Heat Shock Protein 90 Inhibitor Ganetespib Research Articles

HDAC6 modulates the cognitive behavioral function and hippocampal tissue pathological changes of APP/PS1 transgenic mice through HSP90-HSF1 pathway.

The aim of this study was to investigate histone deacetylase 6 (HDAC6) modifies the heat shock protein 90 (HSP90) and heat shock transcription factor 1 (HSF1) affect the levels of pathological markers such as Aβ oligomers (Aβo) and Tau phosphorylation (p-Tau) in APP/PS1 double transgenic mice hippocampal tissues or HT22 neurons as well as the changes in cognitive behavioral functions of mice. (1) APP/PS1 transgenic mice (6 months old, 25 ~ 30g) were randomly assigned to 5 experimental groups, C57BL/6J mice (6 months old, 25 ~ 30g) were used as 4 control groups, with 8 mice in each group. All mice underwent intracerebroventricular (i.c.v.) cannulation, and the experimental groups were administered with normal saline (APP + NS group), HDAC6 agonist tubastatin A hydrochloride (TSA) (APP + TSA group) or HDAC6 agonist theophylline (Theo) (APP + Theo group), HSP90 inhibitor Ganetespib (Gane) (APP + Gane group), or a combination of pre-injected Gane by TSA (APP + Gane + TSA group); the control group received i.c.v. injections of Gane (Gane group), TSA (TSA group), Theo (Theo group) or NS (NS group), respectively. (2) Mouse hippocampal neurons HT22 were randomly divided into a control group (Control) and an Aβ1-42 intervention group (Aβ). Within the Aβ group, further divisions were made for knockdown HSP90 (Aβ + siHSP90 group), overexpression HSP90 (Aβ + OE-HSP90 group), knockdown HSF1(Aβ + siHSF1 group) and knockdown HSF1 followed by overexpression HSP90 (Aβ + siHSF1 + OE-HSP90 group), resulting in a total of 6 groups. Morris water maze test was used to evaluate the cognitive behavior of the mice. Western blot and immunohistochemistry or immunofluorescence were performed to detect the levels of HDAC6, HSP90, HSF1, Aβ1-42, Tau protein, and p-Tau in the hippocampal tissue or HT22 cells. qRT-PCR was used to measure the levels of hdac6, hsp90, and hsf1 mRNA in the hippocampus or nerve cells. (1) The levels of HDAC6, Aβ1-42 and p-Tau were elevated, while HSP90 and HSF1 were decreased in the hippocampal tissue of APP/PS1 transgenic mice (all P < 0.01). Inhibiting HDAC6 upregulated the expressions of HSP90 and HSF1 in the hippocampal tissue of APP/PS1 mice, while decreasing the levels of Aβ1-42 and p-Tau as well as improving the spatial cognitive behavior in mice (P < 0.05 or P < 0.01). The opposite effects were observed upon HDAC6 activation. However, inhibiting HSP90 reduced the expression of HSF1 (P < 0.01) and increased the levels of Aβ1-42 and p-Tau (P < 0.05 or P < 0.01) but did not significantly affect the expression of HDAC6 (P > 0.05). No significant changes were observed in the aforementioned indicators in the 4 control groups (P > 0.05). (2) In the Aβ1-42 intervention group, HDAC6 and Aβ1-42, p-Tau expression levels were elevated, while HSP90 and HSF1 expressions were all decreased, and cell viability was reduced (P < 0.05 or P < 0.01). Overexpression of HSP90 upregulated HSF1 expression, decreased the levels of Aβ1-42 and p-Tau, and increased cell viability (P < 0.05 or P < 0.01). Knocking down HSP90 had the opposite effect; and knocking down HSF1 increased the levels of Aβ1-42 and p-Tau and decreased cells viability (all P < 0.01), but did not result in significant changes in the expression levels of HSP90 (P > 0.05). Inhibiting HDAC6 can upregulate the expressions of HSP90 and HSF1 but reduce the levels of Aβ1-42 and p-Tau in the hippocampus of APP/PS1 mice and improvement of cognitive behavioral function in mice; Overexpression of HSP90 can increase HSF1 but decrease Aβ1-42 and p-Tau levels in the hippocampal neurons and increase cell activity. It is suggested that HDAC6 may affect the formation of Aβ oligomers and the changes in Tau protein phosphorylation levels in the hippocampus of AD transgenic mouse as well as the alterations in cognitive behavioral functions by regulating the HSP90-HSF1 pathway.

