Search

Fibroblast activation includes differentiation to myofibroblasts and is a key feature of organ fibrosis. The Notch pathway has been involved in myofibroblast differentiation in several tissues, inc...

Metabolic alterations in cancer represent convergent effects of oncogenic mutations. We hypothesized that a metabolism-restricted genetic screen, comparing normal primary mouse hematopoietic cells and their malignant counterparts in an ex vivo system mimicking the bone marrow microenvironment, would define distinctive vulnerabilities in acute myeloid leukemia (AML). Leukemic cells, but not their normal myeloid counterparts, depended on the aldehyde dehydrogenase 3a2 (Aldh3a2) enzyme that oxidizes long-chain aliphatic aldehydes to prevent cellular oxidative damage. Aldehydes are by-products of increased oxidative phosphorylation and nucleotide synthesis in cancer and are generated from lipid peroxides underlying the non–caspase-dependent form of cell death, ferroptosis. Leukemic cell dependence on Aldh3a2 was seen across multiple mouse and human myeloid leukemias. Aldh3a2 inhibition was synthetically lethal with glutathione peroxidase-4 (GPX4) inhibition; GPX4 inhibition is a known trigger of ferroptosis that by itself minimally affects AML cells. Inhibiting Aldh3a2 provides a therapeutic opportunity and a unique synthetic lethality to exploit the distinctive metabolic state of malignant cells.

Extracellular vesicles transfer complex biologic material between cells, whose role in in-vivo organismal physiology is poorly defined. Here, we demonstrate that osteoblastic cells in the bone marrow elaborate extracellular vesicles that are taken up by hematopoietic progenitor cells in vivo. Genotoxic or infectious stress rapidly increased stromal-derived extracellular vesicle transfer to granulocyte-monocyte progenitors. Stimulating osteoblastic cells with parathyroid hormone or activating its receptor enhanced extracellular vesicle transfer, myeloid recovery post radiation and improved animal survival from Candida sepsis. The extracellular vesicles contained tiRNAs known to modulate protein translation. 5’-ti-Pro-CGG-1 was preferentially abundant in osteoblast-derived extracellular vesicles and when transferred to granulocyte macrophage progenitors, increased protein translation, cell proliferation and myeloid differentiation. Therefore, EV-mediated tiRNA transfer provides a stress modulated signaling axis distinct from conventional cytokine-driven stress responses.One sentence summaryStress regulated tiRNA transfer alters hematopoiesis

Loss of protein quality is a driving force of aging1. The accumulation of misfolded proteins represents a vulnerability for long-lived cells, such as haematopoietic stem cells. How these cells, which have the ability to reconstitute all haematopoietic lineages throughout life2, maintain their regenerative potential and avert the effects of aging is poorly understood. Here, we determined the protein content in haematopoietic stem and progenitor cells to identify prevalent chaperones that support proteome integrity. We identified Peptidyl-Prolyl Isomerase A (PPIA or Cyclophilin A) as the dominant cytosolic foldase in this cell population. Loss of PPIA accelerated aging in the mouse stem cell compartment. In an effort to define targets of PPIA, we found that RNA- and DNA-binding proteins are common substrates of this chaperone. These proteins are enriched in intrinsically disordered regions (IDRs), which can catalyse protein condensation3. Isomerized target prolines are almost exclusively located within IDRs. We discovered that over 20% of PPIA client proteins are known to participate in liquid-liquid phase separation, enabling the formation of supramolecular membrane-less organelles. Using the poly-A binding protein PABPC1 as an example, we demonstrate that PPIA promotes phase separation of ribonucleoprotein particles, thereby increasing cellular stress resistance. Haematopoietic stem cell aging is associated with a decreased expression of PPIA and reduced synthesis of intrinsically disordered proteins. Our findings link the ubiquitously expressed chaperone PPIA to phase transition and identify macromolecular condensation as a potential determinant of the aging process in haematopoietic stem cells.

Stem cells reside in specialized anatomical locations called niches where supportive stromal cells and the extracellular matrix (ECM) regulate their self-renewal and differentiation. This review explores the critical roles of the ECM in stem cell maintenance in tissue homeostasis, aging, and disease. It is well established that ECM proteins and their biomechanical properties control stem cell fate. In addition to specific molecular interactions, the ECM composition determines the topology and stiffness of the substrate, which also regulate stem cell behavior. Changes in the ECM during aging and disease can impair cell-ECM interactions and ultimately contribute to aging and disease pathogenesis. A deeper understanding of the mechanisms by which the ECM regulates stem cell behavior in health, as well as during aging and in disease states, will facilitate the development of therapeutic strategies. These therapies should focus on recovering normal matrix synthesis and deposition aiming at promoting endogenous repair.

Extracellular vesicles (EV) transfer complex biologic material between cells. However the role of this process in in-vivo organismal physiology is poorly defined. Here, we demonstrate that osteoblastic cells in the bone marrow elaborate extracellular vesicles that are taken up by hematopoietic progenitor cells in vivo. Genotoxic or infectious stress rapidly increased stromal-derived extracellular vesicle transfer to granulocyte-monocyte progenitors. Stimulating osteoblastic cells with parathyroid hormone or activating the parathyroid hormone receptor enhanced extracellular vesicle transfer, myeloid recovery post radiation and improved animal survival from Candida sepsis. The extracellular vesicles contained tiRNAs known to modulate protein translation. 5’-ti-Pro-CGG-1 was preferentially abundant in osteoblast-derived extracellular vesicles and when transferred to granulocyte- monocyte progenitors, increased protein translation, cell proliferation and myeloid differentiation. Therefore, EV-mediated tiRNA transfer provides a stress modulated signaling axis distinct from conventional cytokine-driven stress responses.

