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Overcoming EGFR T790M-based Tyrosine Kinase Inhibitor Resistance with an Allele-specific DNAzyme.

ABSTRACT: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are the main therapeutic agents used to treat non-small-cell lung cancer patients harboring EGFR-activating mutations. However, most of these patients will eventually develop resistance, 50% of which are due to a secondary mutation at T790M in the EGFR. In this paper, we describe the development of an allele-specific DNAzyme, DzT, that can specifically silence EGFR T790M mutant messenger RNA while leaving wild-type EGFR intact. Allele-specific silencing of EGFR T790M expression and downstream signaling by DzT triggered apoptosis in non-small-cell lung cancer cells harboring this mutant. Adding a cholesterol-triethylene glycol group on the 3'-end of DzT (cDzT) improved drug efficacy, increasing inhibitory effect on cell viability from 46 to 79% in T790M/L858R-harboring H1975(TM/LR) non-small-cell lung cancer cells, without loss of allele specificity. Combined treatment with cDzT and BIBW-2992, a second-generation EGFR-tyrosine kinase inhibitor, synergistically inhibited EGFR downstream signaling and suppressed the growth of xenograft tumors derived from H1975(TM/LR) cells. Collectively, these results indicate that the allele-specific DNAzyme, DzT, may provide an alternative treatment for non-small-cell lung cancer that is capable of overcoming EGFR T790M mutant-based tyrosine kinase inhibitor resistance.Molecular Therapy-Nucleic Acids (2014) 3, e150; doi:10.1038/mtna.2014.3; published online 4 March 2014.

SUBMITTER: Lai WY

PROVIDER: S-EPMC3982196 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Overcoming EGFR T790M-based Tyrosine Kinase Inhibitor Resistance with an Allele-specific DNAzyme.

Lai Wei-Yun WY Chen Chi-Yuan CY Yang Shuenn-Chen SC Wu Jer-Yuarn JY Chang Cheng-Ju CJ Yang Pan-Chyr PC Peck Konan K

Molecular therapy. Nucleic acids 20140304

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are the main therapeutic agents used to treat non-small-cell lung cancer patients harboring EGFR-activating mutations. However, most of these patients will eventually develop resistance, 50% of which are due to a secondary mutation at T790M in the EGFR. In this paper, we describe the development of an allele-specific DNAzyme, DzT, that can specifically silence EGFR T790M mutant messenger RNA while leaving wild-type EGFR in ...[more]

PMID: 24594844

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Project description:Activating double mutations L858R/T790M in the epidermal growth factor receptor (EGFR) region are often observed as the cause of resistance to tyrosine kinase inhibitors (TKIs). Third-generation EGFR-TKIs, such as osimertinib and rociletinib (CO-1686), was developed to target such resistance mutations. The detection of activating L858R/T790M mutations is necessary to select sensitive patients for therapy. Hence, we aimed to develop novel radiobromine-labeled CO-1686 as a positron emission tomography (PET) imaging probe for detecting EGFR L858R/T790M mutations. Nonradioactive brominated-CO1686 (BrCO1686) was synthesized by the condensation of N-(3-[{2-chloro-5-(trifluoromethyl)pyrimidin-4-yl}amino]-5-bromophenyl) acrylamide with the corresponding substituted 1-(4-[4-amino-3-methoxyphenyl]piperazine-1-yl)ethan-1-one. The radiobrominated [77Br]BrCO1686 was prepared through bromodestannylation of the corresponding tributylstannylated precursor with [77Br]bromide and N-chlorosuccinimide. Although we aimed to provide a novel PET imaging probe, 77Br was used as an alternative radionuclide for 76Br. We fundamentally evaluated the potency of [77Br]BrCO1686 as a molecular probe for detecting EGFR L858R/T790M using human non-small-cell lung cancer (NSCLC) cell lines: H1975 (EGFR L858R/T790M), H3255 (EGFR L858R), and H441 (wild-type EGFR). The BrCO1686 showed high cytotoxicity toward H1975 (IC50 0.18 ± 0.06 µM) comparable to that of CO-1686 (IC50 0.14 ± 0.05 µM). In cell uptake experiments, the level of accumulation of [77Br]BrCO1686 in H1975 was significantly higher than those in H3255 and H441 upon 4 h of incubation. The radioactivity of [77Br]BrCO1686 (136.3% dose/mg protein) was significantly reduced to 56.9% dose/mg protein by the pretreatment with an excess CO-1686. These results indicate that the binding site of the radiotracers should be identical to that of CO-1686. The in vivo accumulation of radioactivity of [77Br]BrCO1686 in H1975 tumor (4.51 ± 0.17) was higher than that in H441 tumor (3.71 ± 0.13) 1 h postinjection. Our results suggested that [77Br]BrCO1686 has specificity toward NSCLC cells with double mutations EGFR L858R/T790M compared to those in EGFR L858R and wild-type EGFR. However, the in vivo accumulation of radioactivity in the targeted tumor needs to be optimized by structural modification.