Hepatic Microwave Ablation-Induced Tumor Destruction and Animal End Point Survival Can Be Improved by Suppression of Heat Shock Protein 90.

To investigate the effect of heat shock protein 90 (HSP90) modulation on tumor necrosis, apoptosis, tumor growth delay, and end point survival by combining microwave ablation (MWA) with an HSP90 inhibitor in a nude mouse model. This study was approved by the Ethics Committee. Forty mice with HepG2 subcutaneous xenograft tumors (10 ± 1 mm) were randomized into 4 groups: (1) no treatment, (2) MWA only, (3) the HSP90 inhibitor ganetespib only, and (4) ganetespib combined with MWA. Tumors were harvested 24 hours after treatment, and gross coagulation diameters were measured. The effect of ganetespib on HSP90 and caspase 3 expression in the periablational rim was assessed. Another 40 mice with the same tumors and groupings were observed after treatment. Tumor growth curve and Kaplan-Meier survival analyses were performed with a tumor diameter of 2.2 cm and 40 days of survival as the defined survival end points. Combination treatment significantly increased the coagulation size compared to tumors treated with MWA or ganetespib alone (P < 0.05). The combination of MWA and ganetespib decreased HSP90 expression and increased cleaved caspase 3 expression 24 hours after treatment. Compared with MWA or ganetespib only, combination treatment could lengthen the end point survival and reduce the tumor growth rate. Modulation of HSP production can improve MWA-induced tumor apoptosis and destruction, reduce residual tumor growth rates, and prolong end point survival.

HSP90 Inhibition Drives Degradation of FGFR2 Fusion Proteins: Implications for Treatment of Cholangiocarcinoma.

About 15% of intrahepatic cholangiocarcinomas (ICCs) express constitutively active fibroblast growth factor receptor 2 (FGFR2) fusion proteins (FFs) generated by chromosomal translocations. FFs have been nominated as oncogenic drivers because administration of FGFR tyrosine kinase inhibitors (F-TKIs) can elicit meaningful objective clinical responses in patients carrying FF-positive ICC. Thus, optimization of FF targeting is a pressing clinical need. Herein, we report that three different FFs, previously isolated from ICC samples, are heat shock protein 90 (HSP90) clients and undergo rapid degradation upon HSP90 pharmacological blockade by the clinically advanced HSP90 inhibitor ganetespib. Combining catalytic suppression by the F-TKI BGJ398 with HSP90 blockade by ganetespib suppressed FGFR2-TACC3 (transforming acidic coiled-coil containing protein 3) signaling in cultured cells more effectively than either BGJ398 or ganetespib in isolation. The BGJ398 + ganetespib combo was also superior to single agents when tested in mice carrying subcutaneous tumors generated by transplantation of FGFR2-TACC3 NIH3T3 transformants. Of note, FF mutants known to enforce clinical resistance to BGJ398 in ICC patients retained full sensitivity to ganetespib in cultured cells. Conclusion: Our data provide a proof of principle that upfront treatment with the BGJ398 + ganetespib combo improves therapeutic targeting of FGFR2 fusions in an experimental setting, which may be relevant to precision medicine approaches to FF-driven ICC.

Inhibiting heat shock protein 90 and the ubiquitin-proteasome pathway impairs metabolic homeostasis and leads to cell death in human pancreatic cancer cells.

