Alexander Gozman

Targeting Hsp90: small-molecule inhibitors and their clinical development

The Hsp90 multichaperone complex has important roles in the development and progression of malignant transformation. Several small-molecule inhibitors of Hsp90 of diverse chemotypes have shown potent antitumor activity in a wide-range of malignancies, and are currently in clinical or late-stage preclinical investigation. This review intends to update the reader on advances made over the past two years in the clinical development of Hsp90 inhibitors in advanced cancers. It will refer to the two 17-AAG formulations, tanespimycin and IPI-504, and to synthetic small molecules, among which are the purine-scaffold Hsp90 inhibitor CNF2024/BIIB021, the isoxazole derivative VER-52296/NVP-AUY922, and the carbazol-4-one benzamide derivative SNX-5422, and will present our current knowledge on their clinical performance.

Hsp90 inhibitor PU-H71, a multimodal inhibitor of malignancy, induces complete responses in triple-negative breast cancer models

Triple-negative breast cancers (TNBCs) are defined by a lack of expression of estrogen, progesterone, and HER2 receptors. Because of the absence of identified targets and targeted therapies, and due to a heterogeneous molecular presentation, treatment guidelines for patients with TNBC include only conventional chemotherapy. Such treatment, while effective for some, leaves others with high rates of early relapse and is not curative for any patient with metastatic disease. Here, we demonstrate that these tumors are sensitive to the heat shock protein 90 (Hsp90) inhibitor PU-H71. Potent and durable anti-tumor effects in TNBC xenografts, including complete response and tumor regression, without toxicity to the host are achieved with this agent. Notably, TNBC tumors respond to retreatment with PU-H71 for several cycles extending for over 5 months without evidence of resistance or toxicity. Through a proteomics approach, we show that multiple oncoproteins involved in tumor proliferation, survival, and invasive potential are in complex with PU-H71-bound Hsp90 in TNBC. PU-H71 induces efficient and sustained downregulation and inactivation, both in vitro and in vivo, of these proteins. Among them, we identify downregulation of components of the Ras/Raf/MAPK pathway and G2-M phase to contribute to its anti-proliferative effect, degradation of activated Akt and Bcl-xL to induce apoptosis, and inhibition of activated NF-κB, Akt, ERK2, Tyk2, and PKC to reduce TNBC invasive potential. The results identify Hsp90 as a critical and multimodal target in this most difficult to treat breast cancer subtype and support the use of the Hsp90 inhibitor PU-H71 for clinical trials involving patients with TNBC.

Identification of an Allosteric Pocket on Human Hsp70 Reveals a Mode of Inhibition of This Therapeutically Important Protein

Hsp70s are important cancer chaperones that act upstream of Hsp90 and exhibit independent anti-apoptotic activities. To develop chemical tools for the study of human Hsp70, we developed a homology model that unveils a previously unknown allosteric site located in the nucleotide binding domain of Hsp70. Combining structure-based design and phenotypic testing, we discovered a previously unknown inhibitor of this site, YK5. In cancer cells, this compound is a potent and selective binder of the cytosolic but not the organellar human Hsp70s and has biological activity partly by interfering with the formation of active oncogenic Hsp70/Hsp90/client protein complexes. YK5 is a small molecule inhibitor rationally designed to interact with an allosteric pocket of Hsp70 and represents a previously unknown chemical tool to investigate cellular mechanisms associated with Hsp70.

Synthesis of reblastatin, autolytimycin, and non-benzoquinone analogues: potent inhibitors of heat shock protein 90.

