A GATA-2-GPR65 Regulatory Module Controls Hematopoietic Stem Cell Generation

Abstract The generation of hematopoietic stem cells (HSCs) via endothelial-to-hematopoietic transition within the aorta-gonad-mesonephros (AGM) region of the mammalian embryo is crucial for development of the adult hematopoietic system. Many questions remain unanswered regarding the molecular program in hemogenic endothelium that promotes the budding of hematopoietic cell clusters containing HSCs. We demonstrated that a deletion of a Gata2 cis-element reduced GATA-2 levels in the AGM and abrogated the capacity of hemogenic endothelium to generate HSCs. Consistent with the defective HSC generator, the mutant fetal livers were deficient in hematopoietic stem and progenitor cells (HSPCs). Using an ex vivo intact AGM culture system, we demonstrated that retrovirus-mediated GATA-2 expression in the +9.5-/- AGM rescues its hematopoietic defect. Thus, the reduced GATA-2 levels in the +9.5-/- AGM cause the HSC generation defect, and this rescue assay provides a unique system to decipher the downstream genetic network. To discover novel druggable regulators in the GATA-2 pathway to promote HSC generation, we profiled the expression pattern of all G-protein-coupled-receptors, which represent the most successful class of pharmaceutical targets, in the AGM using our RNA-seq dataset (+9.5+/+ vs. +9.5-/- AGM). This global GPCR analysis revealed four GATA-1 and GATA-2 co-regulated genes, Adora3, Gpr65, Ltb4r1, and Adora2b. Database mining revealed that only the Gpr65 expression pattern resembled that of Gata2. To evaluate GPR65 functions during HSC generation, we conducted an shRNA-based loss-of-function analysis in the AGM. While downregulating Gpr65 did not alter the abundance of the CD31+ c-Kit+ hematopoietic cell population, it significantly increased the CD31+ c-Kit+ Sca1+ HSC-containing cell population (1.4 fold, p<0.05), indicating that GPR65 suppresses HSC generation. To validate the involvement of GPR65 during the HSC generation process in vivo, we conducted a morpholino oligonucleotide (MO)-based loss-of-function study in zebrafish. In situ hybridization analysis revealed high Runx1/c-Myb expression (labeling definitive HSCs and progenitors) in 48% of embryos injected with Gpr65 MOs compared with 11% of wild type embryos. Consistent with the ex vivo AGM analysis, this increase in Runx1/c-Myb expression upon Gpr65 MO treatment suggests GPR65 is a negative regulator of HSC emergence in vivo. To dissect the molecular mechanism governing GPR65-suppressed HSC generation, we tested whether lowering Gpr65 levels altered the expression of key HSC regulators. Quantitative RT-PCR analysis revealed that downregulating Gpr65 by 60-70% in AGM CD31+ c-Kit- endothelialcells increased Gata2 mRNA by 2.9 fold (p<0.05), Gata2 primary transcripts by 3.9 fold (p<0.05), and elevated expression of the GATA-2 target gene Runx1 (2.9 fold, p<0.05). These results support a mechanism whereby GPR65-mediated Gata2 repression is an important determinant of GPR65-suppressed HSC generation. In addition to this important function in the AGM, Gpr65 knockdown studies in primary fetal liver HSPCs revealed GPR65 suppression of Gata2 transcription to the same magnitude as in the AGM. To determine if GPR65-mediated Gata2 repression requires the +9.5 site, we infected freshly isolated HSPCs from fetal livers heterozygous for the +9.5 site with retrovirus expressing shRNA targeting Gpr65. Quantitative RT-PCR with allele-specific primers revealed that Gpr65 knockdown significantly upregulates Gata2 primary transcripts from the wild type (3.1 fold, p<0.01), but not the 9.5 mutant, allele. These results establish a requirement of the +9.5 site for GPR65 to repress Gata2 transcription. As we reported that SetD8, the only enzyme known to monomethylate H4K20, represses Gata2 expression via the +9.5 site, we tested whether GPR65 represses Gata2 expression through SetD8. H4K20me1 ChIP revealed that downregulating Gpr65 significantly reduces H4K20me1 levels at the +9.5 site by 30% (p<0.005), suggesting that GPR65 repression of Gata2 transcription involves SetD8. Our studies indicate that a G-protein coupled receptor, GPR65, is negative regulator of HSC generation and establish a GATA-2-GPR65 Type Iincoherent feedforward loop that controls HSC generation, providing a foundation to develop new targets for expanding HSCs for transplantation therapies and a new druggable target to treat hematologic disorders. 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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Use of Genome-Wide Expression Analysis to Optimize An Ex Vivo clinical Protocol for 16,16-Dimethyl Prostaglandin E2 Enhancement of Umbilical Cord Blood In Hematopoietic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v116.21.1451.1451 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1451-1451

