LATE-BREAKING ORAL PRESENTATIONS

2006 – VIAGRA ENABLES EFFICIENT, SINGLE-DAY HEMATOPOIETIC STEM CELL MOBILIZATION Stephanie Smith-Berdan; Bryan Petkus; Alyssa Bercassio; Camilla Forsberg UCSC, Santa Cruz, United States

Despite the use of hematopoietic stem cells (HSCs) in clinical therapy for over half a century, the mechanisms regulating HSC trafficking, engraftment, and life-long persistence after transplantation are unclear. Findings over the past several years have demonstrated that HSC function within bone marrow (BM) niches depends on vascular and peri-vascular cells. We recently showed that the vascular endothelium not only regulates HSC maintenance within BM niches, but also trafficking between the vasculature and the BM space. We found that the vascular endothelium, via the guidance molecule ROBO4, reinforces HSC localization to BM niches both by promoting HSC extravasation from blood-to-BM and by forming vascular barriers that prevent BM-to-blood escape. As ROBO4 promotes vascular stability, we hypothesized that vascular permeability regulates HSC location. We found that vascular permeability induced by intravenous injection of recombinant VEGF led to a 2-fold increase in HSCs in the peripheral blood. Here, we utilized a tet-inducible VEGF overexpression mouse model to show that sustained VEGF expression and robust vascular permeability leads to unprecedented HSC mobilization. VEGF-mediated mobilization was transient and reversible and did not irrevocably damage BM vascular niches. These findings revealed that vascular integrity is an important regulator of HSC location and that manipulation of vascular integrity can be employed to control HSC mobilization. We therefore tested whether FDA-approved vasodilators are capable of mobilizing HSCs. We found that a single, oral dose of Viagra (sildenafil citrate) in conjunction with a single dose of the CXCR4 antagonist AMD3100 leads to efficient HSC mobilization at levels rivaling a 4- day GCSF regimen. Our findings solidify vascular integrity as an essential regulator of HSC trafficking and provide an attractive new, single-day regimen for HSC mobilization using already FDA-approved drugs.

2016 – HUMAN HEMATOPOIETIC STEM CELL SELF-RENEWAL AND ENGRAFTMENT ARE ENHANCED BY THE TRANSCRIPTIONAL REGULATOR MLLT3 Vincenzo Calvanese1; Anastasia Vavilina2; Andrew Nguyen2; Timothy Bolan2; Fides Lay2; Siavash Kurdistani2; Mattias Magnusson2; Hanna Mikkola2 1University of California, Los Angeles, Los Angeles, United States; 2UCLA, Los Angeles, United States

Hematopoietic stem cells (HSC) regenerate the blood and immune system upon transplantation, providing treatment for several blood diseases. However, HSC transplantation is available for only a fraction of patients due to limitations in availability and/or histocompatibility. To achieve robust HSC expansion for transplantation we need to understand the mechanisms governing self-renewal, and why this program fails in culture. Through transcriptional profiling of highly self-renewing human HSC and their differentiated or cultured progeny, we identified MLLT3 as a critical HSC regulator, enriched in human fetal and adult HSC, but downregulated during culture. Knockdown of MLLT3 disrupted the expansion and engraftment of hematopoietic stem/progenitor cells (HSPC). Conversely, sustaining MLLT3 expression in culture led to expansion of HSPC with multilineage differentiation ability and superior engraftment capacity. Engraftment of MLLT3-expressing cultured HSPC was 14-30 fold higher than controls; it was detected for 24 weeks and further maintained in secondary transplantation. MLLT3-expanded HSPC displayed repopulation of the HSPC compartment and all differentiated lineages in bone marrow and peripheral hematopoietic organs. Moreover, HSPC cultured for 2 weeks in presence of MLLT3 showed more effective human engraftment compared to those transplanted before culture expansion. Importantly, MLLT3 did not reprogram progenitors to HSC, or cause oncogenic transformation. Similar to endogenous MLLT3, overexpressed MLLT3 localized to promoters of expressed in HSPC, and stabilized HSC regulatory program in culture. MLLT3 maintained other key HSC regulators, such as HLF and MECOM, through direct binding, and indirectly suppressed abnormal activation of immune response and apoptosis genes in cultured HSPC. HSC genes in MLLT3 expanded HSPC featured higher levels of the transcriptional activation mark H3K79me2, deposited by the MLLT3 interaction partner Dot1L. MLLT3 thus represents a novel HSC maintenance factor that links histone reader and modifying activities to fine-tune HSC genes, and introduces a promising approach to culture HSC for therapeutic applications.

LATE-BREAKING POSTER PRESENTATIONS

3207 – CD97 IS ASSOCIATED WITH POOR OVERALL SURVIVAL IN Houda Alachkar,1; Vijaya Vaikari; Sharon Wu USC, Los Angeles, United States

In the era of precision medicine, the treatment of Acute Myeloid Leukemia (AML) remains a significant challenge with fewer than 50% of patients having long-term disease-free survival. Here we take advantage of the large genomics data to explore the expression of particular genes to establish the rationale for further investigation of these genes as viable therapeutic targets in AML. CD97, a member of the adhesion G -coupled receptor (GPCR) is expressed on leukocytes and smooth muscles. CD97 is also expressed in a variety of solid cancers and support a more aggressive metastatic phenotype. In this report, we analyzed 170 patients from the TCGA AML dataset and found that CD97 was higher in cytogenetically normal (CN-AML) patients compared with cytogenetically abnormal (CA-AML) patients (p=0.008). We dichotomized patients based on their CD97 mRNA expression Z-score (RNA Seq V2 RSEM) into CD97 high (Z≥1) and CD97 low (Z<1). Patients with high CD97 expression had significantly higher WBC count (median: 52.9 vs 12.6, p=0.005) and higher % bone marrow blasts (median: 83 vs 71%, p=0.025). We also analyzed CD97 expression according to mutational status and found that CD97 was significantly higher in patients with NPM1 mutation (n=47) compared with that in patients with NPM1 wild- type (n=123) (1.6 fold, p<0.000). Survival analysis showed that the overall survival (OS) of the CD97 high group (Z >1) was significantly shorter than those of the CD97 low patients (median: 7.2 vs. 21.1 months; p=0.0003; Figure 4A). Patients with high CD97 expression had significantly shorter event-free survival (EFS) than patients with low CD97 (median: 5.3 vs. 12 months; p=0.0003). In multivariate survival analysis, CD97 high expression (Z>1) was associated with shorter overall survival when adjusted for age, white blood count, cytogenetic risk, transplant status, DNMT3A mutation status, and TP53 mutation status (HR= 1.86; 95% Cl: 1.10-3.13; p=0.020). Altogether, our results suggest that high CD97 expression is associated with poor clinical outcome and indicate a need for future functional and mechanistic for the role of CD97 in AML. 3208 – REPROGRAMMED ADULT HUMAN ENDOTHELIUM INTO HEMATOPOIETIC STEM CELLS YIELDS FUNCTIONAL T CELLS IN VIVO Jose Gabriel Barcia Duran; Tyler Lu; Raphael Lis; Shahin Rafii Weill Cornell Medicine, New York, United States

During development, the hematopoietic stem cells that go on to populate the bone marrow and give rise to all blood cell lineages emerge from a specialized endothelial subpopulation. We have previously exploited this vestigial identity to achieve the direct conversion of adult mouse or human endothelial cells (ECs) into long- term engraftable hematopoietic stem and progenitor cells (rEC-HSPCs); however, to date, we had only detected and characterized functional T cells that result from the transplantation, engraftment, and differentiation of mouse rEC-HSPCs. Here, we make use of doxycycline-inducible vectors encoding our four reprogramming transcription factors (FOSB, GFI1, SPI1, and RUNX1; FGRS). Additionally, transplantation is performed in transgenic substrains of NSG mice (one carrying human stem cell factor, granulocyte/macrophage colony-stimulating factor, and interleukin 3; the other, human major histocompatibility complex class I as well as beta-2 microglobulin). We show that human rEC-HSPCs obtained via our established in vitro system yield phenotypically and functionally mature T cells in vivo at 24 weeks post-transplantation (primary and secondary) when FGRS expression is suspended upon transplantation and the transplant recipients express transgenes for human cytokines or MHC class I . Notably, the resulting T cells undergo TCR rearrangement and are able to clear viral particles one week post- LCMV infection. This achievement demonstrates that our direct conversion strategy generates bona fide human hematopoietic stem cells from adult endothelial cells.

3209 – SOMATIC MUTATIONS IN HEMATOPOIETIC STEM CELLS REVEAL LINEAGE RELATIONSHIPS AND AGE-RELATED MUTAGENESIS Arianne Brandsma1; Fernando Osorio2; Axel Rosendahl Huber1; Rurika Oka1; Mark Verheul1; Sachin Patel2; Lisanne de la Fonteijne3; Ignacio Varela4; Fernando Camargo2; Ruben van Boxtel1 1Princess Maxima Center for pediatric cancer, Utrecht, Netherlands; 2Stem Cell Program, Bosten Children's Hospital, Boston, United States; 3Center for Molecular Medicine, UMC Utrecht, Utrecht, Netherlands; 4IBBTEC, CSIC-University of Cantabria, Santander, Spain

Mutation accumulation during human life can contribute to hematopoietic dysfunction; however, the underlying dynamics are unknown. In addition, as people age clonal expansions of mutated stem cells within the blood more commonly occur, which is associated with increased risk of developing hematological malignancies. To obtain increased understanding into the processes driving mutation accumulation and the clonal composition of hematopoietic tissues within the normal human bone marrow, we catalogued and studied somatic mutations in individual hematopoietic stem cells and multipotent progenitors from healthy individuals of different ages. For this, we performed whole genome sequencing of clonally expanded primary cells derived from bone marrow and umbilical cord blood. We found that mutations accumulate gradually during life at a similar rate in these two populations with approximately 17 base substitutions per year, while insertions and deletions occur sporadically and at low numbers. The majority of mutations in adult stem and progenitor cells were acquired after birth and could be explained by the constant activity of various endogenous processes, which also explains the mutation load in acute myeloid leukemia (AML). For one donor, we sequenced 10 clones allowing us to construct a developmental lineage tree of human hematopoiesis by assessing mutations that are shared between each stem and progenitor cell. This analysis revealed a polyclonal architecture of the stem/progenitor compartment and provided evidence that developmental clones exhibit multipotency, though lineage biases can be detected. Our approach highlights novel features of human native hematopoiesis and its implications for leukemogenesis. 3210 – INHIBITION/LOSS OF ENDOTHELIAL MTOR DRIVES HEMATOPOIETIC STEM CELL AGING Jason Butler1,2,3; Pradeep Ramalingam4; Michael Poulos5; Elisa Lazarri5; Jason Butler6 1Hackensack University Medical Center, Nutley, United States; 2Georgetown University, Nutley, United States; 3Weill Cornell Medical College, Nutley, United States; 4Weill Cornell Medical College, New York, United States; 5HUMC, Nutley, United States; 6HUMC/Georgetown University/Weill Cornell, Nutley, United States

