genes involved in DNA damage sensing as well as homologous recombination (HR) and non-homologous end joining (NHEJ) DNA repair pathways compared to unexposed controls. Immunofluorescence analysis on exosome-exposed cells also revealed an increase in the formation of γH2AX foci in cells exposed to HL-60 exosomes, indicating an increase in DNA damage burden. Next, we tested if increased ROS might account for DNA damage and the resulting progenitor frequency. Using a flow cytometric analysis of ROS (DCF-DA) revealed a clear upward shift in median fluorescence intensity of exosome-treated c-kit+ cells compared to untreated controls. To further confirm the involvement of ROS, we treated exosome-exposed cells with the antioxidant NAC. While this did not result in a substantial reduction in ROS levels as measured by flow cytometry, analysis of the transcriptional DNA damage response revealed a dose-response pharmacological rescue of HR and NHEJ pathway gene expression. Our work in aggregate suggests that AML exosomes have a direct suppressive effect on HSPCs that involves, at least in part, gains in ROS that promote DNA damage accumulation and genomic instability. We propose a model whereby the paracrine trafficking of exosomes plays an active role in the erosion of HSPC activity in the AML niche that leads to characteristic cytopenias even at low leukemic burden. Disclosures No relevant conflicts of interest to declare.

Description: Evidence in several organ systems demonstrates that pregnancy presents a window of vulnerability for establishing a foundation for health or chronic disease. Overnutrition and the complex metabolic changes that can accompany it can result in permanent phenotypic changes and a predisposition to metabolic syndrome, inflammatory or immune-mediated diseases. We previously reported that prenatal overnutrition stunted fetal liver size. Herein, we hypothesize that this might perturb hematopoietic stem and progenitor cell (HSPC) expansion. To test the effects of a high-fat diet (HFD) and maternal obesity on offspring hematopoiesis, we used a mouse model of diet-induced obesity, feeding female mice a HFD or control diet starting at 5-7 weeks of age and keeping them on the respective diet during subsequent breeding and pregnancy. We then studied offspring at gestational day 14.5 by immunophenotyping, gene expression analysis, qRT-PCR, and transplantation. Fetal livers from HFD offspring had 51% fewer c-Kit+ Sca-1+ Linlo/- and 27% fewer AA4.1+ Sca-1+ Linlo/- (ASL) hematopoietic stem and progenitor cells (HSPC) than controls. This restriction in HSPC numbers was not due to apoptosis or increased reactive oxygen species, as tested by flow cytometry. To determine whether there might be an increase in hematopoietic differentiation to account for relative HSPC deficiencies in HFD livers, we examined hematopoietic lineage subsets. HFD fetal livers had a relative increase in myeloid (Gr-1+/Ter119+) and B220+ lymphoid cells, with comparable proportion of CD3+ cells to controls. Taken together, these results suggest that chronic HFD fetal programming skews fetal liver HSPCs toward differentiation. When we examined global gene expression of male HFD fetal livers versus controls by RNA-seq, we found differential expression of 125 genes. Among the upregulated transcripts, several were involved in hematopoietic regulation, stress response, and HSPC migration. We then used qRT-PCR to test for expression of several of these genes, along with genes critically involved in fetal HSPC self-renewal, within an HSPC-enriched (Sca-1+) population of chronic HFD fetal liver cells. As in RNA-seq, Matrixmetalloproteinase-8 and 9 (Mmp8, Mmp9), which are involved in cell mobilization, were upregulated in HFD-programmed cells. Early growth response-1 (Egr-1) was downregulated as well, further suggesting premature migration of HSPCs from HFD fetal liver. Hmga2, which is implicated in fetal stem cell self-renewal, and its direct target, Igf2bp2, were significantly downregulated in chronic HFD Sca-1+ cells. Along with the immunophenotyping data, these findings suggest that maternal obesity and HFD bias HSPCs toward differentiation, at the expense of self-renewal. To dissect the direct metabolic impact, we studied fetal livers from timed pregnancy cohorts after acute HFD exposure or diet reversal in obese dams, which partially ameliorated several molecular and immunophenotypic endpoints. Finally, we performed a functional test of chronic HFD fetal liver cells by transplantation. A non-competitive transplant into irradiated male recipients yielded no difference in chimerism between HFD or control fetal liver-engrafted animals. Next, we preconditioned a cohort of female and male animals on HFD (or control diet) for 11 weeks, irradiated them, and then competitively transplanted them with a 1:1 ratio of HFD and control fetal liver cells. HFD-programmed fetal liver HSPCs engrafted HFD-conditioned male recipients at significantly lower rates than in HFD-conditioned female or control recipients of either sex. HFD-programmed donor cells retained the significant bias toward the myeloid (Gr-1+/Mac-1+) lineage, noted in the primary graft cells, and away from the B220+ B cell lineage in HFD-conditioned males. In aggregate, prenatal HFD and maternal obesity suppress self-renewal in favor of HSPC differentiation during a time of critical developmental expansion. This suggests an HSPC defect that appears at least partly specified by the stem cell microenvironment. Our work is the first to demonstrate metabolic vulnerability of the hematopoietic stem and progenitor cell compartment and establishes the hematopoietic system as a target for in utero developmental programming. Disclosures No relevant conflicts of interest to declare.