Author(s):

Caroline Desponts ◽

David Robbins ◽

Thuy Le ◽

Annie Chi ◽

Scott Thies ◽

...

Keyword(s):

Ex Vivo ◽

Treatment Protocol ◽

Employment Equity ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Biologic Activity ◽

Genome Wide ◽

Cd34 Cells ◽

Equity Ownership ◽

Genome Wide Expression

Abstract Abstract 1451 A systematic investigation was performed to optimize the treatment protocol for ex vivo incubation of human hematopoietic stem cells (HSCs) with 16,16-dimethyl prostaglandin E2 (FT1050) prior to transplantation. This protocol is part of an ongoing Phase Ib clinical trial of FT1050-enhanced double cord blood (CB) transplantation after reduced intensity conditioning. FT1050 has been previously shown in vertebrate models to improve the engraftment potential of HSCs from bone marrow (BM) and CB after a brief ex vivo treatment. In these models, treatment of BM or CB with FT1050 was performed for 1 to 2 hours at 4 °C, followed by a wash and subsequent cell infusion into the recipient (North et al. Nature 2007, Hoggatt et al. Blood 2009). Several groups have demonstrated that under these conditions, FT1050-treated cells have an engraftment advantage over vehicle treated cells. The objective of the current investigation was to identify a set of conditions that maximizes the biologic activity of FT1050. Genome-wide expression analysis and cAMP assays were used to optimize the ex vivo FT1050 treatment protocol with respect to concentration, time and temperature. Using this approach, hundreds of up- and down-regulated genes were identified in FT1050-treated CD34+ cells. These signature genes include upregulation of CXCR4, a known mediator of HSC homing via SDF-1a, and CREB, a key gene involved in cAMP signaling. Results from these experiments demonstrated that FT1050 concentrations above 10 μM did not result in increased levels of biologic activity. In terms of duration of incubation, cAMP activity reached maximal levels within 30 minutes of exposure while a 2 hour treatment period was necessary to maximize the changes in gene expression. Finally, the biologic activity of FT1050 was highly sensitive to temperature, with treatment of cells at 37 °C yielding larger changes in cAMP production and gene expression as compared to incubation of cells at 25 °C and 4 °C. The biological effects of FT1050 on subsets of CD34+ cells isolated from CB were also determined. Interestingly, the stem/progenitor subsets of CD34+ cells (Lin-CD34+CD38-CD90+CD45RA-, Lin-CD34+CD38-CD90-CD45RA- and Lin-CD34+) had a greater response to FT1050 relative to the lineage positive cells. The different conditions were also evaluated using CFU-C and 7-AAD assays. No evidence of adverse effects were observed. Based upon these findings, the ongoing clinical trial incorporates the optimized FT1050 ex vivo treatment protocol (10 μM for 120 minutes at 37 °C). Disclosures: Desponts: Fate Therapeutics, Inc.: Employment, Equity Ownership. Robbins:Fate Therapeutics, Inc.: Employment, Equity Ownership. Le:Fate Therapeutics, Inc.: Employment, Equity Ownership. Thies:Fate Therapeutics, Inc.: Employment, Equity Ownership. Mendlein:Fate Therapeutics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Grayson:Fate Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Multani:Fate Therapeutics, Inc.: Employment, Equity Ownership. Shoemaker:Fate Therapeutics: Employment, Equity Ownership.

