To test its effect on HSC function, we conditionally deleted Pten from adult HSCs [12]. Loss of Pten in HSCs led to myeloproliferative disease within days and transplantable AMLs and acute lymphoblastic leukemias (ALLs) within weeks. Pten-deficiency had no discernable effect on HSC differentiation or survival but caused HSCs to go into cycle. This caused a transient increase in HSC numbers; however, by three weeks after Pten deletion HSCs became depleted. Consistent with this, Pten-deficient whole bone marrow cells or purified HSCs were able to engraft in irradiated mice and give rise to all types of blood cell lineages, but only for the first several weeks after injection. The levels of reconstitution declined over time, and recipient mice were rarely long-term multilineage reconstituted by Pten-deficient cells. Longitudinal studies of mice that were chimeric for Pten-deficient and wild-type HSCs showed that the loss of HSCs over time reflected a cell-autonomous requirement for Pten in the maintenance of HSCs [12]. Similar results were independently obtained by Li and colleagues [26]. HSCs thus require Pten to maintain quiescence and to self-renew over time.

In contrast to this requirement for Pten in the maintenance of HSCs, LSCs arose and expanded in number after Pten deletion. The LSCs were transplantable and could be enriched among cells that expressed HSC markers [12]. Most mice died with AML and ALL within 6 weeks of Pten deletion.

The observation that Pten deletion had opposite effects on normal HSCs and LSCs raised the possibility that by targeting this pathway it would be possible to eliminate LSCs without affecting normal HSCs. Pten deletion leads to increased activation of Akt and mTor, the mammalian Target of rapamycin. These signaling kinases have multiple roles within the cell that include promoting proliferation, survival, protein translation, ribosome biogenesis, and glycolysis [27]. To test whether the effects of Pten deletion were mediated by mTor activation, we administered rapamycin, a potent and specific inhibitor of mTor, to Pten-deleted mice. Rapamycin not only eliminated LSCs and maintained the health of mice, but it also rescued the depletion of Pten-deficient HSCs. Pten-deficient HSCs could even give long-term multilineage reconstitution of irradiated mice as long as the mice were maintained on rapamycin. This demonstrated that both the expansion of LSCs and the depletion of normal HSCs were mediated by increased mTor activation. Thus, many of the effects of Pten-deletion were mediated by increased mTor activation. By targeting mTor, LSCs could be eliminated while normal HSC function was rescued.

Although Pten is frequently deleted in many kinds of cancer and the PI-3kinase pathway is usually over-activated in leukemia, Pten is rarely deleted in leukemia [28; 29; 30]. Moreover, inherited germline mutations in Pten [31] are associated with hamartomas and a high risk for breast, thyroid, and endometrial cancers but not an increased risk of leukemia [32]. These observations raise the question of why Pten is rarely deleted in leukemia and why inherited mutations in Pten predispose to many other cancers but not leukemia. Our results offer a potential explanation: HSCs are efficiently depleted after Pten deletion. This means that spontaneous mutations that lead to a loss of Pten from HSCs will lead to the depletion of these HSCs before they have an opportunity to progress to leukemia. As a result, Pten deletion would not represent an efficient path to leukemic transformation. What remains unknown is whether downstream hematopoietic progenitors are also eliminated after Pten deletion, or whether this response is stem cell specific. Nonetheless, it may not be as necessary to deplete downstream progenitors that lose Pten, as the half-lives of these cells are generally much shorter than HSCs, limiting their opportunity to accumulate the additional mutations required for leukemogenesis.

One prediction of this hypothesis is that tissues that give rise to cancers that commonly exhibit Pten deletion should also have stem cells that can tolerate Pten deletion without being eliminated. There is some evidence that this is the case. Pten deletion in both neural stem cells [33] and prostate stem cells [34] leads to sustained increases in self-renewal and stem cell numbers, in contrast to the effect we observed in the hematopoietic system. Furthermore, Pten deletion is frequently observed in brain tumors [35] and prostate cancer [36]. These observations suggest that Pten deletion is an efficient path to transformation only in tissues in which the stem/progenitor cells can tolerate Pten deficiency. Possible mechanisms by which Pten deletion might lead to the loss of certain stem cells are addressed in the next section.

Our observation that rapamycin selectively eliminated LSCs while not harming HSCs suggests that rapamycin and its analogues may be used to treat cancers that exhibit increased PI-3kinase pathway activation. A subset of patients with refractory/relapsed AML responded favorably to a rapamycin analogue [37; 38]. Nonetheless, trials that have tested rapamycin analogues as single agents in a variety of cancers have generally yielded disappointing results [39; 40; 41]. This raises the question of whether our studies of Pten deficient mice can provide any insight into how rapamycin analogues can be used more effectively against cancer. When rapamycin was administered immediately after Pten deletion, it was extremely effective at preventing the generation or maintenance of LSCs: as long as these mice were maintained on rapamycin, they remained healthy with no histological evidence of hematopoietic neoplasms [12]. However, when rapamycin was started weeks after Pten deletion when mice already had leukemia, rapamycin was effective at reducing LSCs and prolonging the life of mice; however, all of these mice eventually died with AML and ALL [12]. It is not clear whether this reduced response to rapamycin in advanced leukemias reflects the accumulation of additional mutations that reduce rapamycin sensitivity, or whether rapamycin simply has a better opportunity to decimate the LSC pool when the pool size is much smaller. Nonetheless, one possibility raised by these results is that rapamycin analogues will yield more encouraging results when used in patients with minimal residual disease.

Our data suggest that whatever pathways lead to the depletion of HSCs after Pten deletion, that this response occurs downstream of mTor because it can be rescued by rapamycin treatment. However, the PI-3kinase pathway is a highly branched pathway, with many outputs and feedback loops, making it difficult to predict exactly which signal induces HSC depletion. mTor exists in two distinct complexes that function downstream and upstream of Akt: mTorc1 and mTorc2 [45; 46]. mTorc1 is downstream of Akt, controls the downstream effectors S6 kinase (S6K, which regulates ribosome biogenesis) and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1, which regulates protein translation), and is rapamycin sensitive [46]. mTorc2 activates Akt and is rapamycin insensitive [46]. Although Akt activation leads to mTorc1 activation, mTorc1 activation can reduce Akt activation by negative feedback mechanisms. Rapamycin treatment increases Akt signaling by disrupting the negative feedback inhibition of activated S6K on the PI-3kinase-Akt pathway ([47]; Figure 1). Rapamycin treatment can therefore activate Akt and the many effectors that lie downstream of Akt on pathways that parallel mTorc1. However, recent data suggest that in a small subset of cancers, including some AMLs, prolonged rapamycin treatment not only inhibits the mTorc1 complex but also prevents the assembly of the mTorc2 complex, thus blocking the activation of Akt [48; 49]. It is possible that prolonged rapamycin treatment inhibits Akt instead of further activating it in Pten-deficient HSCs. This raises the general question of whether HSC depletion is initiated by effectors that lie downstream of mTorc1, such as S6K or 4E-BP1, or parallel pathways that lie downstream of Akt.