ALBERT

Description: Leukemic stem cells (LSCs) are derived from hematopoietic stem or progenitor cells and often share gene expression patterns and specific pathways. Characterization and mechanistic studies of LSCs are critical as they are responsible for the initiation and potential relapse of leukemias, however the overall framework, including epigenetic regulation, is not yet clear. We previously identified microRNA-150 (miR-150) as a critical regulator of mixed lineage leukemia (MLL) -associated leukemias by targeting oncogenes. Our additional results suggest that miR-150 can inhibit LSC survival and disease initiating capacity by suppressing more than 30% of “stem cell signature genes,” hence altering multiple cancer pathways and/or stem cell identities. MLL-AF9 cells derived from miR-150 deficient hematopoietic stem/progenitor cells displayed significant proliferating advantage and enhanced leukemic colony formation. Whereas, with ectopic miR-150 expression, the MLL-AF9 associated LSC population (defined as Lin-ckit+sca1- cells) was significantly decreased in culture. This is further confirmed by decreased blast leukemic colony formation in vitro. Furthermore, restoration of miR-150 levels in transformed MLL-AF9 cells, which often display loss of miR-150 expression in AML patients with MLL-fusion protein expressing, completely blocked the myeloid leukemia development in a transplantation mouse model. Gene profiling analysis demonstrated that an increased level of miR-150 expression down regulates 30 of 114 stem cell signature genes by more than 1.5 fold, partially mediated by the suppressive effects of miR-150 on CBL, c-Myb and Egr2 oncogenes. In conclusion, our results suggest that miR-150 is a potent MLL-AF9 leukemic inhibitor that may act by suppressing the survival and leukemic initiating potency of MLL-AF9 LSCs. Disclosures: No relevant conflicts of interest to declare.