Description: Fanconi Anemia (FA) is a rare, recessively heritable disorder with prominent failure of hematopoiesis. The physiologic role of FA proteins has not been fully resolved to date. While several existing model systems delineate its role in DNA damage response caused by alkylating agents, aldehydes, and inflammatory cytokines, all rely on experimental induction. We previously demonstrated the in utero onset of hematopoietic failure in mice with genetic disruption of Fancc. Herein, we found significant deficits in the fetal liver (FL) hematopoietic stem and progenitor cell (HSPC) pool in Fancd2 mice. Both AA4.1+ Sca-1+ Lin- expressing progenitors (ASL) and CD48- CD150+ Lin- Sca-1+ (SLAM) cells were decreased in frequency in Fancd2-/- versus WT FL. Similarly, we observed a significant decrease in progenitor colony formation and deficits in primary and secondary transplantation among Fancd2-/- FL compared to WT. Fancd2-/- FL cells were characteristically sensitive to mitomycin C and had significantly fewer SLAM cells in the G0 phase of cell cycle and elevated p21 expression, indicating canonical P53 activation. Consistent with prior reports by other groups on embryonic stem cells and our own Fancc-/- FL studies, we found neither exaggerated frequency of apoptotic cells, nor transcriptional induction of Puma or Noxa. We hypothesized that the observed deficits in developmental HSPC pool expansion reflect replication-associated stress. At the transcriptional level, we found activation of the DNA damage response via Rad51 and Prkdc, corroborated by immunofluorescent imaging of Rad51 foci as well as comet assays in FL cells. Next, we tested P38 MAPK as a stress response previously found to confer repopulation deficits in postnatal BM failure among Fancc and Fanca mice; here, our experiments revealed baseline (unprovoked) activation of phospho-p38 and rescue of Fancd2-/- progenitor colony formation using a pharmacological inhibitor, SB203580. Results were further strengthened by transplantation, revealing increased Fancd2-/- donor chimerism after in vivo administration of SB203580. The gains in donor chimerism persisted even after cessation of drug administration. These results suggest that replication-associated stress in the rapidly cycling fetal Fancd2-/- HSPC pool evokes a cellular stress response that constrains physiological expansion. Our work emphasizes the prenatal onset of hematopoietic failure and reveals pharmacological rescue by inhibition of constitutively active P38 MAPK. Furthermore, FA fetal hematopoiesis is an original model of unprovoked hematopoietic failure that allows the study of physiologic role of FA proteins in HSPC. Disclosures No relevant conflicts of interest to declare.

Description: Key PointsFancc −/− mice experience previously unrecognized late gestational lethality. Fancc −/− fetal mouse hematopoiesis is quantitatively and qualitatively deficient.

