Combinatorial inhibition of IGF2BP3-regulated receptor tyrosine kinases offers a new therapeutic strategy for triple-negative breast cancer

Epidermal growth factor receptor (EGFR), characterized by its high positivity rate and strong specificity in triple-negative breast cancer (TNBC), has long been recognized as one of the characteristics of this subtype. However, EGFR inhibitors have demonstrated limited clinical efficacy in TNBC treatments. Previous studies revealed that multiple receptor tyrosine kinases (RTKs) were activated in TNBC. These RTKs cooperate through dimerization to activate downstream pathway phosphorylation, rendering EGFR monotherapy ineffective. This highlights the necessity for combinatorial strategies targeting multiple RTKs to treat TNBC. Published bulk-RNA sequencing datasets from erlotinib resistant TNBC patient-derived xenografts model revealed changes in gene sets in erlotinib resistant tissues. RNA immunoprecipitation and methylated RNA immunoprecipitation sequencing were used to detect co-regulated RTKs by insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3). RNA pull-down and molecular docking clarified the interaction sites between IGF2BP3 and RTKs. Computational virtual docking, hierarchical screening, microscale thermophoresis and cytotoxicity assays were employed to screen small-molecule inhibitors aimed at disrupting IGF2BP3-RTK interactions. The determination of drug synergistic effects was achieved through the calculation of the combination index. In this study, we identified IGF2BP3 could coordinately regulate multiple RTKs’ mRNA through an m6A-dependent manner and activate RTKs expression, and thus could be the key point to address the drug resistance problem. After clarifying the interaction sites between IGF2BP3 protein and RTK mRNAs, we identified cynaroside, a small-molecule inhibitor capable of disrupting IGF2BP3-RTK interactions, as a promising enhancer of tyrosine kinase inhibitor (TKI) efficacy. Moreover, we proposed a novel therapeutic strategy by combining cynaroside with TKI and chose the EGFR/MET bispecific antibody amivantamab for the best candidate in TNBC target treatment. Our study provide a mechanistic insight into IGF2BP3-mediated EGFR inhibitor resistance and present an emerging combinatorial approach to overcome therapeutic limitations in TNBC.