Abstract In 20% – 30% of all glioblastomas (GBMs), the most common and deadly form of glioma, gene rearrangements in amplified epidermal growth factor receptor (EGFR) cause amplification of a highly oncogenic variant of wild-type EGFR called EGFRvIII. A number of studies demonstrate that EGFRvIII amplification is detected predominantly in GBMs harboring wild-type EGFR amplification or those expressing the wild-type isoform of the tumor suppressor p53. However, it remains unclear why such genetic interactions occur or whether they are advantageous for tumor growth. Our limited understanding of these genetic interactions likely stems from an inability to study the function of amplified EGFRvIII in vitro using currently available model systems. Indeed, there are no immortalized GBM cell lines that express EGFRvIII because prolonged in vitro culture of EGFRvIII-positive GBM tumor cells invariably selects against EGFRvIII amplification. Moreover, the commonly used ectopic EGFRvIII expression models are unlikely to fully recapitulate the function, regulation, and downstream signaling activities of amplified EGFRvIII. Herein, we validate the use of short-term cell cultures derived from GBM xenografts expressing amplified EGFRvIII to study the endogenous function of amplified EGFRvIII. These cells exhibit competency in EGFR signaling, and maintain EGFRvIII expression for up to five months. Further characterization of these cells using small molecule inhibitor and short hairpin RNA approaches reveals three novel observations. First, loss of EGFRvIII expression and activity results in growth arrest and cellular senescence, indicating that EGFRvIII activity is required for cell growth. Second, specific depletion of wild-type EGFR significantly decreases EGFRvIII phosphorylation, demonstrating that high-level wild-type EGFR expression is required for maximal EGFRvIII activation. Third, p53 depletion inhibits growth arrest induced by loss of EGFRvIII activity, strongly suggesting that the growth-promoting activity of EGFRvIII is strictly dependent on p53 function. By showing that EGFRvIII functionally interacts with wild-type EGFR and wild-type p53 in a manner that promotes cell growth, these data provide a functional basis for the commonly observed genetic interactions between amplified EGFRvIII and amplified wild-type EGFR or wild-type p53. Furthermore, our data imply that with help from amplified wild-type EGFR, amplified EGFRvIII functions specifically to overcome the growth inhibitory activity of wild-type p53 in order to promote cell growth. Consistent with this insight, we provide preliminary evidence for the efficacy of a combination therapy involving EGFR and MDM2 inhibitors, which permits concomitant inhibition of EGFR signaling and potentiation of p53 function. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4417. doi:10.1158/1538-7445.AM2011-4417