p53 Dependent and Dose Dependent Effects of Rps29 Mutation In the Zebrafish Embryo.

Abstract Abstract 1170 Diamond Blackfan anemia (DBA) is a rare congenital disease characterized by red cell aplasia and craniofacial abnormalities. Ribosomal protein genes are often mutated in patients with this disease, but the mechanism of action is still being investigated. To elucidate the effect of mutations in ribosomal proteins, we are studying a zebrafish rps29 mutant with hematopoietic and endothelial defects. Hematopoietic stem cells (HSCs) in rps29-/- embryos are significantly decreased, as assayed by runx1 and cmyb expression. Although the aorta and posterior cardinal vein form in the mutant, intersomitic vessel formation is affected. To test whether decreased p53 levels can rescue these defects, we crossed fish with mutated p53 into the rps29 background. In rps29-/-;p53-/- embryos, the vascular and HSC phenotypes are rescued, demonstrating that p53 may be required for these effects of rps29 knockdown. We performed a microarray comparing rps29-/- embryos and their siblings to identify genes that are differentially expressed in the mutant. Using gene set enrichment analysis (GSEA), we determined that the list of genes up-regulated in the rps29 mutant is enriched for genes up-regulated by p53 in response to irradiation. Many of the genes identified have known roles in apoptosis and stress response. We have also identified genes whose expression correlates with the number of wildtype copies of rps29. Orthopedia homolog a (otpa), which is specifically expressed in forebrain and hindbrain tissues at 24 hours post fertilization (hpf), is decreased in heterozygous siblings and further decreased in homozygous siblings. In addition, p53 knockdown partially increases otpa levels in the mutant. These data support a model where p53 activation is one of the critical downstream mediators of rps29 knockdown in several tissues, but the mechanism of tissue specificity remains unclear. The otpa phenotype suggests that regulation of some genes is dependent on rps29 levels. The zebrafish rps29 mutant will be a useful model for understanding how a decrease in ribosomal protein levels can cause specific defects in hematopoietic and neural tissues. Disclosures: Zon: FATE, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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Epigenetic Activation of the pH Regulator MCT4 in Acute Myeloid Leukemia Exploits a Fundamental Metabolic Process of Enhancing Cell Growth through Proton Shifting

Blood ◽

10.1182/blood-2019-123604 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3765-3765

Author(s):

Cheuk-Him Man ◽

David T. Scadden ◽

Francois Mercier ◽

Nian Liu ◽

Wentao Dong ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Growth ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Monocarboxylate Transporter ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Equity Ownership ◽

Acute Myeloid

Acute myeloid leukemia (AML) cells exhibit metabolic alterations that may provide therapeutic targets not necessarily evident in the cancer cell genome. Among the metabolic features we noted in AML compared with normal hematopoietic stem and progenitors (HSPC) was a strikingly consistent alkaline intracellular pH (pHi). Among candidate proton regulators, monocarboxylate transporter 4 (MCT4) mRNA and protein were differentially increased in multiple human and mouse AML cell lines and primary AML cells. MCT4 is a plasma membrane H+and lactate co-transporter whose activity necessarily shifts protons extracellularly as intracellular lactate is extruded. MCT4 activity is increased when overexpressed or with increased intracellular lactate generated by glycolysis in the setting of nutrient abundance. With increased MCT4 activity, extracellular lactate and protons will increase causing extracellular acidification while alkalinizing the intracellular compartment. MCT4-knockout (MCT4-KO) of mouse and human AMLdid not induce compensatory MCT1 expression, reduced pHi, suppressed proliferation and improved animal survival. Growth reduction was experimentally defined to be due to intracellular acidification rather than lactate accumulation by independent modulation of those parameters. MCT4-KOmetabolic profiling demonstrated decreased ATP/ADP and increased NADP+/NADPH suggesting suppression of glycolysis and the pentose phosphate pathway (PPP) that was confirmed by stable isotopic carbon flux analyses. Notably,the enzymatic activity of purified gatekeeper enzymes, hexokinase 1 (HK1), pyruvate kinase M2 isoform (PKM2) and glucose-6-phosphate dehydrogenase (G6PDH) was sensitive to pH with increased activity at the leukemic pHi (pH 7.6) compared to normal pHi (pH 7.3). Evaluating MCT4 transcriptional regulation, we defined that activating histonemarks, H3K27ac and H3K4me3, were enriched at the MCT4 promoter region as were transcriptional regulators MLL1 and Brd4 by ChIP in AML compared with normal cells. Pharmacologic inhibition of Brd4 suppressed Brd4 and H3K27ac enrichment and MCT4 expression in AML and reduced leukemic cell growth. To determine whether MCT4 based pHi changes were sufficient to increase cell proliferation, we overexpressed MCT4 in normal HSPC and demonstrated in vivo increases in growth in conjunction with pHi alkalization. Some other cell types also were increased in their growth kinetics by MCT4 overexpression and pHi increase. Therefore, proton shifting may be a means by which cells respond to nutrient abundance, co-transporting lactate and protons out of the cell, increasing the activity of enzymes that enhance PPP and glycolysis for biomass generation. Epigenetic changes in AML appear to exploit that process by increasing MCT4 expression to enforce proton exclusion thereby gaining a growth advantage without dependence on signaling pathways. Inhibiting MCT4 and intracellular alkalization may diminish the ability of AML to outcompete normal hematopoiesis. Figure Disclosures Scadden: Clear Creek Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Sponsored research; Editas Medicine: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bone Therapeutics: Consultancy; Fog Pharma: Consultancy; Red Oak Medicines: Consultancy, 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; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy, Equity Ownership.

