Human CD34-positive Cells Research Articles

LSD1 Inhibition Synergizes with Venetoclax in Acute Myeloid Leukemia By Targeting Cellular Metabolism

Introduction: Acute myeloid leukemia (AML) is a hematological neoplasm with poor clinical outcomes. The introduction of the BCL2 inhibitor venetoclax in combination with hypomethylating agent has improved response rates in older patients with AML by targeting the metabolism of leukemic stem cells (LSCs). Resistance, however, is unfortunately still common. Thus, there remains a need to enhance the clinical efficacy of venetoclax by combining it with novel agents. In diverse AML models, lysine-specific demethylase 1 (LSD1) inhibition promotes cellular differentiation and reduces LSCs. We therefore asked if LSD1 inhibition could enhance the efficacy of venetoclax in the treatment of AML. Methods: We have studied the effect of single-agent and co-treatment with venetoclax and LSD1 inhibitor bomedemstat on cell proliferation in human AML cell lines (N=3), Hoxa9/ Meis1(H9M)- and MN1-transformed murine myeloid progenitor lines as well as primary AML patient samples (N=5). We have analyzed the effect of co-treatment with these two compounds on functional cellular bioenergetics in MOLM-13 cells using Seahorse extracellular flux analyzer and followed up by performing NMR spectrometry to study changes in metabolite abundance. Further, we have studied the efficacy of this drug combination in vivo in patient-derived xenograft (PDX) AML models (N=2). Results: Human AML cell lines showed a differential cytocidal activity at 96 hours (h) to venetoclax with MOLM-13 (IC 50 0.04 µM) being most sensitive and OCI-AML3 most resistant (IC 50 9.81 µM). A similar range (between 0.15 µM and 7.3 µM) was also observed in the primary AML samples. For bomedemstat, we observed a time-dependent response where the IC 50 decreased over time, in particular, in the most sensitive model systems. In murine H9M cells, the IC 50 decreased from 30.43 µM at 48 h to 0.034 µM at 96 h and further down to 0.0068 µM at 168 h. Combining bomedemstat and venetoclax, the IC 50 for venetoclax decreased; and synergism was observed in MOLM-13 cells, the most sensitive AML model, but no synergism was observed in venetoclax-resistant cells. Synergism was also observed in MN1 cells as well as 3 of the 5 primary AML samples studied (Table 1). To investigate the mechanism of the observed synergism, we explored the effect of these treatments on metabolic pathways using the Seahorse extracellular flux analyzer. LSD1 inhibition had no effect on oxidative phosphorylation (measured as oxygen consumption rate (OCR)). However, glycolysis, measured as proton efflux rate (PER), was significantly reduced from 102.88 ± 11.03 pmol/min to 54.17 ± 6.18 pmol/min (P <0.01) after 96 h of treatment with 1μM bomedemstat. With the combined treatments, we observed a reduction in both OCR and PER. This synergistic effect was further investigated by studying metabolite abundance using NMR spectrometry where co-treatment showed a reduction in metabolite abundance across a broad spectrum of metabolic pathways (Fig. 1). To determine if the observed combined effect of bomedemstat and venetoclax could enhance activity against AML cells in vivo, we employed a PDX model with two primary AML samples. In the first model system (monosomy 7), co-treatment resulted in a significant reduction in human CD45-positive cells as compared to vehicle (P <0.001) and single-agent treatments with venetoclax (P= 0.011) and bomedemstat (P <0.001). Similarly, in the second PDX model ( NPM1-mutated, FLT3-ITD positive), the co-treatment exhibited a significant reduction in leukemic burden in bone marrow as compared to vehicle (P=0.003) and single-agent treatments with venetoclax (P= 0.024) and bomedemstat (P < 0.001) (Fig. 1). Conclusion: The combination of bomedemstat and venetoclax had synergistic cytocidal effects on AML cell line and primary AML cells in vitro. It significantly reduced the leukemic burden in PDX AML models and synergistically downregulated cellular energy metabolism. These findings suggest that combining venetoclax with LSD1 inhibition holds promise as a combination treatment in AML and warrants further clinical investigation.

Single and Combinational Multiplex Base-Edited 'Universal' CAR T Cells in a Humanised Model of Primary CD7+CD33+ AML

Introduction Universal allogeneic donor CAR T cells are being developed as part of a 'deep conditioning' strategy against AML to secure disease clearance ahead of allogeneic stem cell transplantation (Allo-SCT). CD33 is widely expressed on AML, sometimes on the entire leukemia-associated populations, but is also present on healthy precursors; CD7 may be expressed on AML subpopulations and is present on normal T and NK cells, resulting in problematic fratricide effects during CAR T manufacture. Healthy donor derived CAR T cell banks generated using cytidine deamination base-editing and lentiviral transduction with CARs against both CD33 and CD7 circumvent such issues and could offer cell based anti-leukemic therapy alongside preparative effects ahead of allo-SCT. Here we investigated single and combinational BE-CAR T cells against primary human AML in vivo. Methods: Healthy donor lymphocytes were activated by anti-CD3/28 stimulation ahead of electroporation to deliver codon optimised base editor mRNA and guide RNAs against CD7, CD52 and TRBC, and lentiviral transduction to express either an anti-CD7 or anti-CD33 CAR. Magnetic bead depletion removed residual TCRab T cells, and disruption of CD7 ensured cells avoided fratricide. In vitro, antigen specific function was verified in cytotoxicity and cytokine release assays, and in vivo potency was confirmed against luciferase modified Molm14 and Kasumi lines in NSG mice. Responses against primary AML were investigated in a patient derived xenograft model (PDX) from a KIT mutated AML patient, established by serial transplantation, with full expression of CD33 (100% of blasts) and partial expression of CD7 (30% of blasts). Cells were injected into irradiated NSG mice (n=10 per group). On day 24 at a mean leukemic engraftment of 15% human CD45 positive cells in peripheral blood, mice were treated with either 10x106 BE-CAR33, BE-CAR7 or a combination of both or with anti-CD19-CAR-T cells (CAR19) as a control group (in total 20x106 T cells per mouse). AML engraftment, T cell persistence and expression of checkpoint markers were monitored by multicolour flow cytometric analysis. Results: On days 14 and 28 after CAR-T cell infusion, AML cells were readily detected in peripheral blood of control CAR19 animals (42.8% and 90% respectively) and the BE-CAR7 group (27.8% and 67.6%), but were notably reduced in the BE-CAR33 alone group (2.3% and 8.3%) and the BE-CAR33 and BE-CAR7 combination group (2.5% and 3.4%) (Figure 1A). Survival was significantly prolonged for BE-CAR33 and BE-CAR33/BE-CAR7 treated mice compared to the other groups (P<0.001) (Figure 1B). CD7+ AML cells were depleted to a mean of 1% and 4.5% in the BE-CAR33 group, to 0.6% and 0.7% in the BE-CAR33/BE-CAR7 and to 2.6% and 2.9% in the BE-CAR7 group, while CD7+ AML cells increased to 12% and 30% in the CAR19 group at day 14 and 28 after CAR-T cell infusion. Total CD2+CAR-T cell detection in peripheral blood ranged from 1.6 to 9.8% at day 14, 1 to 3.7% at day 28, and 0.8 to 2.7% at day 42, indicating durable persistence over the investigation period. Interestingly, PD-L1 on AML cells was present in all groups while checkpoint markers PD1, GAL9, TIM3, and LAG3 were significantly upregulated in BE-CAR33 cell treated mice, but not BE-CAR7 or CAR19 cell treated mice (P<0.001 for all comparisons). Conclusions: Lentiviral transduction and multiplexed base editing to remove TCR, CD52 and the shared AML/T lineage antigen CD7, enabled the generation of universal donor CAR T cells for combinational use. This data demonstrates robust activity of BE-CAR33 alone and in combination with BE-CAR7 against human CD7+CD33+AML in mice and highlights the importance of strategies that ensure complete targeting of leukemic populations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Preclinical Evaluation of a Novel Orally Bioavailable Menin-MLL Interaction Inhibitor, DSP-5336, for the Treatment of Acute Leukemia Patients with MLL-Rearrangement or NPM1 Mutation