Understanding the regulation of normal and malignant human hematopoiesis requires a comprehensive cell atlas of the hematopoietic stem cell (HSC) regulatory microenvironment. Recent studies using scRNA-seq technologies have shed light on the organization of the hematopoietic regulatory microenvironment in the mouse. These studies have resolved some of the controversies regarding the overlap of stromal populations, the description of certain discrete stromal cells as professional, hematopoietic cytokine-producing populations, but also helped to delineate the relationship between specific stromal cell types in the murine BM. Nevertheless, these studies are limited by the number of cells sequenced, potentially hampering our ability to resolve the full spectrum of cellular states and differentiation stages that define the stromal BM microenvironment. Further, knowledge on the conservation of the cellular composition in the human BM stroma is in its infancy due to the difficulty of obtaining high-quality samples with sufficient stromal cell numbers from healthy individuals. This leaves us with two outstanding challenges; how to piece together such different fragments towards a comprehensive molecular atlas and to what extent such an atlas in mice is conserved in the human bone marrow. Here, we dissect the intrinsic organization and the heterogeneity within the endothelial (EC) and mesenchymal cell populations (MSC) governing the BM microenvironment in mouse and human. This was accomplished through customized bioinformatics integration of multiple scRNA-seq datasets along with the inclusion of over 50.000 murine and human bone marrow stromal cells. By these means, we were able to identify new subsets of MSC and EC, but more importantly, to define new molecular markers to identify highly specialized subpopulations of cells in the murine BM microenvironment. Pathway enrichment analysis unveiled multiple, potentially transient cell states defined by differential gene expression and the enrichment of specific functional characteristics. Importantly, 14 EC subsets were characterized by enrichment in pathways known to be essential for endothelial homeostasis maintenance, demonstrating a high degree of specialization in the endothelium. Similarly, 11 transient cell states in the MSC compartment were defined and characterized by their differentiation capacity. Importantly, our deep deconvolution of the heterogeneous mesenchymal and endothelial compartments became feasible only by integrating multiple datasets. Furthermore, based on the knowledge generated in the mouse, we were able to describe how much of the information and targets from the mouse can be of interest in human characterization. This analysis identified the expression of the human orthologs to the murine cluster-defining genes with different degrees of enrichment in the endothelium and mesenchyme. Moreover, some of these shared genes in mice and human stromal cells corresponded to the GO-defining genes of the different clusters identified in the mouse. These findings suggest a significant degree of conservation regarding the cellular states that define the stromal microenvironment in mouse and human. Although additional studies and improved processing of human samples will be required for deep characterization of the human BM microenvironment, these preliminary results validate our integrative cross-species approach. Taken together, our study provides a deeper understanding of the composition and specialization of the BM microenvironment and point towards a significant degree of conservation between species. Moreover, we demonstrate the usefulness of the multi-dataset integration and the customized clustering approach used in our study to improve the resolution of complex tissues and organs. This approach promises to aid in the construction of cell atlases by reducing the resources associated with sequencing that a single lab will need to invest in order to obtain meaningful depth in single-cell analysis. Future studies integrating genome, transcriptome, epigenome, proteome, and anatomical positioning together with functional assays to correlate descriptive phenotypes with functional data will help fully resolve the composition, regulation, and connectivity in the BM microenvironment in health and disease. Figure 1 Figure 1. Disclosures Paiva: Adaptive, Amgen, Bristol-Myers Squibb-Celgene, Janssen, Kite Pharma, Sanofi and Takeda: Honoraria; Bristol-Myers Squibb-Celgene, Janssen, and Sanofi: Consultancy; Celgene, EngMab, Roche, Sanofi, Takeda: Research Funding. Saez: Magenta Therapeutics: Patents & Royalties. Prosper: BMS-Celgene: Honoraria, Research Funding; Janssen: Honoraria; Oryzon: Honoraria.

Production of the cells that ultimately populate the thymus to generate α/β T cells has been controversial, and their molecular drivers remain undefined. Here, we report that specific deletion of bone-producing osteocalcin (Ocn)-expressing cells in vivo markedly reduces T-competent progenitors and thymus-homing receptor expression among bone marrow hematopoietic cells. Decreased intrathymic T cell precursors and decreased generation of mature T cells occurred despite normal thymic function. The Notch ligand DLL4 is abundantly expressed on bone marrow Ocn+ cells, and selective depletion of DLL4 from these cells recapitulated the thymopoietic abnormality. These data indicate that specific mesenchymal cells in bone marrow provide key molecular drivers enforcing thymus-seeding progenitor generation and thereby directly link skeletal biology to the production of T cell–based adaptive immunity.

The glycosyltransferase gene, Ext1, is essential for heparan sulfate production. Induced deletion of Ext1 selectively in Mx1-expressing bone marrow (BM) stromal cells, a known population of skeletal stem/progenitor cells, in adult mice resulted in marked changes in hematopoietic stem and progenitor cell (HSPC) localization. HSPC egressed from BM to spleen after Ext1 deletion. This was associated with altered signaling in the stromal cells and with reduced vascular cell adhesion molecule 1 production by them. Further, pharmacologic inhibition of heparan sulfate mobilized qualitatively more potent and quantitatively more HSPC from the BM than granulocyte colony-stimulating factor alone, including in a setting of granulocyte colony-stimulating factor resistance. The reduced presence of endogenous HSPC after Ext1 deletion was associated with engraftment of transfused HSPC without any toxic conditioning of the host. Therefore, inhibiting heparan sulfate production may provide a means for avoiding the toxicities of radiation or chemotherapy in HSPC transplantation for nonmalignant conditions.

SummaryD-type cyclins (D1, D2, and D3) are components of the mammalian core cell-cycle machinery and function to drive cell proliferation. Here, we report that D-cyclins perform a rate-limiting antiapoptotic function in vivo. We found that acute shutdown of all three D-cyclins in bone marrow of adult mice resulted in massive apoptosis of all hematopoietic cell types. We demonstrate that adult hematopoietic stem cells are particularly dependent on D-cyclins for survival and that they are especially sensitive to cyclin D loss. Surprisingly, we found that the antiapoptotic function of D-cyclins also operates in quiescent hematopoietic stem and progenitor cells. Our analyses revealed that D-cyclins repress the expression of the death receptor Fas and its ligand, FasL. Acute ablation of D-cyclins upregulated these proapoptotic genes and led to Fas- and caspase 8-dependent apoptosis. These results reveal an unexpected function of cell-cycle proteins in controlling apoptosis in normal cell homeostasis.