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Plerixafor (Mozobil ®)Plus G-CSF Is Effective in Mobilizing Hematopoietic Stem Cells in Patients with Concurrent Thrombocytopenia Undergoing Autologous Hematopoietic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v114.22.3229.3229 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3229-3229 ◽

Cited By ~ 1

Author(s):

Ivana N Micallef ◽

Eric Jacobsen ◽

Paul Shaughnessy ◽

Sachin Marulkar ◽

Purvi Mody ◽

...

Keyword(s):

Stem Cell ◽

Platelet Count ◽

Board Of Directors ◽

Research Funding ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Platelet Counts ◽

Cd34 Cells ◽

Low Platelet Count ◽

Equity Ownership

Abstract Abstract 3229 Poster Board III-166 Introduction Low platelet count prior to mobilization is a significant predictive factor for mobilization failure in patients with non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (HD) undergoing autologous hematopoietic stem cell (HSC) transplantation (auto-HSCT; Hosing C, et al, Am J Hematol. 2009). The purpose of this study is to assess the efficacy of HSC mobilization with plerixafor plus G-CSF in patients with concomitant thrombocytopenia undergoing auto-HSCT. Methods Patients who had failed successful HSC collection with any mobilization regimen were remobilized with plerixafor plus G-CSF as part of a compassionate use program (CUP). Mobilization failure was defined as the inability to collect 2 ×106 CD34+ cells/kg or inability to achieve a peripheral blood count of ≥10 CD34+ cells/μl without having undergone apheresis. As part of the CUP, G-CSF (10μg/kg) was administered subcutaneously (SC) every morning for 4 days. Plerixafor (0.24 mg/kg SC) was administered in the evening on Day 4, approximately 11 hours prior to the initiation of apheresis the following day. On Day 5, G-CSF was administered and apheresis was initiated. Plerixafor, G-CSF and apheresis were repeated daily until patients collected the minimum of 2 × 106 CD34+ cells/kg for auto-HSCT. Patients in the CUP with available data on pre-mobilization platelet counts were included in this analysis. While patients with a platelet count <85 × 109/L were excluded from the CUP, some patients received waivers and were included in this analysis. Efficacy of remobilization with plerixafor + G-CSF was evaluated in patients with platelet counts ≤ 100 × 109/L or ≤ 150 × 109/L. Results Of the 833 patients in the plerixafor CUP database, pre-mobilization platelet counts were available for 219 patients (NHL=115, MM=66, HD=20 and other=18.). Of these, 92 patients (NHL=49, MM=25, HD=8 and other=10) had pre-mobilization platelet counts ≤ 150 × 109/L; the median platelet count was 115 × 109/L (range, 50-150). The median age was 60 years (range 20-76) and 60.4% of the patients were male. Fifty-nine patients (64.1%) collected ≥2 × 109 CD34+ cells/kg and 13 patients (14.1%) achieved ≥5 × 106 CD34+ cells/kg. The median CD34+ cell yield was 2.56 × 106 CD34+ cells/kg. The proportion of patients proceeding to transplant was 68.5%. The median time to neutrophil and platelet engraftment was 12 days and 22 days, respectively. Similar results were obtained when efficacy of plerixafor + G-CSF was evaluated in 29 patients with platelet counts ≤ 100 × 109/L (NHL=12, MM=10, HD=3 and other=4). The median platelet count in these patients was 83 × 109/L (range, 50-100). The median age was 59 years (range 23-73) and 60.4% of the patients were male. The minimal and optimal cell dose was achieved in 19(65.5%) and 3(10.3%) patients, respectively. The median CD34+ cell yield was 2.92 × 106 CD34+ cells/kg. The proportion of patients proceeding to transplant was 62.1%. The median time to neutrophil and platelet engraftment was 12 days and 23 days, respectively. Conclusions For patients mobilized with G-CSF alone or chemotherapy ±G-CSF, a low platelet count prior to mobilization is a significant predictor of mobilization failure. These data demonstrate that in patients with thrombocytopenia who have failed prior mobilization attempts, remobilization with plerixafor plus G-CSF allows ∼65% of the patients to collect the minimal cell dose to proceed to transplantation. Thus, in patients predicted or proven to be poor mobilizers, addition of plerixafor may increase stem cell yields. Future studies should investigate the efficacy of plerixafor + G-CSF in front line mobilization in patients with low platelet counts prior to mobilization. Disclosures Micallef: Genzyme Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding. Jacobsen:Genzyme Corporation: Research Funding. Shaughnessy:Genzyme Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Marulkar:Genzyme Corporation: Employment, Equity Ownership. Mody:Genzyme Corporation: Employment, Equity Ownership. van Rhee:Genzyme Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Role of Polycomb Repressive Complex 1 (PRC1) In Runx1 Mediated Gene Regulation

Blood ◽

10.1182/blood.v116.21.3863.3863 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3863-3863

Author(s):

Ming Yu ◽

Tali Mazor ◽

Hui Huang ◽

Emily Huang ◽

Katie Kathrein ◽

...

