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Background: Heat shock protein 70 family members play an important role in cancer. They are up-regulated in wide variety of tumors and the increased Hsp70 protein expression correlates with metastases, resistance to treatment and poor prognosis. Multiple mechanisms explain cancer cells dependence on Hsp70, such as inhibition of apoptosis by Hsp70, induction of autophagy and control of stability of onco-proteins. These Hsp70 activities are mediated in cancer by its ability to chaperone and interact with a large number of proteins in a cell-specific, context dependent manner. Hypothesis: Reagents that enable the capture of tumor-specific Hsp70 complexes facilitate the identification of context-dependent Hsp70 interactomes. Results: Our laboratory recently reported the identification of a novel allosteric site located in the nucleotide binding domain of Hsp70 (Chem Biol 2013). It has also reported the discovery of ligands that bind to the allosteric pocket of Hsp70, inhibit its function in cancer cells and result in anti-cancer activity (J Med Chem 2013). Structure-activity relationship studies in this ligand series gave insights on the attachment of specific linkers for the design of Hsp70-related chemical tools. Here we present the design of Hsp70-directed reagents and use biochemical and cell-based methods to validate Hsp70-directed affinity purification probes. We demonstrate that these tools lock Hsp70 in complex with onco-client proteins and effectively isolate Hsp70 complexes for identification through biochemical techniques. Using these tools we provide proof-of-concept analyses that glimpse into the complex roles played by Hsp70 in maintaining a multitude of cell-specific malignancy-driving proteins. Significance: The knowledge derived from the use of such reagents will be extremely valuable not only to understand tumor-specific roles of Hsp70 and associated mechanisms but also to develop rational strategies for the clinical implementation of these agents to cancer treatment. They may also provide clues on the altered functional proteome in individual tumors, a quest yet elusive by today's proteomics methods. Citation Format: Anna A. Rodina, Tony Taldone, Yanlong Kang, Pallav Patel, John Koren, Pengrong Yan, Erica DaGama Gomes, Chenghua Yang, Maulik Patel, Liza Shrestha, Stefan Ochiana, Ronnie Maharaj, Alexander Gozman, Marc Cox, Hediye Erdjument-Bromage, Ronald Hendrickson, Leandro Cerchietti, Ari Melnick, Monica Guzman, Gabriela Chiosis. Development of chemical tools to study the endogenous Hsp70 interactome in malignant cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1733. doi:10.1158/1538-7445.AM2015-1733

Background: Hsp70, a molecular chaperone responsible, in part, for the folding of nascent peptides following translation, has been implicated as a survival factor and a poor prognostic marker in cancer cells. These pro-cancer mechanisms originate in the ability of Hsp70 to preserve and maintain oncogenic and transformative proteins responsible for the cancer phenotype. Hsp70 is a stress response protein such that expression increases under proteomic/proteotoxic stress events. This increased expression is also evident in cancer cells, an environment under proteomic stress brought on by transformation, and is known to be protective against programmed cell death. Hypothesis: We predict that by interrupting the chaperoning capacity of Hsp70 through allosteric inhibition, we can destabilize oncogenic proteins dependent on Hsp70 for structure and function. Additionally, as Hsp70 is a survival factor, we believe that a loss of Hsp70 activity will result in cancer specific cell death both in vitro and in vivo. Approach: Using chemical tools, primarily novel allosteric Hsp70 inhibiting small molecules, we will determine the fate of oncogenic proteins dependent on Hsp70 for stability. We will also examine the cellular response to the disruption of cancer-specific networks which require Hsp70. In vivo tumor models will be used to examine the pharmacokinetics and pharmacodynamics of small molecule Hsp70 inhibitors and to evaluate the therapeutic potential of Hsp70 inhibition. Significance: The stress response machinery is responsible for the stability of proteins, maintenance of signaling pathways, and evasion from programmed cell death while a cell is under stress; stressors including oncogenic transformation. These pathways maintained by the molecular chaperones have been directly linked to deleterious cancer phenotypes including aggressiveness and the emergence of therapeutic resistances. Targeted therapeutics, and uniquely those targeting the molecular chaperone system, can provide therapeutic options capable of ablating the specific mechanisms involved in proteomic stability and cell survival unique to each tumor cell; effects which can be delivered with minimal effect to normal tissue. This work highlights the potential for therapeutics targeting Hsp70 as anti-cancer agents. Citation Format: John Koren, Chao Xu, Anna Rodina, Liza Shrestha, Tony Taldone, Gabriela Chiosis. Allosteric Hsp70-family inhibitors as targeted anticancer therapeutics. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1740. doi:10.1158/1538-7445.AM2015-1740