The bone marrow (BM) niche, is an extrinsic regulator of hematopoietic stem and progenitor cells (HSPCs) (Morrison and Scadden 2014). The niche can drive disease and loss of HSPC function (Raaijmakers et al., 2010; Kode et al., 2014; Dong et al., 2016). Of particular interest, deletion in specific niche cells of the small RNA processing enzymes, Dicer1 and Angiogenin (Ang1) resulted in hematopoietic transformation (Raaijmakers et al., 2010) and loss of HSC quiescence (Goncalves et al., 2016), respectively. We tested mechanisms by which small non-coding RNAs (sncRNA) from niche cells might contribute to HSPC regulation. The transfer of cytoplasmic contents from specific mesenchymal subsets to wild type (WT) transplanted hematopoietic cells was evaluated by GFP. Osteoblastic cell reporter mice (Ocn-GFP, Col1-GFP) transferred 40-fold more GFP to HSPC than mesenchymal stem cells (Nes-GFP) or osteoprogenitors (Osx-GFP). GFP cannot transfer through gap junctions, however 100nM extracellular vesicles (EVs) labeled with the endocytic marker TSG-101 and GFP were found in Ocn-GFP bone marrow plasma. Granulocyte macrophage progenitor (GMPs) cells were the major hematopoietic recipients of osteoblast derived EVs (Fig. 1A). Small RNA analysis of mouse BM EVs demonstrated >85% were tRNA (Fig. 1B) while Northern blot (NB) revealed both tRNAs and stress induced RNAs (tiRNAs) (Fig. 1C). tRNAs were evident in EVs and in EV generating and recipient cells, while tiRNAs were highly enriched in EVs compared to cells (Fig. 1C). Extracellular vesicle recipient and non-recipient GMPs (GMP[GFP+] and GMP[GFP-]) respectively demonstrated a 2-fold enrichment in reads mapping to tRNAs in GMP[GFP+]. Principle component analysis demonstrated that the two populations are distinct in their tRNA content. In addition, twelve specific tRNAs were present at a significantly higher levels in GMP[GFP+]. Gene expression analysis of GMP[GFP+]and GMP[GFP-] revealed an enrichment of protein translation and cell cycle related functions in GMP[GFP+] which were validated using an in vivo protein translation assay and cell cycle analyses. Synthetic oligos corresponding to the sequence of top ten tiRNAs that are enriched in GMP[GFP+] were transfected into primary GMPs. 5'-Pro-CGG-1 and 5'-Cys-GCA-27 increased the rate of protein translation and cellular proliferation in transfected GMPs (Fig. 1D-E). The physiologic relevance of EV transfer was assessed using two stress states, systemic Candida albicans infection and low dose gamma irradiation. The frequency of GMP[GFP+] increased in both states in concert with increased cell proliferation. Therefore, EV transfer of sncRNAs from niche to hematopoietic progenitors in vivo is a rapid means of conveying information between cells. It alters progenitor protein translation and proliferation in a manner responsive to stress. The transfer of 5'tiRNA EV from specific niche cells to specific hematopoietic cells is an additional mechanism for niche regulation of blood cell production. Disclosures Sykes: Clear Creek Bio: Equity Ownership, Other: Co-Founder. Scadden:Clear Creek Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Fog Pharma: Consultancy; Agios Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; LifeVaultBio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Editas Medicine: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bone Therapeutics: Consultancy; Novartis: Other: Sponsored research; Fate Therapeutics: Consultancy, Equity Ownership; Red Oak Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Stem cells determine homeostasis and repair of many tissues and are increasingly recognized as functionally heterogeneous. To define the extent of—and molecular basis for—heterogeneity, we overlaid functional, transcriptional, and epigenetic attributes of hematopoietic stem cells (HSCs) at a clonal level using endogenous fluorescent tagging. Endogenous HSC had clone-specific functional attributes over time in vivo. The intra-clonal behaviors were highly stereotypic, conserved under the stress of transplantation, inflammation, and genotoxic injury, and associated with distinctive transcriptional, DNA methylation, and chromatin accessibility patterns. Further, HSC function corresponded to epigenetic configuration but not always to transcriptional state. Therefore, hematopoiesis under homeostatic and stress conditions represents the integrated action of highly heterogeneous clones of HSC with epigenetically scripted behaviors. This high degree of epigenetically driven cell autonomy among HSCs implies that refinement of the concepts of stem cell plasticity and of the stem cell niche is warranted.

Somatic mutations in spliceosome genes are detectable in ∼50% of patients with myelodysplastic syndromes (MDS). We hypothesize that cells harbouring spliceosome gene mutations have increased sensitivity to pharmacological perturbation of the spliceosome. We focus on mutant U2AF1 and utilize sudemycin compounds that modulate pre-mRNA splicing. We find that haematopoietic cells expressing mutant U2AF1(S34F), including primary patient cells, have an increased sensitivity to in vitro sudemycin treatment relative to controls. In vivo sudemycin treatment of U2AF1(S34F) transgenic mice alters splicing and reverts haematopoietic progenitor cell expansion induced by mutant U2AF1 expression. The splicing effects of sudemycin and U2AF1(S34F) can be cumulative in cells exposed to both perturbations—drug and mutation—compared with cells exposed to either alone. These cumulative effects may result in downstream phenotypic consequences in sudemycin-treated mutant cells. Taken together, these data suggest a potential for treating haematological cancers harbouring U2AF1 mutations with pre-mRNA splicing modulators like sudemycins., Spliceosome mutations occur in approximately 50% of patients with myelodysplastic syndromes. Here, the authors show that tumour cells harbouring the S34F mutation in the U2AF spliceosome gene is sensitive to compounds that further perturb the spliceosome.

Tissue homeostasis in complex organisms is accomplished through interactions between cells, communication through circulating or locally distributed chemicals, and biophysical properties of their environment. Stem cells are preserved in specific anatomical sites known as niches where these cellular, biochemical, and biophysical interactions drive tissue maintenance and regeneration. Our understanding of mammalian stem cell niches has advanced greatly in recent years; yet, as available technology becomes more sophisticated, several well-accepted paradigms are being discovered to be less than generalizable or being challenged altogether. In this chapter we summarize what is known about the three components of the niche to which stem cells and their neoplastic counterparts are native: cellular, biochemical, and biophysical. As we apply improving technologies to our age-old questions and ask new ones, our understanding of this complex ecosystem is likely to evolve and will hopefully yield insights that are of benefit to patients.

Alternative splicing generates a diversity of messenger RNA (mRNA) transcripts from a single mRNA precursor and contributes to the complexity of our proteome. Splicing is perturbed by a variety of mechanisms in cancer. Recurrent mutations in splicing factors have emerged as a hallmark of several hematologic malignancies. Splicing factor mutations tend to occur in the founding clone of myeloid cancers, and these mutations have recently been identified in blood cells from normal, healthy elderly individuals with clonal hematopoiesis who are at increased risk of subsequently developing a hematopoietic malignancy, suggesting that these mutations contribute to disease initiation. Splicing factor mutations change the pattern of splicing in primary patient and mouse hematopoietic cells and alter hematopoietic differentiation and maturation in animal models. Recent developments in this field are reviewed here, with an emphasis on the clinical consequences of splicing factor mutations, mechanistic insights from animal models, and implications for development of novel therapies targeting the precursor mRNA splicing pathway.