Description: Recent studies have uncovered a specific function of thrombopoietin (TPO) in the regulation of hematopoietic stem/progenitor cell (HSPC) DNA damage response. Eltrombopag, an oral non-immunogenic TPO receptor agonist, has recently received FDA approval for the treatment of patients with refractory severe aplastic anemia, but its mode of action is incompletely understood and a role in HSPC DNA repair has not been investigated. G-CSF mobilized human CD34+ cells from 5 independent healthy donors were cultured in the presence of SCF and Flt3-L (SF), SF and TPO (SFT), or SF and Eltrombopag (SFE) for 24 hours before exposure to 2Gy γ-irradiation, and then cultured for an additional 5 to 24 hours. DNA damage was quantified by flow cytometric determination of γH2AX expression, a marker of irradiation-induced DNA double-strand breaks (DSB), and CD34+ cell survival was measured by flow cytometry using Annexin V and a viability dye. There were significantly fewer γH2AX+ cells 5 hours post-irradiation when the culture included TPO or Eltrombopag than with SF alone (Figure A, n=5). Five hours post-irradiation, cultures containing TPO or Eltrombopag had significantly increased percentages of live cells (Figure B, n=5), as well as decreased percentages of cells undergoing apoptosis compared to cultures with SF alone (SFT 12.6 ± 0.5% p=0.003; SFE 12.4 ± 2.1% p=0.012; SF 21.5 ± 3.7%, n=5). RT-qPCR arrays performed at 5 hours after irradiation on CD34+ cells cultured as above with SFT or SFE showed a significant decrease (p≤0.05) of at least two-fold in several pro-apoptotic or cell cycle arrest genes (BBC3, CCNO, GADD45G, PPM1D) compared to CD34+ cells cultured with SF alone. Twenty-four hours post-irradiation, cells cultured with TPO or Eltrombopag had significantly increased percentages of live cells (Figure B, n=3), and decreased percentages of dead cells compared to cells cultured with SF alone (SFT 9.75 ± 1.0% p=0.013; SFE 16.3 ± 0.6% p=0.032; SF 36.5 ± 6.2%, n=3). Progenitor cell survival was assessed using the CFU assay. The number of colony-forming cells was 5.9 (± 0.4) and 3.6 (± 0.2) fold higher when cultured with TPO or Eltrombopag, respectively, before γ-irradiation than when cultured with SF alone (p=0.005 and 0.006, respectively, n=2). Survival of long-term repopulating HSCs was assessed by quantifying human CD45+ cell engraftment at least 2 months after intravenous injection of NSG mice with irradiated human CD34+CD38- cells pre-cultured for 24 hours with SF, SFT or SFE. Engraftment of cells cultured with TPO or Eltrombopag was significantly higher than engraftment obtained after injection of cells cultured with SF alone before γ-irradiation (Figure C). We conclude that, analogous to TPO, Eltrombopag favors DNA DSB repair and, consequently, survival of both hematopoietic stem and progenitor cells after γ-irradiation. These pre-clinical data suggest that Eltrombopag may be of benefit in the treatment of patients with Fanconi Anemia (FA), an inherited bone marrow failure syndrome in which patients have increased susceptibility to DNA damage due to defects in the FA DNA repair pathway. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Activation of Notch signaling in human hematopoietic stem/progenitor cells (HSPCs) by treatment with Notch ligand Delta1 has enabled a clinically relevant ex vivo expansion of short-term HSPCs. In vitro studies have also revealed a role of low O2 tension in HSPC regulation. A molecular link has been demonstrated in several stem/progenitor cell populations between Notch and hypoxia pathways but their interaction has not been investigated in human HSPCs. G-CSF mobilized human CD34+ cells from 4 healthy subjects were cultured in the presence of cytokines (SCF, FLT3L and TPO) in hypoxia (1.5-2% O2) or normoxia (21% O2) in vessels coated with fibronectin alone or combined with increasing concentrations of the immobilized ligand Delta1 (2.5, 5, 10 and 20 µg/mL). After 21 days in culture, cells were counted and characterized using CFU assays, flow cytometry for lineage (Glycophorin A+, CD13+, CD20+, CD3+ and CD41+ cells) and HSC (CD34+ CD38- CD45RA- CD90+ CD49f+ Rholow) phenotypes, and transplantation in immunodeficient (NSG) mice. In normoxia, the total number of cells increased 118-fold compared to baseline in the absence of Delta1 with limited residual CD34+ cells (1.5 ± 0.7%), extensive differentiation toward the myeloid lineage (96.3 ± 0.3% CD13+ cells) and minimal engraftment potential in NSG mice (0.2 ± 0.2% human CD45+ cells). With increasing concentrations of Delta1 in normoxia, consistent with the hypothesis that Delta1 delays differentiation, the total number of cells increased less (41-, 25-, 11- and 7-fold relative to baseline, respectively) CD34+ cells expanded more (4-, 4-, 3- and 2-fold relative to baseline, respectively), and CFU numbers increased more (8-, 7-, 4- and 3-fold relative to baseline, respectively) than without Delta1. However, phenotypically defined HSCs were undetectable or markedly decreased at the lowest Delta1 concentrations used (2.5 and 5 µg/mL) and their numbers were maintained or only minimally increased at the highest Delta-1 concentrations tested (10 and 20 µg/mL) relative to uncultured CD34+ cells. Accordingly, only cells cultured with 10 and 20 µg/mL Delta1 resulted in levels of engraftment in NSG mice (5.5 ± 5.4% and 5.4 ± 0.9% human CD45+ cells, respectively) comparable to uncultured cells (7.0 ± 0.1% human CD45+ cells). In hypoxia, total cell counts increased less than in normoxia both without (8-fold relative to baseline) and with increasing concentrations of Delta1 (11-, 11-, 9-, 9-fold relative to baseline, respectively) due to diminished myeloid differentiation. Total CD34+ cells decreased 1.7-fold in hypoxia in the absence of Delta1, but expanded modestly in the presence of Delta1 (3-, 3-, 2- and 2-fold, respectively). CFU numbers followed a similar trend. However, in hypoxic cultures with 2.5, 5 and 10 µg/mL Delta1, phenotypically defined HSCs increased 2.5-, 6.6- and 1.3-fold, respectively, compared to uncultured cells. Importantly, hypoxia combined with 2.5, 5 and 10 µg/mL Delta1 concentrations resulted in increased human cell engraftment in NSG mice (21.2 ± 4.4%, 29.3 ± 11% and 11.8 ± 5.4% human CD45+ cells, respectively) compared to uncultured cells (7.0 ± 0.1% human CD45+ cells). When 20 µg/mL Delta1 was used in hypoxia, engraftment potential in NSG mice was decreased (1.1 ± 0.6% human CD45+ cells). We next performed limiting dilution analysis to measure the frequencies of long-term repopulating HSCs (LT-HSCs) within the CD34+ cell compartment at baseline and after 21 days in hypoxic or normoxic cultures supplemented with the optimized concentrations of Delta1 (10 µg/mL in normoxia and 5 µg/mL in hypoxia). LT-HSCs in uncultured CD34+ cells were measured at the expected frequency (1 in 7,706; 95% CI of 3,446 to 17,232). When analyzed at 3 months post-transplantation, a limited (1.5-fold) increase in LT-HSC frequency (1 in 5,090; 95% CI 2.456 to 10,550) was obtained from Delta1 normoxic cultures compared to uncultured cells. In contrast, the frequency of LT-HSCs (1 in 1,586; 95% CI 680 to 3,701) was 4.9-fold higher in hypoxic Delta1 cultures compared to uncultured cells, and 4.2-fold higher than in normoxic Delta1 cultures. Similarly, absolute numbers of LT-HSCs per 100,000 Day 0 equivalent CD34+ cells increased from 13 (baseline) to 216 (normoxia) and 694 (hypoxia). Our data indicate that hypoxia potentiates Notch-induced expansion of human HSPCs and may be of benefit in stem cell transplantation and gene therapy applications. Disclosures Cheruku: Novartis: Research Funding. Larochelle:Novartis: Research Funding.

Description: Thrombopoietin (TPO) is the main regulator of hematopoietic stem and progenitor cell (HSPC) self-renewal and survival. Upon binding to its receptor, c-MPL, TPO activates cell signaling, through JAK-STAT and other pathways, which is tightly balanced by negative regulatory signaling processes. Recent studies indicate that chronic exposure of HSPCs to IFNγ, as exemplified in subjects with severe aplastic anemia (SAA), impairs self-renewal by perturbing TPO signaling pathways. Despite elevated levels of TPO in subjects with SAA, the TPO receptor agonist Eltrombopag (Epag) improves trilineage hematopoiesis in refractory SAA, suggesting that it may activate signaling within HSPC in a way that is distinct from TPO under inflammatory conditions. To address the paradox of Epag efficacy despite high endogenous TPO levels in bone marrow failure, G-CSF mobilized human CD34+ cells from 6 healthy donors were cultured in the presence of SCF, FLT3 and either TPO 5 ng/ml (TPO5) or Epag 3 μg/ml (Epag), with or without IFNγ 100 ng/ml. After 7 days in culture, cells were characterized via flow cytometry, CFU assay and transplantation in immunodeficient (NSG) mice. The percentages of CD34+ cells in cultures containing TPO5 or Epag alone were similar (83.3 ± 9.7% and 87.6 ± 7.1%, respectively), but were better preserved with Epag than TPO5 in the presence of IFNγ (46.7 ± 16.1% and 24.6 ± 15.0% respectively, p