TNF-α induces leukemic clonal evolution ex vivo in Fanconi anemia group C murine stem cells
June Li, … , Grover C. Bagby, Qishen Pang
June Li, … , Grover C. Bagby, Qishen Pang
Published October 25, 2007
Citation Information: J Clin Invest. 2007;117(11):3283-3295. https://doi.org/10.1172/JCI31772.
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Research Article Hematology

TNF-α induces leukemic clonal evolution ex vivo in Fanconi anemia group C murine stem cells

Abstract

The molecular pathogenesis of the myeloid leukemias that frequently occur in patients with Fanconi anemia (FA) is not well defined. Hematopoietic stem cells bearing inactivating mutations of FA complementation group C (FANCC) are genetically unstable and hypersensitive to apoptotic cytokine cues including IFN-γ and TNF-α, but neoplastic stem cell clones that arise frequently in vivo are resistant to these cytokines. Reasoning that the combination of genetic instability and cytokine hypersensitivity might create an environment supporting the emergence of leukemic stem cells, we tested the leukemia-promoting effects of TNF-α in murine stem cells. TNF-α exposure initially profoundly inhibited the growth of Fancc–/– stem cells. However, longer-term exposure of these cells promoted the outgrowth of cytogenetically abnormal clones that, upon transplantation into congenic WT mice, led to acute myelogenous leukemia. TNF-α induced ROS-dependent genetic instability in Fancc–/– but not in WT cells. The leukemic clones were TNF-α resistant but retained their characteristic hypersensitivity to mitomycin C and exhibited high levels of chromosomal instability. Expression of FANCC cDNA in Fancc–/– stem cells protected them from TNF-α–induced clonal evolution. We conclude that TNF-α exposure creates an environment in which somatically mutated preleukemic stem cell clones are selected and from which unaltered TNF-α–hypersensitive Fancc–/– stem cells are purged.

Authors

June Li, Daniel P. Sejas, Xiaoling Zhang, Yuhui Qiu, Kalpana J. Nattamai, Reena Rani, Keaney R. Rathbun, Hartmut Geiger, David A. Williams, Grover C. Bagby, Qishen Pang

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Figure 5

The role of TNF-α–induced ROS in genetic instability.

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The role of TNF-α–induced ROS in genetic instability. (A) 1 × 106 TNF-α–...
(A) 1 × 106 TNF-α–treated WT cells or Fancc–/– preleukemic cells were injected i.v. into lethally irradiated recipients, which after 10 days were injected i.p. with 1 dose of TNF-α (100 μg/kg) followed by NAC (100 mg/kg/d) administration for 10 days. Control mice were injected with PBS only. The mice were sacrificed 24 hours after the last NAC injection, and BM cells were analyzed for DNA strand breaks by the comet assay. The mean tail moment of WT control sample is expressed as 100%. Larger tail moments represent higher levels of DNA damage. Three recipient mice from each group were analyzed, and 50 cells per mouse were scored from random sampling. (B) Tissue sections of liver and lung from recipient mice described in A were stained with anti–8-oxodG antibody and counterstained with H&E. Original magnification, ×40. (C) WT and Fancc–/– preleukemic cells were cultured in the presence of TNF-α (10 ng/ml), and protein extracts were prepared 0, 1, 2, and 4 hours after TNF-α treatment and analyzed by immunoblotting with anti–phospho-p53Ser20, anti-γH2AX, and anti-actin antibodies. Extracts were also prepared from cells 2 hours (2–) after TNF-α withdrawal after the cells had been treated with TNF-α for 4 hours. (D) BM cells from recipient mice described in A were analyzed for p53Ser20 and γH2AX expression. (E) Examples of metaphase chromosomes prepared from donor-derived WT and Fancc–/– BM cells. Chromosomal aberrations (arrows) were noted in Fancc–/– cells but rarely in WT cells.