Aging leads to a decline in hematopoietic stem cell (HSC) function. In addition to a loss of their self-renewal potential, old HSCs exhibit a myeloid biased differentiation and an increased propensity to develop hematologic malignancies. Recent findings suggest that signals from the bone marrow (BM) microenvironment, in particular the BM vascular niche, play crucial roles in regulating HSC aging. In support of this idea, we recently discovered that aging of BM endothelial cells (BMECs) leads to an altered molecular crosstalk between the BMECs and HSCs that instructs HSC aging. We have previously demonstrated that AKT/mTOR activation within young BMECs supports balanced HSC self-renewal and differentiation. Based on these observations, we hypothesized that deficient AKT/mTOR signaling within endothelium will deprive the hematopoietic system of proper BMEC-derived instructive signals resulting in HSC aging. We have generated preliminary data demonstrating that aged BMECs, in contrast to aged HSCs, display decreased AKT/mTOR signaling. Furthermore, our data indicates that pharmacological inhibition of mTOR signaling using Rapamycin, a widely regarded rejuvenating agent and clinically utilized immunosuppressant, has deleterious effects on the hematopoietic system, in part due to its adverse impact on BMECs. To formally determine whether EC-specific inhibition of mTOR signaling can drive hematopoietic aging, we conditionally deleted mTOR in ECs (mTOR(ECKO)) and observed that HSCs from young mTOR(ECKO) mice displayed all the phenotypic and functional attributes of an aged HSC. expression profiling of HSCs isolated from young mTOR(ECKO) mice revealed that their intrinsic signature resembled aged HSCs, both at steady state and following primary transplantation, confirming the role of endothelial-derived instructive signals in governing HSC aging. Utilizing our model system, we have identified candidate pro-aging and pro-rejuvenation factors that we anticipate will lay the foundation for designing therapeutic strategies to rejuvenate the HSC microenvironment and improve overall healthspan.

3211 – NAD BOOSTING STRATEGIES ENHANCE HEMATOPOIETIC STEM CELL FUNCTION BY MODULATING MITOCHONDRIAL METABOLISM AND STRESS RESPONSE Vasco Campos1; Nicola Vannini2; Mukul Girotra3; Simone Ragusa2; Evangelos Stefanidis2; Dongryeol Ryu4; Aikaterini Semilietof2; Yannick Yersin5; Pernille Rainer4; Aimable Nahimana6; Wan-Chen Cheng7; Loic Tauzin8; Eija Pirinen4; Tatiana Petrova9; Ping-Chih Ho7; Michel Duchosal6; Dominique Vanhecke7; Bart Deplancke4; George Coukos10; Johan Auwerx4; Matthias Lutolf4; Olaia Naveiras11 1Institute of Bioengineering, EPFL, Lausanne, Lausanne, Switzerland; 2Ludwig Center for Cancer Research, UNIL, Lausanne, Switzerland; 3Ludwig Center for Cancer Research of the University of Lausanne, UNIL, Lausanne, Switzerland; 4Institute of Bioengineering, EPFL, Lausanne, Switzerland; 5Institute of Bionegineering, EPFL, Lausanne, Switzerland; 6Department of Oncology, Centre Hospitaler Universitaire Vaudois (CHUV), Lausanne, Switzerland; 7Ludwig Center for Cancer Research, Lausanne, Switzerland; 8FACS facility, Life Sciences (SV), EPFL, Lausanne, Switzerland; 9Swiss Institute for Experimental Cancer Research (ISREC), EPFL, Lausanne, Switzerland; 10Ludwig Cancer Center and Department of Oncology, Centre Hospitalier Universitaire Vaudois (CHUV), UNIL, Lausanne, Lausanne, Switzerland; 11Institute of Bionengineering, EPFL and Department of Oncology, CHUV, Lausanne, Switzerland Cellular metabolism is recently emerging as a potential regulator of stem cell fate (Sahin & Depinho, 2010; Suda, Takubo, & Semenza, 2011; Zhang et al., 2011), constituting a crucial regulator of the HSC pool (Gan et al., 2010; Gurumurthy et al., 2010; Nakada, Saunders, & Morrison, 2010). We have previously demonstrated that mitochondrial activity is a functional predictor of HSC engraftment and fate. Indeed, we found that overall function of the hematopoietic progenitor and stem cell compartment can be resolved by mitochondrial activity alone, as illustrated by the fact that cells having low mitochondrial activity (TMRMlow) can provide efficient long-term engraftment, while those having high mitochondrial activity (TMRMhigh) cannot engraft in lethally irradiated mice (Vannini et al., 2016).

Here we show that the modulation of mitochondrial metabolism, through the administration of the newly developed NAD boosting agent nicotinamide riboside (NR) (Belenky et al. 2007; Canto et al., 2012), expands hematopoietic progenitors while maintaining the LT-HSC pool in vivo. Both NAD boosters, NR and nicotimamide mononucleotide (NMN), improved stem cell function by decreasing the mitochondrial membrane potential, contrarily other NAD boosters, nicotinic acid (NA) and nicotinamide (NAM), did not affect hematopoietic progenitor compartments suggesting a major involvement of the NR/Nrk/NMN axis. We herewith demonstrate that this is due to an activation of the unfolded protein response (UPRmt) and mitophagy, as represented by an increased mito-nuclear protein imbalance in LT-HSCs and mCherry/GFP shift in Mito-QC mice supplemented with NR. Finally, we show how the NR-induced hematopoietic progenitor cell expansion dramatically improves survival and accelerates the blood recovery of HSC- transplanted mice.

Our work establishes for the first time a link between HSC self-renewal- UPRmt-mitophagy and unveils the potential of NAD boosting strategies to enhance the outcome of patients suffering from bone marrow insufficiency.

3212 – ACTIVATION OF Β-CATENIN SIGNALING IN MATURE OSTEOBLASTS VERSUS OSTEOBLAST PROGENITORS DEFINES A TRANSCRIPTIONAL AND MUTATIONAL PROFILE FOR THE TRANSFORMATION OF MDS TO AML Álvaro Cuesta-Domínguez1; Ioanna Mosialou1; Junfei Zhao1; Akihide Yoshimi2; Konstantinos Panitsas1; Richard Friedman1; Omar Abdel-Wahab3; Raúl Rabadán1; Stavroula Kousteni4 1Columbia University Medical Center, New York City, United States; 2Memorial Sloan Kettering Cancer Center, New York City, United States; 3Memorail Sloan Kettering Cancer Center, New York City, United States; 4Columbia University Medical Center, New York City , United States

Cells of the bone marrow stroma can have a pathogenetic role in the development of myelodysplasia (MDS), acute myeloid leukemia (AML) and transformation of MDS to AML. β-catenin activation in osteoblasts induces MDS progressing to AML in mice, seen in more than 30% of MDS and AML patients. We constitutively activated β-catenin in mature osteoblasts (βcat(ex3)OCN mice) and osteoblast progenitors (βcat(ex3)2.3Col1a1 mice). βcat(ex3)2.3Col1a1 mice develop MDS which rapidly transforms to AML characterized by multi-organ infiltration with myeloid blasts, hematopoietic deregulation and lethality by 6 weeks of age. However, βcat(ex3)OCN mice presented with thrombocytopenia, anemia and lymphocytopenia along with dysplasia in the erythroid and megakaryocyte lineage in the blood and bone marrow, and although they never developed more than 10% of blasts in the marrow they die by 6 weeks of age. These characteristics indicate the development of MDS in βcat(ex3)OCN mice. In agreement with this diagnosis, high throughput, targeted-DNA-sequencing of CD11b+/Gr-1+ myeloid cells from βcat(ex3)OCN mice, identified MDS-relevant mutations such as splice region variants in the histone methyltransferase Setdb1, and the tyrosine kinases Flt3 and Met. RNA-seq analysis revealed that genes related to cell cycle (cycD), and the oncogene c-myc regulatory cascade (Jund, Ccnd2, Max, Ddx3x and Id2) were potently upregulated in LSK cells from βcat(ex3)OCN mice. Spliceosome regulators, the most common genetic alterations in MDSpatients, Srsf5 and Prpf8 were downregulated in βcat(ex3)OCN LSK versus wild type LSK cells. Expression of the methyltransferases Kmt2a, Kmt2b and Ash1, which induce MLL rearrangement in AML, was increased in LSK cells from leukemic βcat(ex3)2.3Col1a1 mice as compared to LSK cells from MDS βcat(ex3)OCN mice. Notably, spliceosomal deregulation is associated with MLL-rearranged AML, suggesting that increased expressionof Kmt2a, Kmt2b and Ash1 may be crucial in the transformative mechanism leading from MDS in βcat(ex3)OCN mice to AML in βcat(ex3)2.3Col1a1 mice.

