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, we previously showed (see annual report 2016) that IFN, a key proinflammatory cytokine implicated in the destruction of HSPCs in SAA, inhibited TPO signaling in human CD34+ HSPCs cultured in the presence of both cytokines. In contrast, eltrombopag could evade this inhibition in vitro, resulting in improved maintenance of progenitors in clonogenic CFU assays, and long-term repopulating cells in NSG transplantation models compared with TPO-containing cultures (Cheng et al., ASH abstract 2016). In FY17, we sought to characterize the mechanisms by which eltrombopag evades IFN blockade of c-MPL signaling. Because activation of both c-MPL and IFN receptor by their respective ligands induces negative regulatory feedback mechanisms from the SOCS family of proteins, we first measured SOCS expression in CD34+ cells exposed to IFN and TPO or eltrombopag. SOCS expression was equally upregulated by TPO and eltrombopag in the presence of IFN, suggesting an alternative explanation for eltrombopags ability to escape IFN-induced perturbation of TPO signaling. Because TPO and eltrombopag distinctively bind to the extracellular or juxtamembrane domain of c-MPL, respectively, we hypothesized that IFN may decrease binding affinity of TPO, but not eltrombopag, to c-MPL. We used microscale thermophoresis (MST) to assess the impact of IFN on TPO binding to its receptor. As previously reported, we showed that TPO and c-MPL interact via both a high affinity (KD, app<0.11 0.04 nM) and a low affinity (KD, app=1100 130 nM) binding site. In contrast, IFN did not bind to c-MPL. Remarkably, addition of 100-molar excess IFN prevented binding of TPO to c-MPL at the low affinity site, but had no impact on the high affinity binding site (KD, app<0.20 0.04 nM). To explain this result, we hypothesized that TPO and IFN may directly interact and form heterodimeric complexes that hinder binding to c-MPL, whereas the non-peptide small molecule eltrombopag could conceivably evade that process. Strikingly, MST assays revealed a specific, one-site heterodimeric interaction between TPO and IFN (KD, app=540 30 nM), while no heterodimer formation was observed between either TPO or IFN and other early-acting cytokines (SCF and Flt3L). These data suggest that TPO:IFN heterodimers may be responsible for the hindered TPO:c-MPL low-affinity interaction observed in the presence of IFN. To confirm this finding, we investigated whether TPO-induced c-MPL dimerization was interrupted in the presence of IFN. Using raster image correlation spectroscopy (RICS) to determine average diffusion coefficients of GFP-tagged c-MPL in live cells, we have preliminarily found that IFN negatively affects c-MPL dimerization in cells cultured with TPO, but not with eltrombopag. Further studies are underway. Taken together, our data provide a new and provocative paradigm to explain the observed negative impact of the proinflammatory cytokine IFN on human HSPC maintenance in bone marrow failure syndromes, and the ability of a small molecule (eltrombopag) to evade this inhibition. We propose that, under chronic inflammatory conditions, IFN specifically heterodimerizes with TPO, resulting in (i) occlusion of the low-affinity binding site of TPO to c-MPL, (ii) impaired receptor dimerization, (iii) perturbation of TPO-induced signaling pathways, and (iv) decreased survival of human HSPCs. This new understanding could also have far-reaching clinical implications for other disorders of chronic inflammation This work will be presented at the Annual Meeting of the American Society of Hematology (ASH) in December 2017 and a manuscript is in preparation. Ojective 2: Demonstrate that Eltrombopag has DNA repair activity in human HSPCs To assess DNA repair activity of epag in FA CD34+ HSPCs, a population that is markedly reduced in these patients, CD34+ HSPCs from 6 healthy individuals were subjected to CRISPR/Cas9-induced knockout mutations in FANCA, the most commonly mutated gene in FA. These FA HSPCs were cultured for 24hrs in the presence of early-acting cytokines (SCF and Flt3-L, SF) alone or supplemented with epag (SFE) or TPO (SFT), prior to induction of DSBs by exposure to 2Gy -irradiation (IR). Cells were then cultured for an additional 1, 5 or 24hrs to assess the kinetics of DNA repair, as measured by decreases in H2AX expression, a marker of IR-induced DNA DSBs. Maximal H2AX expression was measured 1hr after IR of FA HSPCs and was similar for all culture conditions (>90% H2AX+ cells), indicating that epag and TPO do not prevent DNA damage. Five hours after IR, most FA HSPCs cultured with epag or TPO had already resolved the IR-induced DNA DSBs, but much higher percentages of H2AX+ cells were still detected in the control SF group. The observed effect was specific to epag and TPO; removal of SCF had no significant impact on DNA repair. By 24hrs after exposure to IR, FA HSPCs cultured with and without epag or TPO had similarly resolved DNA breaks. These findings indicate that epag and TPO increase the kinetics of DNA DSB repair in FA HSPCs. To gain insights into the mechanisms of DNA repair, we inhibited DNA-PK, an essential component of the NHEJ pathway. Addition of a DNA-PK inhibitor (NU7441) had no impact on DNA DSB formation measured at 1 hour or on DNA repair at 24hrs, but completely abrogated the enhanced kinetics of DSB repair observed at 5hrs with epag and TPO. These data indicate that epag and TPO favor the fast-acting DNA-PK dependent classical NHEJ (C-NHEJ) DNA repair mechanism in FA HSPCs, a pathway known to promote genomic stability. In contrast, cells cultured without epag or TPO resolved DSBs using a slower DNA-PK-independent alternative NHEJ (alt-NHEJ) mechanism in FA HSPCs, a pathway known as the primary mediator of genomic instability. Shunting of DSB repair in rapid C-NHEJ with epag or TPO was associated with substantial increase in survival of -irradiated FA HSPCs compared with control (SF) groups. In contrast, when C-NHEJ DNA repair was inhibited with NU7441, the cell survival benefit observed with epag or TPO was abolished. In colony forming unit (CFU) progenitor assays, -irradiated HSPCs cultured with epag or TPO yielded 4-6-fold more CFUs than control SF groups. Importantly, when -irradiated HSPCs were tested in NSG transplantation assays, a 2-fold increase in human cell engraftment was observed in cultures containing epag or TPO compared to controls (p<0.01), suggesting activation of DNA repair activity by these cytokines in cells with long-term repopulating capacity. Overall, our data indicate that epag and TPO enhance DNA DSB repair in HSPCs by promoting the fast-operating C-NHEJ pathway. A phase II clinical trial has been approved to assess safety and efficacy of epag at improving the hematological manifestations of FA. Enrollment is expected to begin in FY18.
| 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 |
