Mehdi Mollapour - US grants

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.

PROJECT SUMMARY The molecular chaperone Heat Shock Protein-90 (Hsp90) is essential for the folding and activity of an array of `client' proteins involved in signal transduction pathways. They are also responsible for many maladies including cancer, neurodegenerative, autoimmune and inflammatory diseases. Hsp90 inhibition strategies are currently being explored in these diseases in pre-clinical studies and clinical trials, however the optimal use of Hsp90-targeted therapeutics remains unknown. This is partly due to our limited knowledge of Hsp90 regulation in cells. Unraveling the detailed regulatory mechanisms of Hsp90 function in cells can provide new strategies to increase the cellular potency of Hsp90 inhibitors. Hsp90 chaperone function is coupled to its ATPase activity, which is regulated by co-chaperones and posttranslational modifications (PTMs). However, it is unclear how these regulatory components work together to fine tune Hsp90 function and also contribute towards drug sensitivity. During the past five years we have made major contributions towards the understanding of Hsp90 regulation by co-chaperones and PTMs. i) New co-chaperones: We have identified three new co-chaperones, FNIP1, 2 (collectively FNIPs) and Tsc1, that decelerate the chaperone cycle and facilitate chaper- oning of both kinase and non-kinase clients. They are regulated by PTMs (phosphoryla- tion, O-GlcNAcylation, SUMOlyation and ubiquitination). Their expression also enhances Hsp90 binding to drugs and consequently sensitizes cells to Hsp90 inhibitors. ii) Post- translational modification of Hsp90: Our work during the past decade on Hsp90 PTMs has redefined the regulation of its chaperone activity and revealed the reciprocal regula- tory mechanisms between client proteins, co-chaperones, and Hsp90. We have recently shown that loss of TSC1 co-chaperone leads to hypoacetylation of Hsp90 and elevated its ATPase activity. It also subsequently decreased Hsp90 binding to its inhibitors. Our long-term goal is to unravel the molecular mechanism of Hsp90 chaperone regulation in cells and regulatory factors enhancing cellular potency of Hsp90 inhibitors. Our strategy is to use biochemical, biophysical and cell-based assays to decipher the interconnectivi- ty and compensatory mechanisms between the co-chaperones and PTMs. Our vision is to utilize this information to dissect the intricate network of regulatory signals involved in fine tuning Hsp90 function and their impact towards cellular sensitivity to Hsp90 inhibi- tors.