3213 – HIF1/2-EXERTED CONTROL OVER GLYCOLYSIS BUT NOT OXPHOS PATHWAYS IN HUMAN LEUKEMIC STEM/PROGENITOR CELLS IS NOT CRITICALLY IMPORTANT FOR THEIR METABOLIC STATE Alan Cunningham1,2; Albertus Wierenga1; Ayşegül Erdem1; Nuria Vilaplana-Lopera3; Ulrich Günther 3; Joost Martens4; Gerwin Huls1; Edo Vellenga1; Jan Jacob Schuringa1 1Department of Experimental Hematology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands, Groningen, Netherlands; 2Department of Cancer and Genomic Sciences, University of Birmingham, Birmingham, United Kingdom, Groningen, Netherlands; 3Department of Cancer and Genomic Sciences, University of Birmingham, Birmingham, United Kingdom, Birmingham, United Kingdom; 4Department of Molecular Biology, Faculty of Science and Medicine, Radboud Institute for Molecular Life Sciences, Radboud University Nijmegen, Nijmegen, The Netherlands, Nijmegen, Netherlands

Hypoxia inducible factors 1 and 2 (HIF1, 2) are transcription factors which regulate the homeostatic response to low oxygen conditions. Data relating to the importance of HIF1 and HIF2 in murine and human hematopoietic stem and progenitors is conflicting, in particular in hematological malignancies such as Acute Myeloid Leukemia (AML). We and others have shown that HIF1 and HIF2 control overlapping as well as distinct gene expression signatures in human CD34+ cells related to glycolysis and cell cycle control. Here, we investigated the chromatin binding profiles of HIF1 and HIF2 and linked that to transcriptional networks and the cellular metabolic state, both in healthy and leukemic human CD34+ cells. Chromatin immunoprecipitation (ChIP)-sequencing performed on oxygen-insensitive HIF1α and HIF2α mutants in K562 cells revealed that overlapping HIF1- and HIF2-bound loci were highly enriched for reactome pathway terms related to metabolism, particularly those involved in glucose metabolism, as well as chromatin organization and cellular response to stress. HIF1-specific binding profiles were enriched for the terms signalling by GPCR, cell cycle, and mRNA splicing. ChIPseq data was confirmed by ChIP-qPCRs indicating that glycolysis-related genes were controlled by both HIF1 and HIF2 in leukemic cell lines and primary AMLs, while in healthy CD34+ cells these loci were predominantly controlled by HIF1 but not HIF2. Transcriptome analysis on healthy CD34+ cells and leukemic cell lines cultured under hypoxia, expressing HIF1α and HIF2α mutants or HIF/ARNT CRISPR/Cas9 knockouts revealed that HIFs can control the vast majority of glycolysis-related genes, but not genes related to the TCA cycle or glutaminolysis. However, and in contrast to our initial hypotheses, knockout of HIF signalling in K562 cells did not affect growth, internal metabolite concentrations (1D NMR and 2D NMR flux analysis), glucose consumption or lactate production (spectrophotometry-based assays) under hypoxia. These data indicate that, while HIFs exert control over glycolysis but not OxPHOS pathways in human leukemic cells, this is not critically important for their metabolic state. 3214 – SELECTIVE HSC-ABLATION USING ANTI-CD117 ANTIBODY DRUG CONJUGATE ENABLES SAFE AND EFFECTIVE AUTOLOGOUS AND ALLOGENIC HEMATOPOIETIC STEM CELL TRANSPLANTATION Agnieszka Czechowicz1,2,3,4; Rahul Palchaudhuri5; Amelia Scheck1; Zhanzhou Li6; Yu Hu7; Jonathan Hoggatt8; Borja Saez8; Wendy Pang1; Michael Mansour9; Tiffany Tate8; Yan Yi Chan1; Emily Walck1; Gerlinde Wernig1; Judith Shizuru1; Philip Murphy10; Florian Winau7; David Scadden8; Derrick Rossi7 1Stanford University, Stanford, United States; 2Boston Children's Hospital, Stanford, United States; 3Dana Farber Cancer Institute, Stanford, United States; 4Harvard Medical School, Stanford, United States; 5Magenta Therapuetics, Cambridge, United States; 6National Institutes of Health, Bethesda, United States; 7Boston Children's Hospital, Boston, United States; 8Harvard University, Cambridge, United States; 9Massachusetts General Hospital, Boston, United States; 10National Institute of Allergy and Infectious Disease, Bethesda, United States

Hematopoietic stem cell transplantation(HSCT) can be used to replace virtually any blood or immune system, however the procedure has significant toxicity which comes primarily from the irradiation/chemotherapy conditioning needed to enable donor HSC engraftment. We have previously shown that competition with host HSC limits donor HSC engraftment, and that antibodies depleting host HSC can be safe alternatives. Over the years we and colleagues have generated several antibody-based conditioning strategies including antagonistic anti-CD117 antibodies and anti-CD45 antibody-drug conjugates(ADCs), however each has unique limitations hindering use and translatability. To overcome this, here we generated anti-CD117-ADCs by linking anti-CD117 antibodies to protein synthesis toxin saporin and show they can be optimal agents that enable near complete hematopoietic replacement. Specifically we found this conditioning was effective in syngeneic HSCT and in combination with transient immunosuppression in allogeneic HSCT. The CD117-ADC led to >99.9% depletion of host HSCs and enabled>99.9±0.1% engraftment of syngeneic donor murine bone marrow cells and>69.0±12.8% engraftment of syngeneic donor murine HSCs. The CD117-ADC in combination with transient immune suppression also induced robust mixed chimerism even in HLA-mismatched HSCT settings. Importantly and uniquely, this agent did not cause any significant toxicities and grossly spared red blood cells, platelets, and all major immune cells. No gross tissue toxicity was observed apart from mild transient transaminitis, and animals remained fertile and did not require transplantation to maintain hematopoiesis. CD117-ADCs provide the possibility of safe and effective autologous and allogeneic transplantation with a single-agent, one-time injection without major toxicities. As multiple anti-CD117 antibodies and ADCs are in development and being tested in clinical trials, such an approach may be rapidly translatable a range of patients with malignant and non-malignant diseases. Importantly, such agents also enable improved study of hematopoiesis as they allow HSC replacement without microenvironment injury.

3215 – MUTATIONS CONTRIBUTING TO CLONAL HEMATOPOIESIS ARE ASSOCIATED WITH POOR PROGNOSIS IN CHRONIC ISCHEMIC HEART FAILURE Lena Dorsheimer1; Birgit Assmus1; Tina Rasper2; Christina Ortmann1; Andreas Ecke1; Khalil Abou-El- Ardat1; Tobias Schmid3; Bernhard Brüne1; Sebastian Wagner1; Hubert Serve1; Jedrzej Hoffmann4; Florian Seeger1; Stefanie Dimmeler1; Andreas M. Zeiher1; Michael A. Rieger1 1University Hospital Goethe University Frankfurt, Frankfurt, Germany; 2Goethe University Frankfurt, Frankfurt, Germany; 3University Hospital Goethe University Frankfur, Frankfurt, Germany; 4German Center for Cardiovascular Research DZHK, Frankfurt, Germany Somatic mutations causing clonal expansion of hematopoietic cells (clonal hematopoiesis of indeterminate potential, CHIP) are increased with age and associated with atherosclerosis and inflammation. Age and inflammation are the major risk factors for heart failure, yet the impact of CHIP on heart failure in humans is entirely unknown. To assess the potential prognostic significance of CHIP in patients with chronic heart failure due to ischemic origin (CHF), we analyzed bone marrow mononuclear cells from 200 patients (pts) with CHF by deep targeted amplicon sequencing to detect the presence of CHIP and associated such with long-term prognosis in patients with CHF. The median age of the patients was 65 years, NYHA class 2 and left ventricular ejection fraction 31%. 45 mutations with a variant allele fraction >2% were found in 38 out of the 200 pts with CHF (18.5%). The somatic mutations most commonly occurred in the genes DNMT3A (13 cases), TET2 (9), KDM6A (4), BCOR (3) followed by ASXL, SF3B1, TP53 (2 each) and 10 other genes. None of the classical CHF baseline parameters differed significantly between CHIP and Non-CHIP carriers, except that CHIP carriers were older and suffered more frequently from hypertension. During a median clinical follow-up of 4.4 years, a total of 53 pts died, and 23 pts required hospitalization for heart failure. There was a significantly worse long-term clinical outcome for pts with either DNMT3A or TET2 mutations compared to Non-CHIP carriers. By multivariable Cox proportional regression analysis, death and rehospitalization for heart failure remained independently associated with the presence of TET2 or DNMT3A mutations (HR 2.11, 95% confidence interval 1.12;4.00), in addition to patient age (HR 1.04, 95% CI 1.01;1.07). Somatic mutations in hematopoietic cells, specifically in the most commonly mutated CHIP driver genes TET2 and DNMT3A, significantly contribute to the progression and poor prognosis of CHF. Future studies will have to address whether targeting specific inflammatory pathways may be valuable for precision medicine in pts with CHF carrying specific mutations encoding for CHIP.

3216 – MODELING THE EARLIEST EVENTS IN ACUTE MYELOID LEUKAEMIA USING HUMAN PLURIPOTENT STEM CELL DIFFERENTIATION Andrew Elefanty1,2,3; Freya Bruveris1; Elizabeth Ng1; Ali Motazedian1; Peter Kearne4; Edouard Stanley1; Constanze Bonifer4 1Murdoch Childrens Research Institute, Parkville, Australia; 2The University of Melbourne, Parkville, Australia; 3Monash University, Parkville, Australia; 4University of Birmingham, Birmingham, United Kingdom

Acute myeloid leukaemia is a highly heterogeneous disease caused by recurrent mutations in the transcription regulatory machinery that interfere with normal blood cell development. Besides the original driver mutation, that in many cases generates a pre-leukaemic cell type, other mutations are acquired during evolution of the disease. Such tumour heterogeneity leads to a scenario in which the major clone at relapse may have been only a minor component at diagnosis or may derive from remaining pre-leukaemic cells. We do not know at the global level (i) how the initial mutation sets the cells on the path to malignancy and (ii) how different types of mutations cooperate to generate overtly leukaemic cells. However, this knowledge is essential if we want to identify specific pathways required for tumour maintenance that could be therapeutically targeted. Therefore, we are engineering inducible leukaemogenic mutations into normal human pluripotent stem cell lines, and using haematopoietically differentiated progeny to address leukaemia pathogenesis. As a first step, we have generated a doxycycline-inducible RUNX1-ETO stem cell line that also carries SOX17 mCHERRY and RUNX1C GFP reporter genes, to enable the simultaneous observation of endothelia and haematopoietic progenitors. We demonstrate that dose-dependent induction of RUNX1- ETO increases the proportion of immature, clonogenic hematopoietic progenitors, with prolonged in vitro survival. This suggests an explanation for the long term persistence of cells carrying a RUNX1-ETO driver mutation. RNA seq analysis of these haematopoietic progenitors confirmed that RUNX1-ETO induction reduces myelopoiesis by down-regulating myeloid regulator genes such as CEBPA, shuts down cell cycle genes as well as repair genes such as BRCA1. Gratifyingly, a large number of up- and down-regulated genes identified by RNA seq are known RUNX1-ETO targets in patients. In future experiments we will examine the effects of introducing additional inducible secondary mutations into the RUNX1-ETO cell line.