Anemia of inflammation develops in settings of chronic inflammatory, infectious, or neoplastic disease. In this highly prevalent form of anemia, inflammatory cytokines, including IL-6, stimulate hepatic expression of hepcidin, which negatively regulates iron bioavailability by inactivating ferroportin. Hepcidin is transcriptionally regulated by IL-6 and bone morphogenetic protein (BMP) signaling. We hypothesized that inhibiting BMP signaling can reduce hepcidin expression and ameliorate hypoferremia and anemia associated with inflammation. In human hepatoma cells, IL-6–induced hepcidin expression, an effect that was inhibited by treatment with a BMP type I receptor inhibitor, LDN-193189, or BMP ligand antagonists noggin and ALK3-Fc. In zebrafish, the induction of hepcidin expression by transgenic expression of IL-6 was also reduced by LDN-193189. In mice, treatment with IL-6 or turpentine increased hepcidin expression and reduced serum iron, effects that were inhibited by LDN-193189 or ALK3-Fc. Chronic turpentine treatment led to microcytic anemia, which was prevented by concurrent administration of LDN-193189 or attenuated when LDN-193189 was administered after anemia was established. Our studies support the concept that BMP and IL-6 act together to regulate iron homeostasis and suggest that inhibition of BMP signaling may be an effective strategy for the treatment of anemia of inflammation.

SummaryFollowing injury, stem cells restore normal tissue architecture by producing the proper number and proportions of differentiated cells. Current models of airway epithelial regeneration propose that distinct cytokeratin 8-expressing progenitor cells, arising from p63+ basal stem cells, subsequently differentiate into secretory and ciliated cell lineages. We now show that immediately following injury, discrete subpopulations of p63+ airway basal stem/progenitor cells themselves express Notch pathway components associated with either secretory or ciliated cell fate commitment. One basal cell population displays intracellular Notch2 activation and directly generates secretory cells; the other expresses c-myb and directly yields ciliated cells. Furthermore, disrupting Notch ligand activity within the basal cell population at large disrupts the normal pattern of lineage segregation. These non-cell-autonomous effects demonstrate that effective airway epithelial regeneration requires intercellular communication within the broader basal stem/progenitor cell population. These findings have broad implications for understanding epithelial regeneration and stem cell heterogeneity.

Following myocardial infarction (MI), myeloid cells derived from the hematopoietic system drive a sharp increase in systemic leukocyte levels that correlates closely with mortality. The origin of these myeloid cells, and the response of hematopoietic stem and progenitor cells (HSPCs) to MI, however, is unclear. Here, we identify a CCR2(+)CD150(+)CD48(-) LSK hematopoietic subset as the most upstream contributor to emergency myelopoiesis after ischemic organ injury. This subset has 4-fold higher proliferation rates than CCR2(-)CD150(+)CD48(-) LSK cells, displays a myeloid differentiation bias, and dominates the migratory HSPC population. We further demonstrate that the myeloid translocation gene 16 (Mtg16) regulates CCR2(+) HSPC emergence. Mtg16(-/-) mice have decreased levels of systemic monocytes and infarct-associated macrophages and display compromised tissue healing and post-MI heart failure. Together, these data provide insights into regulation of emergency hematopoiesis after ischemic injury and identify potential therapeutic targets to modulate leukocyte output after MI.

Background The bone marrow microenvironment (BMEV) regulates the highly regenerative hematopoietic system. However, there are a limited number of BMEV-derived molecules with a definitive role in maintaining hematopoietic stem and progenitor cells (HSPCs). Extracellular vesicles (EVs) encapsulate bioactive molecules, and may modify the physiology of their target cells. In hematopoiesis, EVs derived from culture-expanded mesenchymal cells can rescue irradiation damage, expand human umbilical cord blood cells and support HSPCs in vitro . However, in vivo evidence of EV function is lacking. We therefore sought to investigate the role of EVs in the interaction between the BMEV and the hematopoietic system and took advantage of existing mice bearing genetic reporters of key mesenchymal cell types. Results While analyzing the bone marrow (BM) of different mesenchymal cell-GFP reporter mice, we unexpectedly found CD45+ GFP+ cells. These were confirmed as single cells with intracellular GFP as demonstrated by imaging flow cytometry and confocal microscopy (Fig. 1A). Moreover, their hematopoietic identity was confirmed by their ability to form myeloid colonies in methylcellulose. Transplanted CD45.1 BM into Osteocalcin-Topaz (Ocn-Topaz) and Collagen1-GFP (Col1-GFP) mice that label osteoblasts, as well as Nestin-GFP (Nes-GFP) that labels mesenchymal stem cells demonstrated that donor cells are comparably labeled with GFP in Ocn-Topaz and Col1-GFP (2.2%) but at a much lower frequency (0.05%) in Nes-GFP. We therefore decided to proceed with the Ocn-Topaz model to investigate the role of osteoblast derived EVs in hematopoietic communication. Within the lineage negative compartment, the frequency of GFP+ cells increased with maturation. The highest frequency found in GMPs (0.06% of live cells were GFP+), followed by CMPs (0.01%), MEPs (0.002%) and LKS (0.004%) (Fig. 1B). Of particular interest, Lin- GFP+ cells formed ~5 fold more colonies as compared to their GFP- counterparts. However, transplantation assays demonstrated that the GFP+ cells possessed a decreased ability for long term reconstitution. Given the molecular weight of GFP, we hypothesized that EVs were the basis for transfer. Transmission electron microscopy coupled with immunogold staining revealed microvesicular structures of ~100 nm in size that contained GFP and that were labeled with the exosome marker TSG101 (Fig. 1C). Western blotting and flow cytometry detected labeling with exosome markers CD81 and CD9. Heparin sulfate proteoglycans (HSPGs) have been implicated in the biogenesis and uptake of EVs. Osteoblast-specific disruption of HSPGs by the knock out of the glycosyl transferase EXT1 resulted in a (40%) drop in the frequency of GFP+ cells in the GMP compartment. These findings demonstrate the EV-dependent transfer of GFP from osteoblasts to BM hematopoietic cells, and confirm GFP as a marker for the isolation and characterization of EV target cells. Exosomes from the BM of Ocn-Topaz mice in addition to GFP+ and GFP- GMPs were isolated for small RNA sequencing. In parallel, GMP populations were collected for mRNA sequencing. Global analysis of small RNA libraries from EVs and GMPs demonstrated that piRNAs was the most abundant species in both EVs (30%) and GMPs (18%). Surprisingly, EVs had low miRNA content (1.4%) compared to GMPs (9.2%) (Fig. 1D). When comparing GFP+ GMPs to GFP- ones, 6 miRNAs (mir-143, mir-122, mir-423-5p, mir-451, mir-206, mir-146b*) showed at least 100% increase in the GFP+GMPs. Predicted targets of mir-143, mir-206, mir-146 emerged as enriched sets when comparing gene expression of GFP+ and GFP- GMPs. In contrast, tRNAs was the most enriched species in EVs (10.5%) when compared to GMPs (2.5%) (Fig. 1D) and interestingly, GFP+ GMPs had higher content of tRNA when compared to GFP- (3.3% vs 1.7%) respectively. Given the role of tRNAs in translation and the emerging role of tRNA fragments (tRFs) in translation regulation and stress signaling, it was of interest to see translation and ribosome genesis among the top enriched gene sets when comparing GFP+ and GFP- GMPs. In conclusion, we present evidence for the in vivo transfer of bioactive EVs from osteoblasts to BM progenitor populations, and that this transfer alters hematopoietic cell function and gene expression. Moreover, we identify piRNAs and tRNAs as the most enriched species of small RNAs within BM derived EVs. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

