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Most cancers are characterized by multiple molecular alterations, but identification of the key proteins involved in these signaling pathways is currently beyond reach. We show that the inhibitor PU-H71 preferentially targets tumor-enriched Hsp90 complexes and affinity captures Hsp90-dependent oncogenic client proteins. We have used PU-H71 affinity capture to design a proteomic approach that, when combined with bioinformatic pathway analysis, identifies dysregulated signaling networks and key oncoproteins in chronic myeloid leukemia. The identified interactome overlaps with the well-characterized altered proteome in this cancer, indicating that this method can provide global insights into the biology of individual tumors, including primary patient specimens. In addition, we show that this approach can be used to identify previously uncharacterized oncoproteins and mechanisms, potentially leading to new targeted therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1263. doi:1538-7445.AM2012-1263

To maintain homeostasis, cells employ intricate molecular machineries comprised of thousands of proteins programmed to execute well-defined functions. Dysregulation of these pathways, through protein mis-expression or mutation, provides biological advantages that confer the malignant phenotype. At the molecular level, this requires cells to invest energy in maintaining the stability and function of these proteins, and for this reason cancer cells co-opt molecular chaperones, including Hsp90. Hsp90 is recognized to play important roles in maintaining the transformed phenotype - the chaperone and its associated co-chaperones assist in the correct folding of cellular proteins, collectively referred to as “client proteins,” many of which are effectors of signal transduction pathways controlling cell growth, differentiation, the DNA damage response, and cell survival. Tumor cell addiction to these proteins (i.e. through mutations, aberrant expression, improper cellular translocation, etc) thus makes them critically reliant on Hsp90. While Hsp90 is expressed in all cells and tissues, it was shown that tumors preferentially contain Hsp90 that is in a higher order multi-chaperone complex with high affinity for certain Hsp90 inhibitors, while normal tissues harbor a latent, uncomplexed Hsp90 that has low affinity for these inhibitors. We here extend this model and propose that Hsp90 forms biochemically distinct complexes in cancer cells. In this view, a major fraction of cancer cell Hsp90 retains “house keeping” chaperone functions similar to normal cells, whereas a functionally distinct Hsp90 pool enriched or expanded in cancer cells specifically interacts with oncogenic proteins required to maintain tumor cell survival. Perhaps this Hsp90 fraction represents a cell stress specific form of chaperone complex that is expanded and constitutively maintained in the tumor cell context. Our data suggest that it may execute functions necessary to maintain the malignant phenotype. One such role is to regulate the folding of mutated (i.e. mB-Raf) or chimeric proteins (i.e. Bcr-Abl). We here also present experimental evidence for an additional role; that is, to facilitate scaffolding and complex formation of molecules involved in aberrantly activated signaling complexes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3029. doi:1538-7445.AM2012-3029