Ojective 1: Demonstrate that Eltrombopag can bypass the TPO signaling defect induced by IFN 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 g/ml (Epag), with or without IFN 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 IFN (46.7 16.1% and 24.6 15.0% respectively, p<0.05). Accordingly, when comparing 7-day cultures with and without IFN, the absolute numbers of CD34+ cells were markedly reduced with TPO5 (average 7.6-fold, p<0.005) but only minimally decreased with Epag (average 1.6-fold, p = n.s.). The adjusted numbers of CFUs after 7 days in the presence of IFN similarly decreased 2.7-fold with TPO5 but remained unchanged with Epag compared to cultures without IFN. When the 7-day expanded progeny of an equal starting number of CD34+ cells was transplanted in NSG mice, human cell engraftment was superior with Epag (34 3.8% human CD45+ cells) than with TPO5 (21 1.8% human CD45+ cells, p<0.05) cultures in the presence of IFN, suggesting an impact of Epag on the most primitive long-term repopulating HSPCs. To investigate potential mechanisms by which Epag positively affects maintenance of HSPCs under inflammatory conditions, we examined cell signaling pathways induced upon binding of TPO, Epag and IFN to their respective receptors in human CD34+ cells. At a concentration of 5ng/mL, TPO induced a rapid (peak < 1 hour) and high potency rise in STAT5 phosphorylation followed by a rapid (< 2 hours) decay in signal. In contrast, Epag induced a slow (peak 4 hours) low potency rise in STAT5 phosphorylation, and the signal persisted for at least 10 hours. The difference in cell signaling potency and kinetics between TPO and Epag is likely related to their binding to distinct regions of c-MPL, resulting in alternate receptor conformational changes. We next investigated the impact of IFN on TPO and Epag-induced STAT5 phosphorylation at the signal peak (<1 and 4 hours, respectively). As previously shown in murine HSPCs, IFN impaired TPO signaling in human HSPCs. In contrast, Epag-induced STAT5 phosphorylation was preserved or increased in the presence of IFN. When Epag and TPO were combined, inhibition of TPO signaling by IFN was partially restored. By reducing the dose of TPO from 5 to 1ng/mL, and therefore reducing the potency of signaling to levels similar to Epag, the inhibitory effect of IFN on TPO signaling was abolished. Activation of IFN receptor by its ligand induces phosphorylation of STAT1 and subsequent expression of suppressor of cytokine signaling-1 (SOCS-1), a negative regulator of both IFN and c-MPL receptors via inhibition of STAT1 and STAT5 phosphorylation, respectively. We found that IFN-induced phosphorylation of STAT1 was increased in the presence of TPO 5ng/mL (1.5-fold increase, p<0.05) but unaffected by Epag (1.1-fold increase, p = n.s.) or TPO 1ng/mL (1.1-fold increase, p = n.s.). Our data indicate that Epag counters IFN-induced perturbation of TPO signaling in human HSPCs. Epag produces an unopposed low potency, slow kinetic positive signal and activates c-Mpl above a threshold level critical for HSPC self-renewal. Epags evasion of IFN blockade of a critical pathway of growth factor cell signaling may explain its efficacy in improving hematopoiesis in SAA. This work will be presented at the Annual Meeting of the American Society of Hematology (ASH) in December 2016 and a manuscript is in preparation. Ojective 2: Demonstrate that Eltrombopag has DNA repair activity in human HSPCs 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 -irradiation, and then cultured for an additional 5 to 24 hours. DNA damage was quantified by flow cytometric determination of H2AX 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 H2AX+ cells 5 hours post-irradiation when the culture included TPO or Eltrombopag than with SF alone (n=5). Five hours post-irradiation, cultures containing TPO or Eltrombopag had significantly increased percentages of live cells (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 (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 -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 -irradiation. To gain insights into the mechanisms of DNA repair, the non-homologous end joining (NHEJ) and homologous recombination (HR) DNA repair pathways were examined by transfecting a linearized reporter plasmid which, upon repair by NHEJ or HR, expresses GFP. A tdTomato plasmid was used for normalization and efficiency was calculated by the ratio of GFP+tdTomato+ to total tdTomato+ cells. TPO and eltrombopag significantly increased NHEJ efficiency (mean 0.26 0.01 and 0.25 0.02, respectively) compared with cells cultured with SF alone (mean 0.18 0.01, p=0.002 and p=0.02, respectively, n=3). Because CD34+ cells do not use HR, the c-Mpl expressing HEL 92.1.7 cell line was used to investigate HR repair pathways. No improvement in HR was observed with TPO or Eltrombopag. We conclude that, analogous to TPO, Eltrombopag favors NHEJ DNA DSB repair and, consequently, survival of both hematopoietic stem and progenitor cells after -irradiation. These pre-clinical data suggest that Eltrombopag may be of benefit in the treatment of patients with Fanconi Anemia (FA). In FY17, we will also confirm the utility of Eltrombopag in FA by directly testing DNA repair activity in FA HSPCs. The latter, inaccessible in FA patients due to bone marrow failure, will be obtained via CRISPR/Cas9-knockdown of FANCA gene in normal CD34+ cells. A clinical trial testing Eltrombopag in patients with FA is also in preparation and will also be initiated in FY17.
| Townsley, Danielle M; Scheinberg, Phillip; Winkler, Thomas et al. (2017) Eltrombopag Added to Standard Immunosuppression for Aplastic Anemia. N Engl J Med 376:1540-1550 |
| Ito, Sawa; Barrett, A John; Dutra, Amalia et al. (2015) Long term maintenance of myeloid leukemic stem cells cultured with unrelated human mesenchymal stromal cells. Stem Cell Res 14:95-104 |
| Wu, Chuanfeng; Hong, So Gun; Winkler, Thomas et al. (2014) Development of an inducible caspase-9 safety switch for pluripotent stem cell-based therapies. Mol Ther Methods Clin Dev 1:14053 |
| Sellers, Stephanie E; Dumitriu, Bogdan; Morgan, Mary J et al. (2014) No impact of lentiviral transduction on hematopoietic stem/progenitor cell telomere length or gene expression in the rhesus macaque model. Mol Ther 22:52-8 |