R loop, a transcription intermediate containing RNA:DNA hybrids and displaced single-stranded DNA (ssDNA), has emerged as a major source of genomic instability. RNaseH1, which cleaves the RNA in RNA:DNA hybrids, plays an important role in R loop suppression. Here, we show that replication protein A (RPA), a ssDNA-binding protein, interacts with RNaseH1 and colocalizes with both RNaseH1 and R loops in cells. In vitro, purified RPA directly enhances the association of RNaseH1 with RNA:DNA hybrids and stimulates the activity of RNaseH1 on R loops. An RPA binding-defective RNaseH1 mutant is not efficiently stimulated by RPA in vitro, fails to accumulate at R loops in cells, and loses the ability to suppress R loops and associated genomic instability. Thus, in addition to sensing DNA damage and replication stress, RPA is a sensor of R loops and a regulator of RNaseH1, extending the versatile role of RPA in suppression of genomic instability.

Stem cells integrate inputs from multiple sources. Stem cell niches provide signals that promote stem cell maintenance, while differentiated daughter cells are known to provide feedback signals to regulate stem cell replication and differentiation. Recently, stem cells have been shown to regulate themselves using an autocrine mechanism. The existence of a 'stem cell niche' was first postulated by Schofield in 1978 to define local environments necessary for the maintenance of haematopoietic stem cells. Since then, an increasing body of work has focused on defining stem cell niches. Yet little is known about how progenitor cell and differentiated cell numbers and proportions are maintained. In the airway epithelium, basal cells function as stem/progenitor cells that can both self-renew and produce differentiated secretory cells and ciliated cells. Secretory cells also act as transit-amplifying cells that eventually differentiate into post-mitotic ciliated cells . Here we describe a mode of cell regulation in which adult mammalian stem/progenitor cells relay a forward signal to their own progeny. Surprisingly, this forward signal is shown to be necessary for daughter cell maintenance. Using a combination of cell ablation, lineage tracing and signalling pathway modulation, we show that airway basal stem/progenitor cells continuously supply a Notch ligand to their daughter secretory cells. Without these forward signals, the secretory progenitor cell pool fails to be maintained and secretory cells execute a terminal differentiation program and convert into ciliated cells. Thus, a parent stem/progenitor cell can serve as a functional daughter cell niche.

Mutation or epigenetic silencing of the transcription factor C/EBP alpha is observed in similar to 10% of patients with acute myeloid leukemia (AML). In both cases, a common global gene expression profile is observed, but downstream targets relevant for leukemogenesis are not known. Here, we identify Sox4 as a direct target of C/EBP alpha whereby its expression is inversely correlated with C/EBP alpha activity. Downregulation of Sox4 abrogated increased self-renewal of leukemic cells and restored their differentiation. Gene expression profiles of leukemia-initiating cells (LICs) from both Sox4 overexpression and murine C/EBP alpha mutant AML models clustered together but differed from other types of AML. Our data demonstrate that Sox4 overexpression resulting from C/EBP alpha inactivation contributes to the development of leukemia with a distinct LIC phenotype.

Bone marrow/hematopoietic stem cell transplantation (BMT/HSCT) holds the remarkable ability to correct any blood or immune disease. Unfortunately, despite the tremendous potential of this procedure, BMT remains fairly limited in part due to the severe risks associated with the toxic conditioning regimens, such as irradiation and chemotherapy that are currently employed to enable donor HSC engraftment. Although significant work has been done to dose reduce the amount of these preparative agents, patients still experience many side effects including neutropenia/infections, anemia, mucositis, infertility, organ damage and secondary malignancies. Complete elimination of these toxic conditioning regimens could dramatically improve the safety profile of BMT and expand the potential applications to include many more non-malignant hematologic disorders, a wide variety of autoimmune disorders including diabetes, as well as facilitate solid organ tolerance. We have previously shown that competition with host HSC limits donor HSC engraftment, and that in immunocompromised hosts antagonistic anti-ckit monoclonal antibodies deplete host HSC and are an effective and safe alternative conditioning approach (Czechowicz, Science 2007). However, this modality of conditioning is not effective in hosts with competent immune systems. To further understand efficacy of antagonistic anti-ckit conditioning, we tested its functionality in multiple strains of immunocompromised mice and show that inhibition of SCF signaling is not sufficient to deplete host HSC in mouse strains with competent B-cells or T-cells, and that the addition of these cells interferes with the ability of antagonistic anti-ckit antibodies to effectively condition. In an attempt to overcome this hurdle, wildtype mice were immune-depleted with a variety of regimens but none enabled antagonistic anti-ckit conditioning in the immunocompetent setting. To strengthen the potency of anti-ckit mAbs we linked them to protein synthesis toxins, which when internalized by host HSC led to their rapid decline in vitro and in vivo. Administration of anti-ckit-saporin to wild-type mice resulted in >99% depletion of host HSC (Ckit+Lin-Sca1+CD150+CD48-), and lack of residual host HSC activity in the bone marrow was confirmed by CFC assays and competitive transplantation into lethally irradiated recipients. Interestingly, although ckit is expressed by a majority of HSPC, LT-HSC were most significantly affected and no cellularity changes in the bone marrow were observed. Uniquely this regimen was entirely non-peripheral blood ablative unlike other more broadly targeted conditioning regimens such as CD45 immunotoxins (Palchaudhuri, Nat Biotech 2016), and treated animals did not experience any significant depletion of myeloid, lymphoid, or erythroid cells. Figure 1 Figure 1. Treatment with anti-ckit-saporin effectively conditioned wild-type animals and near complete donor granulocyte chimerism was rapidly achieved post transplantation of whole bone marrow cells (99.54 ± 0.35 % vs. 6.79 ± 0.57 %, p25-fold increase compared to unconditioned controls. Similarly, anti-ckit-saporin conditioning enable efficient engraftment of FACS purified donor HSC (Ckit+Lin-Sca1+CD150+CD48-). In both settings, donor HSC chimerism matched donor granulocyte chimerism further confirming replacement of host HSC. Importantly, host immunity was entirely intact in these animals throughout, with slower recalibration of the longer-lived immune cells given the lack of their direct depletion. Figure 2 Figure 2. This work sets the stage for redefining the way BMT/HSCT is performed, as it opens up the possibility for entirely safe, quick and easy transplantation that potentially could be done in the outpatient setting with no perturbation to host immunity. Extrapolation of these methods to humans may enable efficient yet gentle conditioning regimens for transplantation, which is especially exciting in the gene-therapy settings where no immune suppression is required, allowing for simple, safe and curative treatment of a wide magnitude of grievous blood and immune diseases ranging from sickle cell to hemophilia to HIV. As multiple anti-ckit mAbs are currently in development and being tested in clinical trials, such an approach may be rapidly translatable to patients. Disclosures Czechowicz: Third Rock Ventures: Consultancy; Global Blood Therapeutics: Equity Ownership; Editas Medicines: Equity Ownership, Patents & Royalties; Decibel Therapeutics: Equity Ownership; Magenta Therapeutics: Consultancy, Equity Ownership, Patents & Royalties; Forty Seven Inc: Patents & Royalties. Palchaudhuri:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hoggatt:Magenta Therapeutics: Consultancy, Equity Ownership, Research Funding. Scadden:Teva: Consultancy; Apotex: Consultancy; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Dr. Reddy's: Consultancy; GlaxoSmithKline: Research Funding; Fate Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Bone Therapeutics: Consultancy. Rossi:Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Intellia Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Moderna Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Haematopoietic stem cells (HSCs) can convert between growth states that have marked differences in bioenergetic needs. Although often quiescent in adults, these cells become proliferative upon physiological demand. Balancing HSC energetics in response to nutrient availability and growth state is poorly understood, yet essential for the dynamism of the haematopoietic system. Here we show that the Lkb1 tumour suppressor is critical for the maintenance of energy homeostasis in haematopoietic cells. Lkb1 inactivation in adult mice causes loss of HSC quiescence followed by rapid depletion of all haematopoietic subpopulations. Lkb1-deficient bone marrow cells exhibit mitochondrial defects, alterations in lipid and nucleotide metabolism, and depletion of cellular ATP. The haematopoietic effects are largely independent of Lkb1 regulation of AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling. Instead, these data define a central role for Lkb1 in restricting HSC entry into cell cycle and in broadly maintaining energy homeostasis in haematopoietic cells through a novel metabolic checkpoint.