Heat shock protein 90 (HSP90) and the ubiquitin-proteasome pathway play crucial roles in the homeostasis of pancreatic cancer cells. This study combined for the first time the HSP90 inhibitor ganetespib (Gan) and the proteasome inhibitor carfilzomib (Carf) to target key mechanisms of homeostasis in pancreatic cancer. It was hypothesized that Gan plus Carf would elicit potent antitumor activity by modulating complementary homeostatic processes. In vitro and in vivo effects of this combination on mechanisms of cell growth and viability were evaluated with human pancreatic cancer cell lines (MIA PaCa-2 and HPAC). Combined treatment with Gan and Carf significantly decreased cell viability. The mechanism varied by cell line and involved G2 -M cell-cycle arrest accompanied by a consistent reduction in key cell-cycle regulatory proteins and concomitant upregulation of p27. Further studies revealed increased autophagy markers, including the upregulation of autophagy related 7 and light chain 3 cleavage, and evidence of apoptosis (increased Bax expression and processing of caspase 3). Immunoblot analyses confirmed the modulation of other pathways that influence cell viability, including phosphoinositide 3-kinase/Akt and nuclear factor κB. Finally, the treatment of athymic mice bearing HPAC tumors with Gan and Carf significantly reduced tumor growth in vivo. An immunoblot analysis of freshly isolated tumors from animals at the end of the study confirmed in vivo modulation of key signaling pathways. The results reveal Gan plus Carf to be a promising combination with synergistic antiproliferative, apoptotic, and pro-autophagy effects in preclinical studies of pancreatic cancer and will further the exploration of the utility of this treatment combination in clinical trials. Cancer 2017;123:4924-33. © 2017 American Cancer Society.

Abstract 4516: Dual inhibition of CDK1 and HSP90 destabilizes HIF1α and synergistically induces cancer cell death

Abstract Solid tumors are often characterized by intratumoral hypoxia. Hypoxia-inducible factor 1α (HIF1α) has been implicated in cell proliferation, survival, angiogenesis, invasion and migration. The expression of HIF-1α is increased in a variety of cancers but this is not restricted to hypoxic regions. We have previously shown that cyclin-dependent kinase 1 (CDK1) stabilizes HIF1α through direct phosphorylation of its Ser668 residue in a Von Hippel-Lindau (VHL)-independent manner both under hypoxia and at G2/M under normoxia (Warfel et al., Cell Cycle, 2013). Heat shock protein 90 (HSP90) is also an acknowledged VHL-independent HIF1α stabilizer. We sought to explore the link between CDK1-mediated and HSP90-mediated HIF1α stabilization. Administration of the CDK1 inhibitor, Ro-3306, reverses the heat shock (40°C)-increased level of HIF1α in normoxia suggesting that CDK1 activity may contribute to HSP90-mediated HIF1α stabilization. Under hypoxia, combination treatment with HSP90 inhibitor geldanamycin and CDK1 inhibition/knockdown decreases HIF1α levels more robustly than either treatment alone. This led us to investigate whether combined CDK1 and HSP90 inhibition results in synergistic anti-cancer effects. Indeed, dual inhibition of CDK1 and HSP90 synergistically decreases HCT116 colon cancer cell viability. PARP cleavage and sub-G1 analysis indicate the combinational treatment triggers cancer cell death through apoptosis. Consistent with these findings, we observed that inhibition of cell viability by the combination is impaired in HCT116 Bax-/- cells. Furthermore, the dual inhibition also suppresses cell migration in vitro. We further validated our results with CDK1 and HSP90 inhibitors that have entered clinical trials. Second-generation HSP90 inhibitor ganetespib and Ro3306 synergistically inhibit cell viability in HCT116 cells. We have previously demonstrated that CDK4 also contributes to the stabilization of HIF1α under hypoxia (Warfel et al., Cell Cycle, 2013). Interestingly, the combination of CDK4 and HSP90 inhibitors is also able to improve downregulation of HIF1α as compared to either agent alone in various cancer cell lines (eg. colon cancer, gliobastoma, etc). The FDA-approved CDK4 inhibitor palbociclib along with ganetespib synergistically inhibits cell viability in HCT116 cells under hypoxia. Such effect is not observed in WI38 normal cells under normoxia at similar concentrations. Our findings demonstrate that CDK1 inhibition disrupts the interaction between HSP90 and HIF1α providing a rationale for therapeutic targeting of HIF1α through the combination of CDK1/4 and HSP90 inhibitors in cancer. Ongoing studies are examining the effects of CDK1/4 plus HSP90 dual inhibition in other tumor types and in vivo. Citation Format: Shuai Zhao, David T. Dicker, Wafik S. El-Deiry. Dual inhibition of CDK1 and HSP90 destabilizes HIF1α and synergistically induces cancer cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4516. doi:10.1158/1538-7445.AM2017-4516

Targeting the cell cycle for cancer therapy in rhabdomyosarcoma.