3217 – LARGE DNA METHYLATION NADIRS ANCHOR CHROMATIN LOOPS MAINTAINING HEMATOPOIETIC STEM CELL IDENTITY Mira Jeong1; Xingfan Huang2; Xiaotian Zhang3; Jianzhong Su4; Muhammad Shamim5; Ivan Bochkov1; Jaime Reyes1; Haiyoung Jung1; Emily Heikamp1; Aviva Aiden1; Wei Li5; Erez Aiden1; Margaret Goodell1 1Baylor College of Medicine, Houston, United States; 2Rice University, Houston, United States; 3Van Andel Institute, Grand Rapids, United States; 4Baylor College mEDICINE, Houston, United States; 5Baylor College Medicine, Houston, United States

Higher order chromatin structure and DNA methylation are implicated in multiple developmental processes, but their relationship to cell state is unknown. Here, we found that large (~10kb) DNA methylation nadirs can form long loops connecting anchor loci that may be dozens of megabases apart, as well as interchromosomal links. The interacting loci comprise ~3.5Mb of the . The data are more consistent with the formation of these loops by phase separation of the interacting loci to form a genomic subcompartment, rather than with CTCF-mediated extrusion. Interestingly, unlike previously characterized genomic subcompartments, this subcompartment is only present in particular cell types, such as stem and progenitor cells. Further, we identify one particular loop anchor that is functionally associated with maintenance of the hematopoietic stem cell state. Our work reveals that H3K27me3-marked large DNA methylation nadirs represent a novel set of very long-range loops and links associated with cellular identity.

3218 – MPP3 IS A SECRETORY MULTIPOTENT PROGENITOR THAT REGULATES MYELOID DIFFERENTIATION Yoon-A Kang1; Jonathan Chen2; Hyojung Paik3; Siyi Zhang4; Matt Warr5; Rong Fan2; Emmanuelle Passegué1 1Columbia University Medical Center, New York, United States; 2Yale University, New Haven, United States; 3Korea Institute of Science and Technology Information , Daejeon, Republic of Korea; 4King's College London, London, United Kingdom; 5Gilead Sciences, Seattle, United States

The proliferation and differentiation rates of hematopoietic stem cells (HSCs) and lineage-biased multipotent progenitors (MPPs) MPP2, MPP3, and MPP4 tailor appropriate blood production on demand. Numerous studies have documented the critical functions of cytokines in regulating myeloid differentiation and the function of the hematopoietic stem and progenitor cell (HSPC) compartment. Recently, HSPCs were shown to secrete cytokines during stress hematopoiesis, although the producing population was not identified. Here, we show that myeloid-biased MPP3 has the most robust secretory activity among all HSPCs, correlating with a high volume of rough endoplasmic reticulum and up-regulation of unfolded protein response genes. MPP3 secretion is triggered by a variety of inflammatory stimuli acting through both NF- B and calcium signaling pathways. Importantly, we find that conditioned media containing stimulated MPP3-secreted cytokines promotes myeloid differentiation from HSCs, MPP3, and MPP4. Moreover, imaging of the bone marrow (BM) niche reveals that MPP3 locates nearby HSCs and MPP4, suggesting an autocrine and paracrine effect of MPP3-secreted cytokines. We also show that MPP3 expansion is a common feature of diverse myeloid malignancies and that leukemic MPP3 constitutively secrete cytokines that are different from the ones secreted upon inflammatory stimuli, suggesting a context-dependent MPP3 secretory activity. However, leukemic MPP3-secreted cytokines also promote HSCs, MPP3, and MPP4 proliferation, and stimulate myeloid differentiation from HSCs, indicating a conserved mechanism of MPP3 secretion in triggering myeloid cell production. Together, our results identify myeloid-biased MPP3 as the main secretory population in the HSPC compartment and discover a novel regulatory function of MPP3 in controlling myeloid differentiation. They reinforce the idea that HSPCs actively regulate their own functionality through the autocrine and paracrine effects of locally secreted cytokines, hence highlighting a previously underestimated role of the BM niche microenvironment in regulating myeloid cell production in stress and leukemic contexts.

3219 – ACCOUSTIC TWEEZER METHOD TO ASSESS DEFORMABILITY OF PATIENT- DERIVED ACUTE LYMPHOBLASTIC LEUKEMIA CELLS Hsiao-Chuan Liu1,2; Eun-Ji Gang3; Hye Na Kim4; Hae Gyun Lim5; Hayong Jong5; Ruimin Chen6; Kirk Shung7; Yong-Mi Kim4 1Department of Biomedical Engineering and NIH Ultrasonic Transducer Resource Center,, University of Southern California, Los Angeles, United States; 2Department of Pediatrics, Division of Hematology, Oncology, Blood and Marrow Transplantation, Children's Hospital Los Angeles, University of Southern California, Los Angeles, United States; 3Department of Pediatrics, Division of Hematology, Oncology, Blood and Marrow Transplantation, Children's Hospital Los Angeles, University of Southern California , Los Angeles, United States; 4Department of Pediatrics, Division of Hematology, Oncology, Blood and Marrow Transplantation, Children's Hospital Los Angeles, University of Southern California , Los Angeles, United States; 5Department of Biomedical Engineering and NIH Ultrasonic Transducer Resource Center, University of Southern California, Los Angeles, United States; 6Department of Biomedical Engineering and NIH Ultrasonic Transducer Resource Center, University of Southern California, Los Angeles, United States; 7Department of Biomedical Engineering and NIH Ultrasonic Transducer Resource Center, Biomedical Engineering, Los Angeles, United States

The role of cell mechanics in cancer cells is a novel research area revealing new mechanisms of therapy resistance. Previous studies have shown that solid tumor cells display great deformability during drug resistance. However, mechanical properties of ALL cells in particular in the context of chemotherapy resistance have not been widely studied. Single beam acoustic (SBA) tweezer is a novel and promising technology to quantify the mechanical phenotype of cells. Here, we describe a non-contact acoustic trapping method using a SAB tweezer for quantification of deformability of chemotherapy resistant ALL cells. We found that patient-derived (primary) drug resistant ALL cells change their deformability with increasing voltages of the SAB tweezer. Secondly, primary ALL cells resistant to chemotherapy treatment consisting of Vincristine, Dexamethasone and L-Asparaginase, are more deformable than untreated control ALL cells. We demonstrated that the acoustic trapping technology based on SBA tweezers is a promising label free tool for non-contact quantitative discrimination of mechanical phenotypes of cells.

3220 – CRISPR-DRIVEN MODELING OF CLINICALLY-RELEVANT GENETIC VARIANTS IN HEMATOPOIETIC STEM AND PROGENITOR-DERIVED ERYTHROID CELLS Josee Laganiere1,2; Yelena Boccacci1; Nellie Dumont1; Yannick Doyon2 1Hema-Quebec, Quebec, Canada; 2Laval University, Quebec, Canada Genome editing with engineered nucleases allows targeted genetic manipulations and holds great promise for the treatment of inherited and acquired disorders. In addition, faithful in vitro modeling of erythropoiesis to study specific genetic variants would be a useful tool for investigating gene function and help guide the management of complex clinical cases in the field of transfusion medicine. However, the relatively low efficiency of editing in primary cells often hampers the creation of such models as well as the development of therapies.

In this work, we combined an HSC-erythroid differentiation protocol with a robust genome editing protocol to develop a cellular model showcasing desired genetic variants.

We used a three-step culture method to efficiently produce cultured red blood cells (cRBC) from hematopoietic stem and progenitor cells (HSPC). This erythroid-differentiation procedure yielded an average of 20, 000 fold expansion and ~90% enucleation by day 18 when using mobilized peripheral blood HSPCs as a cell source. In this setting, we achieved up to 70% of precise gene modification at the HBB in cell populations with 40% of the cells harboring the sickle cell anemia-causing mutation (HbS) at both alleles.

In summary, the virus-free CRISPR-based editing strategy presented here reached high efficiencies for the desired gene modification in HPSC-derived cultured RBCs. This proof-of-concept paves the way for the modeling of other genetic variants of clinical significance and may help decipher their specific role in physiology and disease.

3221 – TWIST1/DNMT3A COMPLEX REGULATES METHYLATION OF KEY CELL CYCLE GENES THAT CONTRIBUTE DAC SENSITIVITY OF MDS CELLS Xiang Li1; Hongjiao Li2 1College of Life Science, Northwest University, Xi'an, China (People's Republic); 2 Northwest University , Xi'an, China (People's Republic)

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of myeloid neoplasms disorders which characterize by peripheral cytopenia, dysplasia, and a variable disease course. Increasing evidences suggest that epigenetic mechanisms, such as DNA hypermethylation, have been invoked as a central factor in MDS pathogenesis, however, the underlying mechanisms are unclear. Our previous work had showed TWIST1, a basic helix-loop-helix transcription factor, is increased in advanced MDS precursor cells and negatively regulates apoptosis by inhibiting the pro-apoptotic protein p53, meanwhile TWIST1 expression can be regulated by microRNA10a/10b and inhibition of miR10a/10b in clonal cells interfered with proliferation and enhanced sensitivity to apoptosis, which all demonstrate that TWIST1 plays a significant role in MDS pathogenesis and progression. In this study, we found that up-regulation of TWIST1 correlated with the increased expression of DNMT3a, a de novo DNA methyltransferase which commonly contributed to DNA methylation in hematological tumor cells. Co-immunoprecipitation (Co-IP) and pull-down assay showed that TWIST1 directly interacted with DNMT3a both in vivo and in vitro. Methylation microarray analysis showed the TWIST1 targeting genes containing E-boxes are significantly highly methylated in KG1a-TWIST1 cells. We indicated that TWIST1 can recruit DNMT3a and enhance DNA methylation on the promoter of CDK inhibitors CDK1A and CDK1C, two targeting gene of TWIST1, resulting in less arrest of G0/G1 phase in KG1a-TWIST1 cells under DAC treatment. The clinical data also indicated TWIST1 negatively regulated CDK1A and CDK1C in the hematopoietic cell from patients with MDS compared to healthy donors. Overexpression of TWIST1 in primary CD34+ cells from cord blood resulted in lower expression level of CDK1A and CDK1C, and more clonal expansion and more drug resistance to DAC. Taken together, our data clearly indicated TWIST1/DNMT3a complex is involved in alteration of DNA methylation status of CDK1A and CDK1C that interfere the arrest of G0/G1 phase to change the sensitivity to DAC treatment. 3222 – THE PLACENTA IS A SITE FOR DEFINITIVE HSC EMERGENCE Guixian Liang1; Yifang Zhang2; Suwei Gao2; Yuanyuan Xue2; Lu Wang3; Feng Liu3 1Institute of Zoology, Chinese Academy of Sciences, beijing, China (People's Republic); 2 Institute of Zoology, Chinese Academy of Sciences, Beijing, China (People's Republic); 3Institute of Zoology, Chinese Academy of Sciences, Beijing, China (People's Republic)

