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.