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Blockade of XBP1 Splicing by Inhibition of IRE1α Is a Promising Therapeutic Option in Multiple Myeloma

Blood ◽

10.1182/blood.v118.21.133.133 ◽

2011 ◽

Vol 118 (21) ◽

pp. 133-133 ◽

Cited By ~ 2

Author(s):

Naoya Mimura ◽

Mariateresa Fulciniti ◽

Gullu Gorgun ◽

Yu-Tzu Tai ◽

Diana D. Cirstea ◽

...

Keyword(s):

Growth Inhibition ◽

Er Stress ◽

Board Of Directors ◽

Tumor Cells ◽

Therapeutic Option ◽

Rt Pcr ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership

Abstract Abstract 133 Multiple myeloma (MM) cells are characterized by high protein synthesis resulting in chronic endoplasmic reticulum (ER) stress, which is adaptively managed by the unfolded protein response (UPR). Therefore blockade of UPR could provide a novel therapeutic option in MM. Upon UPR, inositol-requiring enzyme 1α (IRE1α) is activated by auto-phosphorylation, resulting in activation of its endoribonuclease domain to cleave XBP1 mRNA from XBP1 unspliced form (XBP1u: inactive) to generate the XBP1 spliced form (XBP1s: active). XBP1s protein in turn regulates genes responsible for protein folding and degradation, playing a pro-survival signaling role in the UPR. In this study, we specifically examined whether IRE1α-XBP1 pathway is a potential therapeutic target in MM. We first examined the biologic significance of IRE1α by knockdown using lentiviral shRNA and observed significant growth inhibition in IRE1α knockdown cells. We next examined the impact of inhibition of XBP1 splicing using a novel small molecule IRE1α endoribonuclease domain inhibitor MKC-3946 (MannKind, Valencia CA). MKC-3946 blocked not only the basal level, but also inducible (by tunicamycin) XBP1s, evidenced by RT-PCR analysis in RPMI8226 cells, without affecting phosphorylation of IRE1α. Importantly, MKC-3946 also inhibited XBP1s in primary tumor cells from MM patients. We also confirmed functional inhibition of XBP1s, with target genes including SEC61A1, p58IPK, and ERdj4 downregulated by MKC-3946 treatment. Importantly, MKC-3946 triggered growth inhibition in MM cell lines, without toxicity in normal mononuclear cells. Furthermore, it significantly enhanced cytotoxicity induced by bortezomib or 17-AAG in RPMI8226 and INA6 cells, as well as primary tumor cells from MM patients. Both bortezomib and 17-AAG induced ER stress with XBP1s, which was markedly blocked by MKC-3946. Moreover, apoptosis induced by bortezomib or 17-AAG was enhanced by MKC-3946, associated with increased CHOP mRNA and protein, a proapoptotic factor triggered by ER stress. We next demonstrated that XBP1s was induced by bortezomib in INA6 cells co-cultured with bone marrow (BM) stromal cells, which was inhibited by MKC-3946, associated with enhanced cytotoxicity induced by the combination. Finally, MKC-3946 inhibited XBP1s in a model of in vivo ER stress induced by tunicamycin. To evaluate the anti-MM effect of MKC-3946, we used the subcutaneous RPMI8226 xenograft model in mice. MKC-3946 significantly reduced MM tumor growth in the treatment versus control group, associated with prolonged overall survival. We also confirmed that MKC-3946 treatment significantly inhibited XBP1s in excised tumors, assessed by RT-PCR. In order to examine the activity of MKC-3946 on MM cell growth in the context of the human BM microenvironment in vivo, we used the SCID-hu model, in which INA6 cells are directly injected into a human bone chip implanted subcutaneously in SCID-mice. MKC-3946 treatment significantly inhibited tumor growth compared with vehicle control. Moreover, XBP1s in excised tumor cells was inhibited, evidenced by RT-PCR. In conclusion, these data demonstrate that blockade of XBP1s by MKC-3946 triggers MM cell growth inhibition in vivo and prolongs host survival. Taken together, our results demonstrate that blockade of XBP1 splicing by inhibition of IRE1α endoribonuclease domain is a potential novel therapeutic option in MM. Disclosures: Tam: MannKind Corporation: Employment, Equity Ownership. Zeng:MannKind Corporation: Employment, Equity Ownership. Patterson:MannKind Corporation: Employment, Equity Ownership. Richardson:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Munshi:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; MannKind: Membership on an entity's Board of Directors or advisory committees.