Mutations in spliceosome genes are detectable in ~50% of patients with myelodysplastic syndromes (MDS), making this cellular pathway the most commonly mutated in MDS and providing a novel target for therapeutic intervention. Spliceosome gene mutations are mutually exclusive, implying they are either redundant in pathogenic function or are not tolerated in a cell when they co-occur. Our group and others identified recurrent heterozygous missense mutations in the splicing factor gene U2AF1 in 11% of MDS patient samples. The most common U2AF1 mutation results in a conversion of serine to phenylalanine at position 34 (S34F) of the U2AF1 protein. We previously reported that expression of mutant U2AF1(S34F) in vivo using doxycycline-inducible U2AF1(S34F) transgenic mice revealed an expansion of hematopoietic bone marrow progenitor cells and leukopenia following transgene induction; both phenotypes are seen in patients with MDS. We also identified mutant U2AF1-specific alterations in pre-mRNA splicing in transgenic mouse bone marrow progenitor cells, primary AML patient samples, and CD34+ cells by RNA sequencing. We hypothesize that cells harboring spliceosome gene mutations have increased sensitivity to pharmacological perturbation of the spliceosome by splicing modulator drugs, providing a new treatment approach for patients with U2AF1 mutations. For our studies, we utilize sudemycins, which are compounds that bind the SF3B1 spliceosome protein and modulate pre-mRNA splicing in non-hematopoietic tissues. We examined the effects of sudemycin treatment on pre-mRNA splicing in primary hematopoietic cells by treating CD34+ cells isolated from human umbilical cord blood with sudemycin in vitro. We performed whole transcriptome (RNA-seq) analysis following 6 hours of sudemycin treatment (1µM) of CD34+ cells and identified robustly altered pre-mRNA splicing patterns that are sudemycin-specific (26,120 splice junctions by DEXSeq, FDR Disclosures No relevant conflicts of interest to declare.

Hoxa9 and Meis1 are overexpressed in >70% of acute myeloid leukemia (AML) and associated with poor prognosis and survival. Hoxa9 and Meis1 interact with DNA and PBX to achieve transcription of differentiation-blocking genes. We tested transcriptional repression at Hoxa9-PBX-Meis1 genomic binding sites to induce differentiation in a model of human AML We designed a DNA-recognition strategy based on the known structure of the Hoxa9-PBX-DNA complex by fusing the DNA binding helices of Hoxa9 and PBX to create concise homeodomain fusion proteins that target the Hoxa9-PBX DNA recognition sequence. To confer transcription-repressing properties to the proteins, we attached a transcriptional repressor (sin3 interacting) domain and ectopically expressed this protein in Hoxa9-Meis1 immortalized murine progenitors. Introduction of this transcription repressor protein significantly enabled cell differentiation versus control (51.2% Mac-1high Gr-1high cells versus 11.3% for control). Multiple gene transcripts indicative of differentiation, such as GCSFR, myeloperoxidase, neutrophil elastase, and the calcium binding protein, S100A8, were also elevated in repressor-expressing cells. Furthermore, direct transcriptional targets of Hoxa9 (e.g. SOX2, CD34, FOXP1, FLT3R, DNAJC10) were down regulated in repressor-expressing cells. Importantly, a mutant repressor lacking the DNA-interacting amino acids did not affect transcription of Hoxa9 targets, demonstrating on-target specificity. Repressor-expressing cells also exhibited lower surface expression of c-Kit and Flt3 receptors and when transplanted into mice resulted in a significant increase in disease latency with a 94 day median latency versus 62 day latency for the control group (p value = 0.002). Our results demonstrate that site-specific DNA-targeting using homeodomain fusion proteins can enable AML cell differentiation and significantly increase disease latency. Disclosures: Scadden: Fate Therapeutics: Consultancy, Equity Ownership.

