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PDF file - 79K, Fold-changes in protein expression following ganetespib treatment in KRAS mutant NSCLC lines using reverse phase protein array analysis

Mutant KRAS is a feature of more than 25% of non–small cell lung cancers (NSCLC) and represents one of the most prevalent oncogenic drivers in this disease. NSCLC tumors with oncogenic KRAS respond poorly to current therapies, necessitating the pursuit of new treatment strategies. Targeted inhibition of the molecular chaperone Hsp90 results in the coordinated blockade of multiple oncogenic signaling pathways in tumor cells and has thus emerged as an attractive avenue for therapeutic intervention in human malignancies. Here, we examined the activity of ganetespib, a small-molecule inhibitor of Hsp90 currently in clinical trials for NSCLCs in a panel of lung cancer cell lines harboring a diverse spectrum of KRAS mutations. In vitro, ganetespib was potently cytotoxic in all lines, with concomitant destabilization of KRAS signaling effectors. Combinations of low-dose ganetespib with MEK or PI3K/mTOR inhibitors resulted in superior cytotoxic activity than single agents alone in a subset of mutant KRAS cells, and the antitumor efficacy of ganetespib was potentiated by cotreatment with the PI3K/mTOR inhibitor BEZ235 in A549 xenografts in vivo. At the molecular level, ganetespib suppressed activating feedback signaling loops that occurred in response to MEK and PI3K/mTOR inhibition, although this activity was not the sole determinant of combinatorial benefit. In addition, ganetespib sensitized mutant KRAS NSCLC cells to standard-of-care chemotherapeutics of the antimitotic, topoisomerase inhibitor, and alkylating agent classes. Taken together, these data underscore the promise of ganetespib as a single-agent or combination treatment in KRAS-driven lung tumors. Mol Cancer Ther; 11(12); 2633–43. ©2012 AACR.

PDF file - 251K, Chemical structures of ganetespib and crizotinib.

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PDF file - 486K, The addition of ganetespib to crizotinib does not result in added toxicity in H3122 xenografts.

PDF file - 2538K, Crizotinib-resistant H3122 CR1 cells express an EMT phenotype.

PDF file - 890K, Combinations of ganetespib with MEK and PI3K/mTOR inhibitors in vitro

PurposexDECIDE is a clinical decision support system, accessed through a web portal and powered by a “Human-AI Team”, that offers oncology healthcare providers a set of treatment options personalized for their cancer patients, and provides outcomes tracking through an observational research protocol. This article describes the xDECIDE process and the AI-assisted technologies that ingest semi-structured electronic medical records to identify and then standardize clinico-genomic features, generate a structured personal health record (PHR), and produce ranked treatment options based on clinical evidence, expert insights, and the real world evidence generated within the system itself.MethodPatients may directly enroll in the IRB-approved pan-cancer XCELSIOR registry (NCT03793088). Patient consent permits data aggregation, continuous learning from clinical outcomes, and sharing of limited datasets within the research team. Assisted by numerous AI-based technologies, the xDECIDE team aggregates and processes patients’ electronic medical records, and applies multiple levels of natural language processing (NLP) and machine learning to generate a structured case summary and a standardized list of patient features. Next a ranked list of treatment options is created by an ensemble of AI-based models, called xCORE. The output of xCORE is reviewed by molecular pharmacologists and expert oncologists in a virtual tumor board (VTB). Finally a report is produced that includes a ranked list of treatment options and supporting scientific and medical rationales. Treating physicians can use an interactive portal to view all aspects of these data and associated reports, and to continuously monitor their patients’ information. The xDECIDE system, including xCORE, is self-improving; feedback improves aspects of the process through machine learning, knowledge ingestion, and outcomes-directed process improvement.ResultsAt the time of writing, over 2,000 patients have enrolled in XCELSIOR, including over 650 with CNS cancers, over 300 with pancreatic cancer, and over 100 each with ovarian, colorectal, and breast cancers. Over 150 VTBs of CNS cancer patients and ∼100 VTBs of pancreatic cancer patients have been performed. In the course of these discussions, ∼450 therapeutic options have been discussed and over 2,000 consensus rationales have been delivered. Further, over 500 treatment rationale statements (“rules”) have been encoded to improve algorithm decision making between similar therapeutics or regimens in the context of individual patient features. We have recently deployed the xCORE AI-based treatment ranking algorithm for validation in real-world patient populations.ConclusionClinical decision support through xDECIDE is available for oncologists to utilize in their standard practice of medicine by enrolling a patient in the XCELSIOR trial and accessing xDECIDE through its web portal. This system can help to identify potentially effective treatment options individualized for each patient, based on sophisticated integration of real world evidence, human expert knowledge and opinion, and scientific and clinical publications and databases.