Keyword(s):

Gene Regulation ◽

Board Of Directors ◽

Target Genes ◽

Polycomb Repressive Complex ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Human T Cell ◽

Equity Ownership ◽

Polycomb Repressive Complex 1

Abstract Abstract 3863 The transcription factor Runx1 is required for the generation of all definitive hematopoietic stem cells (HSCs), and for normal megakaryocyte, lymphocyte and granulocyte terminal maturation. Runx1 and its cofactor CBF-β are also the most common targets of chromosomal translocations in human leukemias. Somatic and germline point mutations in Runx1 occur in myelodysplastic syndrome and undifferentiated leukemias, and are associated with a poor prognosis. Despite the key roles that Runx1 plays in normal and malignant hematopoiesis, its transcriptional mechanisms remain incompletely understood. In this study, we purified Runx1 containing multiprotein complexes from megakaryocytic cells and identified several associated chromatin-remodeling complexes, including Polycomb Repressive Complex 1 (PRC1), NuRD, SWI/SNF and MLL/TrxG. Interactions were validated by independent biochemical assays and demonstrate a direct interaction between Runx1 and the PRC1 component Bmi1. ChIP-seq studies identified a large overlap between Runx1/CBF-β and Ring1b (another PRC1 core component) occupied sites, with 45% of the peaks at these genes < 200 bp from each other. ShRNA mediated gene knockdown of CBF-β shows differential gene expression of many of the co-occupied genes. Among the direct CBF-β/Ring1b co-occupied targets are other key hematopoietic transcription factors including FOG-1, SCL and Lyl1, and a number of cell adhesion related genes. ShRNA knockdown of Ring1b impairs megakaryocyte endomitosis, partially phenocopying Runx1 deficient megakaryocytes. Morpholino mediated knockdown of Ring1b or Bmi1 in zebrafish embryos reduces the number of phenotypic definitive HSCs, also partially phenocopying Runx1 morphants. We also show that Runx1/CBF-β interact with Ring1b in the human T cell line Jurkat, and that Ring1b occupies Runx1/CBF-β bound sites of key direct target genes in primary murine thymocytes, including CD4, TCRβ, and Th-POK. Surprisingly, we did not find enrichment for histone 2A monoubiquitination at most of the megakaryocytic and T-lymphocyte co-occupied sites examined, suggesting that PRC1 acts through alternate mechanisms at these genes. Collectively, these data provide evidence for a broad role of PRC1 in Runx1 mediated gene regulation. Disclosures: Zon: FATE, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Cantor:Amgen, Inc: Consultancy.

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Epoxyeicosatrienoic Acids Regulate Hematopoietic Stem/Progenitor Cell Fate Decision During Stress Response and Embryonic Hematopoiesis

Blood ◽

10.1182/blood.v118.21.860.860 ◽

2011 ◽

Vol 118 (21) ◽

pp. 860-860

Author(s):

Pulin Li ◽

Emily K Pugach ◽

Elizabeth B Riley ◽

Dipak Panigrahy ◽

Garrett C Heffner ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Marrow Transplantation ◽

Mouse Bone Marrow ◽

Zebrafish Embryos ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Equity Ownership ◽

