Increased cancer rates that result from exposure to radiation or other carcinogens are generally attributed to the mutagenic actions of these agents via increasing frequencies of mutations in oncogenes and tumor suppressor genes (3
). Surprisingly, evidence to support a role for γ-radiation in the direct induction of oncogenic mutations is largely lacking (1
). We and others have previously proposed an alternative explanation: DNA damaging carcinogens might induce growth inhibitory conditions which lead to selection for oncogenic mutations that confer at least partial resistance to the defect (6
). Of course, mutagenic and selective effects of carcinogens need not be mutually exclusive. Ultraviolet light exposure of the skin represents perhaps the best example of a carcinogenic agent that has been shown to both directly result in oncogenic mutations (such as in p53
) and to promote the clonal expansion of progenitors with these mutations (31
). The studies described here tested the long-term effects of X-irradiation on the fitness of hematopoietic progenitor populations and on selection for different oncogenic mutations.
Previous studies have shown that the accumulation of DNA damage, resulting either from γ-irradiation or DNA repair deficiency, leads to reductions in the repopulating ability of HSC (34
). Here, we show that sub-lethal irradiation impacts on HSC fitness both by the permanent loss of repopulating activity and by reducing the per-cell fitness of HSC that retain some repopulating capacity. We further demonstrate that persistent ROS induction that follows irradiation is partially responsible for this reduction of fitness. These results raise the possibility that people who are exposed to γ-irradiation might benefit from treatment with anti-oxidants, even if treatment is initiated after exposure.
The key finding of our study is that previous irradiation substantially alters the selective impact of certain oncogenic mutations, augmenting (ICN), inhibiting (Ras and Bcr-Abl) or not affecting (Akt and Myc) the ability of oncogenes to drive clonal expansion and leukemogenesis. These results argue against the prevailing paradigm whereby irradiation promotes leukemogenesis by inducing the accumulation of cooperating oncogenic events, as one would need to surmise that irradiation induced oncogenic events cooperated with some oncogenes, while inhibiting leukemia induction by others. Our results instead provide support for the importance of selection in oncogenesis: different oncogenic events should be adaptive for different contexts of reduced cellular fitness (or an otherwise altered environment). Alteration of selective pressures following irradiation is consistent with the idea that cancers arise within a fairly tight trajectory determined by the adaptive landscape specific for a particular tissue, developmental stage and carcinogenic exposure (37
), which could underlie the association of particular oncogenic events with cancers initiated by particular carcinogenic contexts.
Activating mutations in Notch1
are very common in human T-cell acute lymphoblastic leukemias (T-ALL) (28
mutations that generate N-terminally truncated proteins, predicted to functionally mimic the ICN oncoprotein, are found in over half of T-lymphomas induced in mice by γ-irradiation (39
). While the authors suggested that irradiation causes T-lymphomas by generating strand breaks at the Notch1
locus, our data indicate that previous irradiation creates a context that potently selects for activating Notch1
mutations in progenitor pools.
Our experiments indicate that this selection occurs within early multipotent progenitors, since we detect substantial expansion of ICN-expressing cells in HSC-enriched pools of previously irradiated BM, and increased representation of ICN-expressing cells is evident within multiple hematopoietic lineages. Of relevance, a recent study demonstrated that ICN expression is insufficient to confer self-renewal to T-precursors, as transfer of the initial polyclonal ICN-expressing CD4+
population into secondary recipient mice resulted in loss of these cells within a week (40
). In contrast, cells with properties of committed T-progenitors from monoclonal ICN-induced leukemias did efficiently transfer the disease. Thus, should an activating Notch1
mutation occur in a T-precursor, in all likelihood this clone would be lost long before other events could accumulate. But if the Notch1
mutation happens in an HSC and
is selected for, a pool of ICN-expressing T-precursors could be maintained, which would then have the potential to accumulate additional events leading to leukemia development.
We hypothesize that irradiation can promote carcinogenesis by decreasing the fitness of progenitor cells, thereby increasing selection for oncogenic mutations that can provide some adaptation in the face of this defect (6
). While the results presented here support this hypothesis, they do not provide definitive proof. Increased selection for ICN-expressing cells could also be influenced by non-cell-autonomous effects of irradiation on hematopoiesis. For example, irradiated hematopoietic cells could produce higher levels of inflammatory cytokines, and the transplanted non-irradiated BM could partially alleviate this environmental change by replacement of irradiated hematopoiesis. Irradiation-induced senescence of fibroblasts dramatically boosts cytokine production (41
), which could be envisioned to promote tumorigenesis either by selective pro-proliferative effects of cytokines on initiated cells or by selectively impairing the fitness of non-initiated cells. Regardless, in the irradiated background, ICN mutations clearly confer a greater advantage relative to a normal background. Thus, ICN-expressing progenitors must have a fitness advantage relative to other irradiated progenitors, supporting the idea that irradiation increases cancer predisposition at least in part by altering the environment and thus the adaptive landscape.
In summary, our results argue that irradiation substantially changes the selective impact of different oncogenic mutations, either inhibiting or promoting oncogene-driven expansion. The ability of non-irradiated competitors to limit ICN-driven expansion from irradiated cells supports the idea that protecting or restoring the fitness of a stem cell population might limit the onset of cancer even when oncogenic mutations cannot be avoided.