Background Rapid mobilization of hematopoietic stem and progenitor cells (HSPCs) from the bone marrow (BM) to the peripheral blood by anti-CXCR4 agents such as AMD3100 is a complex process, which requires CXCL12 secretion, activation of proteolytic enzymes and supporting cellular compartments (Dar et. al, Leukemia 2011). Notably, components of innate immune system were also shown to be involved (Ratajczak et. al, Leukemia 2010). Human β-defensin-3 (hBD3) is an antimicrobial peptide possessing also anti-CXCR4 effects on human T cells in vitro (Feng et. al, JI 2006), suggesting its HSPC mobilizing potential. Here, we describe a novel approach for targeting CXCR4 in vivo by utilizing β-defensin-derived peptides, resulting in rapid HSPC mobilization. Results While AMD3100 blocked CXCL12-mediated signaling and migration of enriched BM mononuclear cells (MNCs) in vitro, we unexpectedly detected rapid phosphorylation of AKT, p38 and ERK1/2 in BM stromal cells (BMSCs). Interestingly, single administration of AMD3100 to mice resulted in enhancement of CXCR4 phosphorylation within minutes in both BM residing mesenchymal stem/progenitor cells (MSCs) and HSPCs, thus suggesting a CXCR4 agonistic activity. Aiming to test HSPC mobilizing potential of hBD3 and avoiding potential toxicity of systemic administration, we synthesized short linear peptides, comprising the C-terminal parts of hBD3 and the murine ortholog β-defensin-14 (mBD14), as well as a cyclic peptide of hBD3, comprising the same amino acids as the linear one, to serve as a control. All full-length β-defensins and tested peptides (both linear and cyclic) specifically bound CXCR4 (demonstrated by docking approach and anti-CXCR4 antibody competition assay) and efficiently blocked CXCL12-mediated activity of enriched BM MNCs in vitro including cell migration and CXCR4-dependent HIV infection. Intriguingly, full-length β-defensins and derived linear peptides (but not cyclic) revealed a strong stimulatory effect on BMSCs in vitro: triggering phosphorylation of AKT, p38 and ERK1/2 along with enhancing secretion of functional CXCL12. Administration of linear peptides to mice led to a fast activation of CXCR4 signaling in BMSCs and MSCs as well as in HSPCs accompanied by CXCL12 release to the circulation, increased activity of proteolytic enzymes and consequent rapid mobilization of progenitors as well as long-term repopulating stem cells. In addition, linear peptides augmented AMD3100-induced rapid mobilization. Importantly, the control cyclic peptide, which bound CXCR4 but failed to activate BMSCs in vitro, did not induce HSPC mobilization in vivo. Moreover, it inhibited both steady-state egress and AMD3100-induced mobilization of HSPCs. A series of in vivo inhibitory analyses confirmed dependence of hBD3- and mBD14-derived peptide-induced HSPC mobilization on the activation of CXCL12/CXCR4 axis and revealed involvement of uPA and JNK signaling as well as ROS generation. Conclusions Our study demonstrated for the first time the capability of β-defensin-derived peptides to activate in vivo CXCL12/CXCR4 signaling in both hematopoietic and non-hematopoietic BM cells, leading to rapid HSPC mobilization. We suggest that activation of CXCR4 signaling in non-hematopoietic BM cells is crucial for inducing HSPC mobilization. Accordingly, CXCR4-binding agents capable of triggering CXCR4 signaling in non-hematopoietic BM cells in vitro, would induce rapid HSPC mobilization. The results presented here help to better understand the mechanisms of rapid HSPC mobilization and have the potential of improving existing clinical protocols to increase the yield of HSPC harvest for transplantation. Disclosures: Scadden: Fate Therapeutics: Consultancy, Equity Ownership.

Abstract 208 Altered metabolism has long been recognized as a signature finding of malignant cells with changes in glucose handling termed the ‘Warburg effect’. Now recognized as an adaptation of metabolic pathways to meet the distinctive demands of malignant cells, metabolism represents a potential area of opportunity for developing novel therapeutics. We established a system by which a wide spectrum of metabolic pathways could be examined for differential activity in tumor cells and their normal counterparts in the context of acute Myeloid Leukemia (AML). We studied metabolism using a biased shRNA library comprising 1000 shRNAs on a well-defined population of leukemia stem or initiating cells (LICs) in a model of AML driven by the MLL-AF9 oncogene (Krivtsov, 2006) and performed identical assays on the normal counterparts of LICs called normal granulocyte monocyte precursors (N-GMPs). The shRNAs chosen, targeted canonical ‘rate limiting enzymes’ as well as those enzymes which were found to be differentially expressed in LICs and N-GMPs by gene expression profiling. Both LICs and N-GMPs were maintained in the presence of murine bone marrow mesenchymal cells in order to mimic the native micro-environment in which these cells grow. To maximize physiologic relevance we used primary mouse LICs, N-GMPs and bone marrow mesenchymal cells, instead of cell lines, which may have distinct metabolic dependencies secondary to their adaption to culture conditions. Our screen has identified Aldehyde Dehydrogenase 3a2 (Aldh3a2 or Fatty Aldehyde Dehydrogenase, FALDH) as a key target gene. Aldh3a2 depletion in LICs with three independent validated shRNAs resulted in improvement in survival of transplanted mice (p value of 0.0004 to 0.0018) and significantly decreased growth of cells in methylcellulose soft agar assays and in co-culture with primary bone marrow stroma. In a knock-out mouse model of Aldh3a2, normal hematopoiesis as assessed by immunophenotype seems unperturbed. Using Tumorscape, the Broad Institute's database on gene copy number variation in tumors, we showed that Alh3a2 is significantly focally amplified (Q value =1.39−5) across the entire dataset of 3,131 tumors, including hematologic malignancies and is located within a focal peak region containing 11 other genes (chromosome 17:19117656–19932585). We have also probed a gene expression dataset of 436 samples obtained from two different multicenter clinical trials: the AMLSG HD98a trial (patients < 60 years of age) and the AMLSG HD98b trial (patients > 60 years of age). Analysis revealed significant overexpression of Aldh3a2 in MLL-AF9 samples compared to all other AML subtypes (p< 0.0006 by ANOVA). Patients in the highest quartile of Aldh3a2 expression had a significantly prolonged overall survival compared to those in all other quartiles (p=0.038). Whether this survival association is due to increased dependency on Aldh3a2 and sensitivity to chemotherapy is unclear, but an issue for future experiments. We have observed increased apoptosis, decreased cycling and altered ROS in L-GMPs with knock-down of Aldh3a2. Metabolomic experiments to identify the precise metabolites which are responsible for the differential sensitivity of LICs and normal hematopoietic cells to Aldh3a2 depletion are ongoing. Our work is aimed at confirming the metabolic dependency of leukemia on Aldh3a2 and dissecting the biology of this enzyme in the context of this disease. We hope this will lead to the development of novel therapeutic strategies and provide the groundwork for the further study of leukemia metabolism. Disclosures: No relevant conflicts of interest to declare.