Androgen ablation therapy represents the first line of therapeutic intervention in men with advanced or recurrent prostate tumors. However, the incomplete efficacy and lack of durable response to this clinical strategy highlights an urgent need for alternative treatment options to improve patient outcomes. Targeting the molecular chaperone heat shock protein 90 (Hsp90) represents a potential avenue for therapeutic intervention as its inhibition results in the coordinate blockade of multiple oncogenic signaling pathways in cancer cells. Moreover, Hsp90 is essential for the stability and function of numerous client proteins, a number of which have been causally implicated in the pathogenesis of prostate cancer, including the androgen receptor (AR). Here, we examined the preclinical activity of ganetespib, a small molecule inhibitor of Hsp90, in a panel of prostate cancer cell lines. Ganetespib potently decreased viability in all lines, irrespective of their androgen sensitivity or receptor status, and more effectively than the ansamycin inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). Interestingly, while ganetespib exposure decreased AR expression and activation, the constitutively active V7 truncated isoform of the receptor was unaffected by Hsp90 inhibition. Mechanistically, ganetespib exerted concomitant effects on mitogenic and survival pathways, as well as direct modulation of cell cycle regulators, to induce growth arrest and apoptosis. Further, ganetespib displayed robust antitumor efficacy in both AR-negative and positive xenografts, including those derived from the 22Rv1 prostate cancer cell line that co-expresses full-length and variant receptors. Together these data suggest that further investigation of ganetespib as a new therapeutic treatment for prostate cancer patients is warranted.

Background: Ganetespib is a fully synthetic and selective inhibitor of heat shock protein 90 (HSP90), a molecular chaperone recognized as a key facilitator of breast cancer initiation, progression and metastasis. Methods: Preclinical activity of ganetespib across the four major breast cancer subtypes and inflammatory breast cancer was assessed in vitro and in vivo. Modulation of cell proliferation and viability was determined both in monolayer and three-dimensional cultures. HSP90 client protein expression and activity was monitored by Western blot and protein array. To recapitulate clinical dosing, kinetics of client protein destabilization were measured following short exposures to drug in vitro. Anticancer activity of ganetespib was further investigated in vivo using breast cancer xenografts. Results: Ganetespib displayed potent, low nanomolar activity in luminal (A and B), basal (A and B) and inflammatory breast cancer cell lines grown as monolayers in vitro. BT-474 (HER2 amplified) luminal cells grown as mammospheres in 3D were equally as sensitive to ganetespib as those grown in monolayer. In luminal cells, ganetespib simultaneously disrupted multiple signaling components including the estrogen and progesterone receptor, several receptor and non-receptor tyrosine kinases, as well as the MAPK pathway. Further, ganetespib effectively inhibited AKT, PDK1 and SGK3 activity in PIK3CA mutant cells suggesting that HSP90 is essential for both AKT-dependent and AKT-independent signaling. Clinically relevant exposure times to ganetespib in vitro resulted in potent, long term destabilization of HER2. In the basal-like breast cancer cell line MDA-MB-231, enriched in CD44+CD24- stem like cells that commonly display chemotherapeutic resistance and activated JAK2/STAT3 signaling, ganetespib (50 nM) induced significant degradation of JAK2 concordant with loss of both tyrosine and serine phosphorylation of STAT3, followed by cell death. The potent anticancer activity in vitro translated in vivo, where ganetespib was effective in modulating breast cancer xenograft growth as a single agent in both luminal and basal-like breast cancer models. Finally, ganetespib has demonstrated encouraging signs of clinical activity in breast cancer patients, including confirmed partial responses in both a triple negative breast cancer patient and a HER2 positive breast cancer patient. Conclusions: Ganetespib is a highly potent HSP90 inhibitor that displays preclinical activity in breast cancer due to its ability to simultaneously perturb multiple oncogenic signaling pathways. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-17-05.

