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Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide great opportunities for mechanistic dissection of human cardiac pathophysiology; however, hiPSC-CMs remain immature relative to the adult heart. To identify novel signaling pathways driving the maturation process during heart development, we analyzed published transcriptional and epigenetic datasets from hiPSC-CMs and prenatal and postnatal human hearts. These analyses revealed that several components of the MAPK and PI3K-AKT pathways are downregulated in the postnatal heart. Here, we show that dual inhibition of these pathways for only 5 days significantly enhances the maturation of day 30 hiPSC-CMs in many domains: hypertrophy, multinucleation, metabolism, T-tubule density, calcium handling, and electrophysiology, many equivalent to day 60 hiPSC-CMs. These data indicate that the MAPK/PI3K/AKT pathways are involved in cardiomyocyte maturation and provide proof of concept for the manipulation of key signaling pathways for optimal hiPSC-CM maturation, a critical aspect of faithful in vitro modeling of cardiac pathologies and subsequent drug discovery.

PDF file - 156K, AML exosomes can alter gene expression and protein secretion

PDF file - 2.9MB, The detection of IGF-1R in HL-60 cell and vesicle lysates by Western blot

PDF file - 143K, All primers were used in semi-quantitative PCR

PDF file - 456K, Co-culture of AML cells with stromal cell can alter gene expression and protein secretion

Skeletal muscle relies on coordination between myogenic and non-myogenic interstitial cells for homeostasis and for regeneration and response to injury. Fibroadipogenic progenitors (FAPs) have recently been recognized as key modulators of signaling to promote myogenesis following injury. FAPs are also responsible for the fibrosis and fatty replacement of muscle tissue seen in many diseased states. While extensive use of surface markers to purify FAPs has been undertaken in the mouse system, in particular PDGFRA, markers for human FAPs are less well understood. Here, we show that CD73 can be used as a single positive marker to purify FAPs from the lineage-negative (CD45-neg, CD31-neg) fraction of skeletal muscle mononuclear cells. Although CD73 was previously found to be expressed in cultured myogenic cells, we find that this marker is only acquired upon culture and that the CD73+ fraction of human skeletal muscle has no myogenic activity. We show that Lin-neg CD73+ cells from human muscle undergo fat differentiation as well as fibrogenesis when exposed to appropriate activating signals in vitro. This simple single positive marker approach effectively enables isolation of human FAPs from fresh human skeletal muscle biopsies., Cell biology; Cell culture; Cell differentiation; Stem cell research; Musculoskeletal system; Human fibroadipogenic progenitors, FAPs, CD73.

SummaryOur mechanistic understanding of Fanconi anemia (FA) pathway function in hematopoietic stem and progenitor cells (HSPCs) owes much to their role in experimentally induced DNA crosslink lesion repair. In bone marrow HSPCs, unresolved stress confers p53-dependent apoptosis and progressive cell attrition. The role of FA proteins during hematopoietic development, in the face of physiological replicative demand, remains elusive. Here, we reveal a fetal HSPC pool in Fancd2−/− mice with compromised clonogenicity and repopulation. Without experimental manipulation, fetal Fancd2−/− HSPCs spontaneously accumulate DNA strand breaks and RAD51 foci, associated with a broad transcriptional DNA-damage response, and constitutive activation of ATM as well as p38 stress kinase. Remarkably, the unresolved stress during rapid HSPC pool expansion does not trigger p53 activation and apoptosis; rather, it constrains proliferation. Collectively our studies point to a role for the FA pathway during hematopoietic development and provide a new model for studying the physiological function of FA proteins.

We recently demonstrated that acute myeloid leukemia (AML) cell lines and patient-derived blasts release exosomes that carry RNA and protein; following an in vitro transfer, AML exosomes produce proangiogenic changes in bystander cells. We reasoned that paracrine exosome trafficking may have a broader role in shaping the leukemic niche. In a series of in vitro studies and murine xenografts, we demonstrate that AML exosomes downregulate critical retention factors (Scf, Cxcl12) in stromal cells, leading to hematopoietic stem and progenitor cell (HSPC) mobilization from the bone marrow. Exosome trafficking also regulates HSPC directly, and we demonstrate declining clonogenicity, loss of CXCR4 and c-Kit expression, and the consistent repression of several hematopoietic transcription factors, including c-Myb, Cebp-β and Hoxa-9. Additional experiments using a model of extramedullary AML or direct intrafemoral injection of purified exosomes reveal that the erosion of HSPC function can occur independent of direct cell–cell contact with leukemia cells. Finally, using a novel multiplex proteomics technique, we identified candidate pathways involved in the direct exosome-mediated modulation of HSPC function. In aggregate, this work suggests that AML exosomes participate in the suppression of residual hematopoietic function that precedes widespread leukemic invasion of the bone marrow directly and indirectly via stromal components.