Description: Hematopoietic failure is the predominant manifestation of Fanconi anemia (FA), a recessively inherited disorder in DNA double strand break (DSB) repair. The etiology underlying the progressive loss of hematopoietic stem and progenitor cells (HSPC) in FA remains to be fully clarified, but is widely considered to be mediated by the “stress activation” of p53. In a murine model of FA (Fancc), we recently showed that the onset of hematopoietic failure precedes the developmentally timed HSPC migration to the bone marrow (BM), corroborating prior reports of reduced cord blood progenitor frequency and work in human ES cell lines. Comparison of genotype frequencies at E14.5 and postnatally (25% vs 19%; p

Description: Abstract 1334 Bone marrow failure is the most common cause of morbidity and mortality from Fanconi anemia (FA), a recessively inherited disorder resulting from mutations in one of 15 known genes that cooperate in a DNA repair pathway. The underlying etiology is thought to reflect an accelerated postnatal exhaustion of the hematopoietic stem and progenitor cell (HSPC) pool. However, laboratory evidence of compromised hematopoietic function in patients generally precedes symptoms of cytopenia, and several other mesodermal-derived organ systems show defects with prenatal onset, including the skeletal system, heart, kidneys, and others. Further, recent experimental evidence in human embryonic stem cell lines suggested that RNA interference-mediated knock-down of FANCD2 and FANCA impairs development of hematopoietic cells. The fetal liver provides a unique microenvironment for development of definitive hematopoietic function and serves as a site of massive HSPC expansion. However, neither the potential developmental onset of bone marrow failure or non-stem cell-autonomous contributions in FA have been systematically clarified to-date. We relied on a murine model of FA with a transgenic disruption of Fancc to test the hypothesis that hematopoietic failure for this disease may have developmental origins. Although spontaneous bone marrow failure does not occur in this FA mouse model, animals recapitulate impaired repopulating ability, characteristic cell cycle abnormalities, and impaired cytokine responses. To determine whether number and function of fetal liver (FL) HSPCs affect postnatal hematopoietic function in FA mice, we plated unfractionated cells from 14.5 days post coitum (dpc) FL in methylcellulose and undertook a chronologic assessment of postnatal bone marrow progenitor clonogenicity. These studies showed that, compared with wild-type (wt) littermates, Fancc−/− animals demonstrate a progressive deficiency in progenitor number and function that increases with age, suggesting that HSPC attrition is developmentally programmed. Fancc−/−fetal mice revealed a 10% reduction in body mass and 33% lower total liver cell count compared with wt littermates. Cytogenetic analysis shows Fancc−/−FL cells exhibit mitomycin-c hypersensitivity characteristic of FA, with increased chromosomal breakage and radial formation. Livers of 14.5±.5 dpc Fancc−/−fetuses contain approximately 43% fewer c-Kit+Sca-1+ progenitor-enriched cells, compared with wt littermates. Cell cycle status of fetal livers revealed a characteristically increased proportion of Fancc−/− fetal liver progenitor-enriched (c-Kit+ Sca-1+) cells in G2-M phase of cell cycle, compared to wt littermate liver. When plated in methylcellulose assays, Fancc−/−FL showed an approximately 20% reduction in progenitor frequency, compared to wt littermates, and plating in mitomycin-c resulted in outgrowth of fewer colonies. Further, studies to determine the relative in vivo repopulating cell frequency were performed using CD45-isotype mismatched, submyeloablatively irradiated (750 cGy) animals. Recipients receiving unfractionated 14.5±.5 dpc Fancc−/−liver cells showed a slight, but consistent reduction in peripheral blood chimerism at serial timepoints (1–5 months) and bone marrow chimerism at sacrifice. We also found a 21% reduction in total Fancc−/−clonogenic bone marrow progenitor frequency by methylcellulose assay in primary recipients, compared to wt-transplanted controls. In sum, these studies suggest a developmental origin of hematopoietic failure in FA, whereby the prenatal onset potentially contributes to disease progression. Results contrast with a conventional model of postnatal stem cell attrition and may impact the development of preemptive therapies for FA patients. Disclosures: No relevant conflicts of interest to declare.