PtdIns3K plays a central role in metabolism, cell growth and survival. Signaling through PtdIns3K is prominent in high-grade GBM brain tumors, due to activated growth factor-receptor signaling, inactivation of PTEN, a negative regulator of PtdIns3K and activating mutations in PtdIns3K itself. PtdIns3K therefore represents an attractive target for small molecule inhibitor therapy. Disappointingly, however, inhibitors of PtdIns3K and of downstream kinases including Akt and mTOR, are generally cytostatic rather than cytotoxic in GBM cell lines and xenografts.
Do inhibitors of this signaling network induce pathways that promote cell survival? Because mTOR is a target of both growth factor and nutrient signaling, its blockade is likely to activate one or more survival pathways that enable cells to endure periods of starvation or stress. Autophagy, a cellular self-digestion process that provides energy and nutrients during stress, is a good candidate for such a survival pathway. The idea that mTOR is involved in the negative control of autophagy is now generally accepted; however, the mechanism by which this occurs remains poorly understood.
We first verified that PI-103, a dual inhibitor of PtdIns3K and mTOR, induces autophagy in glioma. Using both siRNA and small molecule inhibitors, we next demonstrated that mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) independently regulate autophagy, and contribute to induction of autophagy in an additive fashion. Having established that mTOR blockade is necessary to induce autophagy, we examined whether blocking the induction or progression of autophagy could promote cell death in response to PI-103. Combining PI-103 with 3-methyladenine (3MA), which inhibits early steps in autophagosome formation, or with bafilomycin A1 (Baf A1), which inhibits autophagosome maturation, led to significant apoptosis in cells wild type for PTEN. In cells mutant for PTEN, Baf A1 was more effective than 3MA in potentiating apoptosis driven by PI-103.
Using MEFs deficient in the apoptosis regulator Bax, we demonstrated that PI-103 cooperates with inhibition of autophagy to elicit apoptosis through the intrinsic mitochondrial pathway. Furthermore, we demonstrated that PI-103 alone is sufficient to induce apoptosis in MEFs deleted for the essential autophagy gene Atg5 (). Collectively, these studies indicate that dual inhibitors of PtdIns3K and mTOR activate autophagy in glioma, promoting survival. Subsequent blockade of autophagy in the setting of dual inhibition of PtdIns3K and mTOR drives apoptosis.
Figure 1 Autophagy and Akt promote survival in glioma. Induction of apoptosis in response to inhibition of PtdIns3K-Akt and mTOR kinase required inhibition of autophagy. Autophagy is a survival pathway in this setting, as a dual PtdIns3K-mTOR inhibitor induces (more ...)
We showed previously that glioma cells generally are defective in signaling between PtdIns3K-Akt and mTOR. Consistent with this result, inhibition of PtdIns3K or Akt does not significantly block mTOR, induces autophagy weakly, and fails to induce apoptosis in combination with inhibitors of autophagy. The allosteric mTORC1 inhibitor rapamycin does induce autophagy, but also fails to induce apoptosis in combination with inhibitors of autophagosome maturation. In contrast, inhibitors of mTOR kinase, dual inhibitors of PtdIns3K-mTOR and inhibition of PtdIns3K and mTOR in combination all activate autophagy, and induce apoptosis in conjunction with blockade of autophagosome maturation ().
Rapamycin induces autophagy and triggers a negative feedback loop through an IRS-dependent mechanism, resulting in increased phosphorylation of Akt in glioma. In contrast, inhibitors of mTOR kinase, dual inhibitors of PtdIns3K-mTOR and inhibition of PtdIns3K and mTOR in combination, all induce autophagy without activating Akt. Importantly, activation of Akt (using cells carrying an allele of Akt fused to the steroid binding domain of the estrogen receptor: Akt-ER) blocks apoptosis driven by the inhibition of PtdIns3K, mTORC1 and autophagosome maturation and by the inhibition of PtdIns3K, mTOR kinase and autophagosome maturation (). These results suggest that Akt signaling plays a central role in promoting resistance to the combination of rapamycin with inhibitors of autophagy, and confirm that apoptosis in response to combining inhibitors of mTOR with inhibitors of autophagy also requires inhibition of Akt.
To translate these studies to a relevant preclinical setting, we established xenografts from human PTEN-mutant GS2 glioma cells. We combined the PtdIns3K-mTOR inhibitor NVP-BEZ235, which is in clinical use, with the lysosomotropic autophagy inhibitor chloroquine, also in clinical use, demonstrating marked shrinkage of tumors, associated with synergistic decreases in proliferation and increases in apoptosis.
The implication of these studies is that allosteric inhibitors of mTORC1 induce separate autophagy-dependent and Akt-dependent pathways of drug-resistance in glioma. We clarified roles for mTORC1 and mTORC2 as independent regulators of autophagy, and showed that both mTOR and PtdIns3K-mTOR inhibitors activate autophagy in glioma, promoting survival. Next, we demonstrated that a feedback loop linking allosteric inhibitors of mTORC1 to activation of Akt also promotes survival, independently of autophagy. Finally, we showed that the clinical dual PtdIns3K-mTOR inhibitor NVP-BEZ235 cooperates with the clinical lysosomotropic autophagy inhibitor chloroquine to block both survival signals, inducing apoptosis in glioma xenografts in vivo and offering a therapeutic approach translatable to patients.