The malignant gliomas show intrinsic resistance to most medical therapies, contributing to the poor prognosis associated with these tumors (Persson et al., 2007
). The association of EGFR
amplification with high-grade glioblastoma multiforme tumors therefore led to early optimism that EGFR inhibition would be beneficial in glioma. This initial optimism was mitigated however, by the realization that only a subset of patients with EGFR
-amplified glioma actually responded to blockade of EGFR (Nicholas et al., 2006
). The failure of this approach in the majority of patients with EGFR
-amplified glioma could stem from inefficient blockade of the receptor or from inability to reverse signaling abnormalities associated with EGFR
amplification, even in the setting of adequate blockade of p-EGFR. Loss of PTEN
is a likely contributor to this failure, as loss of PTEN
effectively blocks the ability of EGFR inhibitors to impact downstream signaling through PI3K and ultimately through mTOR.
In this communication, we present a preclinical approach aimed at reversing signaling abnormalities associated with EGFR amplification, offering a mechanistic rationale to combine inhibitors of EGFR and of mTOR to effect proliferation blockade in patients with EGFR-amplified, PTENmt glioma. We demonstrated efficacy for inhibitors of EGFR as monotherapy in glioma cells wild-type for PTEN, and that the antiproliferative effect of EGFR inhibitors correlated with the ability of these agents to impact levels of mTOR.
In contrast to PTENwt
cells, erlotinib treatment of PTENmt
cells did not appreciably impact proliferation and specifically did not impact mTOR, even when inhibitors of EGFR were used at doses sufficiently high to block p-Akt (). Although erlotinib had little measurable activity as monotherapy in PTENmt
cells, erlotinib clearly augmented the efficacy of PI-103 as measured both by blockade of mTOR and of proliferation (). Intriguingly, the ability of PI-103 and erlotinib to impact mTOR again was observed in a setting where combination therapy did not appreciably alter levels of p-Akt in comparison with PI-103 alone (). The dissociation of Akt from mTOR in PTENmt
glioma has also been observed by others (Wang et al., 2006
), and suggests the presence of Akt-independent regulators of mTOR.
The failure of inhibitors of EGFR to impact mTOR signaling in PTENmt
glioma also provides a rationale to combine inhibitors of EGFR and mTOR. While targeting both kinases simultaneously led to decreased proliferation in comparison with targeting either EGFR or mTOR alone (), blockade of mTOR by rapamycin actually led to increased levels of p-Akt (, lane 5). The activation of p-Akt by rapamycin and its analogues has been described previously in primary human tumors (O'Reilly et al., 2006
; Shi et al., 2005
; Sun et al., 2005
). Addition of an mTOR inhibitor effectively blocks mTOR, but at the cost of activating other targets of PI3K and Akt.
In response to the failure of EGFR inhibitors to block PI3K, Akt, or mTOR in PTENmt
glioma, and because mTOR inhibitors actually activate the PI3K/Akt axis, we tested inhibitors of EGFR and of mTOR in combination with inhibitors of PI3Kα. The combinatorial inhibition of three targets effectively blocked signaling (), and was more effective than any two targeted therapies in combination as measured both biochemically and through flow cytometric analyses (). It is intriguing in this regard that even using approaches that blocked EGFR, PI3Kα, and mTOR in combination, and in the setting of efficient inhibition of Akt—an important mediator of anti-apoptotic signaling (Luo et al., 2003
)—we failed to observe any appreciable apoptosis in any glioma cell lines tested ( and data not shown). Thus, while the ability to translate our findings to patients awaits the development of isoform-specific inhibitors of PI3K that are well tolerated clinically, the ability to develop targeted agents that induce cytotoxic rather than cytostatic responses in the malignant gliomas represents a more formidable challenge, and one that may be critical to the long-term efficacy of these approaches in patients.
We are grateful to Russ Pieper and Cynthia Cowdry for cell lines, and Lou Chesler, Chris Hackett, and Matt Grimmer for critical review. This work, dedicated to the late Jeffrey P. Weiss, was supported by grants from the Burroughs Wellcome Fund, The Brain Tumor Society, The National Brain Tumor and Samuel G. Waxman Foundations, The Sandler Family, and the NCI SPORE Program.