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Transient Inhibition of Transforming Growth Factor-β1 in Human Blood-Derived CD34+ Cells Enhances Survival and Proliferation in Vitro and Vascular Reparative functions In Vivo.

Blood ◽

10.1182/blood.v114.22.3546.3546 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3546-3546

Author(s):

Stephen Bartelmez ◽

Ashay Bhatwadekar ◽

Patrick Iversen ◽

Francis W Ruscetti ◽

Maria Grant

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Ex Vivo ◽

Cxcr4 Expression ◽

Employment Equity ◽

Advisory Committees ◽

Cd34 Cells ◽

Equity Ownership ◽

Tgf Β1

Abstract Abstract 3546 Poster Board III-483 CD34+ cells from diabetic patients demonstrate reduced vascular reparative function due to decreased proliferation as well as diminished migration prowess which is largely due to lower levels of bioavailable nitric oxide (NO). We asked whether a transient TGF-β1 blockade in CD34+ cells from diabetics would improve their reparative ability given that TGF-β is a key factor modulating stem cell quiescence. Peripheral blood lin-CD34+ cells or lin-CD34+CD38+/− cells were treated ex vivo with antisense phosphorodiamidate morpholino oligomers (TGF-β1 -PMO), demonstrated to inhibit TGF-β1 protein expression in stem cells. Cells were then analyzed for cell surface TGF-β Receptor 2 (TGF-β R2) and CXCR4 expression, their ability to generate NO, their ability to migrate toward SDF-1, their ability to survive in the absence of added growth factors, and tested in vivo for their vascular reparative ability. After TGF-β1-PMO treatment, healthy and diabetic CD34+CD38+ and - cells downregulated TGF-βR2, upregulated CXCR4 expression, survived in the absence of added growth factors ex vivo and migrated more efficiently to SDF-1 compared to controls. TGF-β1-PMO treated diabetic CD34+ cells restored NO production to non-diabetic levels. In contrast, TGF-β1-PMO did not enhance NO generation in CD34+ cells from healthy subjects. Using an in vivo retinal ischemia reperfusion model, we observed that TGF-β1-PMO treatment increased the ability of both healthy and diabetic CD34+ cells to home to injured capillaries compared to control PMO treated cells. As also observed in our current study, a reduction of TGF-β1 levels in murine hematopoietic stem cells correlates with a reduction in TGF-βR2 expression which may induce proliferation in vivo. We also show that both diabetic and healthy lin-CD34+CD38+ cells express TGF-βR2 by FACS. In contrast, only healthy lin-CD34+CD38- cells expressTGF-βR2 while diabetic lin-CD34+CD38 - cells express essentially no cell surface TGF-βR2 (<5 % of cells are TGF-βR2+). Our results suggest that a transient blockade of TGF-β1 may represent a promising therapeutic strategy in restoring vascular reparative function in diabetic CD34+ cells. Disclosures: Bartelmez: BetaStem Therapeutics: Employment, Equity Ownership, Head, SRB, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Iversen:AVI-Biopharma: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

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Targeting Meningeoma-1 Driven AML through Epigenetic Modulation of the Cell of Origin

Blood ◽

10.1182/blood.v124.21.838.838 ◽

2014 ◽

Vol 124 (21) ◽

pp. 838-838

Author(s):

Simone Riedel ◽

Kathrin M Bernt ◽

Jessica Haladyna ◽

Matthew Bezzant ◽

Brett Stevens ◽

...