Skeletal muscle possesses remarkable regenerative potential due to satellite cells, an injury-responsive stem cell population located beneath the muscle basal lamina that expresses Pax7. By lineage tracing of progenitor cells expressing the Twist2 (Tw2) transcription factor in mice, we discovered a myogenic lineage that resides outside the basal lamina of adult skeletal muscle. Tw2+ progenitors are molecularly and anatomically distinct from satellite cells, are highly myogenic in vitro, and can fuse with themselves and with satellite cells. Tw2+ progenitors contribute specifically to type IIb/x myofibers during adulthood and muscle regeneration, and their genetic ablation causes wasting of type IIb myofibers. We show that Tw2 expression maintains progenitor cells in an undifferentiated state that is poised to initiate myogenesis in response to appropriate cues that extinguish Tw2 expression. Tw2-expressing myogenic progenitors represent a previously unrecognized, fiber-type specific stem cell involved in post-natal muscle growth and regeneration.

Objective Recent evidence indicates that the adult hematopoietic system is susceptible to diet-induced lineage skewing. It is not known whether the developing hematopoietic system is subject to metabolic programming via in utero high-fat diet (HFD) exposure, an established mechanism of adult disease in several organ systems. We previously reported substantial losses in offspring liver size with prenatal HFD. As the liver is the main hematopoietic organ in the fetus, we asked whether the developmental expansion of the hematopoietic stem and progenitor cell (HSPC) pool is compromised by prenatal HFD and/or maternal obesity. Methods We used quantitative assays, progenitor colony formation, flow cytometry, transplantation, and gene expression assays with a series of dietary manipulations to test the effects of gestational high-fat diet and maternal obesity on the day 14.5 fetal liver hematopoietic system. Results Maternal obesity, particularly when paired with gestational HFD, restricts physiological expansion of fetal HSPCs while promoting the opposing cell fate of differentiation. Importantly, these effects are only partially ameliorated by gestational dietary adjustments for obese dams. Competitive transplantation reveals compromised repopulation and myeloid-biased differentiation of HFD-programmed HSPCs to be a niche-dependent defect, apparent in HFD-conditioned male recipients. Fetal HSPC deficiencies coincide with perturbations in genes regulating metabolism, immune and inflammatory processes, and stress response, along with downregulation of genes critical for hematopoietic stem cell self-renewal and activation of pathways regulating cell migration. Conclusions Our data reveal a previously unrecognized susceptibility to nutritional and metabolic developmental programming in the fetal HSPC compartment, which is a partially reversible and microenvironment-dependent defect perturbing stem and progenitor cell expansion and hematopoietic lineage commitment.

Extrinsic signaling cues in the microenvironment of acute myelogenous leukemia (AML) contribute to disease progression and therapy resistance. Yet, it remains unknown how the bone marrow niche in which AML arises is subverted to support leukemic persistence at the expense of homeostatic function. Exosomes are cell membrane–derived vesicles carrying protein and RNA cargoes that have emerged as mediators of cell–cell communication. In this study, we examined the role of exosomes in developing the AML niche of the bone marrow microenvironment, investigating their biogenesis with a focus on RNA trafficking. We found that both primary AML and AML cell lines released exosome-sized vesicles that entered bystander cells. These exosomes were enriched for several coding and noncoding RNAs relevant to AML pathogenesis. Furthermore, their uptake by bone marrow stromal cells altered their secretion of growth factors. Proof-of-concept studies provided additional evidence for the canonical functions of the transferred RNA. Taken together, our findings revealed that AML exosome trafficking alters the proliferative, angiogenic, and migratory responses of cocultured stromal and hematopoietic progenitor cell lines, helping explain how the microenvironmental niche becomes reprogrammed during invasion of the bone marrow by AML. Cancer Res; 73(2); 918–29. ©2012 AACR.