During mammalian embryogenesis, hematopoietic stem cells (HSCs) are formed and expanded during a narrow time window at multiple embryonic sites, such as the yolk sac (YS), aorta-gonad-mesonephros (AGM) and fetal liver (FL), which have been well demonstrated to function as hematopoietic organs. Recently, the placenta has been implicated as a newly identified source of hematopoietic stem and progenitors (HSPCs). However, it is still incompletely understood whether and how the placenta contributes to the generation and/or expansion of HSPCs. Here we use surface markers to purify endothelial cells, Type 1 pre-HSCs and Type 2 pre-HSCs in the AGM and placenta and HSCs in the FL. RNA-seq analysis demonstrated that placental pre-HSCs show unique features in signaling pathway and transcription factor network, as well as similar features to AGM pre-HSCs, which implies that the placenta may have the capacity to generate HSCs through the endothelial-to-hematopoietic transition. Moreover, we used LY6a- GFP transgenic mice and three-dimensional (3D) confocal imaging to characterize the newly emerging HSPCs located within the vasculature in chorioallantoic plate. Importantly, we showed that the proportion of placenta HSPCs and their ability of colony formation were decreased in Rbpj deficient mice. Taken together, our data support that the placenta is a de novo site for HSC emergence and that Notch signaling participates in placenta definitive hematopoiesis. These results will inform future efforts to utilize placental blood cells for clinical application. Key words: Placenta, Hematopoietic Stem Cell, Endothelial-to-Hematopoietic Transition, Notch

3223 – PERTURBATION OF DEVELOPMENTAL HEMATOPOIESIS IMPACTS NEONATAL IMMUNE FUNCTION IN THE LUNG Diego Lopez1; Gloria Hernandez2; E. Camilla Forsberg3; Anna Beaudin4 1University of California, Merced, Merced, United States; 2University of California Los Angeles, Los Angeles, United States; 3University of California Santa Cruz, Santa Cruz, United States; 4Univeristy Of California Merced, Merced, United States

During fetal hematopoiesis, transient hematopoietic progenitors produce waves of discrete immune cells that persist into adulthood and contribute to the adult immune system. We have previously identified a developmentally-restricted hematopoietic stem cell (drHSC) that exists within a limited window of perinatal development and specifically gives rise to innate-like lymphocytes, including B1-B cells and γδ-T-cells. The discovery of a transient precursor that generates a distinct component of the adult immune system defines a “critical window” of immune development during which perturbation may result in altered immune function in the adult. As proof of principle of this critical window, we have demonstrated that maternal immune perturbation via a single low-dose injection of poly (i:c) at mid-gestation induces lasting changes to the hematopoietic and immune compartment in offspring. Specifically, maternal immunostimulation causes expansion of the drHSC population in neonates and its inappropriate persistence into adulthood. Furthermore, neonates also exhibit increased cellularity of innate-lymphocytes, including B1-B cells and splenic MZ cells, that are normalized by adulthood. In order to directly investigate how perturbation during this critical developmental window drives susceptibility to immune dysfunction, we examined underlying changes to the lung immune compartment and accompanying susceptibility to airway dysfunction. Developmental perturbation resulted in remodeling of the neonatal lung immune compartment, with specific increases in NKT cells, eosinophils, and most notably, ILC2s. ILC2s have recently been recognized as potent producers of type-2 cytokines, including IL-5 and IL-13, that are important for eosinophil recruitment, activation and goblet cell hyperplasia during allergic airway inflammation. Surprisingly, developmental perturbation also induced heightened production of IL-5 and IL-13 in lung ILC2s in response to in-vitro stimulation with PMA and ionomycin. Current experiments are aimed at assessing how alterations in ILC2 functionality, in response to developmental perturbation, underlie susceptibility to allergic airway disease development.

3224 – TYPE I INTERFERONS DRIVE HSC COLLAPSE DURING SHOCK-LIKE INFECTION VIA INTRINSIC CASPASE-INDEPENDENT CELL DEATH AND ENVIRONMENTALLY- ENFORCED QUIESCENCE Kate MacNamara1,1; Julianne Smith2; Jackson Maloney1; Hui Jin Jo1 1Albany Medical College, Albany, United States; 2Case Western University, Cincinnati, United States

Tick-borne infections represent important emerging infections, and are often associated with severe cytopenias, though mechanisms of hematologic dysfunction are not well understood. In a model of lethal shock-like infection caused by tick-borne ehrlichiae we found type I IFNs induced severe bone marrow (BM) aplasia, reduced hematopoietic stem and progenitor cells (HSC/HSPCs), and blunted emergency myelopoiesis. In the absence of type I IFN signaling, BM and splenic hematopoiesis were increased and HSCs were functionally superior. Type I IFNs impaired infection-induced hematopoiesis by both limiting HSC/HSPC proliferation and increasing HSPC death. Type I IFNs restricted proliferation indirectly, whereas HSPC death occurred via direct IFNαR-mediated signaling. Sterile inflammation drives IFNαR- and caspase- 3-dependent HSPC apoptotic cell death. In contrast, infection caused caspase-independent and RIPK1- dependent cell death. RIPK1 antagonism with Necrostatin 1 rescued both HSPC and HSC numbers during acute infection. Early antibiotic treatment is essential to protect against infection-induced death in this infection model. Despite improved survival antibiotic treated mice exhibited severely reduced HSPCs and HSCs, demonstrating a profound long-term impact of infection on the hematopoietic compartment. Combination therapy with antibiotics and Necrostatin 1 improved HSPC and HSC numbers and reduced bacterial burden in surviving mice. We identify a pathologic role for type I IFNs in infection-induced hematopoietic collapse via increased HSC quiescence and HSPC cell death, and reveal a strategy to ameliorate the long-term effects of infection-induced shock on hematopoiesis, which may improve outcomes in patients recovering from serious infections.

3225 – ENHANCEMENT OF DEFINITIVE HEMATOPOIESIS FROM HUMAN PLURIPOTENT STEM CELLS THROUGH ACTIVATION OF ARTERIAL PROGRAM IN HEMOGENIC ENDOTHELIUM Mi Ae Park1; Akhilesh Kumar2; Ho Sun Jung2; Gene Uenishi2; Oleg Moskvin2; James Thomson2; Igor Slukvin2 1National Primate Research Center, University of Wisconsin, Madison, United States; 2University of Wisconsin, Madison, United States

Our recent studies in human pluripotent stem cells (hPSCs) have demonstrated a direct link between arterial- type hemogenic endothelial (HE) progenitors and definitive hematopoiesis, in addition to the critical role of NOTCH signaling in specification of CD144+CD43-CD73-DLL4+ arterial-type HE (Uenishi et al., 2018). Here, we explore whether arterial program activation can be used as a strategy to enhance definitive hematopoiesis from hPSCs. In embryo, arterial fate is specified following induction of DLL4 expression (Chong et al., 2011), which is initiated by signaling through an arterial-specific enhancer located within the third intron of DLL4 that is controlled by ETS factors (Sacilotto et al., 2013; Wythe et al., 2013). To evaluate whether activation of arterial-specific enhancer affects arterial programming of HE and hematopoiesis from hPSCs, we engineered H1 human embryonic stem cells (hESCs) carrying doxycycline (DOX)-inducible ETS1 transgene and differentiated them to endothelial and hematopoietic cells in chemically defined conditions. We found that ETS1 overexpression lead to arterial HE formation with CD144+CD43-CD73- DLL4+CXCR4+/- phenotype, T and B lymphoid and definitive erythroid potentials. In addition, we revealed that arterial HE specification and definitive hematopoiesis from hPSCs could be enhanced through modulation of MAPK/ERK signaling with small molecules. The effects of ETS1 and MAPK/ERK on arterial programming were primarily mediated through NOTCH signaling. Additionally, we uncovered interplay between ETS1 and MAPK/ERK pathways by demonstrating the role of ERK in controlling ETS1 activity during arterial HE development. Together, these findings demonstrate that promotion of arterial specification in cultures can be used as novel strategy to enhance lymphopoiesis and eventual HSC production from hPSCs.

3226 – BIOLOGIC TO SELECTIVELY TARGET CRLF2+ B CELL PRECURSOR LEUKEMIA WHILE SPARING NORMAL B CELL DEVELOPMENT Kimberly Payne1,2; Cornelia Stoian3; Jacqueline Coats1; Hossam Alkashgari1; Ineavely Baez1; Hannah Choi1; WayAnne Watson1; Omair Kamal1; Benjamin Becerra1; Rishikesh Chavan1; Shadi Farzin-Gohar1; Sinisa Dovat4; Xianmei Meng5 1Loma Linda University, Loma Linda, United States; 2Elf Zone, Inc., Loma Linda, United States; 3Loma Linda University, Loma Linda , United States; 4Penn State University College of Medicine, Hershey, United States; 5Elf Zone, Inc. , Loma Linda, United States