Keyword(s):

Therapeutic Target ◽

Ex Vivo ◽

Cell Of Origin ◽

Loss Of Function ◽

New Paradigm ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Epigenetic Modulation ◽

High Level

Abstract Meningeoma-1 (MN1) overexpression in AML is common and predicts a poor prognosis. Forced expression of MN1 in early murine hematopoietic progenitors (CMP and LSK) but not hematopoietic stem cells (HSC) or committed progenitors (GMP) induces an aggressive myeloid leukemia as a single hit. This leukemia is strictly dependent on the high-level expression of a defined gene expression program in the cell of origin, which includes the key component HoxA9. This “susceptibility program” has been proposed as a therapeutic target in MN1high AML, but means to modulate this program have so far remained elusive. We previously showed that the pharmacologically targetable histone methyltransferase Dot1l is critically required for HoxA9 expression in MLL-rearranged leukemias. We now report dependence on Dot1l of the MN1 “susceptibility-program” in murine MN1-driven leukemia using a genetic loss of function model. Key genes including HoxA9 depend on Dot1l in normal LSK-cells, and in MN1 transduced leukemogenic early progenitors. Inactivation of Dot1l prevents leukemia establishment and abrogates leukemia maintenance in vivo. Phenotypically, deletion of Dot1l in MN1-driven leukemias induces differentiation, cell cycle arrest and apoptosis. RNA-Seq data document that deletion of Dot1l antagonizes the cell of origin-derived susceptibility program in MN1 leukemias, including HoxA9 expression. We also confirm previous reports that MN1-transduced HSC can expand ex vivo but fail to cause leukemia in vivo. This has been linked to a lower expression level of HoxA9 in normal and MN1-transformed HSCs compared to early progenitors. We confirm that HoxA9 expression levels in MN1-transduced HSCs are lower than in MN1 transduced progenitors. Importantly, in contrast to MN1-transduced progenitors, this lower, “HSC-level” expression of HoxA9 is independent of Dot1l. This suggests a developmental switch at the HSC-to-progenitor transition that involves increased expression of HoxA9 and a change from Dot1l-independent to Dot1l-dependent regulation. The high HoxA9 expression necessary to cooperate with MN1 to cause AML is strictly Dot1l-dependent. HOXA9 is co-expressed with MN1 in a subset of clinical MN1high AML. Exposure of MN1highHOXA9high AML primary patient samples to a pharmacologic inhibitor of DOT1L (EPZ4777) demonstrated sensitivity to DOT1L inhibition as expected from the murine model. Taken together, these data point to DOT1L as a potential therapeutic target in MN1high AML. The targeted epigenetic modulation of the interplay between an oncogenic lesion and its cooperating normal developmental program could represent a new paradigm for the treatment of AML. Disclosures Pollyea: Celgene: Membership on an entity's Board of Directors or advisory committees. Armstrong:Epizyme : Consultancy.

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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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Stroma-Free Ex Vivo Expanded, CD34+ Cord Blood-Derived NK Cells Retain Lytic Activity After Long-Term Engraftment in NSGS Mice

Blood ◽

10.1182/blood.v120.21.580.580 ◽

2012 ◽

Vol 120 (21) ◽

pp. 580-580

Author(s):

Mark Wunderlich ◽

Mahesh Shrestha ◽

Lin Kang ◽

Eric Law ◽

Vladimir Jankovic ◽

...