Mesenchymal stromal cells (MSCs) present in the bone marrow microenvironment secrete cytokines and angiogenic factors that support the maintenance and regenerative expansion of hematopoietic stem and progenitor cells (HSPCs). Here, we tested the hypothesis that extracellular vesicles (EVs) released by MSCs contribute to the paracrine crosstalk that shapes hematopoietic function. We systematically characterized EV release by murine stromal cells and demonstrate that MSC-derived EVs prompt a loss of HSPC quiescence with concomitant expansion of murine myeloid progenitors. Our studies reveal that HSPC expansion by MSC EVs is mediated via the MyD88 adapter protein and is partially blocked by treatment with a TLR4 inhibitor. Imaging of fluorescence protein-tagged MSC EVs corroborated their cellular co-localization with TLR4 and endosomal Rab5 compartments in HSPCs. The dissection of downstream responses to TLR4 activation reveals that the mechanism by which MSC EVs impact HSPCs involves canonical NF-κB signaling and downstream activation of Hif-1α and CCL2 target genes. Our aggregate data identify a previously unknown role for MSC-derived EVs in the regulation of hematopoiesis through innate immune mechanisms and illustrate the expansive cell-cell crosstalk in the bone marrow microenvironment.

microRNAs (miRNAs) are small, single-stranded RNAs that down-regulate target genes at the post-transcriptional level. miRNAs regulate target genes by guiding mRNA cleavage or by repressing translation. miRNAs play crucial roles in a broad range of developmental processes in plants. Multiple miRNAs are present in germinating seeds and seedlings of Arabidopsis, some of which are involved in the regulation of germination and seedling growth by plant hormones such as abscisic acid (ABA) and auxin. The involvement of miRNAs in ABA responses is not limited to the early stages of plant development but seems to be important for general stress responses throughout the plant life cycle. This Darwin review summarizes recent progress in miRNA research focusing on seed and stress biology, two topics which were of interest to Charles Darwin.

Germination and early seedling development are critical for successful stand establishment of plants. Following germination, the cotyledons, which are derived from embryonic tissue, emerge from the seed. Arabidopsis seedlings at post-germinative stages are supported mainly by the supply of nutrition from the cotyledons until vegetative leaves emerge and initiate photosynthesis. The switch to autotrophic growth is a significant transition at the post-germinative stage. Here, we provide evidence that down-regulation of SQUAMOSA PROMOTER-BINDING PROTEIN LIKE13 (SPL13) by microRNA156 (miR156) plays an important role in the regulation of the post-germinative switch from the cotyledon stage to the vegetative-leaf stage. Silent mutations created in the SPL13 sequence in the region that is complementary to the miR156 sequence caused the deregulation of the mutant form of SPL13 (mSPL13) mRNA from miR156. Mutant seedlings over-accumulated miRNA-resistant messages and exhibited a delay in the emergence of vegetative leaves compared to wild-type seedlings. The delay was not observed in control transgenic plants expressing non-mutated SPL13, indicating that the phenotype was caused specifically by the silent mutations and deregulation of SPL13 from miR156. Characterization of the SPL13 promoter indicated that this gene is expressed mainly in the hypocotyl and affects leaf primordium development. These results suggest that the repression of SPL13 by miR156 is essential for normal post-germinative growth in Arabidopsis.

MicroRNAs (miRNAs) are involved in developmental programmes of plants, including seed germination and post-germination. Here, we provide evidence that two different miRNA pathways, miR156 and miR172, interact during the post-germination stages inArabidopsis. Mutant seedlings expressing miR156-resistantSQUAMOSA PROMOTER-BINDING PROTEIN-LIKE13(mSPL13), which has silent mutations in the miR156 complementary sequence, over-accumulatedSPL13mRNA and exhibited a delay in seedling development. Microarray analysis indicated thatSCHNARCHZAPFEN(SNZ), anAP2-like gene targeted by miR172, was down-regulated in these mutants. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and miRNA gel blot analyses showed that theMIR172genes were up-regulated inmSPL13mutants. These results suggest that the miRNA regulation cascades (miR156⊣SPL13 → miR172⊣SNZ) play a critical role during the post-germination developmental stages inArabidopsis.

Relapse remains the major cause of mortality for patients with Acute Myeloid Leukemia (AML). Improved tracking of minimal residual disease (MRD) holds the promise of timely treatment adjustments to preempt relapse. Current surveillance techniques detect circulating blasts that coincide with advanced disease and poorly reflect MRD during early relapse. Here, we investigate exosomes as a minimally invasive platform for a microRNA (miRNA) biomarker. We identify a set of miRNA enriched in AML exosomes and track levels of circulating exosome miRNA that distinguish leukemic xenografts from both non-engrafted and human CD34+ controls. We develop biostatistical models that reveal circulating exosomal miRNA at low marrow tumor burden and before circulating blasts can be detected. Remarkably, both leukemic blasts and marrow stroma contribute to serum exosome miRNA. We propose development of serum exosome miRNA as a platform for a novel, sensitive compartment biomarker for prospective tracking and early detection of AML recurrence.