10535 Background: Rhabdomyosarcoma (RMS) is an aggressive malignancy of childhood with a poor prognosis in patients with metastatic or recurrent disease. Inhibitors of Wee1 kinase and heat shock protein 90 (HSP90) have in vitro activity in RMS and have emerged as potential novel treatment strategies. We performed a comprehensive preclinical phase III study to compare the Wee1 inhibitor AZD1775 and HSP90 inhibitor ganetespib (GANET) in combination with irinotecan (IRN) and vincristine (VCR). Methods: Orthotopic xenografts (O-PDXs) were created by injecting luciferase labeled RMS cells into the hind-leg muscle of CD-1 nude mice. Pharmacokinetic studies on RMS O-PDXs were performed to determine matched human AUC-guided dosing. A total of 540 O-PDXs derived from 4 high risk RMS patients, 2 alveolar and 2 embryonal, were randomly enrolled into 14 treatment groups. Six courses of blinded placebo-controlled therapy were given on a clinically relevant schedule. Mice were classified as having progressive disease if tumor approached 20% body weight at any time in the study. For mice completing all 6 courses, bioluminescence was used to determine complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD). Results: The addition of AZD1775 to IRN and VCR demonstrated the most significant response for all 4 O-PDX lines tested; 70% of mice achieved a CR or PR. GANET combined with IRN and VCR had a 54% response (CR + PR) which was not significantly better than IRN plus VCR for most O-PDXs tested. Overall response data for select treatment groups is shown in the table below. Conclusions: Comprehensive preclinical testing using multiple O-PDX models of RMS that represent the clinical spectrum of disease is feasible. Comparison of novel treatment regimens to standard of care at clinically relevant doses is warranted as justification for future clinical trials. Observation to assess durable response following completion of therapy is ongoing to determine if AZD1775 remains a promising treatment strategy. [Table: see text]

Abstract B105: HSP90 inhibitor, ganetespib, enhances responses to cancer immunotherapy through increased expression of interferon response genes

Abstract Recently, T cell based immunotherapies have moved to the forefront of cancer immunotherapy with the success of Adoptive T cell therapy (ACT) and Immune checkpoint blockade. ACT, where patients are treated with tumor infiltrating T cells (TILs), conferred a clinical response rate of ∼50%. Treatment with anti-CTLA4 therapy, Ipilimumab, conferred response rates of 10-20%, greatly improving the overall survival of patients with advanced melanoma. Despite the encouraging outcomes, there are relatively low response rates coupled with the delay of weeks to months before tumor shrinkage can be appreciated. Thus, understanding mechanisms of resistance to immune therapies, to improve response rates, shorten time to treatment effect and developing predictive biomarkers of response are vital to the care of melanoma patients. In order to identify possible resistance mechanisms to immunotherapy, a high-throughput in vitro screen with 850 different bio-active compounds (Selleckchem), was designed to search for agents that could either increase or decrease the resistance of melanoma tumor cells to T cell mediated killing. Paired patient derived human melanoma tumor samples and TILs were used to assess which compounds when used to treat the melanoma cell lines can enhance the cytotoxic activity of the TILs against the paired melanoma sample, using a flow cytometry based assay in which active caspase 3 was used as a read out of apoptosis. We identified heat shock protein 90 (HSP90) inhibitors amongst the top compounds that improved T cell mediated cytotoxicity of treated tumor cells. We show that treatment with the HSP90 inhibitor ganetespib (Synta) greatly improves T cell mediated cytotoxicity of human cancer cells lines in vitro. Furthermore, in vivo murine studies using the MC38/gp100 tumor model show that ganestespib in combination with anti-CTLA4, resulted in superior antitumor effect and survival compared to either treatment alone (Average tumor volume at day 21 of treatment: Vehicle 294.3mm3, α-CTLA4 193 mm3, Ganetespib 237.5 mm3 and Ganetespib + α-CTLA4 105.8 mm3, P &amp;lt; 0.0001). Microarray analysis of human cell lines treated with ganetespib in vitro revealed an increase in interferon response genes including IFIT1, IFIT2, IFIT3. We confirmed these findings with quantitative real time PCR and western blot analyses and found IFIT1, IFIT2 and IFIT3 to be consistently upregulated across multiple melanoma cell lines following treatment with ganetespib. We next sought to verify the importance of the IFIT genes in the synergy observed between ganetespib treatment and T cell killing. First, we overexpressed IFIT1, IFIT2 and IFIT3 in human melanoma cell lines to recapitulate the improved sensitivity of the human melanoma cell lines to T cell killing following treatment with ganetespib. We then co-cultured these cells with their autologous T cells and found that overexpressing IFIT1, IFIT2 and IFIT3 mimicked the effects of ganetespib by increasing the sensitivity to T cell killing over the GFP control. On the other hand, silencing IFIT1, IFIT2 and IFIT3 simultaneously, abrogated the synergy between ganetespib and T cell killing. We are further elucidating the role of these genes in lowering the apoptotic threshold of cancer cells and contributing to the synergy of ganetespib and immunotherapy. This will enable the emergence of a new combination therapy of HSP90 inhibitors and anti-CTLA4 for the treatment of melanoma patients that will increase the percentage of patients responding to immunotherapy and achieving long term responses. Citation Format: Rina M. Mbofung, Jodi A. McKenzie, Shruti Malu, Chengwen Liu, Weiyi Peng, Isere Kuiatse, Leila Williams, Seram Devi, Zhe Wang, Trang Tieu, Tim Heffernan, Richard E. Davis, Rodabe Amaria, Patrick Hwu. HSP90 inhibitor, ganetespib, enhances responses to cancer immunotherapy through increased expression of interferon response genes [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B105.

