PROJECT SUMMARY The molecular chaperone Heat Shock Protein-90 (Hsp90) is considered to be a critical facilitator of oncogene addiction and cancer cell survival. Hsp90 function is coupled to its ATPase activity, which is regulated by co-chaperones and posttranslational modifica- tions (PTMs). Hsp90 inhibitors are currently being evaluated in various stages of clinical trials in cancer patients. However, the optimal use of Hsp90-targeted therapy remains unknown. This is partly due to the limited knowledge of Hsp90 regulation in cancer cells. Folliculin-interacting proteins (FNIP-1 and 2), also known as FNIPs, are the newly dis- covered co-chaperones that decelerate Hsp90 activity. FNIPs compete with the activat- ing co-chaperone Aha1 for binding to Hsp90 and FNIPs expression correlates with the cellular response to Hsp90 inhibitors. It was also shown that PTMs (phosphorylation, O- GlcNAcylation, ubiquitination) of FNIPs and Aha1 affect their binding to Hap90, however, the detailed crosstalk between these PTMs and their impact on drug sensitivity remains unknown. Without this knowledge, optimal Hsp90-targeted therapeutics remains limited. The long-term goal of this proposal is to enhance the activity of the Hsp90 inhibitors in cancer cells by targeting the regulators of Hsp90. The overall objective is to determine the crosstalk between PTMs of co-chaperones in regulating Hsp90 function and how they contribute towards drug sensitivity. The central hypothesis is that PTMs of the FNIPs and Aha1 co-chaperones reciprocally impact their binding to Hsp90 and influence the potency of its drugs in cancer cells. The rationale of this proposal is that, by under- standing the impact of PTMs on co-chaperones association with Hsp90 and consequent- ly drug binding, it could be possible to target those enzymes responsible for the PTMs, therefore increasing the potency of Hsp90 inhibitors in cancer cells. Biochemical, bio- physical and cell-based assays as well as cancer cell lines will be used to test the hy- pothesis in the following specific aims; Aim 1: Determine the impact of crosstalk between phosphorylation and O-GlcNAcylation on co-chaperone activity of the FNIPs. Aim 2: De- termine the reciprocal role of FNIP1 and Aha1 co-chaperones on Hsp90 regulation. Aim 3: Determine the mechanism of FNIP co-chaperones on increasing the activity of Hsp90 inhibitors. In determining the interplay between FNIPs and Aha1 co-chaperones and how PTMs of these two proteins impact their binding to Hsp90, it is expected to unravel new targets to increase the potency of Hsp90 inhibitors in cancer cells.