Eicosanoid-GPCR Signaling Enhances Hematopoiesis and Marrow Transplant

Abstract Small molecule treatment of hematopoietic stem cells ex vivo has the potential to expand these cells or increase their engraftability. Previously, we discovered that ex vivo treatment of marrow with 11,12-epoxyeicosatrienoic acid (EET) enhances the engraftment of hematopoietic stem and progenitor cells in both zebrafish and mammals. Additionally, EET treatment promotes specification of HSPC from the hemogenic endothelium, suggesting a broad pro-hematopoietic role of this molecule. Indeed, bioactive lipids play an important role as signaling molecules both during embryo development and adult tissue homeostasis. However, due to their small-molecule nature, identifying their receptors biochemically has been a long-standing challenge which impedes the understanding of the biological processes they regulate. The identity of the EET receptor remains unknown despite more than a decade of research. Here, we utilized a novel bioinformatic approach to identify candidate EET receptors and identified a candidate functional in cell culture, zebrafish and mouse assays. EET signaling is known to be G-protein dependent, suggesting its receptor is a G-protein coupled receptor (GPCR). We performed RNAseq on U937 monocytes, EaHy endothelial cells, and PC3M-LN4 prostate cancer cells, three human cell lines with clear EET-responsive phenotypes. These three cell lines expressed 37 GPCR in common at a basal level of greater than or equal to 0.3 fragments per kilobase per million reads (FPKM). 27 of these GPCR were also expressed in a non-EET-responsive cell line, HEK293, leaving only 10 candidate EET receptors. We screened 7 of these candidates for EET-responsiveness using a cell-culture based β-arrestin recruitment assay. Of these, only GPR132 exhibited EET-dependent recruitment of β-arrestin to the cell membrane, indicating GPCR activation. GPR132 was previously identified as a receptor for a variety of small oxygenated fatty acids, and we confirmed that these related molecules induce GPR132-dependent β-arrestin recruitment. We additionally treated developing zebrafish embryos with these molecules. Like EET, these GPR132 ligands increased HSPC numbers in the zebrafish aorta-gonad-mesonephros (AGM) and caused ectopic expression of the HSPC marker runx1 in the zebrafish tail, a phenotype that was previously seen only with EET treatment. To test the requirement of GPR132 for EET signaling, we knocked down the zebrafish ortholog of GPR132 by morpholino injection, which prevented the EET-induced increase of runx1in both the AGM and tail. Finally, we performed competitive whole bone marrow transplant using wildtype and GPR132-/- mice as donors and found that while treatment with EET increases engraftment of WT donor cells, no such improvement is seen in GPR132-/- cells. GPR132 is thus required in both zebrafish and mice for EET phenotypes. Combining bioinformatic, biochemical, and genetic approaches, we identified GPR132 as a receptor for EET involved in regulating hematopoiesis and marrow transplant. GPR132 thus represents a therapeutic target for the enhancement of hematopoietic stem cell transplant, and genetic manipulation of GPR132 could help illuminate the endogenous roles of its fatty acid ligands. Disclosures Zon: Fate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Marauder Therapeutics: Equity Ownership, Other: Founder; Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

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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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Simultaneous Kinase Inhibition with Ibrutinib and BCL2 Inhibition with Venetoclax As a Therapeutic Strategy for Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2019-131288 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3950-3950

Author(s):

Christopher A. Eide ◽

Stephen E Kurtz ◽

Andy Kaempf ◽

Nicola Long ◽

Jessica Leonard ◽

...

Keyword(s):

Clinical Trial ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Mononuclear Cells ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Advisory Committees ◽

