Although FA is known as a disorder of cytogenetic instability in which somatic cells are hypersensitive to bifunctional alkylating agents (43
), the major life-threatening complications are commonly confined to hematopoietic tissues and include aplastic anemia and conditions resulting from clonal expansion of cytogenetically unstable stem cells, myelodysplasia, and acute myelogenous leukemia (44
). The unique vulnerability of hematopoietic cells is likely reflective of key prosurvival roles of the FA proteins in hematopoietic cells. For example, while it is known that the FA proteins function to protect against genotoxic stress by forming complexes with each other (19
), some are known to also protect hematopoietic cells, in part by suppressing apoptotic responses to extracellular apoptotic cues, particularly TNF-α (23
). The strongest evidence that FA proteins are likely multifunctional comes from studies on the role of FANCC in suppressing apoptotic responses. In fact, the antiapoptotic functions of FANCC, including mediating resistance to TNF-α, require structural features that differ from those that permit the formation and function of the canonical nuclear core complex (45
), confirming structurally the multifunctionality of FANCC. Because aplastic anemia is nearly universal in FA patients, we suspect that most of the other 12 FA proteins will ultimately prove to be multifunctional. Although most of the FA proteins collaborate in the nuclear pathway to protect the genome (1
), there is no evidence that the mechanisms by which they may promote survival of hematopoietic stem cells are precisely the same. For example, FANCA mutant cells are hypersensitive to TNF-related apoptosis-inducing ligand (TRAIL) (46
), but FANCC mutant cells are not (47
). Therefore, it is quite possible that specific molecular leukemogenic events in FA cells will differ from one complementation group to another because the adaptive responses required may differ. Alternatively, the leukemogenic events might function to suppress a signaling bottleneck for more than 1 apoptotic pathway, in which case the adaptive adjustment may be in a common effector pathway. That is, the leukemogenic events might be the same across the groups. These questions are not answered by this study, because we focused specifically on 1 well-defined complementation group, FA-C.
We argue that the multifunctionality of FANCC explains the high incidence of clonal evolution in FA stem cells of the C complementation group. First, FA stem cells are genetically unstable. Second, owing to their proapoptotic phenotype, the FA stem cells are less fit than normal stem cells (48
). Third, the neoplastic FA clones seem to be resistant to precisely the same cytokines to which their non-neoplastic progenitors were hypersensitive (34
). Therefore, we have proposed that repeated cytokine release responses throughout the life of FA patients provides recrudescent selective sweeps that purge hematopoietic tissues of all but the selected or adapted neoplastic stem cells. Rarely, new stem cell clones can emerge through mutations that specifically correct the inherited mutation, a process leading to mosaicism that can be so complete that hematopoiesis is normalized (50
). More often, emerging clones have not corrected the mutant FA gene. Encouraged by a preliminary report that suggested that certain conditions of ex vivo culture might enhance clonal evolution of Fancc –/–
), we tested the hypothesis that TNF-α exposure in vitro can provide a selective sweep in Fancc–/–
stem cells, resulting in the outgrowth of TNF-α–resistant leukemogenic clones. We specifically chose TNF-α and Fancc–/–
mutant cells as the instruments of our studies because we and others have shown that FA-C cells are hypersensitive to TNF-α (23
), because some of the roles of FANCC in the TNF-α signaling pathway have been defined biochemically (26
), and because we have reported TNF-α resistance in clonally evolved human FA progenitor cells (34
We found, as expected, that TNF-α exposure initially inhibited profoundly the expansion of Fancc–/–
stem progeny but noted that longer-term exposure of these cells promoted the outgrowth of TNF-α–resistant, cytogenetically abnormal clones that upon transplantation into syngeneic WT mice led to acute myelogenous leukemia (Figures and ). That the recipient animals survived transplantation for more than 100 days indicated either that nonclonal stem cells repopulated the animals early or that the clonally derived cells had the potential for multilineage repopulation of the recipient animals and were, therefore, preleukemic. Once leukemia developed, the TNF-α–resistant leukemic cells retained their characteristic hypersensitivity to mitomycin C and exhibited high-level constitutional chromosomal instability but did not require TNF-α–induced NF-κB activation for their survival and proliferation (Figure ). We also noted that TNF-α enhanced ROS-dependent genetic instability in Fancc–/–
but did not induce genetic instability in WT cells (Figures and and Table ). These findings are consistent with prior indications that TNF-α’s effects on other cell types are ROS dependent (35
) and with reports that FA cells are hypersensitive to oxidative stress (51
Expression of FANCC
cDNA in Fancc–/–
stem cells protected them from TNF-α–induced clonal evolution (Figure ). Of potential importance for prevention of clinical leukemogenesis, we also found that inhibition of ROS accumulation in vivo delayed leukemia development in recipients of preleukemic clones (Figure and Table ). We recently reported that prolonged activation of the MAPK kinase JNK contributes to the overproduction of ROS and exacerbated inflammation in Fancc–/–
mice challenged with bacterial lipopolysaccharide (41
). We have demonstrated herein that JNK is persistently activated in the preleukemic Fancc–/–
cells and that inhibition of JNK kinase using either dominant mutant of an upstream kinase or a pharmaceutical JNK inhibitor could: (a) inhibit the activity of the endogenous kinases; (b) reduce TNF-α–induced ROS production; and (c) prevent the outgrowth of Fancc–/–
progenitor cells (Figure ). Mice transplanted with the TNF-α–promoted preleukemic Fancc–/–
BM cells accumulated high levels of ROS, but NAC administration suppressed ROS as well as the levels of secreted proinflammatory cytokines in the recipients (Figure and Table ).
Intriguingly, oxidative DNA damage alone appears to be insufficient for Fancc–/– malignant transformation. We provide evidence that prolonged treatment of the Fancc–/– BM stem/progenitor cells with a sublethal dose of H2O2 failed to transform Fancc–/– cells, whereas combined treatment of the cells with the pan-caspase inhibitor Z-VAD-FMK and H2O2 supported the emergence of Fancc–/– preleukemic stem cells (Figure ).
We reason that exposure of Fancc–/–
stem cells to agents that damage DNA would not be enough to account for evolution of malignant clones. If this were true, one would expect that all somatic cells (all of which are hypersensitive to alkylating agents) would be potential targets for neoplastic transformation. In fact, the vulnerable tissues are limited. One of the key nonstochastic targets is the hematopoietic organ system that in Fancc–/–
cells is inherently hypersensitive to the apoptotic effects of TNF-α. To gain a competitive advantage, clonal neoplasms must overcome any apoptotic hypersensitivities. Indeed, we have found (34
) that TNF-α resistance occurs in clonal FA cells. That these TNF-resistant preleukemic and leukemic cells retain their characteristic DNA cross-linker hypersensitivity confirms that the key adaptive response in leukemogenesis involves TNF resistance, not resistance to cross-linking agents. That the leukemic cells retain genetic instability on the one hand and have a suppressed cytokine-dependent apoptotic response on the other favors rapid 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. Moreover, TNF-α provides 2 functions of relevance to clonal evolution in vitro. First, it inhibits survival of unadapted (constitutionally unfit) cells, and second, the cytokine generates ROS-dependent genetic instability that may enhance the likelihood of adaptive mutations in vitro. The ability to reliably induce clonal evolution in vitro in such a short period of time without expressing oncogenes ectopically provides an important opportunity for the general field of leukemogenesis.