Keyword(s):

Nk Cells ◽

Research Funding ◽

Nk Cell ◽

Ex Vivo ◽

Employment Equity ◽

Cell Product ◽

Equity Ownership ◽

Cellular Therapeutics

Abstract Abstract 580 Generating a large number of pure, functional immune cells that can be used in human patients has been a major challenge for NK cell-based immunotherapy. We have successfully established a cultivation method to generate human NK cells from CD34+ cells isolated from donor-matched cord blood and human placental derived stem cells, which were obtained from full-term human placenta. This cultivation method is feeder-free, based on progenitor expansion followed by NK differentiation supported by cytokines including thrombopoietin, stem cell factor, Flt3 ligand, IL-7, IL-15 and IL-2. A graded progression from CD34+ hematopoietic progenitor cells (HSC) to committed NK progenitor cells ultimately results in ∼90% CD3-CD56+ phenotype and is associated with an average 10,000-fold expansion achieved over 35 days. The resulting cells are CD16- and express low level of KIRs, indicating an immature NK cell phenotype, but show active in vitro cytotoxicity against a broad range of tumor cell line targets. The in vivo persistence, maturation and functional activity of HSC-derived NK cells was assessed in NSG mice engineered to express the human cytokines SCF, GM-CSF and IL-3 (NSGS mice). Human IL-2 or IL-15 was injected intraperitoneally three times per week to test the effect of cytokine supplementation on the in vivo transferred NK cells. The presence and detailed immunophenotype of NK cells was assessed in peripheral blood (PB), bone marrow (BM), spleen and liver samples at 7-day intervals up to 28 days post-transfer. Without cytokine supplementation, very few NK cells were detectable at any time-point. Administration of IL-2 resulted in a detectable but modest enhancement of human NK cell persistence. The effect of IL-15 supplementation was significantly greater, leading to the robust persistence of transferred NK cells in circulation, and likely specific homing and expansion in the liver of recipient mice. The discrete response to IL-15 versus IL-2, as well as the preferential accumulation in the liver have not been previously described following adoptive transfer of mature NK cells, and may be unique for the HSC-derived immature NK cell product. Following the in vivo transfer, a significant fraction of human CD56+ cells expressed CD16 and KIRs indicating full physiologic NK differentiation, which appears to be a unique potential of HSC-derived cells. Consistent with this, human CD56+ cells isolated ex vivo efficiently killed K562 targets in in vitro cytotoxicity assays. In contrast to PB, spleen and liver, BM contained a substantial portion of human cells that were CD56/CD16 double negative (DN) but positive for CD244 and CD117, indicating a residual progenitor function in the CD56- fraction of the CD34+ derived cell product. The BM engrafting population was higher in NK cultures at earlier stages of expansion, but was preserved in the day 35- cultured product. The frequency of these cells in the BM increased over time, and showed continued cycling based on in vivo BrdU labeling 28 days post-transfer, suggesting a significant progenitor potential in vivo. Interestingly, DN cells isolated from BM could be efficiently differentiated ex vivo to mature CD56+CD16+ NK cells with in vitro cytotoxic activity against K562. We speculate that under the optimal in vivo conditions these BM engrafting cells may provide a progenitor population to produce a mature NK cell pool in humans, and therefore could contribute to the therapeutic potential of the HSC-derived NK cell product. The in vivo activity of HSC-derived NK cells was further explored using a genetically engineered human AML xenograft model of minimal residual disease (MRD) and initial data indicates significant suppression of AML relapse in animals receiving NK cells following chemotherapy. Collectively, our data demonstrate the utility of humanized mice and in vivo xenograft models in characterizing the biodistribution, persistence, differentiation and functional assessment of human HSC-derived cell therapy products, and characterize the potential of HSC-derived NK cells to be developed as an effective off-the-shelf product for use in adoptive cell therapy approaches in AML. Disclosures: Wunderlich: Celgene Cellular Therapeutics: Research Funding. Shrestha:C: Research Funding. Kang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Law:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Jankovic:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Zhang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Herzberg:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Abbot:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hariri:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Mulloy:Celgene Cellular Therapeutics: Research Funding.