Equity Ownership

Background: In patients with acute lymphoblastic leukemia (ALL), patient outcomes vary considerably by patient age group, specific genetic subtypes, and treatment regimen. Large-scale sequencing efforts have uncovered a spectrum of mutations and gene fusions in ALL, suggesting that combinations of agents will be required to treat these diseases effectively. Previous preclinical studies have shown efficacy of the BCL2 inhibitor venetoclax alone or in combination in ALL cells (Chonghaile et al., Can Disc 2014; Leonard et al, STM 2018), and the multi-kinase inhibitor ibrutinib (approved for patients with chonic lymphoblastic leukemia (CLL)) has also shown potent activity in subsets of ALL (Kim et al., Blood 2017). However, the combination of ibrutinib and venetoclax has not been evaluated to date in patients with ALL. Methods: We used a functional ex vivo screening assay to evaluate the potential efficacy of the combination of ibrutinib and venetoclax (IBR+VEN) across a large cohort (n=808) of patient specimens representing a broad range of hematologic malignancies. Primary mononuclear cells isolated from leukemia patients were plated in the presence of graded concentrations of venetoclax, ibrutinib, or the combination of both FDA-approved drugs. IC50 and AUC values were derived from probit-based regression for each response curve. A panel of clinical labs, treatment information, and genetic features for tested ALL patient specimens was collated from chart review. Single and combination drug treatment sensitivity were compared within groups by Friedman test, across groups by Mann-Whitney test, and with continuous variables by Spearman rank correlation. Results: Consistent with clinical data and previous literature, IBR+VEN was highly effective in CLL specimens ex vivo (median IC50=0.015 µM). Intriguingly, among specimens from 100 unique ALL patients, we also observed that IBR+VEN demonstrated significantly enhanced efficacy by AUC and IC50 compared to either single agent (p<0.001; median IC50=0.018 µM). In contrast, evaluation of this combination on primary mononuclear cells from two healthy donors showed little to no sensitivity. Breakdown of combination sensitivity (as measured by AUC) by a variety of clinical and genetic features revealed no associations with gender or specimen type. Among continuous variables tested, age was modestly correlated with combination AUC (Spearman r = 0.26) and increased blasts in the bone marrow were associated with increased sensitivity to the combination (Spearman r = -0.41; p = 0.0068). More broadly, specimens from patients with B-cell precursor disease (B-ALL) were more sensitive to IBR+VEN than those with T-cell precursor leukemia (T-ALL) (p = 0.0063). Within the B-ALL patient samples, those harboring the BCR-ABL1 fusion were significantly less sensitive to IBR+VEN than other subtypes of B-ALL (p = 0.0031). Within the T-ALL subset, there was a trend toward reduced sensitivity in patients with evidence of mutations in NOTCH1, though statistical significance was not reached. Evaluation of the combination using an expanded 7x7 concentration matrix in human ALL cell lines revealed varying degrees of sensitivity. For example, IBR+VEN showed enhanced efficacy in RCH-ACV B-ALL cells and showed synergy for the majority of drug-pair concentrations by the highest single agent (HSA) method (ibrutinib, venetoclax, and combination IC50: 0.60, 3.4, and 0.28 uM, respectively). Conclusion: Our findings suggest that the IBR+VEN combination, currently approved for patients with CLL, also demonstrates impressive efficacy against primary leukemia cells from ALL patients, warranting further investigation as a treatment strategy in the clinic to continue to improve outcomes for patients. Disclosures Leonard: Amgen: Research Funding. Druker:Cepheid: Consultancy, Honoraria; Pfizer: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Merck & Co: Patents & Royalties: Dana-Farber Cancer Institute license #2063, Monoclonal antiphosphotyrosine antibody 4G10, exclusive commercial license to Merck & Co; Dana-Farber Cancer Institute (antibody royalty): Patents & Royalties: #2524, antibody royalty; OHSU (licensing fees): Patents & Royalties: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees ; Gilead Sciences: Other: former member of Scientific Advisory Board; Beta Cat: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Aptose Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; ALLCRON: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Patents & Royalties, Research Funding; Pfizer: Research Funding; Aileron Therapeutics: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees , Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Novartis: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Patents & Royalties: Patent 6958335, Treatment of Gastrointestinal Stromal Tumors, exclusively licensed to Novartis, Research Funding; GRAIL: Equity Ownership, Other: former member of Scientific Advisory Board; Patient True Talk: Consultancy; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Beat AML LLC: Other: Service on joint steering committee; CureOne: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; ICON: Other: Scientific Founder of Molecular MD, which was acquired by ICON in Feb. 2019; Monojul: Other: former consultant; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Burroughs Wellcome Fund: Membership on an entity's Board of Directors or advisory committees. Tyner:Petra: Research Funding; Agios: Research Funding; Array: Research Funding; Gilead: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Syros: Research Funding; Takeda: Research Funding; Seattle Genetics: Research Funding; AstraZeneca: Research Funding; Seattle Genetics: Research Funding; Array: Research Funding; Aptose: Research Funding; Incyte: Research Funding; Syros: Research Funding; Takeda: Research Funding; Petra: Research Funding; Agios: Research Funding; Constellation: Research Funding; Aptose: Research Funding; Gilead: Research Funding; Incyte: Research Funding; AstraZeneca: Research Funding; Constellation: Research Funding; Janssen: Research Funding; Genentech: Research Funding.

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Extensive Metabolic Correction of Hurler Disease By Hematopoietic Stem Cell-Based Gene Therapy: Preliminary Results from a Phase I/II Trial

Blood ◽

10.1182/blood-2019-128805 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 607-607 ◽

Cited By ~ 1

Author(s):

Bernhard Gentner ◽

Maria Ester Bernardo ◽

Francesca Tucci ◽

Erika Zonari ◽

Francesca Fumagalli ◽

...

