Mechanism of action of Tarloxotinib, a hypoxia-activated pan-HER inhibitor: Potential for targeting wild-type EGFR in non-small cell lung cancer

Abstract

Strategies that achieve inhibition of wild-type epidermal growth factor receptor (WT EGFR) in non-small cell lung cancer (NSCLC) whilst sparing normal tissues would have broad clinical utility. First and second generation EGFR tyrosine kinase inhibitors (TKIs) originally sought to achieve this goal but lacked the necessary therapeutic index for selective inhibition of WT EGFR in tumours. Third generation EGFR-TKIs are designed to spare WT EGFR and selectively target mutant EGFR including the secondary T790M mutation which mediates resistance in the majority of patients treated with 1st and 2nd generation EGFR-TKIs. TH-4000 (Tarloxotinib bromide) is a hypoxia-activated prodrug designed to release a dose-potent EGFR-TKI (TH-4000E) selectively in hypoxic regions of solid tumours offering, for the first time, the possibility of achieving a therapeutic window for inhibition of oncogenic WT EGFR. This thesis evaluated the mechanism of action and efficacy of TH-4000 in WT EGFR driven NSCLC and heterozygous EGFR-mutant NSCLC; the latter is where the presence of the WT EGFR allele is proposed to have a causative role in resistance to conventional EGFR-TKIs.Mechanism of action studies employing non-fragmenting chemical biology tools confirmed the requirement for one-electron mediated prodrug fragmentation to elicit the desired biological effect. The anti-proliferative activity of TH-4000 is enhanced by as much as 71-fold in cells following anoxic equilibration, and is directly associated with inhibition of EGFR-mediated signal transduction. Accordingly, metabolism of TH-4000 to TH-4000E is dependent on the lack of oxygen in cell lines while hyperbaric oxygen significantly suppresses TH-4000 metabolism in vivo. Consistently, a non-fragmenting isomer of TH-4000 displayed suppressed antitumour activity that was associated with minimal TH4000E release. When assessed for anti-proliferative activity, TH-4000E was significantly more dose-potent than the 1st and 2nd generation EGFR-TKIs, erlotinib and afatinib respectively, in NSCLC cell lines with WT EGFR as the driver kinase. In WT EGFR driven xenografts (A431 and H125), TH-4000 was more efficacious than afatinib when both agents were evaluated at their respective human equivalent doses in mice. The combination of TH-4000 with cetuximab is synergistic in H125 xenografts and provides more antitumour activity in comparison to either agent alone.Hypoxia-induced transforming growth factor α (TGFα) signalling through WT EGFR is proposed to mediate resistance to EGFR-TKIs in heterozygous EGFR-mutant NSCLC cell lines (e.g. PC9). Stimulation of PC9 cells with TGFα led to resistance of all EGFR-TKIs (as much as 7.5 fold with erlotinib) although TH-4000E remained the most resilient following TGFα stimulation. Transfection and stable overexpression of WT EGFR in PC9 cells led to a strong induction of EGFR signalling following TGFα stimulation and further decreased the sensitivity of EGFR-TKIs, an effect that was most profound for osimertinib. In vivo evaluation of daily osimertinib treatment showed a decreased sensitivity in WT EGFR transfected PC9 xenografts (relative to parental PC9) which also progressed on treatment at approximately 40 days after treatment initiation; intervention with TH-4000 reversed progression of these tumours resulting in a sharp decline in tumour volumes. Efficacy of TH-4000 will be dependent on activation by one-electron reductases. To identify TH-4000 reductases, microarray-based gene expression profiling was performed for a panel of 30 human cancer cell lines and correlated with rates of TH-4000 metabolism measured by mass spectrometry. This led to the identification of the metalloreductase STEAP4 as the sole candidate reductase whose knockdown significantly decreased TH-4000 metabolism in two cell lines (HCC1954 and SiHa). Transfection of STEAP4 in C33A and H1299 cells led to as much as a 52-fold increase in the rate of TH-4000 metabolism. Notably, lung adenocarcinomas (a subtype of NSCLC) are reported as having high STEAP4 expression although highest STEAP4 expression is noted in prostate cancer. Identification of cancer types that are not only WT EGFR-dependent but also positive for STEAP4 expression is likely to determine the success of TH-4000 in the clinic.