Abstract 2491: Effect of HSP90 inhibitor ganetespib on stemness in small cell lung cancer

Abstract Small cell lung cancer (SCLC) is the most aggressive and metastatic form of lung cancer with a 5 year survival rate of less than 7%. Typically, patients are diagnosed with SCLC when the tumor has already widely metastasized. Combination chemotherapy, generally platinum-based plus etoposide is the mainstay first-line treatments for metastatic SCLC. Despite being highly sensitive to first-line therapy treatments, most patients with SCLC experience relapse with resistant disease within 2 years and die from systemic metastasis. Since there are no approved therapies for the treatment of SCLC, discoveries of therapies that can counteract the spread and relapse of SCLC are needed. Therapy resistance in SCLC can occur through a variety of mechanisms, one possible mechanism might be through the activities of cancer stem-like cells (CSCs) within the SCLC cell population. Different markers, such as ALDH+, CD133+, and uPAR+, are considered as markers of CSCs in SCLC. We used flow cytometry and the Aldefluor kit to separate cells with high and low ALDH activity; flow cytometry and fluorescently- labeled antibody against CD133 and uPAR to separate CD133+ and uPAR + cells. We confirmed that ALDH+/CD133+ and ALDH+/uPAR+ cells were capable of generating tumor spheres in the second and third generations. When we compared SCLC cell lines that were generated from untreated tumors (DMS-53 cell line) with those generated from tumors that were heavily treated (H69 and DMS-114 SCLC cell lines), we found that the percentage of CSCs in DMS-53 cell was significantly lower than that found in H69 and DMS-114 SCLC cell lines. CSCs in SCLC express a distinctive pattern of receptors, signaling molecules, and transcription factors such as ALDHA1, CD133, uPAR, OCT-4, SOX2, STAT3, PKM2, DNA-PK, HIF-1a, and HIF-2a; the upregulation of these factors was determined through analysis of genes and protein expression levels. These proteins are associated with cancer stemness, resistance to conventional therapies, proliferation, and metastasis. Many of these proteins are known to interact with the molecular chaperone heat shock protein 90 (HSP90) and therefore can be targeted simultaneously with HSP90 inhibitors. We used a new HSP90 inhibitor ganetespib, which is showing considerable promise in late stage clinical trials for treatment of other cancer types. Ganetespib used as monotherapy induced cell cycle arrest and apoptosis and effectively inhibited growth of bulk SCLC cells and CSCs growing as tumor spheres. Ganetespib at low nanomolar concentrations significantly potentiates the effect of cisplatin and etoposide in vitro. Further in vivo studies are needed to unravel role played by HSP90 in therapy resistance and metastasis in SCLC. Citation Format: Roberto Gomez-Casal, Peng Zhang, Hong Wang, Paul Gardner, James Herman, Vera Levina. Effect of HSP90 inhibitor ganetespib on stemness in small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2491.