Malignant transformation and multidrug resistance are linked to resistance to apoptosis, yet the molecular mechanisms that mediate tumor survival remain poorly understood. Because the stroma can influence tumor behavior by regulating the tissue phenotype, we explored the role of extracellular matrix signaling and tissue organization in epithelial survival. We report that elevated (alpha6)beta4 integrin-dependent Rac-Pak1 signaling supports resistance to apoptosis in mammary acini by permitting stress-dependent activation of the p65 subunit of NF-kappaB through Pak1. We found that inhibiting Pak1 through expression of N17Rac or PID compromises NF-kappaB activation and renders mammary acini sensitive to death, but that resistance to apoptosis could be restored to these structures by overexpressing wild-type NF-kappaB p65. We also observed that acini expressing elevated levels of Pak1 can activate p65 and survive death treatments, even in the absence of activated Rac, yet will die if activation of NF-kappaB is simultaneously inhibited through expression of IkappaBalphaM. Thus, mammary tissues can resist apoptotic stimuli by activating NF-kappaB through alpha6beta4 integrin-dependent Rac-Pak1 signaling. Our data emphasize the importance of the extracellular matrix stroma in tissue survival and suggest that alpha6beta4 integrin-dependent Rac stimulation of Pak1 could be an important mechanism mediating apoptosis-resistance in some breast tumors.

Mice unable to synthesize norepinephrine (NE) and epinephrine due to targeted disruption of the dopamine β-hydroxylase gene, Dbh , were used to critically test roles for NE in mediating acute behavioral changes elicited by different classes of antidepressants. To this end, we used the tail suspension test, one of the most widely used paradigms for assessing antidepressant activity and depression-related behaviors in normal and genetically modified mice. Dbh –/– mice failed to respond to the behavioral effects of various antidepressants, including the NE reuptake inhibitors desipramine and reboxetine, the monoamine oxidase inhibitor pargyline, and the atypical antidepressant bupropion, even though they did not differ in baseline immobility from Dbh +/– mice, which have normal levels of NE. Surprisingly, the effects of the selective serotonin reuptake inhibitors (SSRIs) fluoxetine, sertraline, and paroxetine were also absent or severely attenuated in the Dbh –/– mice. In contrast, citalopram (the most selective SSRI) was equally effective at reducing immobility in mice with and without NE. Restoration of NE by using l - threo -3,4-dihydroxyphenylserine reinstated the behavioral effects of both desipramine and paroxetine in Dbh –/– mice, thus demonstrating that the reduced sensitivity to antidepressants is related to NE function, as opposed to developmental abnormalities resulting from chronic NE deficiency. Microdialysis studies demonstrated that the ability of fluoxetine to increase hippocampal serotonin was blocked in Dbh –/– mice, whereas citalopram's effect was only partially attenuated. These data show that NE plays an important role in mediating acute behavioral and neurochemical actions of many antidepressants, including most SSRIs.

The cytoskeleton, integrin-mediated adhesion, and substrate stiffness control a common set of cell functions required for development and homeostasis that are often deranged in cancer. The connection between these mechanical elements and chemical signaling processes is not known. Here, we show that α 5 β 1 integrin switches between relaxed and tensioned states in response to myosin II–generated cytoskeletal force. Force combines with extracellular matrix stiffness to generate tension that triggers the integrin switch. This switch directly controls the α 5 β 1 -fibronectin bond strength through engaging the synergy site in fibronectin and is required to generate signals through phosphorylation of focal adhesion kinase. In the context of tissues, this integrin switch connects cytoskeleton and extracellular matrix mechanics to adhesion-dependent motility and signaling pathways.

