Efficacy of continuous EGFR-inhibition and role of Hedgehog in EGFR acquired resistance in human lung cancer cells with activating mutation of EGFR

Abstract

PurposeThe aim of this work was to investigate the efficacy of sequential treatment with first-, second- and third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors and the mechanisms of acquired resistance occurring during the sequential use of these inhibitors.Experimental designWe developed an in vivo model of acquired resistance to EGFR-inhibitors by treating nude mice xenografted with HCC827, a human non-small-cell lung cancer (NSCLC) cell line harboring EGFR activating mutation, with a sequence of first-generation EGFR tyrosine kinase inhibitors (EGFR-TKIs) (erlotinib and gefitinib), of second-generation EGFR-TKI (afatinib) plus/minus the anti-EGFR monoclonal antibody cetuximab, and of third-generation EGFR-TKI (osimertinib).ResultsHCC827-derived xenografts and with acquired resistance to EGFR-inhibitors were sensitive to the sequential use of first-, second- and third-generation EGFR-TKIs. Continuous EGFR inhibition of first-generation resistant tumors by sequential treatment with afatinib plus/minus cetuximab, followed by osimertinib, represented an effective therapeutic strategy in this model. Whereas T790M resistance mutation was not detected, a major mechanism of acquired resistance was the activation of components of the Hedgehog (Hh) pathway. This phenomenon was accompanied by epithelial-to-mesenchymal transition. Cell lines established in vitro from gefitinib-, or afatinib- or osimertinib-resistant tumors showed metastatic properties and maintained EGFR-TKIs resistance in vitro, that was reverted by the combined blockade of Hh, with the selective SMO inhibitor sonidegib, and EGFR.ConclusionsEGFR-mutant NSCLC can benefit from continuous treatment with EGFR-inhibitors, indepenently from mechanisms of resistance. In a complex and heterogenous scenario, Hh showed an important role in mediating resistance to EGFR-inhibitors through the induction of mesenchymal properties.

Antitumor Efficacy of Dual Blockade of EGFR Signaling by Osimertinib in Combination With Selumetinib or Cetuximab in Activated EGFR Human NCLC Tumor Models

“…Cell proliferation was evaluated by the 3-[4,5dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, as previously described. 16 Concentration that inhibits 50% of cell growth (IC 50 ) was determined by interpolation from the dose-response curves. Results represent the median of three separate experiments, each performed in triplicate.…”

“…Cell migration was assessed using a commercially available chemotaxis assay. 16 The in vitro invasive ability of cells was measured by using Transwell chambers (Corning Life Sciences, Tewksbury, MA) according to the manufacturer's protocol. Assays were performed in triplicate.…”

“…It resulted in substitution of threonine to methionine at the "gatekeeper" amino acid 790 and negatively influenced the sensitivity to firstgeneration EGFR-TKIs through a major adenosine triphosphate affinity and steric effect. 6,7 Other mechanisms of resistance include activation of HER2, 8 MET, 9 PIK3CA, 10 BRAF, 11 NF1, 12 FGFR, 13 AXL, 14 and hedgehog 15,16 or histologic changes to small cell lung cancer (SCLC) or epithelial-to-mesenchymal transition. 17 Various strategies have been proposed to overcome resistance to first-generation EGFR-TKIs, including the use of irreversible EGFR-TKIs.…”

“…23 Recently, our group showed that EGFR-mutated NCLC maintains EGFR dependency after the onset of resistance to EGFR-TKIs and can benefit from continuous treatment with a sequence of EGFR inhibitors: first-generation EGFR-TKIs, second-generation EGFR-TKI afatinib plus cetuximab, and third-generation EGFR-TKI osimertinib, independently from mechanisms of resistance. 16 Considering the fundamental EGFR dependency and the role of the RAS/MAPK pathway as escaping mechanism to EGFR inhibition, 16,23 we designed an investigational strategy of treatment aiming to a more potent inhibition of EGFR signaling in EGFR-mutated NCLC models (both T790M-positive and -negative) by reinforcing the effect on the receptor with the monoclonal antibody cetuximab, or on downstream pathways by inhibiting MAPK with the selective MEK1/2-inhibitor selumetinib in addition to osimertinib in second-line treatment in osimertinibresistant tumors and in first-line treatment. In particular, we investigated the best therapeutic option among these combinational strategies and we explored the mechanisms of acquired resistance to these treatments.…”

Dual blockade of EGFR tyrosine kinase using osimertinib and afatinib eradicates EGFR‑mutant Ba/F3 cells

“…Previously, several combination therapies have been reported to overcome the resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), which depends on EGFR secondary mutations or other mechanisms such as bypass signaling (Table I) (20)(21)(22)(23)(24)(25)(26)(27)(28). Another EGFR-TKI, brigatinib, or allosteric EGFR inhibitor EAI045 could overcome both T790M-and C797S-positive triple-mutant resistant clones, particularly when combined with the anti-EGFR antibody cetuximab (21,22).…”