Interrogating the effects of IGF1R blockade on childhood sarcoma fusion oncoprotein stability

The overall goal of this proposal is to determine how IGF1R supports transformation by dominantly-acting oncoprotiens found in pediatric sarcomas.  Aim 1 will focus specifically on ETV6-NTRK3 (EN), a dominant chimeric tyrosine kinase that we first discovered in pediatric saromas.  We have recently gained valuable insights into how IGF1R supports EN transformation.  First, we confirmed that EN transformation requires the IGF1R.  Second, we found that EN physically co-localizes and interacts with IGF1R at the membrane in live cells.  Third, we unexpectedly found that blocking IGF1R with the small molecule dual specificity IGF1R/insulin receptor (INSR) kinase inhibitor, BMS-536924, dramatically reduces EN protein stability in a proteasome-dependent manner.  This causes robust re-localization of EN into cytoplasmic protein aggregates, where EN is ubiquitinated.  Moreover, another IGF1R kinase inhibitor, BMS-754807, the IGF1R blocking antibody CP-751871, and siRNAs to IGF1R but NOT INSR, have similar effects as BMS-536924.  In exciting very recent studies, we found that the E3 ligase KPC1 preferentially associates with EN in the presence of BMS-536924, and that over-expression of KPC1 induces EN degradation.  Based on these findings, we hypothesize that EN becomes membrane-localized via IGF1R, and that this prevents EN degradation by KPC1/2.  The goal of Aim 1 is to uncover the molecular mechanisms behind these findings.  The goal of Aim 2 is to determine whether similar mechanisms control other fusion proteins found in pediatric sarcomas that have been shown to respond clinically to IGF1R inhibition.  The rationale for studying this process is that a more rigorous understanding of how IGF1R contributes to transformation by such oncoproteins is essential to identify proteins other than IGF1R itself for therapeutic targeting.  This is important as resistance to IGF1R inhibitors in clinical trials has already been observed.  Our studies may therefore identify new pathway targets that can be co-inhibited to overcome IGF1R resistance.