Fanconi anemia (FA) is an inherited multisystem disorder leading to symptomatic hematopoietic defects by early school age in most patients. Observations of lymphoid mosaicism and spontaneous intragenic correction of stem cells leading to clonal hematopoiesis in a small number of patients indicate that phenotypically corrected cells enjoy a survival advantage over FA deficient cells. Accordingly, FA is a leading genetic disorder target for HSPC directed gene therapy. However, reports over the insertional complications of commonly used integrating vectors continue to raise concerns, while available non-integrating lentivectors (NILVs) are generally lost from dividing hematopoietic stem cells. We recently showed that the incorporation of human Scaffold/Matrix Attachment Region (S/MAR) sequences in NILVs results in the cellular retention of NILV episomes in actively dividing cell lines and HSPCs without selection. Southern analysis, Alu PCR and episome specific PCR all confirmed long-term episomal existence of this vector without genomic integration (NAR. 2014; PMID: 24474068). Here, we designed and tested an episome lentivector to phenotypically correct FA cells (aNILV GFP-FANCC). Results showed the vector expressing the human FANCC ORF fused with GFP could be readily packaged, with stable persistence of vector episomes and sustained GFP expression. Taking advantage of the selective expansion of phenotypically corrected cells, we transduced a FANCC -deficient human cell line pD331 by using aNILV-GFP-FANCC vector. Exaggerated sensitivity to DNA alkylating agents (e.g. Mitomycin C, MMC) is a hallmark of FA deficient cells and can be used as a selection agent. At week 2, we observed 15% retention of GFP expressing cells in MMC treated pD331-aNILV-FANCC population versus 3% retention in non-treated pD331-aNILV-FANCC cells. Long-term maintenance of cells up to 6 weeks under MMC selection resulted the enrichment of corrected cells up to 95% that is comparable to the enrichment observed by integrating LV expressingGFP-FANCC in pD331 cells under MMC selection. Molecular studies of aNILV episomes from long-term cultures by episome specific PCR revealed LTR episome persistence. Integration analysis by Alu-PCR from the bulk population with Southern analysis of clones is ongoing. We already reported that episomal S/MAR NILV transduced short-term murine hematopoietic stem and progenitor cells and are currently characterizing this vector in a murine model of Fanconi Anemia. In conclusion, epsiome maintenance under selective pressure and reversal of canonical alkylator sensitivity in FA cells make this novel aNILV-GFP-FANCC vector a consideration for expanded preclinical exploration.

Hematopoietic failure is the predominant clinical manifestation of Fanconi anemia (FA), a rare, recessively inherited disorder. Mutations in 1 of 15 genes that coordinately function in a complex pathway to maintain DNA integrity also predispose patients to constitutional defects in growth and development. The hematologic manifestations have been considered to reflect the progressive loss of stem cells from the postnatal bone marrow microenvironment. Ethical concerns preclude the study of human hematopoiesis in utero. We report significant late gestational lethality and profound quantitative and qualitative deficiencies in the murine Fancc(-/-) fetal liver hematopoietic stem and progenitor cell pool. Fancc(-/-) fetal liver hematopoietic stem and progenitor cells revealed a significant loss of quiescence and decline in serial repopulating capacity, but no substantial difference in apoptosis or levels of reactive oxygen species. Our studies suggest that compromised hematopoiesis in Fancc(-/-) animals is developmentally programmed and does not arise de novo in bone marrow.