Abstract 4360: Inhibition of HSP90 enhances T cell-mediated antitumor immune responses through expression of interferon-alpha response Genes

Abstract Recently, T cell based immunotherapies have moved to the forefront of cancer immunotherapy with the success of Adoptive T cell therapy (ACT) and Immune checkpoint blockade. ACT, where patients are treated with tumor infiltrating T cells (TILs), conferred a clinical response rate of ∼50%. Treatment with anti-CTLA4 therapy, Ipilimumab, conferred response rates of 10-20%, greatly improving the overall survival of patients with advanced melanoma. Despite the encouraging outcomes, there are relatively low response rates coupled with the delay of weeks to months before tumor shrinkage can be appreciated. Thus, understanding mechanisms of resistance to immune therapies, to improve response rates, shorten time to treatment effect and developing predictive biomarkers of response are vital to the care of melanoma patients. In order to identify possible resistance mechanisms to immunotherapy, a high-throughput in vitro screen with 850 different bio-active compounds (Selleckchem), was designed to search for agents that could either increase or decrease the resistance of melanoma tumor cells to T cell mediated killing. Paired tumor samples and TILs from melanoma patients were used to assess which compounds when used to treat the melanoma cell lines can enhance the cytotoxic activity of the TILs against the paired melanoma sample, using a flow cytometry based assay in which active caspase 3 was used as a read out of apoptosis. We identified heat shock protein 90 (HSP90) inhibitors amongst compounds that improved T cell mediated cytotoxicity. We show that treatment with the HSP90 inhibitor ganetespib (Synta) greatly improves T cell mediated cytotoxicity of both human and murine cancer cells lines in vitro. Furthermore, in vivo murine studies using the MC38/gp100 tumor model show that ganestespib in combination with anti-CTLA4, resulted in superior antitumor effect and survival compared to either treatment alone (Average tumor volume at day 21 of treatment: Vehicle 294.3mm3, α-CTLA4 193 mm3, Ganetespib 237.5 mm3 and Ganetespib + α-CTLA4 105.8 mm3, P &amp;lt; 0.0001). Microarray analysis of human cell lines treated with ganetespib in vitro revealed an increase in interferon alpha (IFN-α) response genes including IFIT1, IFIT2, IFIT3 and IFIH1. Silencing IFIT2 abrogated the synergy observed with ganetespib treatment and T cell mediated killing, suggesting that the IFN-α response pathway plays an important role in this combination therapy. We are further elucidating the role of these genes in the synergy observed. This will enable the emergence of a new combination therapy of HSP90 inhibitors and anti-CTLA4 for the treatment of melanoma patients that will increase the percentage of patients responding to immunotherapy and achieving long term responses. Citation Format: Rina M. Mbofung, Jodi A. McKenzie, Shruti Malu, Chengwen Liu, Leila Williams, Weiyi Peng, Zhe Wang, Satyendra Tripathi, Trang Tieu, Shuping Zhao, Seram Devi, Isere Kuiatse, Emily Ashkin, Leah Bailey, Jason Roszik, Samir Hanash, Timothy Heffernan, Richard E. Davis, Rodabe N. Amaria, Patrick Hwu. Inhibition of HSP90 enhances T cell-mediated antitumor immune responses through expression of interferon-alpha response Genes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4360.

Abstract 2942: p90 Ribosomal S6 kinase mediates acquired resistance to ganetespib in KRAS mutant NSCLC