Runx1 Expression

Abstract Abstract 860 During bone marrow transplantation, hematopoietic stem/progenitor cells (HSPCs) are exposed to various stress signals, and undergo homing, rapid proliferation and differentiation in order to achieve engraftment. To explore how fate decisions are made under such stress conditions, we developed a novel imaging-based competitive marrow transplantation in zebrafish. The feasibility of handling hundreds of zebrafish for transplantation per day allowed us to screen a library of 480 small molecules with known bioactivity, aimed at identifying new drugs and pathways regulating HSPC engraftment. Two structurally related eicosanoids, 11,12-epoxyeicosatrienoic acid (EET) and 14,15-EET, were able to enhance GFP+ marrow engraftment compared to DsRed2+ engraftment in zebrafish. This remarkable effect of EETs on adult marrow prompted us to study the effect of EETs in embryonic hematopoiesis. Treating zebrafish embryos with 11,12-EET during definitive hematopoiesis increased the HSPC marker Runx1 expression in the AGM (Aorta-Gonad-Mesonephros), resulting in a significant increase of HSPC in the next hematopoietic site, caudal hematopoietic tissue, the equivalent of fetal liver/placenta in mammals. The same treatment condition also induced ectopic Runx1 expression in the tail mesenchyme, a non-hematopoietic tissue. Microarray analysis on EET-treated zebrafish embryos revealed an upregulation of genes involved in stress response, especially Activator Protein 1 (AP-1) family members. Genetic knockdown experiments confirmed AP-1 members, especially JunB and its binding partners, cFos and Fosl2, are required for Runx1 induction. Motif analysis also predicted several conserved AP-1 binding sites in the Runx1 enhancer regions. To understand how EETs induced AP-1 expression, a suppressor screen was performed in zebrafish embryos. The screen revealed that activation of both PI3K/Akt and Stat3 are required for induced AP-1 expression, and therefore Runx1 upregulation. Similarly, ex vivo treatment of mouse whole bone marrow with 11,12-EET resulted in a 2-fold increase of long-term repopulating units. Microarray data had previously shown that Cyp2j6, one of the cytochrome P450 enzymes involved in EET biosynthesis from arachidonic acid, is enriched in quiescent mouse long-term HSCs. To further increase the EET levels in HSPCs, human CYP2C8 enzyme was over-expressed in transgenic mice using the Tie2 promoter. These transgenic mice have a 4-fold increase of long-term multi-lineage repopulating unit compared to their wild-type siblings. In purified mouse HSPCs, EETs directly and cell-autonomously activate PI3K/AKT pathway. Co-treatment of mouse bone marrow with EET and a PI3K inhibitor, LY294,002, completely blocked EET-induced enhancement of mouse bone marrow engraftment. In conclusion, we performed the first competitive marrow transplantation-based chemical screen, leading to the discovery of arachidonic acid-cytochrome P450-EETs as a novel modulator of HSC cell fate decision. PI3K/Akt and Stat3 pathways activated by EETs are required for adult HSPC engraftment and/or embryonic HSC specification, partially through transcriptional regulation of AP-1. We also demonstrated the requirement of AP-1 family members for Runx1 expression during embryonic development. This discovery may have clinical application in marrow or cord blood transplantation. Disclosures: Daley: iPierian, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Epizyme, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Verastem, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Solasia, KK: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; MPM Capital, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees. Zon:Fate Therapeutics: Founder; Stemgent: Consultancy.

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Diacylglycerol Enhances Hematopoietic Stem/Progenitor Cell Development and Engraftment Via MAP Kinase Activation

Blood ◽

10.1182/blood.v122.21.174.174 ◽

2013 ◽

Vol 122 (21) ◽

pp. 174-174

Author(s):

Vera Binder ◽

Pulin Li ◽

Francesca Barrett ◽

Alex Leung ◽

Leonard I. Zon

Keyword(s):

Cord Blood ◽

Map Kinase ◽

Board Of Directors ◽

Marrow Transplantation ◽

Fluorescent Protein ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Equity Ownership ◽

Marrow Cells

Abstract Hematopoietic stem and progenitor cells (HSPCs) are exposed to a variety of intrinsic and extrinsic factors regulating all processes needed during development, and for successful engraftment after transplantation. In order to decipher the molecular pathways that may promote engraftment of HSPCs after marrow transplantation, we performed a competitive transplantation screen using chemical genetics in zebrafish. Green fluorescent protein-labeled kidney marrow cells (equivalent to mammalian bone marrow cells) were treated ex vivo with single compounds of a chemical library of known biologically active compounds, and administered by retro-orbital venous injection to lethally irradiated recipient zebrafish. About 500 chemicals were screened. Untreated kidney marrow cells labeled with a red fluorescent protein were used as competitors. Imaging-based assessment of short-term engraftment demonstrated that 1,2-Didecanoylglycerol, a membrane permeable but non-physiologic analogue of diacylglycerol (DAG), significantly improved engraftment compared to competitor cells. Follow-up by FACS analysis showed a 3.5 fold increase of long-term repopulating units after DAG treatment. To interrogate whether DAG treatment not only affects HSPCs under transplant conditions, but also during normal embryonic development, we treated zebrafish embryos within the time window of HSC formation in the dorsal aorta. DAG treatment increased expression of the HSPC markers Runx1 and c-myb in the AGM (Aorto-Gonad-Mesonephros). Treatment after HSC specification also led to an upregulation of HSPC markers in the caudal hematopoietic tissue (equivalent to fetal liver in mammals). These data suggest that DAG affects not only HSC formation, but also migration and engraftment of HSPCs as hematopoiesis transitions from the AGM to the CHT during development. To determine whether HSPCs respond to DAG in a cell autonomous manner, and to identify the underlying molecular mechanism, we treated human CD34+ cells from umbilical cord blood with DAG and performed RNA-seq analysis. Ingenuity Pathway Analysis of the 395 differentially expressed genes (q-value < 0.05) implicated the MAP kinase pathway as an upstream regulator. Human Phosphokinase array analysis of treated CD34+ showed ERK 1/2 activation. DAG is known to activate Protein Kinase C (PKC) with subsequent Raf kinase phosphorylation, which has the potential to activate ERK. Co-treatment of CD34+ cells with DAG and the ERK inhibitor PD98059 blocked upregulation of downstream ERK-targets (e.g. AREG, CSF2, EGR1, HMOX, SERPINE1, DUSP4, DUSP6), whereas the PI3K family inhibitor LY294002 and the p38 MAP kinase inhibitor SB202190 did not alter the effect of DAG on expression of these genes. This demonstrates that DAG activates ERK and its downstream targets. Our competitive marrow transplantation-based chemical screen has led to the discovery of 1,2-Didecanoylglycerol as a novel modulator of HSPC development and engraftment after transplantation. This discovery may be of clinical relevance to marrow or cord blood hematopoietic stem cell transplantation. Disclosures: Zon: FATE Therapeutics, Inc: Consultancy, Equity Ownership, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Stemgent, Inc: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Stocks, Stocks Other; Scholar Rock: Consultancy, Equity Ownership, Founder, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

