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

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

The overall goal of this proposal J,s to determine how IGF1 R supports transformation by dominantly-acting oncoproteins found in pediatric sarcomas. Aim 1 will focus specifically on ETV6-NTRK3 (EN), a dominant chimeric tyrosine kinase that we first discovered in pediatric sarcomas. We have recently gained valuable insights into how IGF1 R supports EN transformation. First, we confirmed that EN transformation requires the IGF1 R. Second, we found that EN physically co-localizes and interacts with IGF1 Rat the membrane in live cells. Third, we unexpectedly found that blocking IGF1 R with the small molecule dual specificity IGF1 R/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 IGF1 R kinase inhibitor, BMS-754807, the IGF1 R blocking antibody CP-751871, and siRNAs to IGF1 R but NOT to 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 IGF1 R, 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 IGF1 R inhibition. The rationale for studying this process is that a more rigorous understanding of how IGF1 R contributes to transformation by such oncoproteins is essential to identify proteins other than IGF1 R itself for therapeutic targeting. This is important as resistance to IGF1 R inhibitors in clinical trials has already been observed. Our studies may therefore identify new pathway targets that can be co-inhibited to overcome IGF1 R resistance.

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