Approximately half of high-risk Ph-like B cell precursor acute lymphoblastic leukemia (B-ALL) arises due to overexpression of CRLF2. CRLF2 pairs with the IL-7Ra to form a receptor that is activated by the cytokine, TSLP. Binding of TSLP induces activation of JAK/STAT5 and PI3/AKT/mTOR, two pathways that promote proliferation and survival of leukemia cells. To study the role of this cytokine during in vivo production of normal and malignant B cell production we developed a PDX model that expresses human TSLP (mouse TSLP does not activate human CRLF2). PDX generated from normal cord blood CD34+ cells showed a 3-6 fold increase in B cell precursor production when circulating human TSLP was present at either normal or high physiological levels. Surprisingly, while CRLF2+ B-ALL from patients cells grew robustly in PDX with normal circulating levels of human TSLP, they were essentially eliminated in PDX with high physiological levels of TSLP. A potential mechanism for the anti-leukemia effects of high-dose TSLP is upregulation of the Suppressor of Cytokine Signaling (SOCS) genes. SOCS genes encode a family of proteins (SOCS1-7 and CISH) that regulate cytokine signaling through multiple mechanisms including ubiquitin-mediated cytokine receptor degradation. We found that SOCS1-SOCS3 and CISH mRNA were upregulated in primary CRLF2 B-ALL cells cultured with high dose TSLP. CRLF2+ B-ALL cells cultured with TSLP showed a dose-dependent loss of CRLF2 signaling as indicated by their inability to induce STAT5 or S6 phosphorylation following TSLP stimulation. This loss was correlated with the loss of surface IL-7Ra and maintained for 24-48 hours following a pulse of high-dose (but not low-dose) TSLP. These data provide evidence that TSLP exerts its anti-leukemia effects by shutting down CRLF2-mediated signals and suggest that these effects are at least partially mediated by the loss of the IL-7Ra component, and potentially through SOCS family proteins. These studies identify the human TSLP cytokine as a potential biologic therapy to treat CRLF2 B-ALL while maintaining normal B cell production. Supported by 1R01CA209829 and R43CA224723. 3227 – UBIQUITOUS OVEREXPRESSION OF CXCL12 CONFERS RADIATION PROTECTION AND ENHANCES MOBILIZATION OF HEMATOPOIETIC STEM AND PROGENITOR CELLS Smrithi Rajendiran1,2,3,4; Stephanie Smith-Berdan4; Camilla Forsberg4 1Institute for the Biology of Stem Cells, Santa Cruz, United States; 2Biomolecular Engineering Department, Santa Cruz, United States; 3School of Engineering, Santa Cruz, United States; 4UC Santa Cruz, Santa Cruz, United States

Stromal cell-derived factor alpha, or CXCL12, is one of the most widely studied chemokine niche factors and a major regulator of hematopoietic cell trafficking. There is substantial evidence for roles of CXCL12 in cell proliferation, survival and homing. Most studies have focused on homeostatic levels of CXCL12 or on loss-of-function models. Complete loss of CXCL12 and its receptor CXCR4 leads to both hematopoietic and non-hematopoietic dysfunction and embryonic lethality. Cell type-specific CXCL12 deletion in distinct bone marrow stromal cells has differential effects on hematopoietic stem and progenitor cell (HSPC) maintenance and function. This suggests that HSPC location with respect to CXCL12 gradients, and different CXCL12 producing cells, are important regulators of the HSPC homeostasis. Our lab has previously shown that CXCL12 expression increases robustly after total body irradiation, but the functional consequences of cell- selective CXCL12 upregulation are unknown. To test the role of increased levels of CXCL12, we generated a new dox-inducible transgenic mouse model that enables global or cell-specific manipulation of CXCL12 levels. Ubiquitous CXCL12 overexpression led to an increase in multipotent progenitors in the bone marrow and myeloid progenitors in the blood. Increased CXCL12 expression also conferred radioprotection of HSPCs compared to control mice. Cell cycle analysis showed that a higher fraction of HSPCs in the CXCL12 overexpressing mice reside in the G0-G1 phase; thus, CXCL12 overexpression promoted HSPC quiescence. Additionally, by disrupting the CXCL12-CXCR4 interaction with AMD3100, we observed higher mobilization of HSPCs into the blood in the CXCL12 overexpressing mice compared to controls. Together, our findings demonstrate that ubiquitous overexpression of CXCL12 not only provides radiation protection of HSPCs, but also facilitates AMD3100-mediated mobilization to the blood. Future experiments using cell type-specific CXCL12 expression will provide new insights on how CXCL12 gradients affect hematopoietic function.

3228 – CYTOKINE-INDUCED REGULATORY CIRCUITS IN THE CONTROL OF EARLY HEMATOPOIETIC STEM CELL FATE DECISIONS Michael A. Rieger Goethe University Frankfurt, Department of Medicine, Hematology/Oncology, Frankfurt am Main, Germany

Life-long blood regeneration relies on hematopoietic stem cell (HSC) maintenance, integrity and renewal. However, despite of the importance of a balanced and protected HSC pool, a continuous and adequate differentiation into all blood cell lineages, dependent on the demand, is equally important. Both stem cell fate decisions, self-renewal and differentiation, are orchestrated by extrinsic signals and intrinsic molecular programs. We are interested in the molecular control of these early cell fate decisions, how extrinsic cues are integrated in the maintenance of stable HSC decision programs, and how these programs are altered in malignancy. Long-term time-lapse microscopy-based cell tracking combined with molecular studies and in vivo examinations allow us to link molecular control with future cell fate. Hematopoietic cytokines are essential for hematopoiesis, controlling survival, proliferation, lineage choice and maturation of multipotent cells. We investigated molecular programs that are triggered by cytokine signaling in HSCs, and that switch the fate of HSCs from stemness into differentiation. We have identified several key regulators including members of the Growth Arrest and DNA-damage inducible 45 (Gadd45) family, and microRNAs (e.g. miR-193b) that are downstream of cytokine signaling in HSCs, but can also be induced upon genotoxic stress. These molecular pathways restrict HSC numbers and act as important regulators of excessive HSC self-renewal and leukemia suppressors. Consequently, we recently demonstrated that the absence of miRNA-193b is detrimental for the prognosis and outcome of acute myeloid leukemia, and that miR-193b may serve as advanced therapeutics in the future. Understanding early steps in differentiation will help to manipulate HSC fate for getting closer to the long- sought goal of ex vivo HSC maintenance and expansion for regenerative medicine, and to target misregulated self-renewal in leukemia.

3229 – PIEZO1-SENSITIVE BIOMECHANICAL PULSATION STIMULATES LONG-TERM HEMATOPOIETIC STEM CELL FORMATION Dhvanit Shah1,2; Giorgia Scapin1; Jennier Cillis1 1The Research Institute at Nationwide Children's Hospital, Columbus, United States; 2Ohio State University College of Medicine, Columbus, United States

The birth and development of hematopoietic stem cells (HSCs) remain a mystery. During fetal development, a subset of endothelial cells transitions to become HSCs in the aorta-gonad-mesonephros (AGM) region. Blood flow-mediated shear stress and activation of nitric oxide synthase (NOS) were demonstrated to stimulate the endothelial-to-HSC transition in the AGM. However, we showed that malbec (bw306), a zebrafish mutant for cadherin 5, produces HSCs despite circulation arrest and the inhibition of NOS, suggesting that other biomechanical forces, mechanosensation pathways, or epigenetic mechanisms might regulate HSC formation. Using zebrafish, murine, and human models, we show that Piezo1-sensitive biomechanical stretching of hemogenic endothelial cells enhances Dnmt3b expression for long-term (LT)- HSC formation.

Our microangiography and time-lapse confocal imaging established that cdh5-MO embryos have a heartbeat and pulsation in blood vessels despite the absence of blood flow. We also employed light sheet and time- lapse confocal microscopy followed by Fourier transform analyses to establish that although pulsation is independent of blood flow in the AGM, it is concurrent with the endothelial-to-hematopoietic transition. To establish the functional link between pulsation and HSC formation, we developed a bioreactor simulating the pulsating blood vessel conditions. We found that the biomechanical stretching of hemogenic endothelial cells or the pharmacological activation of Piezo1 yields three times higher amounts of LT-HSC formation; which reconstitute to multi-lineage adult blood upon serial transplantation. Our gene-silencing, explant culture, and computational analyses further demonstrated that biomechanical stretching activates Piezo1; which enhances epigenetic regulator Dnmt3b expression to stimulate the endothelial-to-HSC transition.

Our results demonstrate how pulsation-mediated biomechanical forces stimulate cell-fate transitions and stem cell formation by activating mechanosensitive channels as well as epigenetic machinery. We have established a bioreactor, pharmacological target, and the epigenetic mechanism to stimulate and scale-up LT- HSC formation.

3230 – WNT5A FROM OSTERIX-EXPRESSING OSTEOLINEAGE CELLS REGULATES THE AMPLITUDE OF ACTIN-ASSEMBLY RESPONSES IN HEMATOPOIETIC STEM CELLS Theresa Sippenauer1; Christina Schreck2 1Klinikum rechts der Isar der TU München, Munich, Germany; 2Klinikum rechts der Isar, Munich, Germany Theresa Sippenauer1,8, Christina Schreck1, Rouzanna Istvanffy1, Christoph Ziegenhain2, Beate Vieth2, Steffen Massberg3, Marieke Essers4, Claudia Waskow5, Hartmut Geiger6, Matthias Schiemann7, Christian Peschel1, Wolfgang Enard2, and Robert A.J. Oostendorp1

1. 3rd Department of Internal Medicine, Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany.

2. Anthropology and Human Genomics, Department Biology II, Ludwig-Maximilian- Universität, Munich, Germany.

3. Department of Internal Medicine I, Ludwig-Maximilian-Universität, 81377 Munich, Germany.

4. German Cancer Research Center (DKFZ) and Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM), 69120 Heidelberg, Germany

5. Regeneration in Hematopoiesis and Animal Models in Hematopoiesis, Institute for Immunology, TU Dresden, 01309 Dresden, Germany

6. Institute of Molecular Medicine, University of Ulm, 89081 Ulm, Germany

7. Department of Medical Microbiology, Immunology, and Hygiene, Technische Universität München, 81675 Munich, Germany.

8. Presenting author.

We have previously shown that factors secreted by bone-forming cells regulate the maintenance and behavior of hematopoietic stem cells (HSCs). Here, we show that full expression of one such factor: Wnt5a is required to regenerates functional HSCs, which successfully engraft in secondary recipients. Thus, HSCs regenerated in a Wnt5a-haploinsufficient bone marrow are dysfunctional. RNA sequencing of the donor Lin- SCA1+ KIT+ (LSK) cells regenerated in Wnt5a-mutant recipients shows dysregulated expression of ZEB1- target genes involved in the small GTPase-dependent actin polymerization pathway. Misexpression of DOCK2, WAVE2, and activation of CDC42 results in apolar F-actin localization, leading to defects in adhesion, migration and homing of HSCs regenerated in a Wnt5a haplo-insufficient microenvironment. Similar defects are observed in HSCs isolated from mice in which Wnt5a-expression is knocked out in the context of Osterix+ osteolineage cells. Moreover, in single cell cultures, the dysregulated HSCs cells show increased differentiation in vitro, with rapid loss of HSC-enriched LSK cells. Our study further shows that the Wnt5a-haploinsufficient environment similarly affects BCR-ABLp185 pre-leukemic cells, which fail to develop leukemia in 42% of the studied Wnt5a+/- recipients, or to transfer leukemia to secondary hosts. Thus, we show that expression of Wnt5a in bone marrow-resident osteolineage-like cells is required to regenerate HSCs and generate leukemia-initiating cells with functional ability to assemble F-actin and engraft successfully.