Background: The 11q23 abnormalities involving mixed lineage leukemia (MLL) gene are frequently found in adult and pediatric patients with acute leukemia. MLL rearrangements (MLL-r) are often associated with a poor prognosis and show poor response to currently available therapies, thus developing more effective therapy is urgently required. The leukemogenic activity of MLL fusion proteins, the products of the chimeric genes of MLL and its fusion partners generated by MLL-r, is critically dependent on direct interaction with MENIN, the product of the MEN1 gene. Interaction of MLL fusion proteins with MENIN plays an important role to enhance the proliferation and to block the differentiation of hematopoietic cells by maintaining high expression of hematopoietic stem cell program genes, such as HOXA9 and MEIS1. It has also been reported that the MENIN interaction with wild-type MLL, is required to induce HOXA9 and MEIS1 expression and also crucial for the development of acute leukemia with nucleophosmin (NPM1) mutations. Therefore, the MENIN-MLL interaction inhibitor is expected to have anti-leukemogenic activity against acute leukemia with MLL-r or NPM1 mutation by suppressing the expression of HOXA9 and MEIS1 and inducing terminal differentiation.Results: We generated DSP-5336, a novel, potent, and orally bioavailable MENIN-MLL interaction inhibitor for the treatment of acute leukemia patients with MLL-r or NPM1 mutation. DSP-5336 directly bound to the MENIN protein (Kd = 6.0 nM) and inhibited the MENIN-MLL interaction (IC 50 = 1.4 ± 0.058 nM). DSP-5336 selectively inhibited the cell growth of human leukemia cell lines including MV-4-11, MOLM-13, KOPN-8, and OCI-AML3 (IC 50 = 10, 15, 31 and 15 nM, respectively). These DSP-5336-sensitive cell lines possess a MLL-r or NPM1 mutation. On the other hand, DSP-5336 did not affect the cell growth of human leukemia cell lines such as HL-60, MOLT-4, and Reh (IC 50 > 10 μM), which do not have MLL-r or NPM1 mutations. In a mouse xenograft model using MV-4-11 cells, which express MLL-AF4, DSP-5336 exhibited a significant antitumor activity at the doses of 25 mg/kg and 50 mg/kg, administered twice daily (BID) for 20 days. There were no dose related changes in general condition or body weight. The effects of DSP-5336 on the expression of MENIN-MLL-regulated genes and differentiation marker genes were evaluated using MV-4-11 cells as pharmacodynamics markers in vitro and in vivo. In both cases, DSP-5336 significantly reduced the gene expression of MEIS1 and HOXA9, representative leukemic genes regulated by the MENIN-MLL complex. On the contrary, DSP-5336 significantly increased the gene expression level of ITGAM, a terminal differentiation marker.The efficacy of DSP-5336 was further assessed in acute leukemia patient samples and in mouse AML models. DSP-5336 strongly inhibited blast colony formation and changed the gene expression of the pharmacodynamics markers (HOXA9, MEIS1 and ITGAM ) in an AML patient sample carrying the MLL-AF6 fusion. In patient-derived xenograft (PDX) model with NPM1 mutation, human CD45 positive cells in peripheral blood progressively decreased during and beyond the 28 day period of DSP-5336 administration at doses of 25, 50, and 100 mg/kg BID and achieved a complete remission with no relapse at the doses of 50 and 100 mg/kg BID. At these three dose levels, DSP-5336 also induced a significant prolongation of survival compared to the vehicle control. Similarly, in a PDX model with MLL-AF4, DSP-5336 induced complete remission and significant prolongation of survival at the doses of 100 mg/kg BID compared to the vehicle control. In mouse AML models wherein MLL-ENL- or MLL-AF10-transduced bone marrow cells are transplanted in syngeneic mice, DSP-5336 induced a significant prolongation of survival at the doses of 200 mg/kg once daily (QD) compared to the vehicle control and the standard chemo therapy (cytarabine+daunorubicin) group.Summary: DSP-5336 has a potential as an antitumor drug that provides survival advantages in acute leukemia patients with MLL rearrangement or NPM1 mutation. Currently, a Phase 1/2 clinical study of DSP-5336 is planned in AML and ALL patients. DisclosuresEguchi: Sumitomo Dainippon Pharma: Current Employment. Shimizu: Sumitomo Dainippon Pharma: Current Employment. Kato: Sumitomo Dainippon Pharma: Current Employment. Furuta: Sumitomo Dainippon Pharma: Current Employment. Kamioka: Sumitomo Dainippon Pharma: Current Employment. Ban: Sumitomo Dainippon Pharma: Current Employment. Ymamoto: Sumitomo Dainippon Pharma: Current Employment. Yokoyama: Sumitomo Dainippon Pharma: Research Funding. Kitabayashi: Sumitomo Dainippon Pharma: Research Funding.

Anti-B7-H3 Antibody (T-1A5) Blocks Immunomodulatory Function of B7-H3 and Enhances NK Cell-Mediated Cytotoxicity Against Acute Myeloid Leukemia Cells