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Novel Inhibitors Of CRM1/XPO1 Nuclear Exporter Exhibit Striking Activity Against AML “primagrafts,” Including AML Leukemia Initiating Cells, While Sparing Normal Hematopoietic Cells

Blood ◽

10.1182/blood.v122.21.3932.3932 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3932-3932

Author(s):

Julia Etchin ◽

Bonnie Thi Le ◽

Alex Kentsis ◽

Richard M. Stone ◽

Dilara McCauley ◽

...

Keyword(s):

Board Of Directors ◽

Cell Lines ◽

Nuclear Export ◽

Hematopoietic Cells ◽

Complex Karyotype ◽

Employment Equity ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Nsg Mice ◽

Equity Ownership

Abstract Current treatments for acute myeloid leukemia (AML) often fail to induce long-term remissions and are also toxic to normal tissues, prompting the need to develop new targeted therapies. One attractive cellular pathway with therapeutic potential is nuclear export, which is mediated in part by nuclear exporter CRM1/XPO1. XPO1 mediates the transport of ∼220 proteins and several mRNAs and is the sole nuclear exporter of the major tumor suppressor and growth regulatory proteins p53, p73, FOXO, IkB/NF-kB, Rb, p21, and NPM. Our findings demonstrate that novel irreversible inhibitors of XPO1, termed Selective Inhibitors of Nuclear Export, or SINE, induce rapid apoptosis in 12 AML and 14 T-ALL cell lines with IC50s of 15-474 nM. In the SINE-sensitive cell lines, BCL2 overexpression suppresses SINE-induced apoptosis, indicating its intrinsic pathway mediation. Oral administration of clinical XPO1 inhibitor, Selinexor (KPT-330), at 15 or 25 mg/kg, induced remarkable growth suppression in MV4-11 human AML cells and MOLT-4 human T-ALL cells engrafted in immunodeficient NSG mice with negligible toxicity to normal mouse hematopoietic cells after 35 days of treatment. Bone marrow biopsies of selinexor - treated mice were remarkable in that they showed normal hematopoietic cell morphology and cellularity after 35 days of treatment. Significant survival benefit was observed in mice treated with selinexor, compared to vehicle-treated mice. Selinexor is now in Phase 1 clinical trial in patients with AML and other hematological malignancies (NCT01607892). Recently, we have established primagraft models of AML, using primary leukemia blasts isolated from AML patients at diagnosis transplanted into immunocompromised NSG mice. We demonstrated that selinexor exhibits striking anti-leukemic activity against different subtypes of primary AML, including AML-M4; FLT3-ITD and complex karyotype subtypes of the disease. To determine whether selinexor targets leukemia-initiating cells (LICs) of primary AML, we re-transplanted serial dilutions of human CD45+ cells isolated from leukemic mice treated with either vehicle or selinexor. The preliminary results of our re-population assays indicate that selinexor greatly diminished LIC frequency in AML-M4; FLT3-ITD AML (∼6 fold) and complex karyotype disease (∼100 fold). These findings demonstrate that selinexor may represent a novel targeted therapy for the treatment of AML, which spares normal hematopoietic stem and progenitor cells. Disclosures: McCauley: Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Patents & Royalties. Kauffman:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Shacham:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

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IL-8 and CXCR1 Remodel the Vascular Niche to Promote Hematopoietic Stem and Progenitor Cell Engraftment

Blood ◽

10.1182/blood.v126.23.783.783 ◽

2015 ◽

Vol 126 (23) ◽

pp. 783-783

Author(s):

Bradley Wayne Blaser ◽

Jessica Moore ◽

Brian LI ◽

Owen J. Tamplin ◽

Vera Binder ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Board Of Directors ◽

Progenitor Cell ◽

Transgenic Animals ◽

Employment Equity ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Equity Ownership ◽