A full account of an asymmetric synthesis of reblastatin (1) and the first total synthesis of autolytimycin (2) and related structural compounds is described. The syntheses expand the utility of a highly regio- and diastereoselective hydrometalation aldehyde addition sequence to assemble the fully functionalized ansa chain of the natural products. Also documented is an intramolecular copper-mediated amidation reaction to close the 19-membered macrolactams. The amidation reaction was also employed for the generation of structural derivatives (6-9) of phenolic ansamycins. Ansamycin natural products and selected structural analogues were evaluated in a competitive binding assay to breast cancer cell lysate and a cytotoxicity assay. Both reblastatin (1) and autolytimycin (2) were shown to bind the heat shock protein 90 with enhanced binding activity (approximately 25 nM) than 17-allylamino-17-demethoxygeldanamycin (17-AAG, 4), a geldanamycin (3) derivative currently under evaluation for treatment of cancer (approximately 100 nM).

Heat Shock Protein 70 Inhibitors. 1. 2,5′-Thiodipyrimidine and 5-(Phenylthio)pyrimidine Acrylamides as Irreversible Binders to an Allosteric Site on Heat Shock Protein 70

Heat shock protein 70 (Hsp70) is an important emerging cancer target whose inhibition may affect multiple cancer-associated signaling pathways and, moreover, result in significant cancer cell apoptosis. Despite considerable interest from both academia and pharmaceutical companies in the discovery and development of druglike Hsp70 inhibitors, little success has been reported so far. Here we describe structure–activity relationship studies in the first rationally designed Hsp70 inhibitor class that binds to a novel allosteric pocket located in the N-terminal domain of the protein. These 2,5′-thiodipyrimidine and 5-(phenylthio)pyrimidine acrylamides take advantage of an active cysteine embedded in the allosteric pocket to act as covalent protein modifiers upon binding. The study identifies derivatives 17a and 20a, which selectively bind to Hsp70 in cancer cells. Addition of high nanomolar to low micromolar concentrations of these inhibitors to cancer cells leads to a reduction in the steady-state levels of Hsp70-sheltered oncoproteins, an effect associated with inhibition of cancer cell growth and apoptosis. In summary, the described scaffolds represent a viable starting point for the development of druglike Hsp70 inhibitors as novel anticancer therapeutics.

Heat Shock Protein 70 Inhibitors. 2. 2,5′-Thiodipyrimidines, 5-(Phenylthio)pyrimidines, 2-(Pyridin-3-ylthio)pyrimidines, and 3-(Phenylthio)pyridines as Reversible Binders to an Allosteric Site on Heat Shock Protein 70

The discovery and development of heat shock protein 70 (Hsp70) inhibitors is currently a hot topic in cancer. In the preceding paper in this issue (10.1021/jm401551n), we have described structure–activity relationship studies in the first Hsp70 inhibitor class rationally designed to bind to a novel allosteric pocket located in the N-terminal domain of the protein. These ligands contained an acrylamide to take advantage of an active cysteine embedded in the allosteric pocket and acted as covalent protein modifiers upon binding. Here, we perform chemical modifications around the irreversible inhibitor scaffold to demonstrate that covalent modification is not a requirement for activity within this class of compounds. The study identifies derivative 27c, which mimics the biological effects of the irreversible inhibitors at comparable concentrations. Collectively, the back-to-back manuscripts describe the first pharmacophores that favorably and selectively interact with a never explored pocket in Hsp70 and provide a novel blueprint for a cancer-oriented development of Hsp70-directed ligands.

A Hyperactive Signalosome in Acute Myeloid Leukemia Drives Addiction to a Tumor-Specific Hsp90 Species

SUMMARY Acute myeloid leukemia (AML) is a heterogeneous and fatal disease with an urgent need for improved therapeutic regimens given that most patients die from relapsed disease. Irrespective of mutation status, the development of aggressive leukemias is enabled by increasing dependence on signaling networks. We demonstrate that a hyperactive signalosome drives addiction of AML cells to a tumor-specific Hsp90 species (teHsp90). Through genetic, environmental, and pharmacologic perturbations, we demonstrate a direct and quantitative link between hyperactivated signaling pathways and apoptotic sensitivity of AML to teHsp90 inhibition. Specifically, we find that hyperactive JAK-STAT and PI3K-AKT signaling networks are maintained by teHsp90 and, in fact, gradual activation of these networks drives tumors increasingly dependent on teHsp90. Thus, although clinically aggressive AML survives via signalosome activation, this addiction creates a vulnerability that can be exploited with Hsp90-directed therapy.