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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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Development of a Human Therapeutic L-Cyst(e)Ine-Degrading Enzyme for the Treatment of Hematological Malignancies

Blood ◽

10.1182/blood.v128.22.1587.1587 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1587-1587

Author(s):

Giulia Agnello ◽

Susan Alters ◽

Joseph Tyler ◽

Jinyun Liu ◽

Peng Huang ◽

...

Keyword(s):

Research Funding ◽

Hematological Malignancies ◽

Ex Vivo ◽

Redox Balance ◽

Lymphocytic Leukemia ◽

Employment Equity ◽

Equity Ownership ◽

Antioxidant Mechanisms

Abstract Cancer cells experience higher intrinsic oxidative stress than their normal counterparts and acquire adaptive antioxidant mechanisms to maintain redox balance. This increased antioxidant capacity has been correlated to malignant transformation, metastasis and resistance to standard anticancer drugs. This enhanced antioxidant state also correlates with cancer cells being more vulnerable to additional oxidative insults, therefore disruption of adaptive antioxidant mechanisms may have significant therapeutic implications. Hematological malignancies including Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), Acute Myeloid Leukemia (AML) and Multiple Myeloma (MM) are critically dependent on the cellular antioxidant glutathione (GSH), consistent with the higher intrinsic oxidative stress. L-cysteine is the rate-limiting substrate for GSH biosynthesis and adequate levels of cysteine are critical to maintain the intracellular homeostasis of GSH. CLL and a subset of ALL cells have been reported to rely on the stromal supply of cysteine to increase the synthesis of GSH in order to maintain redox balance, which in turn promotes cell survival and fosters drug resistance. One approach to target this cancer specific dependency is by therapeutic depletion of amino acids via enzyme administration; a clinically validated strategy for the treatment of ALL. Aeglea BioTherapeutics Inc. has developed a bioengineered cysteine and cystine degrading enzyme (Cyst(e)inase, AEB3103) and evaluated its therapeutic efficacy against hematological malignancies in in vitro, ex vivo and in vivo pre-clinical studies. The TCL1-TG:p53 -/- mouse model exhibits a drug resistant phenotype resembling human CLL with unfavorable cytogenetic alterations and highly aggressive disease progression. AEB3103 greatly decreased the viability of TCL1-TG:p53 -/- cells cultured in vitro, whereas the CLL therapeutic, fludarabine, showed minimal cytotoxic effects. In vivo treatment of TCL1-TG:p53 -/- mice with AEB3103 resulted in an increase in median survival time (7 months, p<0.0001) compared to the untreated control group (3.5 months, p<0.001) and a fludarabine treated group (5.3 months, p<0.001). These results indicate a superior therapeutic effect of AEB3103 compared to fludarabine. Additionally, evaluation of AEB3103 in in vitro 2D cultures of patient-derived CLL and MM cells, and in ex vivo 3D cultures of cells derived from ALL and AML PDx models resulted in significant cell growth inhibition with therapeutically relevant IC50 values. Collectively these results demonstrate the sensitivity of hematological malignancies to modulation of GSH levels via AEB3103-mediated cyst(e)ine depletion. Disclosures Agnello: Aeglea BioTherapeutics: Employment. Alters:Aeglea BioTherapeutics: Employment, Equity Ownership. Tyler:Aeglea BioTherapeutics: Employment, Equity Ownership. Huang:Aeglea BioTherapeutics: Research Funding. Stone:Aeglea Biotherapeutics: Consultancy, Equity Ownership, Research Funding; University of Texas at Austin: Employment, Patents & Royalties: I am an inventor of technology related to this abstract. Georgiou:Aeglea Biotherapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Lowe:Aeglea BioTherapeutics: Employment, Equity Ownership. Rowlinson:Aeglea BioTherapeutics: Employment, Equity Ownership.

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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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