Summary Heat shock protein 90 (Hsp90) is a molecular chaperone essential for the stability and function of multiple cellular client proteins, a number of which have been implicated in the pathogenesis of breast cancer. Here we undertook a comprehensive evaluation of the activity of ganetespib, a selective Hsp90 inhibitor, in this malignancy. With low nanomolar potency, ganetespib reduced cell viability in a panel of hormone receptor-positive, HER2-overexpressing, triple-negative and inflammatory breast cancer cell lines in vitro. Ganetespib treatment induced a rapid and sustained destabilization of multiple client proteins and oncogenic signaling pathways and even brief exposure was sufficient to induce and maintain suppression of HER2 levels in cells driven by this receptor. Indeed, HER2-overexpressing BT-474 cells were comparatively more sensitive to ganetespib than the dual HER2/EGFR tyrosine kinase inhibitor lapatinib in three-dimensional culture. Ganetespib exposure caused pleiotropic effects in the inflammatory breast cancer line SUM149, including receptor tyrosine kinases, MAPK, AKT and mTOR signaling, transcription factors and proteins involved in cell cycle, stress and apoptotic regulation, as well as providing combinatorial benefit with lapatinib in these cells. This multimodal activity translated to potent antitumor efficacy in vivo, suppressing tumor growth in MCF-7 and MDA-MB-231 xenografts and inducing tumor regression in the BT-474 model. Thus, ganetespib potently inhibits Hsp90 leading to the degradation of multiple clinically-validated oncogenic client proteins in breast cancer cells, encompassing the broad spectrum of molecularly-defined subtypes. This preclinical activity profile suggests that ganetespib may offer considerable promise as a new therapeutic candidate for patients with advanced breast cancers. Electronic supplementary material The online version of this article (doi:10.1007/s10637-013-9971-6) contains supplementary material, which is available to authorized users.

EML4–ALK gene rearrangements define a unique subset of patients with non–small cell lung carcinoma (NSCLC), and the clinical success of the anaplastic lymphoma kinase (ALK) inhibitor crizotinib in this population has become a paradigm for molecularly targeted therapy. Here, we show that the Hsp90 inhibitor ganetespib induced loss of EML4–ALK expression and depletion of multiple oncogenic signaling proteins in ALK-driven NSCLC cells, leading to greater in vitro potency, superior antitumor efficacy, and prolonged animal survival compared with results obtained with crizotinib. In addition, combinatorial benefit was seen when ganetespib was used with other targeted ALK agents both in vitro and in vivo. Importantly, ganetespib overcame multiple forms of crizotinib resistance, including secondary ALK mutations, consistent with activity seen in a patient with crizotinib-resistant NSCLC. Cancer cells driven by ALK amplification and oncogenic rearrangements of ROS1 and RET kinase genes were also sensitive to ganetespib exposure. Taken together, these results highlight the therapeutic potential of ganetespib for ALK-driven NSCLC. Significance: In addition to direct kinase inhibition, pharmacologic blockade of the molecular chaperone Hsp90 is emerging as a promising approach for treating tumors driven by oncogenic rearrangements of ALK. The bioactivity profile of ganetespib presented here underscores a new therapeutic opportunity to target ALK and overcome multiple mechanisms of resistance in patients with ALK-positive NSCLC. Cancer Discov; 3(4); 430–43. ©2013 AACR. This article is highlighted in the In This Issue feature, p. 363

