Eloisi Caldas-Lopes

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.

Affinity-based proteomics reveal cancer-specific networks coordinated by Hsp90

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. We further show that the abundance of the PU-H71-enriched Hsp90 species, which is not dictated by Hsp90 expression alone, is predictive of the cells sensitivity to Hsp90 inhibition.

HSP90 is a therapeutic target in JAK2-dependent myeloproliferative neoplasms in mice and humans

JAK2 kinase inhibitors were developed for the treatment of myeloproliferative neoplasms (MPNs), following the discovery of activating JAK2 mutations in the majority of patients with MPN. However, to date JAK2 inhibitor treatment has shown limited efficacy and apparent toxicities in clinical trials. We report here that an HSP90 inhibitor, PU-H71, demonstrated efficacy in cell line and mouse models of the MPN polycythemia vera (PV) and essential thrombocytosis (ET) by disrupting JAK2 protein stability. JAK2 physically associated with both HSP90 and PU-H71 and was degraded by PU-H71 treatment in vitro and in vivo, demonstrating that JAK2 is an HSP90 chaperone client. PU-H71 treatment caused potent, dose-dependent inhibition of cell growth and signaling in JAK2 mutant cell lines and in primary MPN patient samples. PU-H71 treatment of mice resulted in JAK2 degradation, inhibition of JAK-STAT signaling, normalization of peripheral blood counts, and improved survival in MPN models at doses that did not degrade JAK2 in normal tissues or cause substantial toxicity. Importantly, PU-H71 treatment also reduced the mutant allele burden in mice. These data establish what we believe to be a novel therapeutic rationale for HSP90 inhibition in the treatment of JAK2-dependent MPN.

Measuring the Pharmacodynamic Effects of a Novel Hsp90 Inhibitor on HER2/neu Expression in Mice Using 89Zr-DFO-Trastuzumab

Background The positron-emitting radionuclide 89Zr (t 1/2 = 3.17 days) was used to prepare 89Zr-radiolabeled trastuzumab for use as a radiotracer for characterizing HER2/neu-positive breast tumors. In addition, pharmacodynamic studies on HER2/neu expression levels in response to therapeutic doses of PU-H71 (a specific inhibitor of heat-shock protein 90 [Hsp90]) were conducted. Methodology/Principal Findings Trastuzumab was functionalized with desferrioxamine B (DFO) and radiolabeled with [89Zr]Zr-oxalate at room temperature using modified literature methods. ImmunoPET and biodistribution experiments in female, athymic nu/nu mice bearing sub-cutaneous BT-474 (HER2/neu positive) and/or MDA-MB-468 (HER2/neu negative) tumor xenografts were conducted. The change in 89Zr-DFO-trastuzumab tissue uptake in response to high- and low-specific-activity formulations and co-administration of PU-H71 was evaluated by biodistribution studies, Western blot analysis and immunoPET. 89Zr-DFO-trastuzumab radiolabeling proceeded in high radiochemical yield and specific-activity 104.3±2.1 MBq/mg (2.82±0.05 mCi/mg of mAb). In vitro assays demonstrated >99% radiochemical purity with an immunoreactive fraction of 0.87±0.07. In vivo biodistribution experiments revealed high specific BT-474 uptake after 24, 48 and 72 h (64.68±13.06%ID/g; 71.71±10.35%ID/g and 85.18±11.10%ID/g, respectively) with retention of activity for over 120 h. Pre-treatment with PU-H71 was followed by biodistribution studies and immunoPET of 89Zr-DFO-trastuzumab. Expression levels of HER2/neu were modulated during the first 24 and 48 h post-administration (29.75±4.43%ID/g and 41.42±3.64%ID/g, respectively). By 72 h radiotracer uptake (73.64±12.17%ID/g) and Western blot analysis demonstrated that HER2/neu expression recovered to baseline levels. Conclusions/Significance The results indicate that 89Zr-DFO-trastuzumab provides quantitative and highly-specific delineation of HER2/neu positive tumors, and has potential to be used to measure the efficacy of long-term treatment with Hsp90 inhibitors, like PU-H71, which display extended pharmacodynamic profiles.