Abstract Approximately 25% of non-small cell lung cancer (NSCLC) patients have KRAS mutations and no current therapies targeting this key oncogenic driver exist. There is a critical need for novel agents targeting KRAS mutant NSCLC. Heat shock protein 90 (HSP90) is an ATP-dependent molecular chaperone required for the stability of its ‘client’ oncoproteins, which include a number of KRAS downstream effectors. Therefore, targeting Hsp90 could be a promising therapeutic intervention against “undruggable” KRAS driven tumors. Although single agent activity has been demonstrated with the HSP90 inhibitor ganetespib in vitro, only transient responses have been observed in the clinic in KRAS mutant patients. Our goal is to characterize the mechanisms of acquired resistance to ganetespib and develop rational combinations to overcome ganetespib resistance. We have generated ganetespib resistant (GR) KRAS mutant NSCLC cells and demonstrated that bypass of G2/M arrest and hyper-activation of RAF/MEK/ERK and PI3K/AKT/MTOR pathways contribute to resistance. We observed increased dependence on these pathways as the GR cells were more sensitive to ERK1/2 or dual PI3K/MTOR inhibition. Moreover, the combination of ganetespib with ERK1/2 or PI3K/MTOR inhibitors was more effective than either single agent alone. Furthermore, several key mediators of G2/M progression were significantly increased in the GR cells compared to sensitive parental cell lines. Most notably, the serine/threonine kinase p90 ribosomal S6 kinase (p90-RSK), an important regulator of G2/M progression and the PI3K/AKT/MTOR pathway and a key target of ERK1/2 and PDK1 phosphorylation, were significantly upregulated both in expression and activity. Downstream p90-RSK targets and critical G2/M regulators - cdc25A, cdc25B, and cdc25C were also significantly elevated in GR cells. The p90-RSK family consists of four closely related isoforms (1-4), that are widely expressed in cancer and demonstrated to increase cell survival and proliferation. We observed that isoform-specific genetic silencing and pharmacological inhibition of p90-RSK re-sensitized GR cells to ganetespib. Conversely, overexpression of distinct p90-RSK isoforms in sensitive parental cell lines induced ganetespib resistance as well as led to bypass of ganetespib-induced G2/M arrest. The combination of ganetespib with p90-RSK inhibitor(s) enhanced cytotoxicity compared with either agent alone. These data suggest that combination of ganetespib with ERK1/2 or dual PI3K/mTOR or p90-RSK inhibitors may prevent ganetespib resistance and/or overcome acquired resistance after single agent treatment. These findings provide preclinical rationale for a potential future clinical study with an HSP90 inhibitor and a dual PI3K/mTOR inhibitor or RSK inhibitor in KRAS mutant NSCLC patients. Citation Format: Suman Chatterjee, Eric H-B. Huang, Timothy F. Burns. p90 Ribosomal S6 kinase mediates acquired resistance to ganetespib in KRAS mutant NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2942.

Abstract B25: Anti-Mullerian hormone (AMH) supports epithelial identity and survival signaling in lung cancer

Abstract The purpose of this study is to characterize resistance mechanisms of non-small cell lung cancer (NSCLC) to heat shock protein 90 (HSP90) inhibition. Unexpectedly, we identified anti-Mullerian hormone (AMH) and its type II receptor (AMHR2) as two resistance associated genes. AMH and AMHR2 are TGF-ß/BMP superfamily members that share common type I receptors with BMP, resulting in overlapping signaling outputs. AMH and AMHR2 have thus far predominantly been studied in the context of gonadal development, in regulation of the female reproductive cycle and in gynecological malignancies, and AMH is not known to be expressed outside of these tissues. Using a focused RNAi library designed to detect genes associated with resistance to the HSP90 inhibitor ganetespib, siRNAs against AMH and AMHR2 sensitized 4 out of 5 and 3 out of 5 NSCLC cell lines to ganetespib, respectively. We then for the first time confirmed expression of AMH and AMHR2 in this non-gonadal tumor environment. Furthermore, analysis of data from The Cancer Genome Atlas (TCGA) indicated that AMH and AMHR2 are significantly upregulated in a subpopulation of NSCLC. Strikingly, depletion of AMH/AMHR2 induced EMT-like features, including downregulation of cadherins, a mesenchymal morphology, increased invasion, and expression of mesenchymal markers: features that are generally associated with resistance to chemotherapy. In contrast, inhibition of HSP90 selected for a more epithelial-like population of cells, as evident by increased E-cadherin or P-cadherin, and downregulation of mesenchymal markers such as VIM and ZEB1. To confirm that mesenchymal-like cells are indeed more responsive to HSP90 inhibition, we depleted E-cadherin or P-cadherin and again observed sensitization to ganetespib. We further found that AMH and AMHR2 did not confer sensitization to cisplatin. Interestingly, depletion of cadherins, and the associated induction of EMT-like properties, also resulted in downregulation of AMH and AMHR2, further supporting the link between AMH/AMHR2 expression and epithelial identity. In vivo studies confirmed that AMH depletion sensitizes NSCLC cells to ganetespib and showed that it also significantly reduces overall tumor volume. Reduction of AMH or AMHR2 also decreased phosphorylation of direct effector SMAD proteins, and depressed activity of NFκB and AKT, both regulators of EMT and survival. These results for the first time indicate the presence of an AMH-AMHR2-NFκB-AKT signaling axis of therapeutic relevance in NSCLC. To further explore the therapeutic potential of this observation, we treated NSCLC with combinations of ganetespib and the proteasome inhibitor bortezomib- an inhibitor previously shown to disrupt NFκB signaling in some cancers - and observed significant synergy between the two drugs. In conclusion, our work for the first time characterizes AMH and AMHR2 in NSCLC. We go on to show that AMH and AMHR2 regulated canonical-SMAD signaling as well as non-canonical AKT-NFκB signaling, in addition to regulating the epithelial identify of cancer cells. Importantly, our results also suggest that AMH and AMHR2 may serve as biomarkers to predict resistance to HSP90 inhibitors, and that it may be beneficial to prime lung tumors with HSP90 inhibitors, to reverse EMT and decrease survival signaling, prior to treatment with chemotherapy. Note: This abstract was not presented at the conference. Citation Format: Tim N. Beck, Vladislav Korobeynikov, Alexander Kudinov, Rachel Georgeopoulos, Emmanuelle Nicolas, Margret B. Einarson, Yan Zhou, Yanis Boumber, David A. Proia, Ilya G. Serebriiskii, Erica A. Golemis. Anti-Mullerian hormone (AMH) supports epithelial identity and survival signaling in lung cancer. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B25.

