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Shwachman-Diamond syndrome (SDS) is a rare and clinically heterogeneous bone marrow (BM) failure syndrome caused by mutations in the Shwachman-Bodian-Diamond syndrome (SBDS) gene. Although SDS was described more than 50 years ago, its molecular pathogenesis is poorly understood due, in part, to the rarity and heterogeneity of the affected hematopoietic progenitors. To address this, we used single-cell RNA sequencing to profile scant hematopoietic stem and progenitor cells from patients with SDS. We generated a single-cell map of early lineage commitment and found that SDS hematopoiesis was left-shifted with selective loss of granulocyte-monocyte progenitors. Transcriptional targets of transforming growth factor beta (TGF-β) were dysregulated in SDS hematopoietic stem cells and multipotent progenitors, but not in lineage-committed progenitors. TGF-β inhibitors (AVID200 and SD208) increased hematopoietic colony formation of SDS patient BM. Finally, TGF-β3 and other TGF-β pathway members were elevated in SDS patient blood plasma. These data establish the TGF-β pathway as a candidate biomarker and therapeutic target in SDS and translate insights from single-cell biology into a potential therapy.

SUMMARY Melanoma is the deadliest form of skin cancer, affecting men more frequently and severely than women. Although recent studies suggest that differences in activity of the androgen receptor (AR) underlie the observed sex bias, little is known about AR activity in melanoma. Here we show that AR and EGR1 bind to the long non-coding RNA SLNCR and increase melanoma proliferation through coordinated transcriptional regulation of several growth-regulatory genes. ChIP-seq reveals that ligand-free AR is enriched on SLNCR-regulated melanoma genes and that AR genomic occupancy significantly overlaps with EGR1 at consensus EGR1 binding sites. We present a model in which SLNCR recruits AR to EGR1-bound genomic loci and switches EGR1-mediated transcriptional activation to repression of the tumor suppressor p21Waf1/Cip1. Our data implicate the regulatory triad of SLNCR, AR, and EGR1 in promoting oncogenesis and may help explain why men have a higher incidence of and more rapidly progressive melanomas compared with women., Graphical Abstract, In Brief Long non-coding RNA function can be understood by defining their interacting proteins. Schmidt et al. demonstrate that the melanoma lncRNA SLNCR binds to AR and complexes with different transcription factors to mediate invasion or proliferation. Thus, lncRNAs regulate distinct transcriptional programs based on specific protein interactions.

The residual presence of integrated transgenes following the derivation of induced pluripotent stem (iPS) cells is highly undesirable. Here we demonstrate efficient derivation of iPS cells free of exogenous reprogramming transgenes using an excisable polycistronic lentiviral vector. A novel version of this vector containing a reporter fluorochrome allows direct visualization of vector excision in living iPS cells in real time. We find that removal of the reprogramming vector markedly improves the developmental potential of iPS cells and significantly augments their capacity to undergo directed differentiation in vitro. We further propose that methods to efficiently excise reprogramming transgenes with minimal culture passaging, such as those demonstrated here, are critical since we find that iPS cells may acquire chromosomal abnormalities, such as trisomy of chromosome 8, similar to embryonic stem cells after expansion in culture. Our findings illustrate an efficient method for the generation of transgene-free iPS cells and emphasize the potential beneficial effects that may result from elimination of integrated reprogramming factors. In addition, our results underscore the consequences of long-term culture that will need to be taken into account for the clinical application of iPS cells.

