Mechanisms and Antitumor Activity of a Binary EGFR/DNA–Targeting Strategy Overcomes Resistance of Glioblastoma Stem Cells to Temozolomide

Abstract

Purpose: Glioblastoma (GBM) is a fatal primary malignant brain tumor. GBM stem cells (GSC) contribute to resistance to the DNA-damaging chemotherapy, temozolomide. 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: ZR2002 is a "combi-molecule" designed to inflict DNA damage through its chlorethyl moiety and induce irreversible EGFR tyrosine kinase inhibition. We assessed its in vitro efficacy in temozolomide-resistant patient-derived GSCs, mesenchymal temozolomide-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 antiproliferative 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 p53dependent 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 temozolomide-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.

Design and Mechanism of Action of a New Prototype of Combi-Molecule “Programed” to Release Bioactive Species at a pH Range Akin to That of the Tumor Microenvironment

“…As shown in Figure 2, the conversion of the combi-molecule AL530 into its two bioactive components (DNA damage species and MEK inhibitor) was largely accelerated at an acidic pH. The pH rate profile also indicated that AL530 is hydrolyzed at a pH range between 5.5-6.5, which is within the range of pH reported for the tumor microenvironment [12][13][14]. The results are in line with the cleavage mechanism proposed in Scheme 1, wherein the pH rate profile clearly suggests that N-protonation of the tetrahedral intermediate is the rate determining step of the reaction.…”

Egfr Glioblastoma Stem Cells Targeting by Cd16158v Chimeric Receptor T Cells and Cetuximab

“…However, resistant tumor cell subsets promoting recurrence, rapidly emerge. Although these cells, presenting CSC features, do frequently express EGFR, treatment with small molecules targeting the EGFR signal transduction pathway has proven largely disappointing, thus suggesting that direct immune-mediated targeting of this marker could be more effective (34). Our data indicate that, indeed, expansion of GBM-derived c-CSC may be prevented by ADCC mediated by CD16 158V -CR T cells and anti-EGFR IgG1 mAb.…”

Simultaneous detection of EGFR amplification and EGFRvIII variant using digital PCR-based method in glioblastoma

“…For one patient, the cost decreases from 40 to 50% when using only the EGFR2/EGFR3 assay. Moreover, in contrast to FISH, dPCR allows the simultaneous detection of the EGFRvIII variant, which has been shown to be a potential therapeutic target [26]. Lower copy number values observed between EGFR2 and EGFR3 amplicons are very likely explained by the presence of the EGFRvIII variant.…”