Antitumor Activity of a Binary EGFR/DNA Targeting Strategy Overcomes Resistance of Glioblastoma Stem cells to Temozolomide

Purpose: Glioblastoma (GBM) is a fatal primary malignant brain tumor. GBM stem cells (GSCs) contribute to resistance to the DNA-damaging chemotherapy, temozolomide (TMZ). The epidermal growth factor receptor (EGFR) displays genomic alterations enabling DNA repair mechanisms in half of GBMs. We aimed to investigate EGFR/DNA combi-targeting in GBM. Experimental Design: We assessed the in vitro efficacy of ZR2002, a 9combi-molecule9 designed to inflict DNA damage through its chlorethyl moiety and induce irreversible EGFR tyrosine kinase inhibition in TMZ-resistant patient-derived GSCs, mesenchymal TMZ-sensitive and resistant in vivo derived GSC sublines, and U87/EGFR isogenic cell lines stably expressing EGFR/wild-type or variant III (EGFRvIII). We evaluated its antitumor activity in mice harboring orthotopic EGFRvIII or mesenchymal TMZ-resistant GSC tumors. Results: ZR2002 induced submicromolar anti-proliferative effects and inhibited neurosphere formation of all GSCs with marginal effects on normal human astrocytes. ZR2002 inhibited EGF-induced autophosphorylation of EGFR, downstream Erk1/2 phosphorylation, increased DNA strand breaks and induced activation of wild-type p53; the latter was required for its cytotoxicity through p53-dependent mechanism. ZR2002 induced similar effects on U87/EGFR cell lines and its oral administration significantly increased survival in an orthotopic EGFRvIII mouse model. ZR2002 improved survival of mice harboring intracranial mesenchymal TMZ-resistant GSC line, decreased EGFR, Erk1/2 and AKT phosphorylation and was detected in tumor brain tissue by MALDI imaging mass spectrometry. Conclusions: These findings provide the molecular basis of binary EGFR/DNA targeting and uncover the oral bioavailability, blood brain barrier permeability and antitumor activity of ZR2002 supporting potential evaluation of this first-in-class drug in recurrent GBM.