Mutant KRAS is a feature of more than 25% of non–small cell lung cancers (NSCLC) and represents one of the most prevalent oncogenic drivers in this disease. NSCLC tumors with oncogenic KRAS respond poorly to current therapies, necessitating the pursuit of new treatment strategies. Targeted inhibition of the molecular chaperone Hsp90 results in the coordinated blockade of multiple oncogenic signaling pathways in tumor cells and has thus emerged as an attractive avenue for therapeutic intervention in human malignancies. Here, we examined the activity of ganetespib, a small-molecule inhibitor of Hsp90 currently in clinical trials for NSCLCs in a panel of lung cancer cell lines harboring a diverse spectrum of KRAS mutations. In vitro , ganetespib was potently cytotoxic in all lines, with concomitant destabilization of KRAS signaling effectors. Combinations of low-dose ganetespib with MEK or PI3K/mTOR inhibitors resulted in superior cytotoxic activity than single agents alone in a subset of mutant KRAS cells, and the antitumor efficacy of ganetespib was potentiated by cotreatment with the PI3K/mTOR inhibitor BEZ235 in A549 xenografts in vivo . At the molecular level, ganetespib suppressed activating feedback signaling loops that occurred in response to MEK and PI3K/mTOR inhibition, although this activity was not the sole determinant of combinatorial benefit. In addition, ganetespib sensitized mutant KRAS NSCLC cells to standard-of-care chemotherapeutics of the antimitotic, topoisomerase inhibitor, and alkylating agent classes. Taken together, these data underscore the promise of ganetespib as a single-agent or combination treatment in KRAS-driven lung tumors. Mol Cancer Ther; 11(12); 2633–43. ©2012 AACR . This article is featured in Highlights of This Issue, [p. 2547][1] [1]: /lookup/volpage/11/2547?iss=12

Previous studies have demonstrated that traumatic brain injury (TBI) increases the vulnerability of the brain to an acute episode of hypoxia-ischemia. The objective of the present study was to determine whether TBI alters the vulnerability of the brain to a delayed episode of ischemia and, if so, to identify contributing mechanisms. Sprague-Dawley rats were subjected to lateral fluid-percussion (FP) brain injury (n = 14) of moderate severity (2.3-2.5 atm), or sham-injury (n = 12). After recovery for 24 h, all animals underwent an 8-min episode of forebrain ischemia, followed by survival for 6 days. Ischemic damage in the hippocampus and cerebral cortex of the FP-injured hemisphere was compared to that in the contralateral hemisphere and to that in sham-injured animals. Remarkably, the number of surviving CA(1) neurons in the middle and lateral segments of the hippocampus in the FP-injured hemisphere was significantly greater than that in the contralateral hemisphere and sham-injured animals (p0.05). Likewise, in the cerebral cortex the number of damaged neurons tended to be lower in the FP-injured hemisphere than in the contralateral hemisphere. These results suggest that TBI decreased the vulnerability of the brain to a delayed episode of ischemia. To determine whether TBI triggers protective metabolic alterations, glycogen levels were measured in cerebral cortex and hippocampus in additional animals 24 h after FP-injury (n = 13) or sham-injury (n = 7). Cortical glycogen levels in the ipsilateral hemisphere increased to 12.9 +/- 6.4 mmol/kg (mean +/- SD), compared to 6.4 +/- 1.8 mmol/kg in the opposite hemisphere and 5.7 +/- 1.3 mmol/kg in sham-injured animals (p0.001). Similarly, in the hippocampus glycogen levels in the FP-injured hemisphere increased to 13.4 +/- 4.9 mmol/kg, compared to 8.1 +/- 2.4 mmol/kg in the contralateral hemisphere (p0.004) and 6.2 +/- 1.5 mmol/kg in sham-injured animals (p0.001). These results demonstrate that TBI triggers a marked accumulation of glycogen that may protect the brain during ischemia by serving as an endogenous source of metabolic energy.

