Insulin-like growth factor 1 receptor stabilizes the ETV6-NTRK3 chimeric oncoprotein by blocking its KPC1/Rnf123-mediated proteasomal degradation.

Many oncogenes, including chimeric oncoproteins, require insulin-like growth factor 1 receptor (IGF1R) for promoting cell transformation. The ETS variant 6 (ETV6)-neurotrophic receptor tyrosine kinase 3 (NTRK3) (EN) chimeric tyrosine kinase is expressed in mesenchymal, epithelial, and hematopoietic cancers and requires the IGF1R axis for transformation. However, current models of IGF1R-mediated EN activation are lacking mechanistic detail. We demonstrate here that IGF-mediated IGF1R stimulation enhances EN tyrosine phosphorylation and that blocking IGF1R activity or decreasing protein levels of the adaptor protein insulin receptor substrate 1/2 (IRS1/2) results in rapid EN degradation. This was observed both in vitro and in vivo in fibroblast and breast epithelial cell line models and in MO91, an EN-expressing human leukemia cell line. Stable isotope labeling with amino acids in cell culture (SILAC)-based MS analysis identified the E3 ligase RING-finger protein 123 (Rnf123, more commonly known as KPC1) as an EN interactor upon IGF1R/insulin receptor (INSR) inhibitor treatment. KPC1/Rnf123 ubiquitylated EN in vitro, and its overexpression decreased EN protein levels. In contrast, KPC1/Rnf123 knockdown rendered EN resistant to IGF1R inhibitor-mediated degradation. These results support a critical function for IGF1R in protecting EN from KPC1/Rnf123-mediated proteasomal degradation. Attempts to therapeutically target oncogenic chimeric tyrosine kinases have traditionally focused on blocking kinase activity to restrict downstream activation of essential signaling pathways. In this study, we demonstrate that IGF1R inhibition results in rapid ubiquitylation and degradation of the EN oncoprotein through a proteasome-dependent mechanism that is reversible, highlighting a potential strategy for targeting chimeric tyrosine kinases in cancer.

ETV6-NTRK3-mediated breast epithelial cell transformation is blocked by targeting the IGF1R signaling pathway.

The insulin-like growth factor (IGF) 1 receptor (IGF1R) is an important therapeutic target under study in many cancers. Here, we describe a breast cancer model based on expression of the ETV6-NTRK3 (EN) chimeric tyrosine kinase that suggests novel therapeutic applications of IGF1R inhibitors in secretory breast cancers. Originally discovered in congenital fibrosarcomas with t(12;15) translocations, EN was identified subsequently in secretory breast carcinoma (SBC) which represent a variant of invasive ductal carcinoma. Because fibroblast transformation by EN requires the IGF1R axis, we hypothesized a similar dependency may exist in mammary cells and, if so, that IGF1R inhibitors might be useful to block EN-driven breast oncogenesis. In this study, we analyzed EN expressing murine and human mammary epithelial cell lines for transformation properties. Various IGF1R signaling inhibitors, including the dual specificity IGF1R/insulin receptor (INSR) inhibitor BMS-536924, were then tested for effects on three-dimensional Matrigel cell growth, migration, and tumor formation. We found that EN expression increased acinar size and luminal filling in Matrigel cultures and promoted orthotopic tumor growth in mice. Tumors were well differentiated and nonmetastatic, similar to human SBC. The known EN effector pathway, PI3K-Akt, was activated in an IGF1- or insulin-dependent manner. BMS-536924 blocked EN transformation in vitro, whereas BMS-754807, another IGIFR/INSR kinase inhibitor currently in clinical trials, significantly reduced tumor growth in vivo. Importantly, EN model systems mimic the clinical phenotype observed in human SBC. Moreover, EN has a strict requirement for IGF1R or INSR in breast cell transformation. Thus, our findings strongly encourage the evaluation of IGF1R/INSR inhibitors to treat EN-driven breast cancers.