Abstract 4684: Afatinib targets glioblastoma stem cells by inhibiting EGFRVIII-cMet co-activation
Authors: Raghupathy Vengoji, Muzafar A. Macha, Ramakrishna Nimmakayala, Satyanarayana Rachagani, Kavita Mallya, Maneesh Jain, Moorthy P. Ponnusamy, Surinder K. Batra, Nicole Shonka
Journal: Cancer Research (2019)

Abstract Background Glioblastoma (GBM) is the aggressive primary brain tumor with a median survival rate of 14.6 months. Currently, first-line treatment includes surgical resection, chemoradiation, and adjuvant chemotherapy with temozolomide (TMZ). However, GBM recurs most often within 6.9 months. Most of the targeted therapies failed in GBM, possibly due to the co-activation of the receptor tyrosine kinases (RTKs). Genetic analysis on GBM tumor revealed that RTKs are dysregulated, with epidermal growth factor receptor (EGFR) representing 57.4% of the deleted/mutated GBM, about 30 - 40% of GBM patients with EGFR amplification carry an oncogenic gene rearrangement EGFR variant III (EGFRvIII) which is constitutively active. In addition, EGFRvIII co-activates cMET RTKs thereby enriches GBM cancer stem like cells (CSCs). CSCs are relatively radio- and chemo- resistant and play a pivotal role in tumor recurrence/progression. We hypothesize that afatinib and TMZ combination would inhibit EGFRvIII co-activation and tumor progression in GBM model. Methods GBM cell lines U87MG, U87MG transfected with EGFRvIII (U87 EGFRvIII) and SP/CSC isolated from U87 EGFRvIII cells were treated with afatinib, TMZ alone or in combination and analyzed. The in vivo efficacy of these drugs were also analyzed on U87 EGFRvIII orthograft mouse model. Results We observed significantly higher proportion of CSCs in U87 EGFRvIII cells compared to U87MG cells (p = 0.03). While afatinib decreased the percentage of CSCs in both U87MG and U87 EGFRvIII cells (p = 0.02), TMZ only decreased CSC population in U87MG cells. However, combination of afatinib with TMZ significantly decreased the CSCs in both U87MG and U87 EGFRvIII cells (p = 0.02). Our clonogenic (tumorsphere) assay revealed significantly more tumorspheres (p=0.01) formed by U87 EGFRvIII CSCs cells than U87MG CSCs. In addition, we also observed that TMZ significantly decreased the self-renewal properties of U87 CSCs compared to U87 EGFRvIII CSCs. Furthermore, afatinib alone or in combination with TMZ significantly abolished the tumorsphere formation by U87 EGFRvIII CSCs. The underlying mechanism revealed inhibition of cMET RTK co-activation by EGFRvIII using afatinib. Our in vivo studies using U87 EGFRvIII orthograft model revealed significant tumor growth inhibition by afatinib and TMZ combination compared to control and either drug alone. Conclusion Our results strongly support the efficacy of afatinib and TMZ combination in inhibiting EGFRvIII-cMET signaling mediated GBM stemness and prevention of tumor progression. This treatment should be tested in EGFR amplified/mutated GBM patients. Note: This abstract was not presented at the meeting. Citation Format: Raghupathy Vengoji, Muzafar A. Macha, Ramakrishna Nimmakayala, Satyanarayana Rachagani, Kavita Mallya, Maneesh Jain, Moorthy P. Ponnusamy, Surinder K. Batra, Nicole Shonka. Afatinib targets glioblastoma stem cells by inhibiting EGFRVIII-cMet co-activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4684.