Keyword(s):

Gene Therapy ◽

Phase I ◽

Board Of Directors ◽

Financial Support ◽

Joint Venture ◽

Ex Vivo ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Equity Ownership

Allogeneic hematopoietic stem cell transplantation (HSCT) performed early in life is the current standard of care for patients with severe type 1 mucopolysaccharidosis (Hurler disease), a metabolic disorder caused by mutations in the alpha-L-iduronidase (IDUA) gene, leading to impaired breakdown of glycosaminoglycans (GAG). Secretion of IDUA by donor-derived hematopoietic cells may cross-correct non-hematopoietic cells, slowing progression of tissue damage and cognitive decline. Nevertheless, Hurler patients undergoing HSCT manifest substantial residual disease burden, e.g. on the skeleton and central nervous system (CNS). We conducted a phase I/II clinical study (NCT03488394) to test whether infusion of autologous CD34+ hematopoietic stem and progenitor cells (HSPC) transduced ex vivo with a lentiviral vector coding for the IDUA gene was feasible, safe and capable of restoring enzymatic activity in the patients' blood and tissues, up to supraphysiologic levels. The trial originally planned to enroll 6 Hurler patients with preserved neurocognitive function (DQ/IQ>70) that had no access to a suitable allogeneic donor. Sample size has recently been increased to 8 patients. By July 2019, six patients have been treated at a median age of 24 months (range: 14-34), with a median follow up of 4 months (range: 1-13). In all patients, we collected a high number of autologous HSPC by leukapheresis following mobilization with lenograstim and plerixafor, resulting in drug products with a median of 21 million CD34+ cells/kg (range: 13-29). Transduction efficiency was high with a median above 80% and a vector copy number (VCN) of 1.7 (range: 1.0-5.2), employing a shortened, 2 day transduction protocol that included prostaglandin E2. All patients showed rapid hematopoietic recovery following myeloablative conditioning with busulfan (targeted to an AUC of 80mg*h/L), fludarabine (160mg/sqm) and rituximab (375mg/sqm). Median duration of grade 4 neutropenia associated with conditioning was 15.5 days (range: 13-19). Also associated with conditioning, Grade 3 thrombocytopenia lasted 4 days, while only 2 out of 6 patients experienced a platelet drop below 20,000/mcL on a single day, in the absence of transfusion support. Adverse events were mild and compatible with myeloablative conditioning, with the exception of patient 3 who experienced an anaphylactic reaction on day+12, which promptly responded to antihistamines, IV fluids and steroids. All evaluable patients showed sustained, supraphysiologic blood IDUA activity (dried blood spot), which was on average 3 fold above the upper limit of normal (evaluable patients: n=5 at 1 month, n=4 at 2 months, n=3 at 3 months). Notably, in n=4 Hurler patients treated with allogeneic HSCT, we detected IDUA activity that ranged within the lowest quartile of normal in spite of full donor chimerism, suggesting substantial gain achieved by overexpressing IDUA in ex vivo genetically-modified autologous HSPC. Urinary GAG excretion fell to normal levels within 3-6 months. IDUA activity was also detected in the cerebrospinal fluid (CSF) of treated patients, accompanied by a logfold reduction in CSF GAGs in the 2 patients with longest follow up. This suggests that gene therapy accomplishes full metabolic correction of tissues, including the CNS. Gene therapy did not induce antibodies against the IDUA protein, while pre-existing antibodies induced by enzyme replacement therapy before gene therapy rapidly disappeared. Patient 1 who reached the 1-year follow-up demonstrated a stable cognitive score, improved findings on brain and spine MRI, resumed growth velocity and an improvement of his skeletal phenotype. The preliminary results from our phase I/II study compare favorably with the standard of care in terms of safety and efficacy, and highlight the potential of genetic engineering of HSPC grafts for therapeutic gain-of-function. Disclosures Gentner: Genenta Science: Consultancy, Equity Ownership, Research Funding. Parini:Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support; BioMarin: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support; Ultragenyx: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support; SOBI: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support; Orphan Europe: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support; Sanofi-Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support. Naldini:San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Wiskott-Aldrich Syndrome (WAS) gene therapy was licensed to GlaxoSmithKline (GSK) in 2014. It was then licensed to Orchard Therapeutics (OTL) in April 2018. OTL is the current sponsor of the clinical trial.; Genenta Science: Consultancy, Equity Ownership; Magenta Therapeutics: Equity Ownership. Aiuti:San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Wiskott-Aldrich Syndrome (WAS) gene therapy was licensed to GlaxoSmithKline (GSK) in 2014. It was than licensed to Orchard Therapeutics (OTL) in April 2018. OTL is the current sponsor of the clinical trial.; San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Study PI.

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

Blood ◽

10.1182/blood.v116.21.1170.1170 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1170-1170

Author(s):

Alison M. Taylor ◽

Jessica M. Humphries ◽

Richard M. White ◽

Ryan D. Murphey ◽

Caroline E. Burns ◽

...

Keyword(s):

Ribosomal Protein ◽

Board Of Directors ◽

Ribosomal Proteins ◽

Enrichment Analysis ◽

Gene Set Enrichment Analysis ◽

Ribosomal Protein Genes ◽

Advisory Committees ◽

Cardinal Vein ◽

Hematopoietic Stem ◽

Equity Ownership

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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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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