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CTNNB1 mutations mediate resistance to EGFR targeted therapies in non-small cell lung cancer

ABSTRACT: CTNNB1 mutations, which cause activation of Wnt target genes, are found to co-occur in EGFR-mutant lung cancer. We hypothesize that these mutations enhance stability of β-catenin protein and upregulate Wnt signaling, thereby promoting resistance to targeted therapy. CTNNB1 mutation spectrum was analyzed in non-small cell lung cancer patients in The Cancer Genome Atlas (TCGA) and Moffitt Cancer Center patient databases to identify prevalent CTNNB1 mutations as well as frequency of co-occurring mutations. EGFR-mutant HCC827 cells were engineered to stably express most frequent CTNNB1 mutations to determine their ability to drive resistance to EGFR TKI. Western blotting and RNA sequencing were performed to assess the molecular differences between cells expressing wild-type and erlotinib resistant CTNNB1T41A. A viability screen was also performed in which these cells were treated with either with β-catenin siRNA or a panel of drugs with known targets, in combination with erlotinib, to identify new vulnerabilities in CTNNB1T41A cells. Our analysis shows that EGFR mutations co-occur in ~45% of CTNNB1 mutant patients. We show that CTNNB1 mutations cause stabilization and higher accumulation of active β-catenin but vary in their ability to cause erlotinib resistance, with T41A, S37F, S45C and D32H mutants being resistance inducers. Of the mutations tested, cells expressing CTNNB1T41A demonstrated the strongest resistance to erlotinib. Transcriptomics data reveals higher levels of β-catenin regulated proteins (axin2, TCF7 and survivin), EMT markers (vimentin and N-cadherin), and receptors/ligands of the FGFR signaling pathway, in HCC827 CTNNB1T41A cells when compared to CTNNB1WT. Pathway analysis of genes differentially expressed between the WT and T41A mutant cells reveal Wnt pathway to be the top upregulated pathway in the CTNNB1 mutant cells. CTNNB1T41A cells also showed sustained ERK, AKT, GSK3β and S6 signaling, and lower PARP cleavage compared to wild type. Combining erlotinib with PI3K/mTOR/MEK inhibitors or CTNNB1 knockdown can partially reverses erlotinib resistance in HCC827 CTNNB1T41A cells. Our data suggests that CTNNB1 may be an important co-occurring alteration in developing resistance to targeted EGFR therapy. We show that the ability of a CTNNB1 mutation to drive resistance to EGFR TKI relates with the activation of CTNNB1 signaling and its downstream targets. Our results suggest that combined EGFR TKI and PI3K/mTOR/MEK or β-catenin inhibition may be beneficial in patients harboring co-occurring EGFR and CTNNB1 mutations.

ORGANISM(S): Homo sapiens

PROVIDER: GSE322498 | GEO | 2026/03/05

REPOSITORIES: GEO

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Project description:Introduction: The clinical benefit of EGFR tyrosine kinase inhibitor (TKI) treatment in non-small cell lung cancer (NSCLC) patients with activating EGFR mutations is temporary, as virtually all patients develop acquired EGFR TKI resistance that occurs via diverse mechanisms. Here, we identified increased FGFR1 expression as such a resistance mechanism and using pathways analysis and drug combination testing we identified a novel combination treatment to control growth of these resistant tumors. Methods: Novel erlotinib-resistant NSCLC cell lines were generated and analyzed by mass spectrometry-based proteomics to identify altered pathways associated with erlotinib resistance. The altered pathways were further analyzed in gefitinib and osibenib resistant cell lines. Small molecule inhibitor combinations were used to block the altered pathways and investigate growth reduction in vitro and in two xenograft mouse models. FGFR1 mRNA levels were examined in pre- and (post?)- EGFR TKI treatment clinical tumor samples. Results: Proteomic analysis revealed increased expression of FGFR1 and AXL as well as increased Akt and ERK1/2 activation in a panel of novel erlotinib-resistant HCC827 cell lines. Combined treatment with erlotinib or osimertinib and a panel of small molecule inhibitors targeting AXL/MET, FGFRs, Akt, PI3K/mTOR, MEK or ERK1/2 showed that the most prominent re-sensitization to EGFR TKI occurred with the pan-FGFR inhibitor, PD173074. Interestingly, simultaneous blockade of components of the Akt pathway using specific Akt or dual PI3K-mTOR inhibitors combined with inhibitors targeting the FGFR family exhibited the most efficient growth inhibition of FGFR1 overexpressing EGFR TKI-resistant cell lines. Phosphorylation of proteins downstream of Akt, including PRAS40, FOXO and S6 ribosomal protein, were completely abrogated by PD173074 combined with the Akt inhibitor GSK2141795 . Combination treatment with PD173074 and an Akt inhibitor exhibited synergistic growth inhibition in vivo in two FGFR1 overexpressing NSCLC EGFR TKI-resistant animal models. Conclusion: The significant growth inhibition in vitro and in vivo observed with PD173074 combined with Akt compared to either drug alone imply that inhibition of several key targets may be beneficial in controlling erlotinib-resistant NSCLC. The complete abrogation of PRAS40, FOXO and S6 phosphorylations by PD173074 combined with an Akt inhibitor indicates that the Akt pathway is no longer active.

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CTNNB1 mutations mediate resistance to EGFR targeted therapies in non-small cell lung cancer

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