Background: The immune checkpoint molecule B7-H3 (CD276) is overexpressed in various solid tumors and hematological malignancies; however, its expression is limited in normal tissue, which makes it an attractive therapeutic target in cancer. Several monoclonal antibodies (mAbs) targeting B7-H3 have shown promising results against solid tumors. However, B7-H3's role in acute myeloid leukemia (AML) remains unexplored. Here, we hypothesized that targeting B7-H3 using mAbs alters the immunomodulatory function of B7-H3 and enhances NK cell-mediated cytotoxicity against AML cells.Methods: B7-H3 protein expression was analyzed in the peripheral blood (PB) and bone marrow of 100 patients with AML and 20 healthy donors by flow cytometry and tested for associations with multiple clinical parameters and disease outcomes. To investigate B7-H3's role in immunomodulation, we stably knocked down B7-H3 in AML cell lines including OCI-AML3, MV4-11, and U937 and co-cultured them with activated human NK cells. NK cell-induced apoptosis was measured by annexin-v binding assay using an IncuCyte live-cell imaging system. B7-H3-blocking mAbs (clones T-1A5, HEK5-1B3, and 58B1) were tested for their effect on NK-cell-mediated cytotoxicity in AML cell lines using live-cell imaging. AML xenograft (OCI-AML3) or patient-derived xenograft (PDX) models were used to determine the effect of anti-B7-H3 antibodies on AML growth. In vivo AML growth was monitored by measuring human CD45 positive cells with flow cytometry. A human-mouse chimeric (ch) antibody was generated based on sequences from the T-1A5 antibody, and its binding site on the B7-H3 protein was characterized by epitope mapping. Further, we evaluated the effect of chT-1A5 on NK cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) in primary AML cells and healthy donor PB-derived mononuclear cells (PBMCs).Results: Expression of B7-H3 was significantly higher in AML patients than in healthy donors (p < 0.01) and was higher in CD34 positive than in CD34 negative AML cells (p < 0.01). High B7-H3 expression was associated with poor overall survival (p = 0.04) and prognostic risk scores (p = 0.05). NK cell-mediated apoptosis was 3-fold higher in all 3 B7-H3-knockdown AML cell lines than in scrambled control cells, suggesting that B7-H3 is an important immunomodulator of NK cells. Moreover, we observed a significant increase in NK cell-mediated killing of AML cells in the presence of anti-B7-H3 mAbs (p < 0.01). In vivo, anti-B7-H3 antibodies significantly inhibited AML growth and extended survival in PDX models compared to IgG control treatment. Among PDX-bearing mice treated with the three anti-B7-H3 antibodies, the T-1A5 antibody-treated group survived for longer than the other groups. In combination with NK cells, T-1A5 treatment also significantly increased the survival of AML xenograft-bearing mice compared to NK + IgG control treatment. These data suggest that the T-1A5 antibody blocks B7-H3 and enhances NK cell-mediated cytotoxicity in AML cells in vivo. Next, we found that a chT-1A5 antibody induced NK cell-mediated ADCC in primary AML cells and cell lines in a dose-dependent manner. In contrast, it did not induce any ADCC activity in healthy donor-derived PBMCs, suggesting that chT-1A5 is not toxic to healthy cells. Moreover, the chT-1A5 antibody combined with human NK cells dramatically inhibited leukemia growth and extended survival in B7-H3 positive AML PDX models (p < 0.001) compared to the control (rituximab-treated) group. Finally, epitope mapping using peptides derived from the B7-H3 protein identified the FG loop region of B7-H3 as the binding site for the T-1A5 antibody, which may be involved in the immunomodulatory function of B7-H3.Conclusion: B7-H3 is overexpressed in AML cells, and its expression is associated with poor overall survival. Anti-B7-H3 antibodies block B7-H3's immunomodulatory function and inhibit AML growth in vivo. A chT-1A5 antibody in combination with NK cells induced ADCC in primary AML cells in vitro and in vivo but had no effect on PBMCs from healthy donors. Therefore, targeting B7-H3 could benefit AML patients, specifically those with a poor clinical prognosis. DisclosuresBattula: Tolero Pharmaceuticals: Research Funding.

Dissecting the Immune Cell Progeny of CAR-Expressing Hematopoietic Stem Cells in a Humanized Mouse Model

Background: Chemotherapy-refractory or recurrent B-lineage leukemias and lymphomas yield less than 50% of chance of cure. Therapy with autologous T-cells expressing chimeric antigen receptors (CAR) have led to complete remissions, but the effector cells may not persist, limiting clinical efficacy. Our hypothesis is the modification of hematopoietic stem cells (HSC) with anti-CD19 CAR will lead to persistent generation of multilineage target-specific immune cells, enhancing graft-versus-cancer activity and leading to development of immunological memory. Design/Methods: We generated second-generation CD28- and 4-1BB-costimulated CD19-specific CAR constructs using third-generation lentiviral vectors for modification of human HSC for assessment in vivo in NSG mice engrafted neonatally with human CD34-positive cells. Cells were harvested from bone marrows, spleens, thymus and peripheral blood at different time points for evaluation by flow cytometry and ddPCR for vector copy numbers. Cohorts of mice received tumor challenge with subcutaneous injection of lymphoma cell lines. Results: Gene modification of HSC with CD19-specific CAR did not impair differentiation or proliferation in humanized mice, leading to CAR-expressing cell progeny in myeloid, NK and T-cells. Humanized NSG engrafted with CAR-modified HSC presented similar humanization rates to non-modified HSC, with multilineage CAR-expressing cells present in all tissues with stable levels up to 44 weeks post-transplant. No animals engrafted with CAR-modified HSC presented autoimmunity or inflammation. T-cell populations were identified at higher rates in humanized mice with CAR-modified HSC in comparison to mice engrafted with non-modified HSC. CAR-modified HSC led to development of T-cell effector memory and T-cell central memory phenotypes, confirming the development of long-lasting phenotypes due to directed antigen specificity. Mice engrafted with CAR-modified HSC successfully presented tumor growth inhibition and survival advantage at tumor challenge with lymphoma cell lines, with no difference between both constructs (62.5% survival for CD28-costimulated CAR and 66.6% for 41BB-costimulated CAR). In mice sacrificed due to tumor development, survival post-tumor injection was directly correlated with tumor infiltration by CAR T-cells. Conclusions: CAR modification of human HSC for cancer immunotherapy is feasible and continuously generates CAR-bearing cells in multiple lineages of immune cells. Targeting of different malignancies can be achieved by adjusting target specificity, and this approach can augment the anti-lymphoma activity in autologous HSC recipients. It bears decreased morbidity and mortality and offers alternative therapeutic approach for patients with no available sources for allogeneic transplantation, benefiting ethnic minorities. Disclosures De Oliveira: National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London: Research Funding; NIAID, NHI: Research Funding; Medical Research Council: Research Funding; CIRM: Research Funding; National Gene Vector Repository: Research Funding.

MTOR Signal Pathway Inhibition Promote the Cytotoxicity of Chemotherapy on SHI-1 Cells