3231 – ONCOGENIC COOPERATION BETWEEN TET2 LOSS-OF-FUNCTION AND RTK ACTIVATION IN HAEMATOLOGICAL DISEASE Erinn Soucie1,2; Aurélien Griffon3; Michael Weber4; Olivier Bernard5; Patrice Dubreuil3 1Cancer Research Centre of Marseille, Inserm U1068, Marseille, France; 2Cedex 09, Marseille, France; 3CRCM, Inserm U1068, Marseille, France; 4CNRS UMR7242 Biotechnology and Cell Signalling ESBS, ILLKIRCH, France; 5IGR, Inserm, Villejuif, France

TET2 mutations are prevalent in a variety of human haematological malignancies. While Tet2 and its role in the dynamic regulation of DNA methylation is thought to be important in mammalian development and during blood cell differentiation, it’s not clear how disruption of this epigenetic function cooperates with different driver mutations that can lead to disparate disease phenotypes. Tet2 loss-of-function mutations and activating mutations in the cKit receptor (KitD816V) are correlated with aggressive mastocytosis in humans. These mutations are also sufficient to drive a similar disease phenotype in mice. Epigenetic and expression profiling show that changes at developmental genes due to Tet2 loss-of-function in mast cells are largely rescued upon expression of KitD816V. In this context, DNA methylation changes appear impact on the regulation immune response genes including those downstream of oncogenic cKit and implicated in the abnormal immune activation of mast cells that underlies the aggressive nature of this disease. Moreover, these same mechanisms may contribute to the Associated Clonal Hematologic Non-Mast Cell-Disease (AHNMD) that often occur in parallel in patients with mastocytosis and where affected myeloid and progenitor cells also carry the TET2 mutation.

3232 – NOTCH SIGNALING IN ARTIFICIAL THYMIC ORGANOIDS RECAPITULATES MURINE T CELL DEVELOPMENT IN VITRO Victoria Sun1; Amelie Montel-Hagen2; Chris Seet3; Gay Crooks4 1Molecular Biology Institute, UCLA, Los Angeles, United States; 2Department of Pathology and Laboratory Medicine, David Geffen School of Medicine (DGSOM), UCLA, Los Angeles, United States; 3Division of Hematology–Oncology, Department of Medicine, DGSOM, UCLA, Los Angeles, United States; 4Department of Pathology and Laboratory Medicine, and Division of Pediatric Hematology–Oncology, Department of Pediatrics, DGSOM, and Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, and Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, United States

In vitro models of T cell differentiation have revolutionized thymopoiesis studies; however, the field continues to seek novel approaches to generate mature T cells efficiently. We recently described the artificial thymic organoid (ATO), a novel serum-free, three-dimensional (3D) culture system that supports efficient in vitro differentiation of human hematopoietic stem and progenitor cells (HSPCs) into mature and functional T cells. Now, we demonstrate that mouse HSPCs cultured in ATOs efficiently progress through the T cell developmental stages observed in the murine thymus. Furthermore, murine ATOs produce mature CD3+TCR-γδ+ cells and CD3+TCR-αβ+ single-positive CD8+ or CD4+ cells after three weeks of culture. Murine ATO-derived single-positive T cells expressed a broad repertoire of TCR Vβ chains similar to that of murine thymic single-positive T cells. As proper Notch signaling is crucial for T cell development, we used a transgenic Notch reporter (TNR) mouse to compare Notch activity during thymopoiesis in the murine thymus and ATO. In both systems, we observed highly active Notch signaling in early thymic progenitors (ETPs) that markedly subsided after cells committed to the T cell lineage. Our results indicate that the murine ATO system induces Notch signaling in a manner similar to that in the thymus, thereby instructing progenitor cells to undergo T cell commitment. Thus, the technically simple and efficient murine ATO system is a promising platform to study T cell engineering and Notch regulation during T cell maturation. 3233 – INHIBITION OF RECEPTOR-MEDIATED ENDOCYTOSIS IN LEUKEMIA- PROPAGATING CELLS AS AN EFFECTIVE TREATMENT FOR ACUTE LEUKEMIA Cedric Tremblay1; Sung Kai Chiu1; Jesslyn Saw1; Stefan Sonderegger1; Ngoc CHAU2; Adam McCLUSKEY3; Phillip Robinson2; Richard Lock4; Stephen Jane5; David Curtis1 1Australian Centre for Blood Diseases (ACBD) - Monash University, Melbourne, Australia; 2Children's Medical Research Institute, The University of Sydney, Sydney, Australia; 3University of Newcastle, Newcastle, Australia; 4Children’s Cancer Institute, The University of New South Wales, Sydney, Australia; 5Central Clinical School, Monash University, Melbourne, Australia

Current therapeutic regimen transiently reduce the tumour burden of patients with T-cell acute lymphoblastic leukemia (T-ALL), but fail to eliminate refractory leukemia-propagating cells (LPCs), which are responsible for relapse. Hence, the eradication of LPCs will be determinant for achieving long-term remission. These resistant LPCs are dependent on the growth factors produced by the microenvironment to develop and survive following chemotherapy treatment. Using the Lmo2 transgenic mouse model, we have shown previously that these LPCs have long-term self-renewal potential and resistance to chemotherapy. We have also shown that growth factors produced by the thymic niche, like IL-7 and Notch1, are crucial for the development and maintenance of these resistant LPCs during the early stage of the disease. In thymocytes, the activation of these signalling pathways is tightly controlled by endocytosis, which is dependent on the GTPase Dynamin 2 (DNM2). We found that treatment of Lmo2 transgenic DN3 T-cell progenitors with the specific DNM2 inhibitor Dynole 34-2 blocked IL-7R and Notch1 internalization, which prevents downstream activation of Stat5 and expression of Hes1, confirming that DNM2 activity is essential for cytokine-mediated response in thymocytes. Inhibition of endocytosis with Dynole 34-2 significantly reduced the frequency and the engraftment of LPCs in transplantation assays. We then treated 2 month-old mice to address the importance of niche-mediated signalling in therapeutic resistance. Accordingly, we found that treatment with Dynole 34- 2 sensitized LPCs to the current induction regimen used in the clinic for T-ALL. Finally, inhibition of DNM2 activity significantly delayed leukemia onset in mice treated with standard therapeutic agents, suggesting that endocytosis is required for the maintenance of treatment-resistant T-ALL. These results provide the most convincing in vivo evidence that endocytosis is crucial for the maintenance and therapeutic resistance of LPCs. Targeting endocytosis may represent an attractive therapeutic strategy for improving cure rates in acute leukemias.

3234 – DNMT1 IS REQUIRED FOR HEMATOPOIETIC STEM CELL DEVELOPMENT THROUGH NEGATIVE REGULATION OF DNMT3 IN ZEBRAFISH Lu Wang1; Feng Liu2 1Institute of Zoology, Chinese academy of Sciences, Beijing, China (People's Republic); 2IOZ, CAS, BEIJING, China (People's Republic)

DNA methylation is important for hematopoietic stem cell (HSC) self-renewal and differentiation, but the mechanism is not yet fully understood. Here, we take advantage of zebrafish dnmt1-deficient embryos that display hypomethylation defects but retain relatively normal early development, to explain the roles of dnmt1, DNA methyltransferase 1, in HSC development. Loss-of-function of dnmt1 in zebrafish embryos displayed reduced population of HSCs and their derivatives, with unaltered primitive hematopoiesis and vessel development. Interestingly, the global DNA methylation level was upregulated and the expression of dnmt3 was increased in the neural tube and AGM in dnmt1-deficient embryos. Knockdown of dnmt3 in dnmt1 morphants showed that both the dysregulated methylation level and the HSC defect could be partially rescued. Taken together, we have demonstrated that dnmt1 is required for HSC development through negative regulation of dnmt3 expression in zebrafish.

3235 – VASCULAR NICHE MEDIATED CHEMO-RESISTANCE IN ACUTE MYELOID LEUKEMIA; CRITICAL ROLE OF EXTRINSIC NICHED-MEDIATED PRO-SURVIVAL SIGNALING VIA E-SELECTIN Ingrid Winkler1; 'Valerie Barbier2; Johanna Erbani1; Joshua Yang Tay1; Corrine Fiveash3; Jean-Pierre Levesque1; John Magnani4 1Mater Research Institute - University of Queensland, Brisbane, Australia; 2Mater Research Institute - University of Queensland, Mater Research Institute - University of Queensland, Australia; 3Mater Research Institute - University of Queensland., Brisbane, Australia; 4GlycoMimetics LTD, Rockville, United States

We have shown the vascular adhesion molecule E-selectin to be a key component of the bone marrow (BM) vascular niche, ‘awakening’ otherwise dormant hematopoietic stem cells (HSC). Now we show vascular niches also mediate malignant cell survival.

We find (1) E-selectin becomes upregulated on BM vasculature during leukemia indicating AML induces BM vascular activation & (2) adhesion of BM AML blasts to E-selectin in vitro and in vivo promotes survival signalling. This appears unique to E-selectin - not observed with adhesion with other vascular adhesion molecules P-selectin or integrin ligands.

In vivo we find absence of E-selectin (in sele-/- mice) or therapeutic blockade by administration of GMI- 1271 induces 9-fold greater chemo-sensitivity in 11q23-rearranged AML Initiating Cells (LIC) to cytarabine with doubling in duration of overall mouse survival over that achieved by chemotherapy alone. Together these data support Phase I/II clinical trial of (NCT02306291).

These data also raise the questions: 1) which AML LIC ligands interact with E-selectin at the vascular niche & 2) what are the pathways initiated by E-selectin-adhesion that mediate chemo-resistance. Both were investigated.

To understand the pro-survival signalling pathways involved & those are dampened by E-selectin blockade in vivo RNAseq revealed down-regulation of several components of the PI3K/AKT/NF-kB signalling pathway –that frequently mediates pro-survival signalling. Indeed AKT (Protein Kinase B Ser473) was found to be rapidly phosphorylated in AML blasts within minutes of E-selectin adhesion in vitro suggesting that direct E-selectin-mediated AKT activation at the vascular niche may be a potential mechanism driving vascular mediated chemo-resistance. In vivo we find significantly less AKT phosphorylation in BM AML blasts following 24hr E-selectin blockade.

In summary, leukemic blasts appear reliant on the two canonical ligands (PSGL1/CD162 & CD44) for E- selectin adhesion in contrast to HSC which appear to utilise a range of other receptor to bind E-selectin. Also pro-survival signalling via PI3K/AKT/NFkB can be dampened in AML blasts in vivo by therapeutic E- selectin blockade.