Cell Niche

Abstract The microenvironment is an important regulator of hematopoietic stem and progenitor cell (HSC/HSPC) engraftment during development and in recipients of hematopoietic stem cell transplantation (HSCT). Factors secreted by the hematopoietic microenvironment that promote HSC/HSPC engraftment in the developing zebrafish may therefore be therapeutic targets for enhancing HSC engraftment in patients undergoing HSCT. We previously described a novel behavior we called endothelial cuddling in which sinuosoidal endothelial cells of the niche make intimate interactions with stem cells. To find candidate extracellular factors regulating this behavior, gene expression profiling was performed on sorted zebrafish endothelial cells. Gene set enrichment analysis showed that expression of chemokines and TNF family members was significantly enriched in all endothelial cells. The leading edge gene sets included 16 chemokines and chemokine receptors. Thirteen of these genes were used as candidates in a gain-of-function screen to test whether overexpression was sufficient to stimulate the hematopoietic niche in favor of HSC engraftment. High level, global gene expression was induced at 36 and 48 hours post fertilization (hpf) using a heat shock-inducible system. One gene, CXCR1, enhanced HSC/HSPC engraftment when globally overexpressed (p=0.03, N=63). CXCR1 is a specific receptor for the chemokine IL-8/CXCL8 in higher vertebrates. Zebrafish IL-8 was used in similar gain of function experiments and was also sufficient to enhance HSC/HSPC engraftment (p=0.003, N=41). CXCR2 is a promiscuous chemokine receptor for IL-8, Gro-α and Gro-β and did not enhance HSC/HSPC engraftment in this system. To further characterize the effects of CXCR1 on HSC engraftment, it was overexpressed in transgenic zebrafish carrying a stem-cell specific reporter gene, Runx1:mCherry. HSC engraftment in the CHT was enhanced when CXCR1 expression was induced beginning at 36 hpf (3.0 +/- 2.0 vs 7.4 +/- 2.6 HSC per CHT) or 48 hpf (4.3 +/- 1.1 vs 9.4 +/- 3.6 HSC per CHT). Inhibition of CXCR1 signaling from 48 to 72 hpf using the selective CXCR1/2 antagonist, SB225002, decreased HSC engraftment in Runx1:mCherry animals (1.2 +/- 0.39 vs 0.4 +/- 0.2 HSC per CHT, p=0.03). We next hypothesized that overexpression of CXCR1 might also have effects on the endothelial cell niche itself. Using FLK1(VEGFR2):mCherry reporter zebrafish and 3-dimensional reconstruction of the CHT, we found that global overexpression of CXCR1 increased the volume of the endothelial cell niche (2.0 +/- 0.09 x 106 vs 2.4 +/- 0.1 x 106 μm3, p=0.005) while treatment with SB225002 reduced its volume (6.3 +/- 0.3 x 105 vs 4.9 +/- 0.5 x 105 µm3, p=0.04). Finally, we asked if CHT remodeling would still be enhanced if CXCR1 were constitutively expressed only within the endothelial cell niche. FLK1:CXCR1; FLK1:mCherry double transgenic animals had significantly increased CHT volume when compared with FLK1:mCherry single transgenic animals (1.1 +/- 0.05 x 106 vs 1.3 +/- 0.06 x 106 um3, p=0.02). These findings suggest a model whereby HSC/HSPCs actively participate in the remodeling of the endothelial niche via CXCR1/IL-8 in order to promote their own engraftment. Further, they suggest that CXCR1/IL-8 is a potential therapeutic target for enhancing HSC/HSPC engraftment in patients undergoing HSCT. Disclosures Zon: FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

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Genome-Wide RNA Tomography of the Hematopoietic Stem Cell Niche in Zebrafish Reveals Unexpected Functional Macrophage-Stem Cell Interactions

Blood ◽

10.1182/blood.v128.22.3882.3882 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3882-3882

Author(s):

Elliott J Hagedorn ◽

Julie R Perlin ◽

Clara Mao ◽

Brian Li ◽

Christopher D'Amato ◽

...

Keyword(s):

