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, 12 (3), 457-462

Inactivation of the NHEJ Activity of DNA-PKcs Prevents Fanconi Anemia Pre-Leukemic HSC Expansion

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Inactivation of the NHEJ Activity of DNA-PKcs Prevents Fanconi Anemia Pre-Leukemic HSC Expansion

Srinivas Chatla et al. Int J Stem Cells.

Abstract

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure and high risk of cancer particularly leukemia. Here we show that inactivation of the non-homologous end-joining (NHEJ) activity of DNA-PKcs prevented DNA damage-induced expansion of FA pre-leukemic hematopoietic stem cells (HSCs). Furthermore, we performed serial BM transplantation to demonstrate that the DNA damage-induced expanded FA HSC compartment contained pre-leukemic stem cells that required the NHEJ activity of DNA-PKcs to induce leukemia in the secondary recipients. These results suggest that NHEJ may collaborate with FA deficiency to promote DNA damage-induced expansion of pre-leukemic HSCs.

Keywords: DNA damage; Fanconi anemia; Leukemic stem cells; Non-homologous end-joining.

Figures

Fig. 1
Fig. 1
Inactivation of the NHEJ activity of DNA-PKcs prevents the expansion of pre-leukemic Fanca−/− HSCs. (A) Inactivation of the NHEJ activity of DNA-PKcs prevents MMC-induced expansion of the Fanca−/− HSCs. 2,000 LSK cells from E14.5 embryos with the indicated genotype, along with 200,000 c-Kit-depleted protector cells, were transplanted into lethally irradiated BoyJ (CD45.1+) recipients. The recipients were i.p. injected with low dose of MMC (0.3 mg/kg) weekly for 6 weeks. Two days after the final injection, donor-derived (CD45.2+) BM LSK cells were gated for analysis of the SLAM (LSKCD150+CD48) cell population. (B) MMC induces a significant increase in apoptosis in DNA-PKcs3A/3A, Fanca−/− and DNA-PKcs3A/3AFanca−/− SLAM cells. Donor-derived (CD45.2+) BM LSK cells were gated for SLAM population and analyzed for apoptosis by Annexin V and 7AAD. (C) MMC causes increased cell cycling in Fanca−/− HSCs. Donor-derived (CD45.2+) BM LSK cells were gated for the SLAM population and analyzed for the cell cycle using Hochest 33342/Ki67 staining. (D) Survival of transplant recipients. 2,000 LSK cells from E14.5 embryos with the indicated genotype, along with 200,000 c-Kit-depleted protector cells, were transplanted into lethally irradiated BoyJ recipients (n=10 per group). MMC (0.3 mg/kg) was then administrated to the recipients weekly for 6 weeks. Survival of the recipients plotted by the Kaplan-Meier curve method and analyzed by the log-rank test. (E~G) Anemia but no leukemia developed in primary recipient mice. The moribund mice described in (D) were subjected to analysis for peripheral blood count (E), and splenomegaly (F) and infiltration of mature myeloid cells (E) in the spleens. *p<0.05; **p<0.01.
Fig. 2
Fig. 2
The NHEJ activity of DNA-PKcs is required for the pre-leukemic Fanca−/− HSCs to induce leukemia in the secondary recipients. (A~C) Inactivation of the NHEJ activity of DNA-PKcs restores normal levels of BM cellularity and phenotypic HSCs in the BM of the secondary recipients. 2,000 LSK cells from E14.5 embryos with the indicated genotype, along with 200,000 c-Kit-depleted protector cells, were transplanted into lethally irradiated BoyJ (CD45.1+) recipients. The recipients were i.p. injected with low dose of MMC (0.3 mg/kg) weekly for 6 weeks. Two days after the final injection, the mice were sacrificed and analyzed for BM cellularity (A), total donor (CD45.2) engraftment (B), and donor-derived SLAM (LSKCD150+CD48) cells (C). (D) Fanca−/− secondary recipients develop lethal leukemia. 2 million BM cells from the primary recipient mice described in (A) were transplanted into sublethally irradiated secondary CD45.1+ recipient mice. Survival of the recipients was monitored and plotted by the Kaplan-Meier curve method. n=8~10 mice for each group. (E, F) Increased myeloid infiltration in the spleen of the leukemic mice. Splenocytes from the recipient mice described in (D) were subjected to flow cytometry analysis for mature myeloid cells (E) and immature myeloid blasts (F). (G) Photomicrographs of Wright–Giemsa stain show increased blast cells in the peripheral blood smear from secondary recipient mice transplanted with Fanca−/− donor cells. *p<0.05; **p<0.01.

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