Abstract
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.
Q-FADD: A mechanistic approach for modeling the accumulation of proteins at sites of DNA damage by free diffusion