Project description:IntroductionMechanisms of resistance to EGFR exon 20 insertion mutation active inhibitors have not been extensively studied in either robust preclinical models or patient-derived rebiopsy specimens. We sought to characterize on-target resistance mutations identified in EGFR exon 20 insertion-mutated lung cancers treated with mobocertinib or poziotinib and evaluate whether these mutations would or would not have cross-resistance to next-generation inhibitors zipalertinib, furmonertinib, and sunvozertinib.MethodsWe identified mechanisms of resistance to EGFR exon 20 insertion mutation active inhibitors and then used preclinical models of EGFR exon 20 insertion mutations (A767_V769dupASV, D770_N771insSVD, V773_C774insH) plus common EGFR mutants to probe inhibitors in the absence/presence of EGFR-T790M or EGFR-C797S.ResultsMobocertinib had a favorable therapeutic window in relation to EGFR wild type for EGFR exon 20 insertion mutants, but the addition of EGFR-T790M or EGFR-C797S negated the observed window. Zipalertinib had a favorable therapeutic window for cells driven by EGFR-A767_V769dupASV or EGFR-D770_N771insSVD in the presence or absence of EGFR-T790M. Furmonertinib and sunvozertinib had the most favorable therapeutic windows in the presence or absence of EGFR-T790M in all cells tested. EGFR-C797S in cis to all EGFR mutations evaluated generated dependent cells that were resistant to the covalent EGFR tyrosine kinase inhibitors mobocertinib, zipalertinib, furmonertinib, sunvozertinib, poziotinib, and osimertinib.ConclusionsThis report highlights that poziotinib and mobocertinib are susceptible to on-target resistance mediated by EGFR-T790M or -C797S in the background of the most prevalent EGFR exon 20 insertion mutations. Furmonertinib, sunvozertinib, and to a less extent zipalertinib can overcome EGFR-T790M compound mutants, whereas EGFR-C797S leads to covalent inhibitor cross-resistance-robust data that support the limitations of mobocertinib and should further spawn the development of next-generation covalent and reversible EGFR exon 20 insertion mutation active inhibitors with favorable therapeutic windows that are less vulnerable to on-target resistance.

Project description:BackgroundResistance to third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) presents a major clinical challenge in advanced non-small cell lung cancer (NSCLC). Here, we report resistance mechanisms to abivertinib, a novel third-generation EGFR TKI, from a phase I dose-escalation/expansion study (NCT02330367).MethodsPatients with EGFR T790M-positive advanced NSCLC and progression on prior EGFR TKIs received abivertinib in dose escalation (50-350 mg twice daily [BID]) or expansion (300 mg BID) cohorts. Patients enrolled at Guangdong Lung Cancer Institute who underwent next-generation sequencing (NGS)-based genomic profiling upon abivertinib progression (prior to October 30, 2018) were enrolled in this exploratory analysis.FindingsThirty of 73 patients enrolled were eligible for resistance analysis. Upon abivertinib progression, 27 patients provided plasma samples (six patients also provided paired samples from the progression sites) and three patients only provided tissue samples from the progression sites for NGS. A heterogeneous landscape of resistance to abivertinib was observed: 15% (4/27) experienced EGFR T790M loss and 13% (4/30) developing EGFR tertiary mutations including C797S. EGFR amplification was observed in 11 patients (37%), and considered a putative resistance mechanism in seven (23%) patients. Other EGFR-independent resistance mechanisms involved CDKN2A, MET, PIK3CA, HER2, TP53, Rb1 and small-cell lung cancer transformation.InterpretationOur findings reveal a heterogenous pattern of resistance mechanisms to abivertinib which is distinct from that previously reported with osimertinib. EGFR amplification was the most common resistance mechanism in this cohort. FUND: The National Key R&D Program of China (Grant No. 2016YFC1303800), Key Lab System Project of Guangdong Science and Technology Department - Guangdong Provincial Key Lab of Translational Medicine in Lung Cancer (Grant No. 2012A061400006/2017B030314120).

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Overcoming EGFR T790M-based Tyrosine Kinase Inhibitor Resistance with an Allele-specific DNAzyme.

Publications

Overcoming EGFR T790M-based Tyrosine Kinase Inhibitor Resistance with an Allele-specific DNAzyme.

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