Abstract A27: PI3K/Akt pathway-dependent and -independent mechanisms of resistance to EGFR tyrosine kinase inhibitors in glioblastoma
Authors: Wykosky Jill , Cavenee Webster , Furnari Frank 
Published: March 2013

EGFR-targeted therapies such as the tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib have had limited success clinically for the treatment of glioblastoma (GBM). Interestingly, some of the most common mechanisms of resistance to these agents in other solid tumors are rarely present in GBM. In an effort to identify and characterize molecular mechanisms of acquired resistance to EGFR TKIs in GBM, we utilized TKI-sensitive, transformed ink4a/arf −/− astrocytes overexpressing the constitutively active mutant ΔEGFR (or EGFRvIII). To model TKI-resistance in vitro, cells were seeded in soft agar in the presence of gefitinib or erlotinib for 2 weeks; during this time, colony formation was inhibited by TKIs. The dose was escalated, and after 3 weeks, TKI-resistant colonies began to emerge and were isolated to generate clonal cell lines. TKI resistance was verified in vitro as well as in vivo using an orthotopic intracranial model. In three clonally derived TKI-resistant lines, ΔEGFR expression was not altered, and gefitinib inhibited receptor phosphorylation. Two of these three cell lines demonstrated decreased PTEN expression and increased, sustained Akt phosphorylation, indicating a failure of TKIs to suppress signaling through the oncogenic PI3K/Akt pathway. Importantly, this phenotype mimics a known mechanism, as PTEN loss has been implicated in resistance to EGFR TKIs in GBM. The third TKI-resistant cell line had no change in PTEN expression, and Akt phosphorylation was completely inhibited by gefitinib. Interestingly, TKI-resistance was irreversible in those cells with decreased PTEN, and reversible in cells having no change in PTEN expression. In fact, resensitized cells demonstrated ΔEGFR and ERK phosphorylation that was several-fold greater than that of parental cells, as well as increased sensitivity to gefitinib and erlotinib. Interestingly, autophagy was dramatically elevated in all of these TKI-resistant lines, and the cell line that was resensitized to TKIs displayed a significant reduction in autophagy compared to its resistant counterpart. Overall, we have generated and characterized a model system for the identification of mechanisms of resistance to EGFR TKIs in GBM. We have identified at least two distinct resistance scenarios in cells with wild-type PTEN: an irreversible mechanism involving decreased PTEN expression, and another mechanism that is reversible and does not appear to directly involve the PI3K pathway. These findings suggest that reversibility of TKI-resistance following drug removal may depend on mechanism of resistance, which has potential clinical implications. Moreover, the fact that increased autophagy is a common feature suggests a potential therapeutic opportunity for targeting the autophagic process that may apply despite apparent differences in molecular mechanism. This work forms the basis for our effort to uncover novel targets or approaches to therapy that may increase the utility of these drugs for treatment of GBM.