Acute myeloid leukemia (AML) is a malignant hematologic disorder. Although current chemotherapy could improve the survival rate of AML patients, some patient would suffer from relapse and had bad therapeutic response. Nowadays, many studies have found that the abnormal expression of mTOR signal pathway is closely related to the occurrence and progression of AML, but the roles of mTOR inhibitor in AML is not elucidated. So, in this paper, we would investigate whether rapamycin, a mTOR inhibitor, could enhance the cytotoxicity of chemotherapy. Rapamycin or chemotherapy (adriamycin plus cytarabine) could inhibit the proliferation and induce apoptosis of SHI-1 cells (the proliferation inhibition rate and apoptosis ratio were 35.4%±2.94% and 13.33%±0.74% respectively in rapamycin group; Chemotherapy group: 49.9%±4.11%, 13.2%±1.11%). When rapamycin were combined with chemotherapy, the proliferation inhibition rate and apoptosis ratio were significantly increased to 60.3%±3.36% and 30%±1.74% respectively (p&lt;0.05) (Fig.1A, Fig.1B). Western blotting shown that chemotherapy could enhance the expression of Bax, Beclin-1 and ATG5, and inhibit the expression of Bcl-2, caspase-3, nevertheless chemotherapy didn't influence the expression of proteins in the mTOR signal pathway. When mTOR signal pathway were inhibited by rapamycin, the proteins of mTOR signal pathway such as mTOR, p-mTOR, P70S6K1, p-P70S61, 4EBP1, p-4EBP1, and the apoptosis related proteins Bcl-2 and caspase3 of SHI-1 cells were decreased, otherwise, the expression of autophagy-related proteins LC3B/II, Beclin-1, ATG5 and apoptosis-related proteins Bax increased. Rapamycin combined with chemotherapy could significantly inhibit the expression of proteins in mTOR signal pathway and enhance the expression of autophagy and apoptosis-related proteins of SHI-1 cells than that treated by rapamycin alone (Fig.1C). In order to explore whether mTOR pathway inhibition could enhance the cytotoxicity of chemotherapy in AML animal model. 1×107 SHI-1 cells were inoculated into NPG mice via tail vein to construct systemic leukemia NPG mice. On the 19th day after inoculation, the ratio of human CD45 and 33 double positive cells in peripheral blood of NPG mice was 1.3%, then NPG mice were randomly divided into four groups, control group didn't had treatment, chemotherapy group was treated by adriamycin (1mg/kg) once every other day for three times and cytarabine (100mg/kg) once a day for five days, rapamycin group was treated by rapamycin (10mg/kg) once a day for five days, and combination group was treated by combination chemotherapy and rapamycin. On the first day (24th day)after treatment, the ratio of human CD33 and CD45 double positive cells in peripheral blood, bone marrow and spleen of NPG mice in each treatment group were: control group (20.3%, 23.6%, 15.6%), chemotherapy group (13.6%, 14.1%, 5.50%), rapamycin group (2.03%, 2.68%, 3.31%), combined group (0.308%, 2.49%, 1.10%) (Fig. 1D). The weight of spleen in NPG mice of combination group were significantly lighter than the weight of spleen of NPG mice of other three groups. Pathological immunohistochemistry showed that the infiltration of human CD45 positive cells in spleen of mice in the combination group was significantly lower than that of the other three groups (Fig. 1E). The median survival time of NPG mice in the control group, chemotherapy group, rapamycin group and combination group were 30.5 days, 35.0 days, 34.0 days and 39.0 days, respectively. The survival time of NPG mice in combination group was significantly longer than that in chemotherapy and rapamycin group (P&lt;0.05), while there was no significant difference between chemotherapy group and rapamycin group (P&gt;0.05) (Fig. 1F). When NPG mice were dead, more neoplasms were found in the NPG mice in control group, the treatment of rapamycin or chemotherapy could significantly decrease the degree of extramedullary infiltration of SHI-1 cells in NPG mice, and fewer neoplasms were found in the NPG mice of combination group(Fig.1G). In conclusion, our studies verified that mTOR signal pathway inhibition by rapamycin could enhance the cytotoxicity of chemotherapy via inhibiting mTOR signal pathway, promoting cell autophagy and inducing apoptosis. mTOR signal pathway may be a potential target for the treatment of AML. Figure 1 Disclosures No relevant conflicts of interest to declare.

Generation and Molecular Characterization of Human Ring Sideroblasts

(Background) Sideroblastic anemias are heterogeneous congenital and acquired refractory anemias characterized by bone marrow ring sideroblasts, reflecting excess mitochondrial iron deposition. While the disease is commonly associated with myelodysplastic syndrome, the congenital forms of sideroblastic anemias comprise a diverse class of syndromic and non-syndromic disorders, which are caused by the germline mutation of genes involved in iron-heme metabolism in erythroid cells. Although the only consistent feature of sideroblastic anemia is the bone marrow ring sideroblasts, evidence on the detailed molecular characteristics of ring sideroblasts is scarce owing to a lack of the biological models. We have recently established ring sideroblasts by inducing ALAS2 gene mutation based on human-induced pluripotent stem cell-derived erythroid progenitor (HiDEP) cells (ASH 2017) and have further extended the molecular characterization of human ring sideroblasts to gain new biological insights.(Method) We targeted the GATA-1-binding region of intron 1 of the human ALAS2 gene in HiDEP cells and established two independent clones [X-linked sideroblastic anemia (XLSA) clones]. A co-culture with OP9 stromal cells (ATCC) was conducted with IMDM medium supplemented with FBS, erythropoietin, dexamethasone, MTG, insulin-transferrin-selenium, and ascorbic acid. To obtain human primary erythroblasts, CD34-positive cells isolated from cord blood were induced in a liquid suspension culture (Fujiwara et al. JBC 2014). Bone marrow glycophorin A (GPA)-positive erythroblasts of patients with XLSA and normal individuals were separated using the MACS system (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) after obtaining written informed consent. For transcription profiling, Human Oligo chip 25K (Toray) was used.(Results) We previously demonstrated that co-culture with OP9 cells in the medium supplemented with 100 uM sodium ferrous citrate (SFC) promoted erythroid differentiation of XLSA clones, which enabled the establishment of ring sideroblasts (ASH 2017). To confirm the importance of SFC in terminal erythroid differentiation, we further demonstrated that the addition of SFC, and not transferrin-loaded iron, induced the frequency of GPA+ cells and TfR1-GPA+ mature erythroid population, based on primary erythroblasts derived from human CD34-positive cells. Subsequently, to reveal the molecular mechanism by which abnormal iron mitochondrial iron accumulation occurs by co-culture with SFC, we evaluated the expressions of various metal transporters, demonstrating that the addition of SFC significantly increased the expressions of mitoferrin 1 (MFRN1; a ferrous iron transporter in mitochondria), divalent metal transporter 1 (DMT1), and Zrt- and Irt-like protein 8 (ZIP8; a transmembrane zinc transporter, recently known as a ferrous iron transporter) in the XLSA clone than the wild-type cells, which would have contributed to the formation of ring sideroblasts.Moreover, we performed expression analyses to elucidate the biochemical characteristics of ring sideroblasts. After co-culture with OP9 in the presence of SFC, ring sideroblasts exhibited more than two-fold upregulation and downregulation of 287 and 143 genes, respectively, than the wild-type cells. Interestingly, when compared with the expression profiling results before co-culture (ASH 2017), we noticed prominent upregulation of gene involved in anti-apoptotic process (p = 0.000772), including HSPA1A, superoxide dismutase (SOD) 1, and SOD2. In addition, we conducted a microarray analysis based on GPA-positive erythroblasts from an XLSA patient and a normal individual. The analysis revealed significant upregulation of genes involved in the apoptosis process, as represented by apoptosis enhancing nuclease, DEAD-box helicase 47, and growth arrest and DNA-damage-inducible 45 alpha, and anti-apoptotic genes, such as HSPA1A and SOD2. Concomitantly, when the XLSA clone was co-cultured with OP9 in the presence of SFC, the apoptotic cell frequency as well as DNA fragmentation were significantly reduced compared with the XLSA clone co-cultured without SFC, indicating that ring sideroblasts avoid cell death by inducing anti-apoptotic properties.(Conclusion) Further characterization of the XLSA model would help clarify its molecular etiology as well as establish novel therapeutic strategies. DisclosuresFukuhara:Celgene: Research Funding; Chugai: Research Funding; Daiichi-Sankyo: Research Funding; Boehringer Ingelheim: Research Funding; Eisai: Honoraria, Research Funding; GlaxoSmithKline: Research Funding; Janssen: Honoraria, Research Funding; Japan Blood Products Organization: Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding; Mitsubishi Tanabe: Research Funding; Mundipharma: Honoraria, Research Funding; MSD: Research Funding; Nippon-shinyaku: Research Funding; Novartis pharma: Research Funding; Ono: Honoraria, Research Funding; Otsuka Pharmaceutical: Research Funding; Pfizer: Research Funding; Sanofi: Research Funding; Symbio: Research Funding; Solasia: Research Funding; Sumitomo Dainippon: Research Funding; Taiho: Research Funding; Teijin Pharma: Research Funding; Zenyaku Kogyo: Honoraria, Research Funding; Takeda: Honoraria; Baxalta: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Bayer Yakuhin: Research Funding; Alexionpharma: Research Funding; AbbVie: Research Funding; Astellas: Research Funding; Nihon Ultmarc: Research Funding.

