Iron Chelation Reinforces DNA Damage Response and Leads to G2/M Checkpoint Activation and Autophagy in Myeloid Bone Marrow Cells of Preleukemia Mouse Model

Iron chelation therapy is commonly used in patients with myelodysplastic syndrome (MDS), to prevent majorcomplications of iron overload. Besides effects on maintaining control of iron stores and preventing iron-induced cardiac disease, the impact of chelation therapy on overall survival and leukemia-free survival in MDS has been documented, but not well understood. Since MDS bone marrow cells are known to activate DNA damage response (DDR) signaling and iron chelators target cancer cells through multiple stress-response mechanisms (endoplasmic reticulum (ER) stress, autophagy), we hypothesized that iron chelation could reinforce DDR signaling and could thus support tumor-suppressing role of DDR. Also nucleotide deficiency was shown to contribute to DDR, and iron chelation is known to inhibit ribonucleotide reductase (RR), an iron-dependent enzyme, which supplies cells with deoxyribonucleotides (dNTPs). Here, we tested the effects of lysosomotropic iron chelator deferoxamine mesylate (DFO) in a preleukemia mouse model, wherein epigenetic oncogene-induced leukemogenesis is preceded with a long-lasting preleukemia stage (Takacova S, et al. Cancer Cell. 2012;21(4):517-31.). Preleukemic, aberrantly proliferating myeloid cells in this model activate a replication checkpoint and ATR-Chk1-mediated DDR (consistent with oncogene-induced replication stress) and attain hallmarks of senescence (with a long latency), resulting in the inhibition of leukemia progression.

A group of 10 preleukemia mice and a group of 10 control mice aged 7 month were treated twice daily with DFO doses adjusted to 88,8 mg/kg (i.p. injection) in order to mimic serum concentrations of the drug achieved in patients. After 6 weeks of chelator administration, the treatment lead to the activation of Chk1(S345) in the bone marrow (BM) of control mice, but did not result in accumulation of γH2AX, a marker of DNA damage, in BM of these mice. In contrast, in preleukemia mice, with already activated threshold of ATR-Chk1 signaling (marker of ongoing oncogene-induced replication stress), Chk1(S345) remained unchanged after DFO treatment. However, we observed significant accumulation of γH2AX foci in oncogene-positive BM cells. These data suggested that iron removal may induce Chk1 activation in vivo, and, in addition, may reinforce activation of DDR in preleukemia cells perhaps due to synthetic effect of iron chelation with oncogene activation resulting in increased levels in DDR signaling (assessment of oxidative DNA damage (8-oxoguanine staining) is ongoing).

Next, we analyzed whether iron chelation in both groups of mice influences DNA replication, in which the limiting step is the availability of dNTPs. The RR activity was significantly decreased in the BM of both groups of DFO-treated mice, however, with no impact on the concentration of BM dNTPs; in fact, dNTPs have accumulated in BM of these mice. We revealed that this was a consequence of the activation of S-phase checkpoint in control mice, and of a decrease of actively replicating myeloid cells and activation of G2/M checkpoint in preleukemia mice.

Cellular iron depletion was shown to activate p38MAPK pathway (Yu Y, Richardson DR. J Biol Chem. 2011;286(17):15413-27.). p38MAPK pathway, and its component MK2, establishes intra-S-phase cell cycle checkpoint and activates G2/M checkpoint (as a part of DDR, in parallel to Chk1 activation (Reinhardt HC, et al. Curr Opin Cell Biol. 2009;21:245-55.)). Indeed, our preliminary result revealed phosphorylated MK2 specifically in preleukemia mouse BM treated with DFO. Since we did not detect increased apoptosis in BM of DFO treated mice, and because p38MAPK pathway is involved in the activation of ER stress and autophagy, we tested whether markers of ER stress and autophagy are detectable in the mice upon DFO treatment. MyD116 (marker of recovery from ER stress) and LC3-II (marker of autophagy), were specifically induced in preleukemia cells upon DFO treatment.

Collectively, these data demonstrate that preleukemia cells exposed to DFO activate distinct but functionally overlapping signaling pathways, resulting in reinforced DDR. Whether this mechanism could increase a barrier against leukemia transformation of chelated MDS patients remains to be investigated.

Authorship: LRK and ZS: equal credit as first authors. Acknowledgment: Supported by the Czech Science Foundation (P301/12/1503) and by IGA_LF_2015_015.