Hematopoietic stem and progenitor cells (HSPC) are responsive to paracrine signaling in the bone marrow microenvironment. We recently showed that leukemia derived extracellular vesicles (EVs) directly regulate HSPC in the leukemic niche and confer a net loss of progenitor function in part through direct action of a highly abundant miRNA on the hematopoietic transcription factor c-Myb. These observations motivated us to explore the potential regulation of HSPC by EVs present during steady state hematopoiesis in the unperturbed bone marrow. Mesenchymal stromal cells (MSC) comprise a diverse set of cells secreting supportive factors central to the maintenance of HSPC. We systematically characterized EVs released by murine MSCs as a candidate population and demonstrated that EVs themselves carry cytokines, predominantly interleukin (IL) -1, -2, -4,-6 and -17A, as well as G-CSF and Interferon g. As a functional corollary, we found that MSC EVs led to preferential granulocyte and monocyte differentiation in co-culture with C-kit enriched HSPCs, when compared to vesicle free condition (termed S100). These observations were further supported by the expansion of KSL (c-kit+,Sca-1+,lin-) and MPP2-4 (CD48+, CD150+/-) progenitor populations. To determine the in vivo relevance of these findings we performed a series of competitive transplantation experiments, that demonstrated that MSC-EV exposed HSPCs outperformed HSPCs cultured with EV depleted media, with striking advantage in early, but a significant deficit in later (week 16) myeloid recovery, compared to the S100 condition. These observations were broadly consistent with recent evidence for HSPC regulation by toll like receptors (TLRs) and we confirmed that the observed deficit in progenitor phenotypes was partially reversed in MSC-EV co-cultures with HSPCs from animals with genetic disruption of TLR4 or MyD88. EV-TLR engagement further induced a NF-kB response with downstream transcriptional activation of canonical transcriptional targets (STAT1, IRF1, -9) and significantly upregulated the release of IL-3, -6, IFNg, and G-CSF in exposed WT HSPC, compared with S100. Finally, we performed deconvolution microscopy studies of co-cultured HSPC and demonstrated the Pearson's spatial correlation between fluorescently tagged MSC-EV and TLR4 as well as the early and late endosomal marker proteins, Rab5 and Rab7, respectively. We conclude that MSC-derived EV trafficking of TLR contributes to the homeostatic regulation of HSPCs in the bone marrow microenvironment as a mechanism of tonic regulation of hematopoiesis. Disclosures No relevant conflicts of interest to declare.

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.

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.

Much of the morbidity and mortality caused by Acute Myeloid Leukemia (AML) is secondary to failure of normal hematopoiesis. Several recent studies indicate that this is not due to "overcrowding" within the bone marrow, suggesting other mechanisms account for the loss of bone marrow regenerative function in the leukemic niche. We recently reported that AML patient leukemia cells produce exosomes – small, membrane-enclosed extracellular vesicles – that are enriched in microRNA (miRNA) content and alter gene expression, cytokine secretion, and homing upon entry into neighboring stromal and progenitor cells. In order to examine the consequences of exosome trafficking in vivo, we conducted a series of experiments in a xenograft model. Here, we show that introduction of either AML cells or isolated AML-derived exosomes into the marrow of immunodeficient mice causes suppression of marrow hematopoietic stem and progenitor cell (HSPC) clonogenicity and a dramatic increase in peripheral circulating HSPC that coincided with a marked decrease in expression of Scf and Cxcl12 in murine stromal cells. Hypothesizing that transferred miRNA mediates the changes observed in recipient cells, we evaluated the miRNA content of two AML cell lines and their exosomes using microarrays paired with qRT-PCR. These experiments revealed stark differences between the total miRNA produced by AML cells and the selective incorporation of miRNA in exosomes. Combining AML exosome-enriched miRNA identified in our screen with a review of miRNA previously identified as significant in AML biology, we selected a panel of highly enriched miRNA, including miR-155 (100-fold enriched in exosomes) for further study. In order to mechanistically identify the mRNA targets of these miRNA, we used the uniquely powerful RISC-Trap method (Cambronne et al, PNAS, 2012), beginning with miR-155. This assay revealed 131 likely targets of miR-155, a set which we compared with several other target detection methods previously attempted with this miRNA, finding both significant overlap and several promising new putative mRNA targets. Of these 131 RISC-Trap targets, 98 were predicted by one or more of six tested target prediction algorithms (miRWalk). We compared the miR-155 targets identified by RISC-Trap to predicted targets for several other miRNA in the panel selected from our microarray studies, identifying both miRNA potentially responsible for the expression changes seen in stroma as well as several potential targets common to multiple miRNA. Using bioinformatics analysis of direct targets of our miRNA of interest and their common interacting partners, we were able to identify a network of targets and cellular processes regulated by AML exosomes, including regulation of transcription (p53, SOX9, CEBPB) and apoptosis (ESR1, TRAF2, CHEK2). These networks may provide insight into the mechanisms by which AML impairs hematopoiesis, both directly, through effects on HSPC, and indirectly, through effects on stroma. Ongoing RISC-Trap studies of other miRNA provide a forward genetic screen to evaluate the paracrine targets of exosomal miRNA in a mechanistic way, in specific niche cell types. By identifying the wider networks in addition to the individual contributors of exosomal miRNA in the AML microenvironment, we hope to uncover new non-leukemia targeted “niche” therapies to relieve hematopoietic suppression in AML patients. Disclosures Cambronne: Clontech/Takara Bio: commercial licensing of the RISC-trap technology Patents & Royalties.