Establishment of Human Acute Monocyte Leukemia Model with Systemic Leukemia in Npg Mice

Although tumor cells are easily to growth in the bodies of immunodeficicent animals such as nude mice and NOD-SCID mice, it's hard for acute leukemia cells to grow in the bone marrow of nude mice or NOD-SCID mice even when mice receive extra immunosuppressive treatment such as splenectomy, cyclophosphamide and irradiation. This study aimed to establish a mice model with systemic leukemia using another highly immunodeficicent NPG mice without immunosuppressive treatment before inoculation. 5-week NPG mice were inoculated with 1x107(Group A) or 5x107 (Group B) SHI-1 cells (a cell line derived from a refractory acute monocytic leukemia patient) via tail vein. One NPG mice in each group was killed by ether randomly at the day 14, 21, 28 after inoculation, other NPG mice were observed the survival time. The leukemic cells engrafted in the NPG mice were detected by the following methods: the blast cells were detected by the blood smear and flow cytometer, the MLL-AF6 fuse gene of SHI-1 cells were detected by PCR amplification, the human CD45 positive cells infiltrated in the organs of NPG mice were detected by histopathological examination and immunohistochemistry. At the day 14 after inoculation with SHI-1 cells, fewer blasts cells were found in the smear of peripheral blood of group B; MLL-AF6 fuse gene could be amplified in the spleen of NPG mice in group A and in spleen and bone marrow in group B (Fig A); histopathological examination had shown that CD45 positive leukemia cell just infiltrated in spleen. At the day 21 after inoculation, more blasts were found in the smear of peripheral blood both in group A and B; MLL-AF6 fuse gene were amplified in the organs of NPG mice such as Spleen, liver, kidney, stomach, lung, heart and bone marrow(Fig A); 5.16% and 0.82% of CD45 and CD33 positive cells were detected in the peripheral blood of NPG mice in group A and B respectively; a green solid neoplasm were found in the kidney of NPG mice in group B, leukemia cells were found in the organ of heart, liver, spleen, stomach, kidney and lung in the NPG mice of both groups by histopathological examination. From the third week, the NPG mice presented anorexia, hunched posture, lethargy and weight loss. For the mice sacrificed in the day 28 after inoculation, the proportion of CD45 and CD33 positive cells in peripheral blood, bone marrow and spleen were 9.60%, 11.4% and 23.20% in group A and were 11.0%,37.80% and 60.5% in group B (Fig B). Green solid tumors were grown in many organs such as kidney, liver, spleen, stomach, heart, lymph node and the soft tissues in the NPG mice killed in day 28 after inoculation and the mice which were dead spontaneous (Fig C); When NPG mice were dead, the weight of spleen in group B is significantly higher than the weight of spleen in group A(P<0.05) (Fig D). The median survival time of NPG in group A and group B is 33 and 30 days respectively. Pathological examination and immunohistochemical staining had shown that leukemic cells could infiltrated to many of the organs of NPG mice and the grade of leukemia infiltration was positively correlated with cells numbers of inoculation and the survival time of NPG mice (Fig E). Altogether, SHI-1 cell could growth in the NPG mice without any pre-immunosuppressive treatment and formed a systemic leukemia in NPG mice as like in acute leukemia patients. This efficient and reproducible model may be a useful tool for the studies of the pathogenesis in acute monocytic leukemia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Maintenance of human haematopoietic stem and progenitor cells in vitro using a chemical cocktail

Identification of effective culture conditions to maintain and possibly expand human HSPCs in vitro is an important goal. Recent advances highlight the efficacy of chemicals in maintaining and converting cell fates. We screened 186 chemicals and found that a combination of CHIR-99021, Forskolin and OAC1 (CFO) maintained human CD34-positive cells in vitro. Efficiency of the culture system was characterized using flow cytometry for CD34-positive cells, a colony-forming assay and xeno-transplants. We found that human CD34-positive cells treated with this combination had enhanced expression of human HSPC markers and increased haematopoietic re-populating ability in immune-deficient mice. Single-cell RNA-seq analyses showed that the in vitro cultured human CD34-positive cells were heterogeneous. We found that CFO supports maintenance of human CD34-positive cells by activating HOXA9, GATA2 and AKT-cAMP signaling pathway. These data have implications in therapies requiring maintenance and/or expansion of human HSPCs.

Morphological and optical properties of human immature platelet-enriched population produced in immunodeficient mice.

Ultrastructure analysis of immature platelets is difficult because of the lack of a suitable marker and their relatively low concentration in total platelets. We investigated the morphological and optical properties of human immature platelets produced and enriched in immunodeficient mice via human CD34-positive cell administration. Immunodeficient mice were injected with human CD34-positive cells and administered eltrombopag orally for 14days (eltro-mice). Some of these mice were maintained for 2-3months (steady-state-mice). Platelets were double-stained with a human CD41 antibody and a nuclear staining dye (Sysmex hematology analyzer XN series reagent), and then analyzed by flowcytometry FCM to identify human immature platelets. Human CD41-positive cells were isolated from citrated blood by magnetic cell sorting with human CD41 antibody, and examined using electron microscopy. Flow cytometric analysis with the XN reagent demonstrated that peripheral blood from eltro-mice had a higher percentage of immature platelet fraction in human platelets than that from steady-state-mice. The geometric mean of XN reagent fluorescence for human platelets, divided with that for mouse platelets, revealed that the ratios in eltro-mice were significantly higher than those in steady-state-mice, thus indicating that immature platelets were highly enriched in eltro-mice. Scanning and transmission electron microscopy revealed that human citrated platelets isolated from eltro-mice tended to be larger (n=15, p=0.276) and contained more mitochondria than those isolated from steady-state-mice (n=10, p=0.0002). Therefore, an increased number of mitochondria, rather than platelet size, is a distinctive feature of immature platelets.