BCL6 repression of EP300 in human diffuse large B cell lymphoma cells provides a basis for rational combinatorial therapy

B cell lymphoma 6 (BCL6), which encodes a transcriptional repressor, is a critical oncogene in diffuse large B cell lymphomas (DLBCLs). Although a retro-inverted BCL6 peptide inhibitor (RI-BPI) was recently shown to potently kill DLBCL cells, the underlying mechanisms remain unclear. Here, we show that RI-BPI induces a particular gene expression signature in human DLBCL cell lines that included genes associated with the actions of histone deacetylase (HDAC) and Hsp90 inhibitors. BCL6 directly repressed the expression of p300 lysine acetyltransferase (EP300) and its cofactor HLA-B-associated transcript 3 (BAT3). RI-BPI induced expression of p300 and BAT3, resulting in acetylation of p300 targets including p53 and Hsp90. Induction of p300 and BAT3 was required for the antilymphoma effects of RI-BPI, since specific blockade of either protein rescued human DLBCL cell lines from the BCL6 inhibitor. Consistent with this, combination of RI-BPI with either an HDAC inhibitor (HDI) or an Hsp90 inhibitor potently suppressed or even eradicated established human DLBCL xenografts in mice. Furthermore, HDAC and Hsp90 inhibitors independently enhanced RI-BPI killing of primary human DLBCL cells in vitro. We also show that p300-inactivating mutations occur naturally in human DLBCL patients and may confer resistance to BCL6 inhibitors. Thus, BCL6 repression of EP300 provides a basis for rational targeted combinatorial therapy for patients with DLBCL.

Targeting heat shock protein 90 with non-quinone inhibitors: a novel chemotherapeutic approach in human hepatocellular carcinoma.

The inhibition of heat shock protein 90 (Hsp90) has emerged as a promising antineoplastic strategy in diverse human malignancies. Hsp90 has been predicted to be involved in hepatocellular carcinoma (HCC) development; however, its role in hepatocarcinogenesis remains elusive. Using chemically distinctive Hsp90 inhibitors, we show that Hsp90 capacitates the aberrant expression and activity of crucial hepatocarcinogenesis‐driving factors (e.g., insulin‐like growth factor receptor 1, hepatocyte growth factor receptor, protein kinase B, v‐raf‐1 murine leukemia viral oncogene homolog 1, and cyclin‐dependent kinase 4). In vitro, Hsp90 inhibition with both geldanamycin analogs (17‐allylamino‐17‐desmethoxygeldanamycin (17‐AAG) and 17‐dimethylaminoethylamino‐17‐desmethoxygeldanamycin (17‐DMAG)) and the non‐quinone compound 8‐(6‐iodobenzo[d][1,3]dioxol‐5‐ylthio)‐9‐(3‐(isopropylamino)propyl)‐9H‐purin‐6‐amine (PU‐H71) reduced the viability of various HCC cell lines, induced the simultaneous degradation of numerous hepatocarcinogenic factors, and caused substantial cell cycle arrest and apoptosis. In contrast, nontumorigenic hepatocytes were less susceptible to Hsp90 inhibition. Because conventional geldanamycin‐derivate Hsp90 inhibitors induce dose‐limiting liver toxicity, we tested whether novel Hsp90 inhibitors lacking the benzoquinone moiety, which has been deemed responsible for hepatotoxicity, can elicit antineoplastic activity without causing significant liver damage. In HCC xenograft mouse models, PU‐H71 was retained in tumors at pharmacologically relevant concentrations while being rapidly cleared from nontumorous liver. PU‐H71 showed potent and prolonged in vivo Hsp90 inhibitory activity and reduced tumor growth without causing toxicity. Conclusion: Hsp90 constitutes a promising therapeutic target in HCC. Non‐quinone Hsp90 inhibitors exhibit tumor‐specific accumulation and exert potent antineoplastic activity without causing significant hepatotoxicity. (HEPATOLOGY 2009.)

The epichaperome is an integrated chaperome network that facilitates tumour survival

