deletion increased Akt, mTORC1, and S6 kinase activation in HSCs () but we could find no evidence for reduced FoxO1 or FoxO3a expression or cytoplasmic sequestration (; Fig. S2
). We observed a clear increase in ROS levels within thymocytes after Pten
deletion but not in HSCs (). Consistent with this, NAC treatment attenuated the increase in ROS levels in thymocytes but did not rescue the changes in hematopoiesis, HSC frequency (), or HSC reconstituting capacity () after Pten
deletion. This contrasted with results from FoxO1/3/4
-deficient mice in which ROS levels clearly increased within HSCs and NAC treatment at least partially rescued HSC depletion (Tothova et al., 2007
deletion and FoxO1/3/4
deletion thus lead to the depletion of HSCs by different mechanisms. HSC depletion after Pten
deletion is mediated largely by mTOR activation with no evidence so far for an important contribution by oxidative stress.
deletion induced a tumor suppressor response in hematopoietic cells, characterized by increased expression of p19Arf
and p53 in splenocytes () and increased expression of p16Ink4a
and p53 in HSCs (). p16Ink4a
deficiency, or p53
deficiency significantly accelerated leukemogenesis after Pten
deletion (). p16Ink4a
deficiency did not significantly affect the rate of leukemogenesis after Pten
deletion, though it did suppress the generation of histiocytic sarcomas (). This suggests hematopoietic cells mainly rely upon the p53 pathway to suppress leukemogenesis after Pten
deletion. This is consistent with results from mouse prostate in which Pten
deletion induces p53-dependent senescence (Chen et al., 2005
). Interestingly, this senescence response is p19Arf
-independent in prostate (Chen et al., 2009
) but p19Arf
did suppress leukemogenesis after Pten
deletion, indicating tissue-specific functions for p19Arf
in tumor suppression.
p16Ink4a deficiency, p16Ink4a/p19Arf deficiency, or p53 deficiency all significantly prolonged the ability of Pten-deficient HSCs to give multilineage reconstitution in irradiated mice (). p19Arf deficiency did not prolong the reconstituting capacity of Pten-deficient HSCs (). Thus p19Arf is critical for the suppression of leukemogenesis but not for HSC depletion after Pten deletion. In contrast, p16Ink4a is critical for HSC depletion but plays a limited role suppressing leukemogenesis. One possible explanation for this distinction is that some leukemias may arise from cells other than HSCs and this process could be inhibited by p19Arf expression in those cells.
Our results thus indicate that Pten deletion induces an mTOR mediated tumor suppressor response in hematopoietic cells, suppressing leukemogenesis and depleting HSCs. This suggests that leukemias likely arise from rare clones of Pten-deficient hematopoietic cells that acquire secondary mutations that attenuate the tumor suppressor response. Consistent with this, we observed loss of p53 heterozygosity in leukemias that arose from Ptenfl/fl;Mx-1-Cre+;p53+/− mice, indicating that Pten deletion imposes a strong selection against the tumor suppressor response (). It is also important to note that we do not know which hematopoietic cells are transformed after Pten deletion. Therefore, the tumor suppressors may act in HSCs themselves to suppress leukemogenesis or they may act in downstream cells.
are both encoded at the Cdkn2a
locus, they are regulated by different promoters, have no sequence homology, and different molecular functions (Sherr, 2001
). The mechanisms by which Pten
deletion or other oncogenic stimuli induce these tumor suppressor expression are not understood. While it is well established that oncogenic stresses such as c-Myc or E1A expression also activate the p19Arf
-p53 pathway (de Stanchina et al., 1998
; Zindy et al., 1998
), the mechanisms behind this activation remain unclear. The mechanisms behind p16Ink4a
and p53 activation in response to Ras activation also remain unknown (Serrano et al., 1997
The best-characterized consequences of p53 or p16Ink4a
activation are senescence and apoptosis. However, these responses have only been characterized in certain non-stem cell populations, and it is possible that p53 or p16Ink4a
activation may have other effects on stem cells. We did not detect any evidence that hematopoietic cells underwent senescence or cell death after Pten
deletion (Fig. S7
). However, HSCs are asynchronously depleted over a 4 to 8 week period after Pten
deletion (). This raises the formal possibility that HSCs asynchronously undergo cell death or senescence over 4 to 8 weeks, such that very few HSCs express markers of cell death or senescence at any single time point, rendering it undetectable. Nonetheless, the simplest interpretation of our data is that p16Ink4a
and p53 expression cause HSCs to prematurely exit the stem cell pool, perhaps by maturing to transit amplifying MPPs. As this would occur asynchronously over time, the number of HSCs that prematurely exit the stem cell pool at any single time point would be imperceptibly small but the cumulative effect of premature maturation over a period of weeks would deplete HSCs.
Consistent with this model, deficiency for p16Ink4a
, and p53
dramatically expands the frequency of long-term multilineage reconstituting cells by conferring long-term self-renewal potential to MPPs which normally only give transient multilineage reconstitution (Akala et al., 2008
; Kiel et al., 2008
). These tumor suppressors thus play a physiological role promoting the transition from HSCs to MPPs and negatively regulating the self-renewal potential of multipotent cells. Increased expression of p16Ink4a
and p53 in dividing HSCs after Pten
deletion may accelerate the normal maturation of cells out of the HSC pool, leading to HSC depletion.
The ability to rescue the hematopoietic phenotypes in Pten
-deficient mice with rapamycin suggests these phenotypes are driven by mTORC1 activation. However, our data indicate only that increased mTORC1 activation is required for HSC depletion and leukemogenesis, not that it is sufficient. This may explain why other genetic backgrounds that activate mTORC1, such as Tsc1
deletion (Chen et al., 2008
; Gan et al., 2008
), do not necessarily lead to leukemogenesis. mTORC1-independent pathways downstream of Pten presumably also contribute to leukemogenesis. Since rapamycin can indirectly inhibit mTORC2 in addition to mTORC1 (Sarbassov et al., 2006
) and mTORC2 is required for the development of prostate cancer after Pten
deletion (Guertin et al., 2009
), mTORC2 may mediate some of the effects of Pten
deletion on HSCs and other hematopoietic cells.
The depletion of HSCs (and other hematopoietic progenitors) after Pten
deletion may explain why few leukemias exhibit Pten
deletion (Aggerholm et al., 2000
; Chang et al., 2006
; Sakai et al., 1998
) other than T-ALL (Gutierrez et al., 2009
). Rare clones of Pten
-deficient hematopoietic stem/progenitor cells would be unlikely to have the opportunity to acquire secondary mutations before being depleted and therefore would be unlikely to progress to leukemia. Leukemias may be more likely to hyper-activate the PI-3kinase pathway by other types of mutations that are better tolerated by hematopoietic cells. Additional studies of the PI-3kinase pathway in stem cells will provide additional insights into stem cell regulation and the development of cancer.