Abstract 1938: Store-operated calcium signaling is an effective therapeutic target in AML

Abstract Acute myeloid leukemia (AML) is the most prevalent adult leukemia characterized by genetic or mutational disruption of myeloid differentiation, growth arrest, and apoptosis. Standard “7 + 3” chemotherapy consisting of cytarabine in combination with an anthracycline such as daunorubicin has been used for more than four decades. However this course only results in 10 year disease free survival in 15% of patients age &amp;lt;60 years and 2% among those &amp;gt; 60 years. Several new successful therapies in AML are shown to work by promoting differentiation, and/or inducing apoptosis of the terminally differentiated cells. However these therapies, while promising, have the limitation of working in only a subset of AML with specific genetic profiles. The discovery of novel agents applicable to a broader patient population still represents a critical unmet need in this disease. Intracellular calcium is a common signaling molecule used in a variety of cellular processes including cell cycle progression. One of the major pathways which regulates calcium entry is store operated calcium entry (SOCE) mediated through calcium release-activated Ca2+ channels (CRAC), which respond to depleted calcium stores in ER lumen. This primarily occurs via interaction of ORAI1 (expressed on the plasma membrane) and STIM1 (expressed on the endoplasmic reticulum). Leukemic cells in particular often rely on the influx of calcium through CRAC for proliferation, therefore targeting these channels are a promising therapeutic option. In the current study, we explore the preclinical use of RP4010, a novel inhibitor of ORAI1 developed by Rhizen Pharmaceuticals, in AML. RP4010 is very effective at inhibiting proliferation of both AML cell lines and primary AML patient samples (IC50 values ranging from 0.2 - 1 μM using a tetrazolium-based colorimetric assay (MTS) assay). Importantly, RP4010 efficacy is not altered in the presence of stromal cell support. RP4010 also decreased colony growth in CFU assays. Finally, we evaluated the in vivo efficacy using an MV4-11 xenograft model. RP4010 treatment at 50 mg/kg once daily by oral gavage significantly prolonged survival compared to the vehicle control (p = 0.019). Furthermore, at the time of sacrifice, splenic tumor burden (percentage of human CD45 positive cells) was reduced in the mice treated with RP4010 compared to the vehicle group (p = 0.019). In conclusion, we provide here compelling evidence that inhibition of ORAI1 with RP4010 is a promising therapeutic strategy in AML. A Phase 1/1b study in R/R Non-Hodgkin Lymphoma (NHL) patients is currently underway in the US (ClinicalTrials.gov Identifier: NCT03119467), and our data would support expansion to other hematological malignancies as well, particularly AML. Senior authors J. C. B and E. H contributed equally to this work. Citation Format: Bridget Carmichael, Amy Lehman, Ola A. Elgamal, Shelley J. Orwick, Jean Truxall, Larry Beaver, Kumar V. Penmetsa, Srikant Viswanadha, John C. Byrd, Erin Hertlein. Store-operated calcium signaling is an effective therapeutic target in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1938.

Development of a new knock-in mouse model and evaluation of pharmacological activities of lusutrombopag, a novel, nonpeptidyl small-molecule agonist of the human thrombopoietin receptor c-Mpl

Lusutrombopag (S-888711), an oral small-molecule thrombopoietin receptor (TPOR) agonist, has gained first approval as a drug to treat thrombocytopenia of chronic liver disease in patients undergoing elective invasive procedures in Japan. Preclinical studies were performed to evaluate its efficacy against megakaryopoiesis and thrombopoiesis. To investigate the proliferative activity and efficacy of megakaryocytic colony formation via human TPOR, lusutrombopag was applied to cultured human c-Mpl-expressing Ba/F3 (Ba/F3-hMpl) cells and human bone marrow-derived CD34-positive cells, respectively. Lusutrombopag caused a robust increase in Ba/F3-hMpl cells by activating pathways in a manner similar to that of thrombopoietin and induced colony-forming units-megakaryocyte and polyploid megakaryocytes in human CD34-positive cells. Because lusutrombopag has high species specificity for human TPOR, there was no suitable experimental animal model for drug evaluation, except for immunodeficient mouse-based xenograft models. Therefore, a novel genetically modified knock-in mouse, TPOR-Ki/Shi, was developed by replacing mouse Mpl with human-mouse chimera Mpl. In TPOR-Ki/Shi mice, lusutrombopag significantly increased circulating platelets in a dose-dependent manner during 21-day repeated oral administration. Histopathological study of the TPOR-Ki/Shi mice on day 22 also revealed a significant increase in megakaryocytes in the bone marrow. These results indicate that lusutrombopag acts on human TPOR to upregulate differentiation and proliferation of megakaryocytic cells, leading to platelet production.

Biological implications of somatic DDX41 p.R525H mutation in acute myeloid leukemia.

The DDX41 gene, encoding a DEAD-box type ATP-dependent RNA helicase, is rarely but reproducibly mutated in myeloid diseases. The acquired mutation in DDX41 is highly concentrated at c.G1574A (p.R525H) in the conserved motif VI located at the C-terminus of the helicase core domain where ATP interacts and is hydrolyzed. Therefore, it is likely that the p.R525H mutation perturbs ATPase activity in a dominant-negative manner. In this study, we screened for the DDX41 mutation of CD34-positive tumor cells based on mRNA sequencing and identified the p.R525H mutation in three cases among 23 patients. Intriguingly, these patients commonly exhibited acute myeloid leukemia (AML) with peripheral blood cytopenias and low blast counts, suggesting that the mutation inhibits the growth and differentiation of hematopoietic cells. Data from cord blood cells and leukemia cell lines suggest a role for DDX41 in preribosomal RNA processing, in which the expression of the p.R525H mutant causes a certain ribosomopathy phenotype in hematopoietic cells by suppressing MDM2-mediated RB degradation, thus triggering the inhibition of E2F activity. This study uncovered a pathogenic role of p.R525H DDX41 in the slow growth rate of tumor cells. Age-dependent epigenetic alterations or other somatic changes might collaborate with the mutation to causeAML.

Sonic Hedgehog signaling regulates vascular differentiation and function in human CD34 positive cells: Vasculogenic CD34+ cells with Sonic Hedgehog

Identification of pivotal factors potentially present in the in situ environment and capable of influencing the function of CD34+ cells, which can be used for autologous cell therapy, is of paramount interest. SHh is one of the morphogens essential for embryonic vascular development as well as postnatal neovascularization, and the activation of SHh signaling with angiogenic and vascular differentiation responses in CD34+ cells by SHh treatment differed depending on the G-CSF treatment or the background disease. SHh enhanced the migration, proliferation, adhesion, and EPC colony forming capacities of G-CSF mobilized CD34+ cells, increasing the vasculogenic/angiogenic potential for neovascularization. An increase in the differentiation potential of CD34+ cells toward vascular lineages was demonstrated with SHh treatment involving TGFβ signaling pathway. The SHh-activated G-CSF mobilized CD34+ cells directly contributed to vascular regeneration while non-activated CD34+ cells showed a lower regenerative capacity in a mouse ischemic hindlimb model. SHh signaling regulates human CD34+ cell fate and function, and may potentiate the therapeutic effect of G-CSF mobilized CD34+ cells on ischemic diseases.