Transient, multi-protein complexes are important facilitators of cellular functions. This includes the chaperome, an abundant protein family comprising chaperones, co-chaperones, adaptors, and folding enzymes—dynamic complexes of which regulate cellular homeostasis together with the protein degradation machinery. Numerous studies have addressed the role of chaperome members in isolation, yet little is known about their relationships regarding how they interact and function together in malignancy. As function is probably highly dependent on endogenous conditions found in native tumours, chaperomes have resisted investigation, mainly due to the limitations of methods needed to disrupt or engineer the cellular environment to facilitate analysis. Such limitations have led to a bottleneck in our understanding of chaperome-related disease biology and in the development of chaperome-targeted cancer treatment. Here we examined the chaperome complexes in a large set of tumour specimens. The methods used maintained the endogenous native state of tumours and we exploited this to investigate the molecular characteristics and composition of the chaperome in cancer, the molecular factors that drive chaperome networks to crosstalk in tumours, the distinguishing factors of the chaperome in tumours sensitive to pharmacologic inhibition, and the characteristics of tumours that may benefit from chaperome therapy. We find that under conditions of stress, such as malignant transformation fuelled by MYC, the chaperome becomes biochemically ‘rewired’ to form a network of stable, survival-facilitating, high-molecular-weight complexes. The chaperones heat shock protein 90 (HSP90) and heat shock cognate protein 70 (HSC70) are nucleating sites for these physically and functionally integrated complexes. The results indicate that these tightly integrated chaperome units, here termed the epichaperome, can function as a network to enhance cellular survival, irrespective of tissue of origin or genetic background. The epichaperome, present in over half of all cancers tested, has implications for diagnostics and also provides potential vulnerability as a target for drug intervention.

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.

Abstract 3029: Biochemical evidence towards the existence of an oncogenic Hsp90 complex

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

Abstract 5444: Development of a noninvasive assay to determine drug concentration in tumor during hsp90 inhibitor therapy

As molecularly targeted agents assume a more prominent role in anticancer therapy there is a growing need to determine in a noninvasive manner whether the target is being engaged and to what extent such drug-target binding results in desirable effects. We address this need in the context of Hsp90, a target of significant value and one in critical need for such assessment tools, by combining a novel chemical tool selective for tumor Hsp90 with PET imaging and mathematical modeling. The chemical tool is [124I]-PU-H71, the iodine-124 radiolabeled analog of the potent Hsp90 inhibitor PU-H71, which can be administered in tracer quantities for PET imaging. The resulting diagnostic, PU-PET, has been optimized and validated preclinically in mouse models of cancer and then translated to the clinic. The exquisite design of this assay is based on three essential concepts as it relates to the target (Hsp90) as well as to the PET tracer (124I-PU-H71). First, the target is “oncogenic” Hsp90 and has been shown by numerous biochemical and pharmacokinetic studies to have a strong affinity for inhibitors and a very low koff resulting in selective and prolonged retention in tumor. Secondly, the tracer incorporates a 124I in place of the naturally occurring 127I in the structure of PU-H71 and therefore there is no change in the chemical structure. This feature in a PET tracer intended as a companion diagnostic is unprecedented and ensures that the PK properties are identical to the therapeutic agent (PU-H71). Finally, the radionuclide 124I has a four-day half-life and thus is well-suited to monitor the extended tumor retention profile observed for Hsp90 inhibitors. We here demonstrate that this PET assay informs on Hsp90 targeting in individual tumors in real time and provides accurate tumor drug concentrations for at least four chemically distinct Hsp90 drugs. In contrast, we find that plasma pharmacokinetics is not predictive of intratumor parameters and therefore provides limited value in estimating target engagement. Using PU-PET we demonstrate that at least one Hsp90 inhibitor exhibits tumor targeting and retention in humans, delivering and retaining therapeutic, micromolar, concentrations at safe doses. PU-PET is currently being evaluated in Phase 0/1 (NCT01269593) clinical trials as a noninvasive companion diagnostic to determine intratumoral concentration as well as to identify those patients who would best benefit from Hsp90 inhibitor therapy. This diagnostic assay is intended to be incorporated into future Phase 2 clinical trials in order to preselect those patients who would most likely benefit from Hsp90 inhibitor treatment. Citation Format: Tony Taldone, Nagavarakishore Pillarsetty, Mark PS Dunphy, John F. Gerecitano, Eloisi Caldas-Lopes, Brad Beattie, Radu I. Peter, Yanlong Kang, Anna Rodina, Pengrong Yan, Erica M. DaGama Gomes, Alexander Bolaender, Christina Pressl, Blesida Punzalan, Anson Ku, Thomas Ku, Smit Shah, Mohammad Uddin, Mei H. Chen, Elmer Santos, Jacek Koziorowski, Adriana Corben, Shanu Modi, Komal Jhaveri, Oscar Lin, Efsevia Vakiani, Yelena Janjigian, Pat Zanzonico, Clifford Hudis, Steven M. Larson, Jason S. Lewis, Gabriela Chiosis. Development of a noninvasive assay to determine drug concentration in tumor during hsp90 inhibitor therapy. [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 5444. doi:10.1158/1538-7445.AM2015-5444