Abstract
Abstract 3486
Granulocyte colony-stimulating factor (G-CSF) is widely utilized in multiple clinical settings to lessen the effects of neutropenia. Although clearly beneficial, there are concerns about the long term effects of G-CSF. 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, there are several lines of evidence to suggest that G-CSF treatment contributes to development of leukemia in these patients. First, the risk of leukemia appears to correlate with the cumulative dose of G-CSF. Second, of all the congenital marrow failure syndromes predisposed to AML, SCN alone does not appear to be a hematopoietic stem cell disorder. Since AML appears to rise from sequential mutations in hematopoietic stem cells, this would suggest that therapy, not the intrinsic cell defect is causal. It has been demonstrated that G-CSF does initiate signaling pathways in hematopoietic stem cell (HSC). We hypothesize that G-CSF induced excessive HSC proliferation can lead 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. Our results demonstrated a 3 fold increase in linage negative, Sca positive and cKit positive (LSK) population and a 2 fold increase in the amount of DNA double strand breaks via the presence of nuclear pH2AX in the LSK population. To determine if the G-CSF induced proliferation lead to chromosome alterations, we performed array-comparative genomic hybridization analyses (CGH). DNA from lineage negative bone marrow cells from animals treated with G-CSF for 4 months were compared to untreated mice. Our results demonstrate variations in gains and losses of several chromosome regions. Fluorescence in situ hybridization (FISH) 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. Genistein is a natural soy isoflavone with excellent bioavalibity that has anti-oxidant and anti-proliferative properties. In this study, we utilized a dose of genistein that can easily be obtained through oral supplementation. Mice were concomitant treated with G-CSF and genistein 3 times a week. Genistein partially blocked the G-CSF induced expansion of LSK cells 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. Our results suggest that genistein's effects are mediated primarily through inhibition of HSC proliferation. 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. Inhibition of GSKβ3 has been demonstrated to delay HSC entry into cell cycle by promoting degradation of β-catenin, while HSCs from the triple cyclin knock out mouse (Cyclins D1, D2, and D3) display delayed cell cycle entry. 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 preferentially utilize non-homologus end joining for DNA repair when progressing from G0 to G1. NHEJ is a relatively error prone DNA repair mechanism. Its preferential use by HSCs has been postulated as reason chromosomal deletions and translocations are often seen and many times are causal in the development of acute leukemia. Importantly, 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.
Disclosures:
No relevant conflicts of interest to declare.
Effect of 5-fluorouracil on “primitive” hematopoietic stem cells that reconstitute whole erythropoiesis of genetically anemic W/Wv mice