ZFN-Driven Gene Editing Prevents HLA-A Expression On Hematopoietic Stem Cells -Improving The Chance Of Finding An HLA-Matched Donor

Hematopoietic stem cells (HSCs) are administered (i) to restore hematopoiesis and immunity in the course of hematopoietic stem-cell transplantation (HSCT), (ii) as a replacement for inherited blood disorders and bone marrow failure, (iii) to regenerate cells of alternative lineages for restorative medicine, and (iv) as a source for generating specific hematopoietic cells (e.g., T cells, NK cells, and dendritic cells). However, the widespread application of allogeneic HSCs for humans is hampered by their immune-mediated destruction by host T cells recognizing mismatched HLA or by HLA-specific antibodies. Despite pre-banking umbilical cord blood (UCB) units and access to adult donors through the National Marrow Donor Program (NMDP), finding a suitable HLA-matched product is challenging for many recipients, especially those from ethnic minorities who are under-represented in the donor pool. The available donor pool would be markedly increased if donor HSCs were edited to eliminate expression of the HLA-A locus. Indeed, modeling from NMDP shows that the chance of an African American recipient finding a HLA-matched donor increases from 18% to 73% when matched for HLA-B, C and DR, instead of HLA-A, B, C and DR. We have previously shown that engineered zinc finger nucleases (ZFNs) can disrupt HLA-A expression in genetically edited T cells (Blood 2013). To extend this proof-of-concept to HSCs, we sought to disrupt HLA-A expression by introducing ZFNs targeting this locus. CD34+lineageneg HSCs (99% purity) were isolated using paramagnetic beads from UCB. Electro-transfer of in vitro transcribed mRNA encoding the HLA-A-specific ZFN generated 30% HLA-Aneg HSCs after one week ex vivo culture with defined cytokines (FLT3-L, SCF, TPO, and IL-6) and an aryl hydrocarbon receptor antagonist (stem reginin-1, SR-1). As expected, SR1 treatment maintained greater numbers of CD34+ cells (also CD34posCD38neg) in culture compared to controls. DNA sequence analysis revealed that HLA-Aneg HSCs encode the expected nucleotide changes at the ZFN target site. An in vivo engraftment assay, using NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, demonstrated that HLA-Aneg HSCs maintain the capability of engraftment and differentiation into HLA-Aneg hematopoietic cells (Figure). Thus, disruption of HLA-A expression in HSCs provides an appealing approach to increasing the chances for of finding HLA-matched donors and may broaden the clinical application of allogeneic HSCT. Furthermore, the ability to genetically edit HSCs has implications for (i) preventing immune-mediated recognition of HLA-disparate HSC and (ii) preventing immune mediated recognition of self-antigens.Engraftment of HLA-A2neg HSCs was evaluated in vivo. Data shown are flow-cytometry analysis of bone marrow obtained from NSG mice 16 weeks after HSC injection. HSC engraftment and HLA-A2 expression in NSG mice injected with un-modified HSCs (left panel) and HSCs treated with the HLA-A specific ZFNs (right panel) are shown. Data are gated on human CD45 positive cells. [Display omitted] Disclosures:Reik:Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment.

Establishment of xenotransplantation model of human CN-AML with FLT3-ITD mut /NPM1 − in NOD/SCID mice

Patients with FLT3-ITD (mut) /NPM1 (-) cytogenetically normal acute myeloid leukemia (CN-AML), as high-risk molecular group in CN-AML, are associated with a worse prognosis than other CN-AML patients. It is beneficial to generate xenotransplantation model of FLT3-ITD (mut) /NPM1 (-) CN-AML to better understand the pathogenesis and therapeutic strategies of such AML subtype. The purpose of present study was to establish the xenotransplantation model in NOD/SCID mice with FLT3-ITD (mut) /NPM1 (-) CN-AML primary cells. The FLT3-ITD (mut) /NPM1 (-) CN-AML primary cells from 3 of 7 cases were successfully transplanted into NOD/SCID mice, and human CD45 positive cells were detected in the peripheral blood, spleen and bone marrow of mice by using flow cytometry. Infiltration of human leukemia cells in various organs of mice was observed by using immunohistochemistry. Gene analysis confirmed sustained FLT3/ITD mutation without NPM1 mutation in mice. By performing serial transplantation, it was found that characteristics of the leukemia cells in secondary and tertiary generation models remained unchanged. Moreover, in vivo cytarabine administration could extend survival of NOD/SCID mice, which was consistent with clinical observation. In conclusion, we successfully established xenotransplantation model of human FLT3-ITD (mut) /NPM1 (-) CN-AML in NOD/SCID mice. The model was able to present primary disease and suitable to evaluate the curative effects of new drugs or therapy strategies.

Role of transcriptional corepressor ETO2 in erythroid cells

Transcriptional corepressor ETO2 is a component of a protein complex containing master regulators of hematopoiesis, including GATA-1, SCL/TAL1, LMO2, and LDB1. To elucidate the role of ETO2 during erythroid differentiation, including the effects of ETO2 on GATA-1 targets, we performed gene expression profiling using K562 cells overexpressed with ETO2. The analysis demonstrated that 667 and 598 genes were upregulated and downregulated (more than twofold), respectively, in ETO2-overexpressing cells. ETO2-repressed genes included those encoding prototypical erythroid proteins. To test what percentages of ETO2-repressed genes could be direct target genes of GATA-1 in K562 cells, we merged the microarray results with ChIP-seq profile (n=5,749), demonstrating that 23.1% of ETO2-repressed genes contained significant GATA-1 in their loci. However, there was no significant enrichment of PU.1 target genes among ETO2-repressed genes. Gene ontology analysis among ETO2-repressed genes revealed significant enrichment of genes related to "oxygen transporter," corresponding to globin genes. Quantitative chromatin immunoprecipitation and ETO2 knockdown analyses confirmed that ETO2 directly regulates globin genes in K562 cells. Next, we evaluated the role of ETO2 in human primary erythroblasts, derived from cord blood CD34-positive cells. In an exvivo model of erythroid differentiation from CD34-positive cells, ETO2 protein level peaked at day 2-4 and almost diminished at the later stage of differentiation. Furthermore, short hairpin RNA-mediated knockdown and retroviral vector-mediated overexpression of ETO2 in primary erythroblasts suggested that ETO2 significantly represses HBB, HBA, and ALAS2 expression. In summary, ETO2 regulates GATA-1 target genes critical for erythroid differentiation, and the decrease of ETO2 levels during erythroid differentiation would contribute to the activation of these targets.