Transcription Factor 3 (TCF3) rearrangements are a recurring chromosomal abnormality in B-cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) occurring in approximately five percent of pediatric ALL. Historically, the majority of these patients carried a poor prognosis, but advances with more intensive cytotoxic chemotherapy have improved the survival rate while exposing patients to increased short and long-term toxicities. Two genetic rearrangements produce the chimeric transcription factors, TCF3-PBX1 t(1;19)(q23;p13) and a much rarer TCF3-HLF t(17;19)(q22;p13). Sadly, TCF3-HLF remains an extremely difficult disease to treat with few, if any known survivors. Although it is unknown how these translocations lead directly to disease, it is established that they do result in diseases arrested in a later stage of B-cell differentiation and pre-B cell receptor (pre-BCR) dependence. Recently, we highlighted the concept of targeting the pre-BCR pathway for therapeutic potential using dasatinib (Sprycel). Here, we further examine dasatinib effectiveness in the murine xenograft model for TCF3-rearranged ALL. Methods: Primary patient samples were obtained with written informed consent approved by the Institutional Review Board of Oregon Health and Science University and processed. Mononuclear cells were separated by Ficoll and exposed to increasing concentrations of dasatinib. Inhibitory Concentration of fifty-percent viability (IC50) was calculated for each sample. The median IC50 for over four hundred acute leukemic samples interrogated by this assay was calculated to approximately 100nM. Samples with IC50 values below 30nM were deemed hypersensitive to dasatinib. For xenografts, frozen viable primary patient samples were thawed and grafted via tail-vein into NOD/SCID/IL-2rgnull(NSG) mice 24 hours after sub-lethal irradiation with 200 cGy. Upon engraftment, and in vivo expansion, animals were euthanized and leukemic cells recovered from the spleen were then injected in secondary recipients. One week after injection the mice were divided into two groups and treated by oral gavage with dasatinib at 50mg/kg/dose daily or citrate control for 5 days per week. Treatment continued until the day of sacrifice (4-20 weeks). Peripheral blood engraftment was monitored weekly starting on week 3 by flow cytometry analysis using anti-human CD19 and CD45 (hCD19-APC, hCD45-FITC) versus anti-murine CD45 (mCD45-PerCP-Cy5.5). Flow cytometric data was analyzed using FACS/AriaIII. Results: Screening over one hundred BCP-ALL samples identified that approximately ten percent of these samples show hypersensitivity to dasatinib. TCF3-rearranged ALL and BCR-ABL1 ALL had a majority of samples with IC50's less than 10nM. Throughout all known subsets of ALL except ETV6-RUNX1, there also appeared to be individual samples that have IC50 values less than 30nM, suggesting significant sensitivity to this drug. Of these, three individual TCF3-rearranged ALL samples were identified and xenografted into NSG mice, expanded and injected into secondary recipients. All dasatinib treated cohorts showed significantly less leukemic peripheral blood chimerisms as compared to their vehicle control counterparts. Further, in vitro treatment of xenografted cells with dasatinib indicated inhibition of the pre-BCR by decrease in pan-phospho-SRC. Intriguingly, dasatinib did not completely abolish disease in all TCF3-rearranged ALL, suggesting other important mechanisms for cell viability. Conclusions: These studies show in vivo therapeutic benefits of dasatinib as treatment for TCF3-rearranged ALL, and open the possibility of adding this drug to their treatment. Further studies are underway to address the mechanisms of dasatinib sensitivity of other subsets of ALL identified in our screen in hopes of adding targeted therapies to their treatment. Figure 1 Figure 1. Disclosures Druker: Molecular MD: Consultancy, Equity Ownership, Scientific Founder. Some clinical trials on which I participate as PI or co-investigator utilize MolecularMD for molecular testing. This potential individual and institutional conflict of interest has been reviewed and managed by OHSU. Other; Bristol-Myers Squibb: Clinical trial funding: PI and co-investigator on ARIAD clinical trials. OHSU has contracts with ARIAD to pay for patient costs, nurse and data manager salaries, and institutional overhead. I do not derive salary, or lab funds from these contracts. Clinical trial funding: PI and co-investigator on ARIAD clinical trials. OHSU has contracts with ARIAD to pay for patient costs, nurse and data manager salaries, and institutional overhead. I do not derive salary, or lab funds from these contracts. Other. Off Label Use: Dasatinib use as potential therapy in ALL.