Stem Cell ◽

Board Of Directors ◽

Cell Interactions ◽

Cell Populations ◽

Rna Seq ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Equity Ownership

Abstract The challenges of visualizing the mammalian bone marrow have precluded a rigorous analysis of the dynamic cell-cell interactions that control hematopoietic stem and progenitor cell (HSPC) engraftment. The transparent zebrafish embryo provides an unparalleled opportunity to directly visualize HSPC-niche cell interactions in live animals. To identify genes expressed in the zebrafish caudal hematopoietic tissue (CHT) - an embryonic niche akin to the mammalian fetal liver - we employed a new technique called tomo-seq (RNA tomography). By pairing cryosectioning with RNA-seq, this technology permits spatial analysis of transcriptome-wide gene expression. Using tomo-seq we identified ~300 genes showing enriched expression in the CHT. In situ hybridization for 75 of 107 tested genes confirmed CHT expression. In parallel we performed RNA-seq on isolated cell populations, including endothelial cells, macrophages, neutrophils and erythrocytes, sorted from whole embryos. By cross-referencing these datasets we determined the cell types in which many of the 300 CHT-enriched genes were expressed. This analysis revealed several cell surface adhesion receptors enriched on macrophages in the CHT, including the integrin heterodimers itgam/itgb2, itgae/itgb7, itga4/itgb1b and itga4/itgb7. We examined whether known ligands for any of these integrins were present on HSPCs. In situ hybridization to vcam1 (ligand for itga4/itgb1b)showed punctate HSPC-like staining in the CHT. We then generated a vcam1:GFP promoter fusion, which we found was expressed in HSPCs. Using spinning disk confocal microscopy we imaged HSPCs and macrophages in the CHT and observed direct and specific physical interactions that preceded the engraftment of HSPCs. In a grooming-like behavior that lasts for 30-45 minutes, the HSPC is engaged by the macrophage, which moves all over the surface of the cell, before disengaging the HSPC, which then remains in the CHT. Between 48-72 hours post fertilization (hpf), 20% of HSPCs were engaged in this behavior with a macrophage. To evaluate the specificity of these interactions we established in vitro co-cultures using purified cell populations. In co-cultures between macrophages (mpeg1:mCherry) and HSPCs (cd41:GFP) we observed cell-cell interactions that were strikingly similar to those observed in vivo. In macrophage-HSPC co-cultures, 25% of cells were found to interact, whereas only 5% of cells were found to interact in macrophage-erythrocyte co-cultures. To functionally evaluate the macrophage-HSPC interactions in vivo, we depleted macrophages from zebrafish embryos at 55 hpf using clodronate liposomes and observed circulating HSPCs with a significant reduction in HSPC engraftment in the CHT (11/15 embryos, compared to the control where 14/14 embryos showed normal CHT engraftment). Together these studies establish a role for macrophages in promoting the niche engraftment of HSPCs. The results of this work could have important implications for the design of new therapies to improve engraftment during stem cell transplantation. Disclosures Zon: Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Fate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Marauder Therapeutics: Equity Ownership, Other: Founder.

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High Resolution Imaging Reveals Hematopoietic Stem Cells in the Perivascular Niche Are Anchored to Mesenchymal Stromal Cells That Orient Their Divisions

Blood ◽

10.1182/blood.v124.21.770.770 ◽

2014 ◽

Vol 124 (21) ◽

pp. 770-770

Author(s):

Owen J. Tamplin ◽

Ellen M. Durand ◽

Logan A. Carr ◽

Sarah J. Childs ◽

Elliott H. Hagedorn ◽

...

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Stem Cell ◽

High Resolution ◽

Board Of Directors ◽

Stromal Cells ◽

Stromal Cell ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Equity Ownership

Abstract Hematopoietic stem cells (HSC) reside in a highly structured microenvironment called the niche. There is two-way communication between a stem cell and its niche that determines important cell fate decisions. HSC must remain quiescent to persist throughout life but also divide and contribute progenitors that will replenish the blood supply. Although there have been a number of elegant studies that have imaged the mammalian bone marrow, we still lack a high-resolution real-time view of endogenous HSC behaviors and interactions within the niche. To overcome these challenges, we developed a transgenic zebrafish line that expresses GFP or mCherry in HSC. We generated this line using the previously described mouse Runx1 +23 kb intronic enhancer. We confirmed the purity of these stem cells by adult-to-adult limiting dilution transplantation with as few as one cell. Based on long-term multi-lineage engraftment, we estimated a stem cell purity of approximately 1/35, which is similar to the KSL (Kit+Sca1+Lin-) population in mouse. Using a novel embryo-to-embryo transplantation assay that is unique to zebrafish, we estimated an even higher stem cell purity of 1/2. These experiments have defined the most pure HSC population in the zebrafish. Using this novel transgenic reporter we have tracked HSC as they migrate in the live zebrafish embryo. This allowed us to image HSC as they interact with other cell types in their microenvironment, including endothelial cells and mesenchymal stromal cells. We have shown that a small group of endothelial cells remodel around a single HSC soon after it lodges in the niche. Recently, we have also found that a single stromal cell can anchor an HSC as it divides. In most cases, we observed that an HSC divides perpendicular to the stromal cell, with one daughter cell remaining attached to the stromal cell and the other migrating away. To gain a much higher resolution view of these cellular events than is possible with confocal microscopy we looked for an alternative approach. A combined method is called “Correlative Light and Electron Microscopy” (CLEM), and involves identification of cells by confocal microscopy, followed by processing of the same sample for EM scanning. We have applied this method by: 1) tracking endogenous HSC in the live embryo; 2) fixing the same embryo for serial block-face scanning EM; 3) reconstructing 3D models from high resolution serial EM sections. We used easily visible blood vessels as anatomical markers that allowed us to pinpoint a single cell in a relatively large block of scanned tissue. As expected, the identified HSC was round, had a distinctive large nucleus, scant cytoplasm, and ruffled membrane. The HSC was surrounded by a small group of 5-6 endothelial cells, as predicted from our confocal live imaging. However at this very high resolution (10 nm/pixel), we could see that only part of the HSC surface was contacted and wrapped by an endothelial cell. Other regions of the HSC surface were contacted by small endothelial cell protrusions. Much of the HSC surface was surrounded by a narrow extracellular space with endothelial and stromal cells lying opposite. Strikingly, we were able to identify the firm anchored attachment between a single stromal cell and HSC that we showed previously oriented the plane of division. By combining confocal live imaging of a novel zebrafish HSC reporter, and serial block-face scanning EM, we have created the first high-resolution 3D model of an endogenous stem cell in its niche. Disclosures Tamplin: Boston Children's Hospital: Patents & Royalties. Zon:FATE Therapeutics, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other; Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other; Stemgent: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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Cleavage and Polyadenylation Specificity Factor 1 Is Required for Definitive Hematopoietic Stem Cell Survival In Zebrafish.