Diamond Blackfan Anemia (DBA) is a congenital red blood cell aplasia usually caused by haploinsufficiency of selected ribosomal protein genes. Approximately 10% of DBA patients are anemic at birth, and 80% of DBA patients present by 6 months. These and other data suggest that the DBA erythroid defect is exacerbated by the switch from fetal to adult hematopoiesis. We hypothesize that unique molecular or cellular features initiate DBA pathogenesis during fetal hematopoiesis that manifests after the switch to adult erythropoiesis as severe anemia. To test this hypothesis, we developed a mouse model with constitutive Rpl11 haploinsufficiency in fetal and adult hematopoietic tissues driven by VavCre. Rpl11 is a commonly mutated ribosomal gene in DBA. In adult mice, reduced Rpl11 expression results in severe anemia by 6 weeks, with markedly reduced hemoglobin, increased red cell volume and elevated erythroid adenosine deaminase. Flow cytometric analysis of the bone marrow (BM) revealed no change in the frequency of megakaryocyte-erythroid progenitors (MEP) but a significant increase in both BFU-E and CFU-E, with a corresponding decrease in maturing Ter119+ cells. Functional progenitor assays showed reduced and delayed proliferation with an impaired ability to produce viable CD71+ Ter119+ cells. Treatment of purified Rpl11+/- BFU-E with dexamethasone rescued this proliferative defect. Thus, our data indicate that a differentiation block and functional impairment at the BFU-E stage of adult erythropoiesis contribute to Rpl11 haploinsufficient anemia. At the molecular level, Rpl11 haploinsufficiency in BM resulted in the accumulation of 32S and 12S pre-rRNA. Polysome profiling of total BM suggested reduced polysome assembly and impaired translation. Transcriptional profiling of normal and Rpl11+/- MEP, BFU-E, CFU-E and Ter119+ cells identified 13 differentially expressed genes across DBA erythropoiesis including known and novel targets such as CDKN1A, TNFSF4 and TSPAN14 . Enrichment of the p53 pathway was unique to BFU-E and CFU-E progenitors. Interestingly, TGFB1 and EGF pathways were also selectively affected within Rpl11+/- BFU-E and CFU-E progenitors, suggesting p53-independent self-renewal and proliferative defects in these populations. In contrast to adult hematopoiesis, fetal hematopoiesis defects are less severe. For example, fetal BFU-E are not significantly increased within E14 fetal livers and functionally generate a similar number of maturing erythroblasts in vitro . Lodish and colleagues have established that fetal liver BFU-E comprise at least two distinct subpopulations, termed "early" and "late". Early BFU-E have high self-renewing capacity whereas late BFU-E have low self-renewing capacity. To determine whether the balance between early and late BFU-E is affected by Rpl11 haploinsufficiency, and to identify gene expression changes in early and late fetal Rpl11+/- BFU-E, we performed single cell RNA-sequencing. Normal fetal livers contained similar proportions of early and late BFU-E, whereas normal adult bone marrows only contained late BFU-E. Surprisingly, Rpl11+/- fetal livers primarily contained late BFU-E. One explanation for this observation is that early BFU-E are depleted from Rpl11+/- fetal livers as this population compensates for reduced function of non-renewing late BFU-E. Interestingly, late BFU-E from Rpl11+/- BM have a stronger "late" signature than their normal counterparts suggesting a reduced self-renewing capacity of BM BFU-E in DBA. We are currently testing this hypothesis by overexpressing early genes associated with BFU-E self-renewal in Rpl11+/- BFU-E and reassessing erythroid phenotypes. In conclusion, our studies show that erythropoiesis is defective in Rpl11+/- fetal liver at the cellular and molecular level, although functional impairment is not as marked as in adult mice. Moreover, single cell RNA-sequencing of BM and fetal BFU-E in our Rpl11+/- mouse model has uncovered a novel hypothesis for DBA pathogenesis that the loss of early, self-renewing BFU-E in fetal liver is a harbinger of adult erythroid failure. Disclosures Glader: Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Neuberg: Synta Pharmaceuticals: Other: Stock shares.

The ability to efficiently transduce hematopoietic stem cells (HSC) represents a powerful methodology by which to study the role of specific genes on HSC function, as well as to broaden the potential of gene therapy for hematopoietic related diseases. While retroviruses have been used extensively to transduce a variety of cell types, HIV-derived lentiviruses prove superior for transduction of quiescent HSC due to their ability to infect non-dividing cells. Quality of lentiviral supernatants and starting cells are vital to obtain reproducible consistent results, and therefore, here we describe the production of concentrated lentiviral preparations, the purification of HSC from total mouse bone marrow, and their transduction to obtain long-term HSC engraftment with persistent gene transfer and expression of the desired transgene.

Hematopoietic stem cells (HSC) rely on a highly regulated molecular network to balance self-renewal and lineage specification to sustain life-long hematopoiesis. Despite a plethora of studies aimed at identifying molecules governing HSC fate, our current knowledge of the genes responsible is limited. We have found insulin-like growth factor 2 (IGF2) to be expressed predominantly within long-term HSCs. This study examines IGF2 expression patterns and the effects of the gene in HSCs. Through the overexpression and knockdown of IGF2 within purified HSCs, we report that IGF2 expression increases HSC-derived multilineage colonies in vitro and enhances hematopoietic contribution in vivo on competitive bone marrow transplantation. The effects of IGF2 are mediated by direct upregulation of the CDKi p57, exclusively within long-term HSCs, via activation of the PI3K-Akt pathway. Increased expression of p57 resulted in a concomitant increase in HSCs in the G0/G1 stage of the cell cycle. Analysis of genomic DNA methylation revealed that HSCs exhibited a hypomethylated state within the promoter region of the CDKN1C (p57) gene, providing a potential mechanism for the exclusive effects of IGF2 within HSCs. Our studies indicate a novel role for IGF2 in regulating HSC cell cycle and illustrate potential novel therapeutic targets for hematologic diseases.