Abstract 637 The hematopoietic stem cell niche is known to govern the function of hematopoietic and leukemic stem cells. One constituent of the niche is the extracellular matrix comprised of several elements including a group of molecules called heparan sulfate proteoglycans (HSPGs). These molecules are present in the microenvironment of all tissue stem cells and are critical in the dynamic interactions between stem cells and niche elements. Heparan sulfate proteoglycan synthesis depends upon the glycosyltransferase, Ext1, a known tumor suppressor with a skeletal phenotype (exostoses) when mutated in affected humans. In order to investigate the role of HSPGs in the hematopoietic stem cell niche we conditionally deleted the Ext1 gene in the recently described Mx1+ osteolineage stem/progenitor cell population (Park et al., 2012). Our studies show that Ext1 and its biological product, HSPGs, play a major role in the maintenance of hematopoietic stem cells (HSCs) by modulating the bone marrow microenvironment. Ext1 deletion in osteolineage cells results in a marked loss of hematopoietic stem and progenitor cells (HSPCs) from the bone marrow and an increase in circulating colony forming units (CFU-Cs) and HSCs (designated as lineage -, c-Kit+, Sca1+, CD 150 + and CD48-) both in the spleen and peripheral blood, suggesting a key role of heparan sulfate proteoglycans in the retention of hematopoietic stem/progenitor cells (HSPCs) in the bone marrow. Importantly, histomorphometry analysis showed no defect in bone remodeling upon Ext1 deletion. In order to investigate whether the above phenotype could be therapeutically exploited in the context of bone marrow stem/progenitor cell mobilization, we administered the heparan sulfate proteoglycan inhibitors, heparin and protamine sulfate to mice in combination with conventional mobilization regimes such as G-CSF. Pharmacological inhibition of HSPG induced a significant improvement of hematopoietic stem/progenitor cell mobilization over G-CSF treatment alone, an effect that was completely abrogated in the absence of endogenous HSPGs. Furthermore, by combining HSPG inhibition with G-CSF, we could mobilize a population of HSPCs with superior in-vivo self-renewing ability. Mechanistically, we observed that Ext1 deletion in stromal cells leads to a constitutive activation of the AKT pathway through the PDK1-mediated phosphorylation of AKT-Thr-308, which in turn phosphorylates and inactivate FOXO1 transcription factor. Expression profiling of genes transcriptionally regulated by FOXO1 revealed that VCAM1, a key molecule driving HSCs adhesion to their niche, was highly down regulated in Ext1 deficient cells. VCAM1 protein levels were also decreased accompanied by a profound reduction in HSC adhesion to Ext1 deficient cells in vitro. Taken together our results suggest that Ext1/HSPGs control hematopoietic stem/progenitor cells retention in the bone marrow through an AKT/FOXO1/VCAM1 dependent pathway and that targeting heparan sulfate proteoglycans is a strategy for mobilizing stem cells with superior self renewing capability to the blood. We are grateful to Lexicon Genetics and the MMRRC for kindly providing the Ext1 mouse model. Disclosures: Wagers: BD Biosciences: Consultancy; iPierian, Inc.: Consultancy; MPM Capital: Consultancy; Novartis: Honoraria. Scadden:Fate Therapeutics: Consultancy, Equity Ownership; Genzyme: Consultancy.

Abstract 759 We hypothesized that metabolic differences between leukemia initiating cells and their normal counterparts represent a vulnerability in the leukemia initiating cell, which can be therapeutically exploited. To test this hypothesis, we used the MLL-AF9 acute myeloid leukemia (AML) model in mice. Actin-DsRed mouse bone marrow transduced with MLL-AF9 expressing retrovirus was used to produce serially transplantable leukemia. Leukemic granulocyte-monocyte precursors (L-GMPs), defined by others to be the leukemia initiating cells were flow sorted from secondary recipient mice and compared with normal GMPs (N-GMPs) from actin Ds-Red mice. Gene expression profiling, metabolomic profiling via liquid chromatography- mass spectrometry and an in vitro shRNA screen were used to identify metabolic pathways preferentially activated in leukemia initiating cells. Of 1574 defined metabolic enzymes, 44 were found to be differentially expressed between L-GMPs and their normal counterparts (N-GMPs). These together with 117 classic rate limiting enzymes were subjected to shRNA knockdown in vitro. Metabolomic profiling of both cell populations was used to corroborate findings from shRNA knockdowns. L-GMPs and N-GMPs were transduced with lentivirus expressing shRNAs of interest (5 shRNAs per gene) in a 384 well format, selected with puromycin and cultured for 72–96 hours in the presence of GFP-positive primary bone marrow stroma. The number of cells in each well at the end of this experiment was quantitated using an Image Xpress microscope. Genes, the knockdown of which by at least two independent shRNAs produced a two fold or more decrease in L-GMPs as compared to control wells and did not similarly decrease N-GMPs, were chosen for in vivo validation. Ten genes in the glycolysis pathway and TCA cycle, fatty acid metabolism and detoxification, and ketohexokinase were identified. Ketohexokinase, a rate-limiting enzyme in fructose metabolism was particularly potent and of interest given its potential to be exploited therapeutically. In vivo assessment of its relative ability to inhibit malignant versus normal hematopoietic cells is ongoing. These studies provide preliminary support for the hypothesis that specific metabolic circuits are differentially active in leukemia initiating cells in MLL-AF9 AML and may represent unique points of vulnerability that can be targeted therapeutically. Authors 1 and 2 contributed equally. Authors 3 and 4 contributed equally. Disclosures: No relevant conflicts of interest to declare.

The remodeling of the extracellular matrix is a central function in endochondral ossification and bone homeostasis. During secondary fracture healing, vascular invasion and bone growth requires the removal of the cartilage intermediate and the coordinate action of the collagenase matrix metalloproteinase (MMP)-13, produced by hypertrophic chondrocytes, and the gelatinase MMP-9, produced by cells of hematopoietic lineage. Interfering with these MMP activities results in impaired fracture healing characterized by cartilage accumulation and delayed vascularization. MMP-10, Stromelysin 2, a matrix metalloproteinase with high homology to MMP-3 (Stromelysin 1), presents a wide range of putative substrates identified in vitro, but its targets and functions in vivo and especially during fracture healing and bone homeostasis are not well defined. Here, we investigated the role of MMP-10 through bone regeneration in C57BL/6 mice. During secondary fracture healing, MMP-10 is expressed by hematopoietic cells and its maximum expression peak is associated with cartilage resorption at 14 days post fracture (dpf). In accordance with this expression pattern, when Mmp10 is globally silenced, we observed an impaired fracture-healing phenotype at 14 dpf, characterized by delayed cartilage resorption and TRAP-positive cell accumulation. This phenotype can be rescued by a non-competitive transplant of wild-type bone marrow, indicating that MMP-10 functions are required only in cells of hematopoietic linage. In addition, we found that this phenotype is a consequence of reduced gelatinase activity and the lack of proMMP-9 processing in macrophages. Our data provide evidence of the in vivo function of MMP-10 during endochondral ossification and defines the macrophages as the lead cell population in cartilage removal and vascular invasion. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).