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.

Insertional oncogene activation and aberrant splicing have proved to be major setbacks for retroviral stem cell gene therapy. Integrase-deficient human immunodeficiency virus-1-derived vectors provide a potentially safer approach, but their circular genomes are rapidly lost during cell division. Here we describe a novel lentiviral vector (LV) that incorporates human ß-interferon scaffold/matrix-associated region sequences to provide an origin of replication for long-term mitotic maintenance of the episomal LTR circles. The resulting ‘anchoring’ non-integrating lentiviral vector (aniLV) achieved initial transduction rates comparable with integrating vector followed by progressive establishment of long-term episomal expression in a subset of cells. Analysis of aniLV-transduced single cell-derived clones maintained without selective pressure for >100 rounds of cell division showed sustained transgene expression from episomes and provided molecular evidence for long-term episome maintenance. To evaluate aniLV performance in primary cells, we transduced lineage-depleted murine hematopoietic progenitor cells, observing GFP expression in clonogenic progenitor colonies and peripheral blood leukocyte chimerism following transplantation into conditioned hosts. In aggregate, our studies suggest that scaffold/matrix-associated region elements can serve as molecular anchors for non-integrating lentivector episomes, providing sustained gene expression through successive rounds of cell division and progenitor differentiation in vitro and in vivo.

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 Disclosures: No relevant conflicts of interest to declare.

Small, non-coding micro RNA (miRNA) are recognized for their potent regulatory capacity. Several recent studies indicate the prognostic value of miRNA profiling in acute myelogenous leukemia (AML), although a more mechanistic understanding of the role miRNA play in AML biology is still lacking. We recently demonstrated that patient-derived AML blasts release exosomes (nanometer-size, extracellular vesicles) that traffic a non-random subset of miRNA to stromal bystander cells, eliciting changes in transcriptional activity and growth factor secretion (Huan et al., Cancer Res. 2013). Here we hypothesized that exosome miRNA provide a candidate mechanism for the adaptation of the bone marrow to a specialized leukemic niche. As oxygen levels in the bone marrow are substantially lower than those commonly used in tissue culture, we undertook a systematic study of miRNA incorporation and exosome trafficking in AML under physiological oxygen conditions. In carefully calibrated tissue culture conditions we initially observed an up to 7-fold net increase in exosome number released by Molm14 (Flt3-ITD+ AML cell line) leukemia cells at 1% O2versus 21% O2. Nanoparticle tracking analysis and RNA bioanalyzer tracings suggested that the decreased O2 did not alter vesicle composition, average RNA amount per exosome, or global RNA profiles. Further emphasizing the critical nature of appropriate compartmental oxygenation in exosome trafficking, both murine and human stromal cells demonstrated increased uptake of Molm14 exosomes under hypoxia. Low-oxygen conditions alter transcriptional profiles, phenotypic behavior and drug resistance in AML. Therefore, we next evaluated the miRNA expression of leukemic cells and their incorporation in exosomes at 1% versus 21% O2, utilizing the Affymetrix microarray platform containing >5,000 human (hsa) miRNA probesets, followed by select qRT-PCR validation. Array experiments showed broad differences between cellular and exosomal miRNA and revealed that certain miRNA were selectively regulated in an oxygen-responsive manner. For example, hematopoiesis relevant hsa-miR-124, -146a, and -155 increased an average of 4.6-, 5.5-, and 4.9-fold, respectively, in exosomes from hypoxia-conditioned cells. Intriguingly, several known, non-AML specific, hypoxia-responsive miRNA substantially increased in cells cultured at 1% O2 (e.g. miR-210 by 33-fold), but changed less than 2-fold in exosomes. Several recent reports show that leukemia cells actively convert the bone marrow microenvironment and contribute to the erosion of hematopoiesis by modulating hematopoietic-stromal interactions, in part via decrease in SDF1a, SCF, and Angpt1. We investigated the ability of AML-derived exosomes to regulate these transcripts, and found a 50% decrease in SCF and over 90% decrease in Angpt1 in murine stromal cells after in vitro exposure to leukemia exosomes, again with relatively greater differences for exosomes from hypoxia-conditioned AML cells. These experiments were complemented by observations of altered clonogenicity (CFU-C) of murine lin-negative hematopoietic cells after AML exosome exposure, whereby hypoxia conditioning prompted a decline in colony count to 46% from vesicle-free media baseline, compared with 31% decrease at 21 % O2. Exosomes equilibrate across biological fluids and can be recovered from serum. To translate our observations to an in vivo setting, we developed a xenograft model using Molm14 cells in immune-deficient NSG mice. Early after grafting animals, exosomes could be reproducibly isolated from as little as 20 microL serum and candidate miRNA (hsa-miR-146, -150, 155, 210) were amplified, allowing us to quantitatively track leukemia progression via a unique miRNA signature even before circulating leukemia cells were detectable in the peripheral blood. A comparison of leukemic animals to NSG controls bearing cord blood MNC grafts revealed that changes in circulating miRNA were disease specific and resembled those in the hypoxia setting in vitro. In sum, our work demonstrates that physiologic oxygen levels not only increase AML exosome trafficking between cells, but selectively alter the miRNA profile contained therein. These changes produce phenotypic alterations in stromal and hematopoietic bystander cells that correlate with the functional conversion of the bone marrow to a leukemic niche. Disclosures: No relevant conflicts of interest to declare.