3236 – CLASSIFICATION OF HUMAN T-ALL USING DEEP TRANSCRIPTOMIC PROFILING ACCORDING TO NORMAL INTRATHYMIC DEVELOPMENTAL STAGE REVEALS PATIENT SUBGROUPS WITH POOR RESPONSE TO STANDARD INDUCTION THERAPY Rachel Wong1; Andrew Nguyen1; Kateryna Tyshchenko1; Scott Brown2; Andrew Weng1 1BC Cancer Research Centre, Vancouver, Canada; 2Michael Smith Genome Sciences Centre, Vancouver, Canada

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy characterized by an accumulation of developmentally arrested lymphoid blasts. Characterization by immune phenotyping using a few select CD markers and TCR rearrangement status previously led to classification according to developmental stage; however, due to increased use of transcriptomic profiling, classification by transcription factor (TF) signature has largely superceded developmental stage-based approaches. While there is rough correlation between TF subgroups and stage of developmental arrest, we posited that a more refined developmental classification based on RNA-seq data might yield novel insights into T-ALL pathogenesis. More specifically, we hypothesized that transcriptional mechanisms responsible for enforcing developmental blockade might serve as targetable vulnerabilities for differentiation-inducing therapies. Here, we present a high-resolution characterization of multi-stage differentiation arrest in T-ALL based on similarity to normal T-cell subsets and reveal 6 novel subgroups which organize T-ALLs along a roughly linear developmental trajectory. Briefly, using publicly available RNA-seq data from normal intrathymic human T-cell subsets as defined by conventional developmental landmarks, we generated a set of stage- specific gene expression signatures comprising 2,824 genes. We then performed unsupervised clustering of RNA-seq data from a cohort of 264 primary human T-ALL diagnostic samples (COG TARGET study) limited to just the developmentally regulated 2,824 gene set. We obtained 6 novel subgroups of T-ALL that recapitulate distinct stages of intrathymic T-cell development. Strikingly, this novel classification scheme is orthogonal to TF signature-based classification, yet correlates with clinical features including response to standard induction chemotherapy, thus confirming the biological relevance of classification by differentiation stage. These results have important implications for patient stratification and assignment to standard vs alternate therapy protocols, and further reveals potential molecular targets for differentiation-inducing therapies.

3237 – POLYCOMB BOUND GRAND DNA METHYLATION CANYON FORMS 3D CHROMATIN INTERACTIONS ESSENTIAL FOR HEMATOPOIETIC STEM CELL SELF-RENEWAL Xiaotian Zhang1,2; Mira Joeng3; Erez Liberman-Aiden4; Margaret Goodell3 1Van Andel Institute, Grand Rapids, United States; 2Baylor College of Medicine, Grand Rapids, United States; 3BCM, Center for gene therapy, Houston, United States; 4Baylor College of Medicine, Houston, United States

DNA methylation Canyons (DMC in short, also referred as DNA methylation Valley) are long unmethylated regions (UMR) over 3.5kb in the mammalian genome. DMCs are associated with Homeotic genes and can be classified into the active DMCs marked by H3K4me3 and the repressive DMCs marked by H3K27me3. We performed high resolution in situ HiC on human Hematopoietic Stem and Progenitor cells (HSPC) and the differentiated red blood cell (RBC) progenitors. We found that DMCs over 7.3kb form significant 3D micro-compartment interactions with each other. These interactions are in extreme long range and can occur between two loci separated by 60Mb. Thus, we name these DMCs over 7.3kb as Grand DNA methylation Canyon (GDMC). GDMCs are repressive DMCs and bear the highest level of H3K27me3 in the HSPC compared with the remaining UMRs under 7.3kb. Additionally, we found that the interacting GDMCs don't have CTCF binding sites in the correct convergent direction to form cohesion extrusion loop. Together, these data suggest GDMC interaction is organized by Polycomb body but not cohesion loop extrusion. We also found GDMC interactions disappear in differentiated RBC progenitors from HSPC. This suggests a function of GDMC interactions in stem cell maintenance. We thus set to test the function of GDMC interactions in stem cell self-renewal by deleting GDMC loci. We found one GDMC that lacks genes and transcription activity, is interacting with repressive part of HOXA cluster only in HSPC. By deleting this GDMC, we found the HSPC self-renewal is impaired significantly. Moreover, expression of active HOXA gene adjacent to the repressive part of HOXA cluster also decreased after deletion. When we check the 3D genomic interactions around HOXA region after deletion, we found the long range interactions with this GDMC disappear, and the enhancer interactions with active HOXA cluster gene promoters also weakened. This suggests that GDMC interactions can act as the scaffold for the enhancer-promoter interactions to maintain the active gene expression. Thus we unveiled a new type of 3D genomic interactions in HSPC and its unusual function in regulating gene expression.

3238 – INFLAMMATION-ASSOCIATED CYTOKINES IGFBP1 AND RANTES IMPAIR THE MEGAKARYOCYTIC POTENTIAL OF HSCS IN PT PATIENTS AFTER ALLO-HSCT Jiaxi Zhou1; Cuicui Liu2; Yiqing Yang2 1Institute of Hematology & Blood Diseases Hospital, Tianjin, China (People's Republic); 2Institute of Hematology & Blood Diseases Hospital, Tianjin, China (People's Republic)

Prolonged isolated thrombocytopenia (PT) is a severe complication in patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Whether the megakaryoctic potential of hematopoietic stem cells (HSCs) in bone marrow is intact and what factors drive the pathological process of PT remain elusive. A retrospective study in patients (n=285) receiving HSC transplantation revealed that the occurrence of PT was approximately 8% and the number of platelets and megakaryocytes in PT patients is much lower compared to the control. To test whether the deficiency of thrombopoiesis was caused by the activities of HSCs, the megakaryocytic differentiation potential of HSCs before or after transplantation was assessed. Interestingly, a substantial decrease of megakaryocytic differentiation was observed two weeks after transplantation of HSCs in all of the allo-HSCT recipients. However, four weeks after transplantation, the ability of HSCs to generate CD41+CD42b+ megakaryocytes in SPE patients recovered to the same level as those of HSCs before implantation. In contrast, HSCs derived from PT patients throughout the post-implantation period exhibited poor survival and failed to differentiate properly. A protein array analysis demonstrated that multiple inflammation-associated cytokines were elevated in allo-HSCT recipients with PT. Among them, IGFBP1 and RANTES were found to significantly suppress the proliferation and megakaryocytic differentiation of HSCs in vitro. Our results suggested that the occurrence of PT might be attributed, at least partially, to the damage to HSC function caused by inflammation-associated cytokines after HSC transplantation. These findings shed light on the mechanism underlying HSC megakaryocytic differentiation in PT patients and might provide potential new strategies for treating PT patients after HSC transplantation.

3239 – MEIS1 REGULATES HEMOGENIC ENDOTHELIAL GENERATION, MEGAKARYOPOIESIS AND THROMBOPOIESIS IN HUMAN PLURIPOTENT STEM CELLS BY TARGETING TAL1 AND FLI1 Jiaxi Zhou1; Hongtao Wang2; Cuicui Liu3; Xin Liu2 1Institute of Hematology & Blood Diseases Hospital, Tianjin, China (People's Republic); 2Institute of Hematology & Blood Diseases Hospital, Tianjin, China (People's Republic); 3Institute of Hematology&Blood Diseases Hospital, Tianjin, China (People's Republic)

Human pluripotent stem cells (hPSCs) provide an unlimited source for generating various kinds of functional blood cells. However, efficient strategies for generating large-scale functional blood cells from hPSCs are still lacking, and the mechanism underlying human hematopoiesis remains largely unknown. In this study, we identified Myeloid Ectopic Viral Integration Site 1 homolog (MEIS1) as a crucial regulator of hPSC early hematopoietic differentiation. MEIS1 is vital for specification of APLNR+ mesoderm progenitors to functional hemogenic endothelial progenitors (HEPs), thereby controlling formation of hematopoietic progenitor cells (HPCs). TAL1 mediates the function of MEIS1 in HEP specification. In addition, MEIS1 is vital for megakaryopoiesis and thrombopoiesis from hPSCs. Mechanistically, FLI1 acts as a downstream gene necessary for the function of MEIS1 during megakaryopoiesis. Thus, MEIS1 controls human hematopoiesis in a stage-specific manner and can be potentially manipulated for large-scale generation of HPCs or platelets from hPSCs for therapeutic applications in regenerative medicine.

3240 – INCREASED RIPK1-MEDIATED BONE MARROW NECROPTOSIS LEADS TO MYELODYSPLASIA AND BONE MARROW FAILURE IN MICE Sandra Zinkel Vanderbilt university school of medicine, Nashville, United States

Abstract

Hematopoiesis is a dynamic system that requires balanced cell division, differentiation, and death. The two major modes of programmed cell death (PCD), apoptosis and necroptosis, share molecular machinery, but diverge in outcome with important implications for the microenvironment: apoptotic cells are removed in an immune silent process, whereas necroptotic cells leak cellular contents that incite inflammation. Given the importance of cytokine directed cues for hematopoietic cell survival and differentiation, the impact on hematopoietic homeostasis of biasing cell death fate to necroptosis is substantial and poorly understood. Here we present a mouse model with increased bone marrow necroptosis. Deletion of the pro-apoptotic Bcl-2 family members Bax and Bak inhibits bone marrow apoptosis. Further deletion of the BH3-only member Bid, to generate BaxBakBid TKO mice, leads to unrestrained bone marrow necroptosis driven by increased Rip1 kinase. TKO mice display loss of progenitor cells leading to increased stem cell proliferation and stem cell exhaustion, and increased cytokine production, culminating in bone marrow failure (BMF). Genetically restoring Rip1 kinase to wild type levels, restores normal hematopoiesis as well as normal cytokine production. TKO bone marrow is hyper cellular with abnormal differentiation, resembling the human disorder Myelodysplastic syndrome (MDS), and we demonstrate increased necroptosis in MDS bone marrow. Finally, we show that Bid impacts necroptotic signaling through modulation of Caspase-8-mediated Rip1 degradation. We thus demonstrate that dysregulated necroptosis in hematopoiesis promotes bone marrow progenitor cell death that incites inflammation, impairs hematopoietic stem cells, and recapitulates the salient features of the bone marrow failure disorder, MDS.