Blood ◽

10.1182/blood.v116.21.1606.1606 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1606-1606

Author(s):

Niccolo Bolli ◽

Elspeth Payne ◽

Jennifer Rhodes ◽

Adam Johnston ◽

Feng Guo ◽

...

Keyword(s):

Board Of Directors ◽

Lymphoid Cells ◽

Erythroid Cells ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Primitive Hematopoiesis ◽

Whole Mount ◽

Specificity Factor ◽

Equity Ownership ◽

Definitive Hematopoiesis

Abstract Abstract 1606 Hematopoiesis is a tightly regulated process requiring the differential activation of specific genetic programs. Inactivating mutations of genes involved in normal hematopoiesis have been implicated in bone marrow failure syndromes, myelodysplastic syndromes and hematopoietic neoplasms. Despite recent advances, many genes regulating normal hematopoiesis are still unknown and innovative gene-discovery approaches can benefit our understanding of the molecular mechanisms underlying this complex developmental process. The combined genetic and embryologic strengths of the zebrafish model system are ideal for studying factors regulating hematopoiesis, which is highly conserved with mammals. We have used ENU-mutated zebrafish lines to screen for genes whose inactivation results in the loss of definitive hematopoiesis. Here, we report the identification and analysis of the zebrafish mutant grechetto, caused by a lethal, recessive, inactivating mutation (grcl8a12) of the cleavage and specificity factor 1 (cpsf1) gene. Cpsf1 encodes a protein required for processing the 3’UTR of a subset of pre-mRNAs. Cpsf1 is maternally expressed ubiquitously through the first five days of development including primitive myeloid and erythroid cells and definitive hematopoietic stem cells (HSC). Although grechetto mutants fail to express cspf1 already at 24 hpf, they appear to develop normally until 72 hpf, when they first become morphologically distinct from wild-type siblings with a smaller head and the absence of a protruding jaw. Importantly, primitive hematopoiesis appears normal, as shown by expression of the myeloid and erythroid markers mpx and band3, respectively, at 24 and 48 hpf by whole mount RNA in situ hybridization (WISH). By 120 hpf the phenotype is more marked and embryos have a curved body, cardiac edema, development defects in the jaw and gut and a reduction in the number of iridophores. While many of these affected tissues represent derivatives of the neural crest (NC), the expression of markers of NC specification (sox10 and foxd3 at 15,5 hpf, crestin at 24 hpf), migration (dlx2 at 36 and 48 hpf) and differentiation (pax9a at 48 hpf) are normal. When definitive hematopoiesis was assayed for markers of mature blood cells by WISH at 120 hpf, grechetto mutants exhibit a loss of myeloid (mpx, lysC, l-plastin), erythroid (band3, gata1) and lymphoid cells (rag1, lck). Since myeloid and erythroid cells are normal at 24 hpf, but severely reduced at 120 hpf, we investigated whether the absence of HSC could explain the loss of definitive hematopoiesis in grechetto mutants. Interestingly, WISH staining for the HSC marker c-myb showed that in mutant embryos HSCs are normally specified and represented in the ventral wall of the dorsal aorta at 36 and 48 hpf. Nevertheless, at 72 hpf the numbers of HSCs are reduced in grechetto mutants upon their migration to the caudal hematopoietic tissue (CHT) and by 120 hpf very few remain. To investigate the fate of these HSCs we crossed the grechetto line with the Tg(c-myb:EGFP) reporter line, that expresses EGFP under the control of the c-myb promoter. By whole mount immunostaining, we were able to find increased co-localization of activated caspase3 and TUNEL staining in the c-myb:EGFP positive cells in the CHT in grechetto mutants compared to WT siblings at 72 and 96 hpf, suggesting that HSCs undergo apoptosis during this stage. Furthermore, PI cell cycle profile of sorted c-myb:EGFP+ cells from 96 hpf mutants shows a sub-G1 peak, representing pyknotic cell nuclei undergoing advanced stages of apoptosis. Importantly, both activated caspase3 staining and TUNEL assay did not show increased apoptosis in the tissue immediately surrounding the CHT. These studies show that cpsf1 is not required for primitive hematopoiesis or definitive HSC specification, however, it is essential for HSC survival in the CHT. The fact that grechetto embryos do not undergo generalized apoptotic cell death suggests that, despite ubiquitous cpsf1 expression and the general development defects of grechetto mutants, the prosurvival pathways in the HSC compartment are particularly dependent on Cpsf1 activity. Disclosures: Zon: FATE, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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