Affinity Purification Probes of Potential Use To Investigate the Endogenous Hsp70 Interactome in Cancer

Heat shock protein 70 (Hsp70) is a family of proteins with key roles in regulating malignancy. Cancer cells rely on Hsp70 to inhibit apoptosis, regulate senescence and autophagy, and maintain the stability of numerous onco-proteins. Despite these important biological functions in cancer, robust chemical tools that enable the analysis of the Hsp70-regulated proteome in a tumor-by-tumor manner are yet unavailable. Here we take advantage of a recently reported Hsp70 ligand to design and develop an affinity purification chemical toolset for potential use in the investigation of the endogenous Hsp70-interacting proteome in cancer. 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.

Abstract 1733: Development of chemical tools to study the endogenous Hsp70 interactome in malignant cells

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 today9s 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

Abstract 5463: YK5, a novel dual Hsc70 and Hsp70 inhibitor, is selective for tumor Hsp70 and has potent but selective activity in cancer cells

Background. The heat shock protein 70 (Hsp70) family members, heat-shock cognate 70 (Hsc70) and inducible Hsp70, are molecular chaperones abundantly expressed in human tumors. These proteins are necessary to maintain the dysregulated function and the anti-apoptotic potential of cancer cells. In this respect, the Hsp70 chaperones inhibit key effectors of the apoptotic machinery and are further involved in regulating kinases, hormone receptors and transcription factors that are directly involved in driving multistep malignancy and of mutated oncogenic proteins required for the transformed phenotype. Elevated Hsp70 expression, is characteristic of many tumor types and is associated with metastasis and poor prognosis, and is one of the major factors leading to resistance to anti-cancer therapy. The Hsp70 isoforms also contribute to tumorigenesis through their role as co-chaperones for the Heat shock protein 90 (Hsp90) molecular chaperone. The Hsp90 multi-chaperone complex, also called the Hsp90 super-chaperone machinery, has important roles in the development and progression of pathogenic cellular transformation through regulation of several malignancy driving and supporting client proteins. These biological functions propose Hsp70s as an important target whose inhibition may result in significant but selective apoptosis in a wide-range of cancer cells, and also in inhibition of signaling pathways involved in tumorigenesis and metastasis. Study design. In order to determine the role of Hsp70s modulation in cancer, we treated cancer cells of different origin with the novel dual Hsp70/Hsc70 inhibitor YK5, which is selective for tumor Hsp70s species. The effect of YK5 on the major cancer hallmarks, namely proliferation, cell cycle, apoptosis and invasiveness was evaluated. Results. Hsp70s modulation by YK5 has substantial and wide-ranging phenotypic consequence in cancer cells, directed through the Hsp90 machinery, and executed through disruption of functional Hsp90 machinery/onco-protein complexes. Modulation of Hsp70s also results in effects distinct from Hsp90, and we point out mechanistic differences between the two chaperones. Namely, unlike direct Hsp90 inhibitors, YK5 does not activate HSF-1 nor induces a protective feed-back heat shock response. When compared to direct Hsp90 inhibition, pharmacologic interference with Hsp70s results in increased, but yet selective cytotoxicity against cancer cells, mainly due to apoptosis. Further, we discover through YK5, that Hsp70s accelerate STAT1 dephosphorylation in breast cancer cells, identifying a novel mechanism that cancer cells use to inhibit the pro-apoptotic effects of this tumor suppressor. Conclusion. Our results propose Hsp70s inhibition by agents that act through an YK5-medited mechanism as a novel potent therapeutic approach in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5463.