Background: Heat shock protein 90 (HSP90) is a molecular chaperone required for the stability of hundreds of client proteins, many of which promote tumor growth. While HSP90 inactivation abrogates multiple oncogenic pathways, it subsequently triggers a heat shock response that may limit its full cytotoxic potential. To overcome this limitation, we sought to identify a clinically feasible method to block heat shock protein synthesis induced by the HSP90 inhibitor ganetespib. Results: An immunoassay was developed to screen a library of >300 late stage or approved drugs for their ability to block the upregulation of HSP70 subsequent to HSP90 inhibition. A number of effective agents were identified, with the most prevalent and effective being those that target PI3K/mTOR signaling. Validating this result, mTOR inhibitors diminished ganetespib induced HSP70 and HSP90 protein levels in multiple cancer cell lines. To determine the mechanism of HSP regulation by mTOR inhibitors the expression of >80 heat shock related genes was analyzed. Inhibitors of mTOR had no effect on HSP RNA levels but suppressed the upregulation of HSP genes induced by ganetespib. Heat shock factor 1 (HSF1) is the major transcriptional regulator of HSP's. Under conditions of stress or HSP90 inhibition, HSF1 is released from repressive cytoplasmic chaperone complexes and translocates into the nucleus to initiate heat shock gene transcription. Nuclear HSF1 levels increased within 1 h of ganetespib exposure but were dramatically reduced in the presence of an mTOR inhibitor. A major function of mTOR is regulating protein synthesis through activation of the cap-binding protein EIF4E, therefore we investigated whether inhibition of protein synthesis would affect HSF1 nuclear accumulation. Cycloheximide or an EIF4E inhibitor reduced ganetespib-mediated HSF1 entry into the nucleus suggesting that inhibition of mTOR can prevent HSP induction by blocking protein synthesis. Combinations of ganetespib with mTOR or dual mTOR/PI3K inhibitors resulted in a significant increase in anticancer activity compared to monotherapy in multiple in vitro and in vivo cancer models. Conclusions: Inhibition of mTOR counteracts ganetespib induced upregulation of HSP's by blocking translocation of HSF1 into the nucleus, an effect potentially mediated by a decrease in mTOR driven protein synthesis. Attenuation of heat shock response may contribute to the synergy observed for ganetespib and PI3K/mTOR inhibitors in preclinical cancer models. Targeting mTOR signaling thus represents a promising, clinically feasible approach to maximize the therapeutic potential of ganetespib. Citation Format: Suqin He, Jaime Acquaviva, Julie C. Friedland, Jim Sang, Donald L. Smith, Manuel Sequeira, Chaohua Zhang, David A. Proia. Inhibition of mTOR enhances the activity of HSP90 inhibitors in part through cessation of heat shock protein synthesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1038. doi:10.1158/1538-7445.AM2013-1038

3090 Background: Ganetespib is a potent inhibitor of heat shock protein 90 (Hsp90) currently being studied in a broad range of clinical trials. Phase II results with ganetespib demonstrated an encouraging objective response rate of 50% in non-small cell lung carcinoma (NSCLC) patients whose tumors contained ALK translocations and had progressed on previous treatments. To further understand the clinical potential of ganetespib in ALK-driven cancers, we evaluated its activity in (1) crizotinib-sensitive and -resistant cancer cells harboring ALK fusions; (2) cells expressing amplified ALK or ROS1 translocations (a kinase structurally similar to ALK); and (3) in combination with crizotinib. Methods: H3122 NSCLC cells, which express EML4-ALK, were treated with ganetespib, crizotinib or the combination, and cell viability and signaling cascades were assessed. Similar experiments were done in cells with amplified, constitutively active ALK (NB-39-nu) or ROS1 kinase fusions (HCC-78, U118MG). To generate a model of crizotinib resistance, NPM-ALK-expressing BaF3 cells were exposed to various crizotinib concentrations. Fifteen different ALK kinase domain substitutions were identified; clonal NPM-ALK/BaF3 cells were made for each resistance mutation and assayed for sensitivity to ganetespib. Results: Ganetespib was 50-fold more potent than crizotinib in killing H3122 cells, and when combined together at sub-lethal doses, displayed strong synergistic anticancer activity. Ganetespib showed similar potency in other cells driven by constitutively active ALK or ROS1 kinase fusions, due to abrogation of their oncogenic kinase activity. All 15 of the crizotinib-resistant NPM-ALK/BaF3 mutant clones were highly sensitive to ganetespib (IC50 values ranging from 14-23 nM), including the 7 mutations reported to date in patients with ALK-driven tumors. Conclusions: Ganetespib effectively inhibits the activity of ALK and ROS1, kinases associated with several tumor types, resulting in marked single agent anticancer activity in cells driven by them. Importantly, ganetespib retains its potency irrespective of the mutational status of ALK. The strong synergy observed between ganetespib and crizotinib warrants further study.