Abstract 1378 Cell membrane-derived vesicles carrying protein and RNA cargo have recently emerged as potent mediators of cell-cell communication. These vesicles traffic from their tissue of origin into the blood stream, providing a serum-based and minimally invasive biomarker platform for cancer-specific RNA and protein signatures. We were interested in evaluating vesicle production by acute myeloid leukemia (AML) cells, with an emphasis on RNA content and cell-cell trafficking. Here we demonstrate for the first time that cell lines and primary cells from AML patients release exosome-sized vesicles (30–100nm) capable of transfer to bystander cells. Analysis of their non-protein content revealed the enrichment of many coding- and non-coding RNAs with relevance to AML biology, including prognostically important transcripts encoding FLT3, NPM-1, IGF-IR, CXCR4 and MMP9. In addition, we demonstrated the presence of a diverse group of transcription factor mRNAs (MYC, MEF2C, GATA1, SHIP1, ID1, E2F1, CEPBA CEPBB) in exosomes. PCR array studies also confirmed the broad representation of cellular miRNAs. Among the 234 miRNAs represented, the levels of several (miR −9, −155 and −223 and LET-7A) were enriched over the levels seen in donor cells (U6 RNA control). When examining cell-cell trafficking of AML exosomes, we noted rapid entry into co-cultured stromal cells, with maximum uptake occurring between 2 and 3 hours. AML-derived mRNA transcripts were transferred to bystander cells without direct cell-cell contact after Transwell co-culture or direct exposure to purified exosome preparations, or via vesicle-rich media. Results show that exosome trafficking induced changes in gene expression (OPN, ANGPT, SCF, SDF-1A) and altered the secretion of cytokines and angiopoietic growth factors by bone marrow stromal cells. Additional proof-of-concept studies provided evidence for the canonical function of transferred RNA. Specifically, we found that transfer of IGF-IR mRNA led to proliferative responses in co-cultured OP9 stromal cells that were abrogated by the IGF-IR specific inhibitor picropodophyllin. Further, exosomes derived from AML cell lines (HL60 and Molm14) altered c-FOS and VEGF expression in OP9 cells as well as improving viability and promoting migration toward SDF-1A by a hematopoietic progenitor cell line (Baf3), respectively. Our observations support the concept that AML exosomes exhibit regulatory potential in the leukemic bone marrow niche via direct cytoplasmic cell-cell transfer of transcripts encoding tyrosine kinase receptors and transcription factor as well as micro- RNAs. We propose that AML exosomes contribute to the extrinsic signaling cues in the microenvironment that promote disease resistance in AML and that they are rich in biomarkers to guide diagnosis and treatment. Disclosures: No relevant conflicts of interest to declare.

SummaryStudying 830 pre-B ALL cases from four clinical trials, we found that human ALL can be divided into two fundamentally distinct subtypes based on pre-BCR function. While absent in the majority of ALL cases, tonic pre-BCR signaling was found in 112 cases (13.5%). In these cases, tonic pre-BCR signaling induced activation of BCL6, which in turn increased pre-BCR signaling output at the transcriptional level. Interestingly, inhibition of pre-BCR-related tyrosine kinases reduced constitutive BCL6 expression and selectively killed patient-derived pre-BCR+ ALL cells. These findings identify a genetically and phenotypically distinct subset of human ALL that critically depends on tonic pre-BCR signaling. In vivo treatment studies suggested that pre-BCR tyrosine kinase inhibitors are useful for the treatment of patients with pre-BCR+ ALL.