Retroviral Ectopic Expression of a Signaling-Defective Thrombopoietin Receptor (Mpl) Induces a Systemic Loss of Hematopoietic Stem Cells in Mice,

Abstract 4175MPL signaling, induced by the binding of Thrombopoietin (THPO), regulates megakaryopoiesis and platelet development, and is essential for hematopoietic stem cell (HSC) maintenance. Murine lineage negative, Sca-1 and c-kit positive bone marrow (BM) cells (LSK cells) and human CD34 positive cells expressing MPL have long term BM reconstitution capacities. In human patients, deficiency of MPL due to inactivating mutations in the MPL gene causes severe thrombocytopenia and progressive aplastic anemia, which is lethal if not treated by BM transplantation (BMT). This rare genetic disorder is termed congenital amegakaryocytic thrombocytopenia (CAMT).With the final aim of developing gene therapy to treat CAMT, we explored the effects of ectopic Mpl expression in wild-type C57Bl/6 mice. The enforced overexpression of Mpl induced a rapid expansion of all three hematological cell lineages, resembling chronic myeloproliferative disease (CMPD) 2 months after BMT (n=25 mice). Surprisingly, mice subsequently developed life threatening pancytopenia with normal Thpo levels despite low platelet counts (167+/−52 × 103/μl). The BM showed reduced LSK cell numbers (0.01+/−0.01 % versus controls 0.05+/−0.015%, p=0.03). Non-transduced, co-transplanted BM cells could not rescue the disturbed hematopoiesis. Based on these observations, we hypothesized that the ectopic overexpression of Mpl in hematopoietic cells induces a dominant-negative effect, dysregulating the Thpo-Mpl balance.To test our hypothesis, we ectopically expressed a signaling-defective truncated dominant-negative Mpl (dnMpl) receptor, lacking the complete intracellular domain, in a BMT model in wild-type C57Bl/6 mice (n=23). Lineage negative cells were transduced with gammaretroviral vectors expressing dnMpl from the SFFV promoter in the LTRs and transplanted into lethally irradiated mice. We observed long term engraftment with 57.6+/−1.9% dnMpl positive mononuclear cells in the peripheral blood (controls 76.7+/−7.8%) after 19 weeks and severe thrombocytopenia (174 +/− 32 x103/μl) in the dnMpl expressing animals (controls 951+/−233×103/μl). After 23 weeks, we detected 15–34% dnMpl positive BM cells (controls 15–99%). To address the question of whether the BM of dnMpl treated mice contained fewer HSCs compared to control transplanted mice, we analyzed the LSK cell number and saw a ∼4.6-fold reduction (0.02+/− 0.005% versus control mice 0.07+/−0.012%; p=0.0016). As Thpo/Mpl signaling is known to induce HSC quiescence, we speculated that the loss of these signals induces HSC cycling. Cell cycle analysis of the LSK cells of dnMpl treated mice showed a transition into G1/S/G2, indicating a loss of quiescent HSCs (G0 3.5 +/−1.6% vs controls 29.8 +/−10.5%). This effect was even more pronounced in CD34 negative LSK cells (5.4+/−4.0% vs. 44.5+/−8.5%). Secondary transplantation of dnMpl BM showed severely reduced repopulation capacity with 5 out of 6 secondary recipients dying due to graft failure.In selected animals we transplanted another batch of BM after 16 weeks without preconditioning of the mice. Long term repopulation of the second graft was seen in 71–79% of dnMpl treated mice (controls 0.2–1.1%) and BM cells further engrafted in secondary recipients.To address changes in HSC gene expression, we sorted LSK cells from dnMpl BM (pool of 4–5 dnMpl mice for each sample) and also separated dnMpl positive and negative cells. Our controls were LSK cells from control transplanted mice (one per sample). RNA was analyzed on an Affymetrix Mouse Genome 430 2.0 array in triplicates. Unsupervised clustering and principle component analysis revealed that dnMpl positive and negative samples clustered, suggesting a systemic effect on HSCs. We tested more than 4000 gene sets from the MySigDB Database (Broad Institute, Cambridge, MA) for enrichment in either of the phenotypes and found a profound downregulation of gene sets containing “stemness” genes in the LSK cells of dnMpl treated mice, irrespective of cellular dnMpl expression.In summary, we show that ectopic expression of dnMpl in a subset of BM cells disturbs Thpo/Mpl-signaling inducing thrombocytopenia and a systemic loss of HSCs. Besides underlining the strict requirement for regulated Mpl expression in gene therapy for CAMT, these data create new hypotheses for the pathogenesis of aplastic anemia and the development of non-cytotoxic conditioning regimens in BMT. Disclosures:No relevant conflicts of interest to declare.

Small Molecule Screens for Stem Cell Expansion

Abstract SCI-42 The identification of safe and effective methods to expand human hematopoietic stem cells (HSC) would have a major impact on the use of HSC in clinical medicine. Several features of human HSC, including the lack of a suitable cell line model and cumbersome methods for quantification, have made the identification of conditions for human HSC expansion challenging. Current culture methods using cytokine cocktails in serum-free media support the robust proliferation of CD34 positive (CD34+) cells but this is accompanied by rapid differentiation such that after 1 week of culture fewer than 20% of cells continue to express CD34. To overcome these limitations we developed a high throughput screen that uses primary human CD34+ cells and multiparameter flow cytometry to identify compounds capable of expanding human CD34 positive cells. By screening &gt;100,000 LMW compounds we identified a molecule (SR1) that enhanced CD34 expression during ex vivo culture. Culture of CD34+ cells with cytokines and SR1 for 3 weeks leads to a &gt;600-fold increase in the number of CD34+ cells, and a &gt;2000-fold increase in the number of CFU compared to starting cell numbers. Importantly, cells expanded in the presence of SR1contain a 17-fold increase in the number of NOD-SCID repopulating cells compared to starting cell numbers. Mechanistic studies reveal that SR1 binds to and antagonizes the aryl hydrocarbon receptor (AHR). Knockdown of the AHR in CD34+ cells using lentiviral transduction also maintains CD34 expression. These findings suggest that AHR normally promotes HSC differentiation during ex vivo culture and that AHR antagonists can be used to promote CD34 cell expansion. To determine the clinical utility of these findings, we have begun to explore the use of SR1 to expand CD34+ cells isolated from umbilical cord blood for clinical transplantation. To this end, we have developed a GMP compatible process to manufacture CD34 positive cells expanded with SR1 for use in cord blood transplantation. In addition, we have also explored the use of SR1 to prevent HSC differentiation during HSC transduction and enable manufacturing of differentiated blood cells. These data reveal AHR antagonism and SR1 treatment as a promising method to promote HSC expansion for clinical use. Disclosures: Cooke: Novartis: Employment. Boitano:Novartis: Employment.

Engineered human tumor xenografts with functional human vascular networks

Animal models of human tumor angiogenesis would have multiple applications. However, they are extremely difficult to standardize. In this work, we tried to generate human tumor xenografts containing a human vascular bed in immunodeficient mice by subcutaneous co-implantation of matrigel-embedded human endothelial cells (EC), human mesenchymal stem cells (MSC) as a source of mural cells and HT1080 human fibrosarcoma cells. Unfortunately, in this context human EC were rapidly substituted by their murine counterparts, and by day 16 post-implantation human CD34 positive cells were hardly detectable in intratumoral vessels. In an attempt to inhibit host EC colonization of human xenografts and promote human EC grafting, we investigated the effect of radiation prior to implantation on the vascularization and growth of tumor xenografts. Nude mice underwent either localized (implantation area) or sublethal whole-body exposure to radiation. Localized radiation inhibited both human and murine neovascularization, and even the tumor growth rate was remarkably decreased when compared to control unirradiated mice and sublethally whole-body irradiated mice. Interestingly, numerous human vessels were detectable in sublethally irradiated mice at day 30, with murine EC only overpassing human EC when spontaneous hematopoietic reconstitution has taken place. This observation strongly suggests the implication of bone marrow-derived murine endothelial precursors in tumor neovascularization. In summary, we have established a model of human tumor neovascularization that is amenable to both the study of molecular aspects in the angiogenic process and the evaluation of potential new antiangiogenic drugs.