Abstract 3739: Genistein protects hematopoietic stem cells against G-CSF induced DNA damage.

Granulocyte colony-stimulating factor (G-CSF) is widely utilized in multiple clinical settings to lessen the effects of neutropenia. Although clearly beneficial, a particular concern is that G-CSF therapy may increase the risk of MDS and or AML. The most striking example is that of Severe Congenital Neutropenia (SCN). While G-CSF clearly improves survival on SCN patients, there are several lines of evidence to suggest that G-CSF treatment contributes to development of leukemia. We hypothesize that G-CSF induced excessive hematopoietic stem cell (HSC) proliferation leads to DNA damage and genome instability. To test our premise, mice were treated with G-CSF for 4 months and bone marrow cells were analyzed. Results demonstrated a 3 fold increase in the LSK population and a 2 fold increase in the amount of dsDNA breaks via the presence of nuclear pH2AX. To determine if the G-CSF induced proliferation lead to chromosome alterations, we performed array CGH analyses. DNA from bone marrow cells from animals treated with G-CSF for 4 months were compared to untreated mice. Our results demonstrate variations in several chromosome regions. FISH analysis of Lin-Sca+ bone marrow cells confirmed loss on regions of chromosome 2 (6%) and 17 (30%). Since prolonged G-CSF exposure promotes genomic instability in HSCs we hypothesize that an alternative strategy would be to co-administer a drug that selectively blocks the effect of G-CSF on HSCs. Previous studies suggested genistein as an attractive compound with excellent bioavalibity that has anti-oxidant and anti-proliferative properties. Mice were concomitant treated with G-CSF and genistein 3 times a week. Genistein partially blocked the G-CSF induced proliferation of LSK cells, measured by BrdU incorporation, and reduced pH2AX levels in this population by 40%. This was also accompanied by a reduction in LSK cells with an abnormal FISH signal (50% reduction). Importantly, genistein did not block the G-CSF driven expansion of mature neutrophils as total number of neutrophils in mice treated with G-CSF and genistein are the same as those treated with G-CSF alone. We demonstrate that G-CSF treatment induces GSK3β phosphorylation and Cyclin D1 and D3 expression. Genistein blocked GSK3β phosphorylation and Cyclin D1 and D3 induction. Collectively, our results imply that prolonged G-CSF treatment induces DNA damage in HSCs by initiating cell cycle progression. HSCs are long lived, quiescent cells that utilize NHEJ, a relatively error prone process, for DNA repair when progressing from G0 to G1. We demonstrate, that genistein, at levels obtainable through oral supplementation, is able to reduce DNA damage by attenuating G-CSF induced HSC proliferation without compromising G-CSFs ability to accelerate terminal neutrophilic differentiation. These results suggest that genistein may be an effective therapeutic agent in patients with SCN who require prolonged G-CSF support Citation Format: Liliana Souza, Erica Silva, Michael Rossi, Omer Kucuk, Morgan L. Mclemore. Genistein protects hematopoietic stem cells against G-CSF induced DNA damage. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3739. doi:10.1158/1538-7445.AM2013-3739