Heat shock protein 90 (HSP90) is overexpressed in p16-negative oropharyngeal squamous cell carcinoma, and its inhibition in vitro potentiates the effects of chemoradiation.

Cisplatin and radiation therapy remain the current standard for treating locally advanced SCCHN. Novel treatment approaches are needed, especially in patients with human papilloma virus (HPV)-negative disease who have worse outcomes despite multimodality therapy. Using our institutional review board approved database, we obtained twenty oropharyngeal squamous cell carcinoma (SCC) tissue samples: ten p16 positive, ten p16-negative. Because p16 expression is strongly associated with HPV positivity in oropharyngeal SCC, p16 status was used as a marker of HPV. We subsequently analyzed, via immunohistochemistry, heat shock protein 90 (HSP90) protein levels. Using HPV-positive and HPV-negative SCC cell lines, we compared baseline HSP90 expression levels and the effect of the HSP90 inhibitor ganetespib on viability and apoptosis. Clonogenic survival of HPV-negative cells treated with ganetespib, radiation therapy, and/or cisplatin was then investigated. We characterize the effects of ganetespib on proteins that are thought to drive DNA damage resistance in HPV-negative cells. HSP90 expression was significantly higher in p16-negative compared with p16-positive samples (p = 0.016) and in HPV-negative cell lines compared with positive cells. Ganetespib increased cytotoxicity and induced apoptosis in HPV-negative more than positive cells. Adding ganetespib to cisplatin and/or radiation therapy in HPV-negative cells further decreased clonogenic survival. Finally, ganetespib downregulated expressions of EGFR, ERK, AKT, p53, and HIF-1α. Ganetespib inhibited HPV-negative SCCHN viability and potentiated cell kill when combined with cisplatin or radiation therapy in vitro. With HSP90 expression higher in HPV-negative cells and in p16-negative patients, further exploration of the clinical activity of HSP90 inhibitors in SCCHN is warranted.

Heat Shock Protein 90 (HSP90) Is Preferentially Overexpressed in P16-Negative Oropharyngeal Squamous Cell Carcinoma, and its Inhibition In Vitro Potentiates the Effects of Chemoradiation

Heat Shock Protein 90 (HSP90) Is Preferentially Overexpressed in P16-Negative Oropharyngeal Squamous Cell Carcinoma, and its Inhibition In Vitro Potentiates the Effects of Chemoradiation

The HSP90 inhibitor ganetespib has chemosensitizer and radiosensitizer activity in colorectal cancer

SummaryThe integration of targeted agents to standard cytotoxic regimens has improved outcomes for patients with colorectal cancer (CRC) over recent years; however this malignancy remains the second leading cause of cancer mortality in industrialized countries. Small molecule inhibitors of heat shock protein 90 (HSP90) are one of the most actively pursued classes of compounds for the development of new cancer therapies. Here we evaluated the activity of ganetespib, a second-generation HSP90 inhibitor, in models of CRC. Ganetespib reduced cell viability in a panel of CRC cell lines in vitro with low nanomolar potency. Mechanistically, drug treatment exerted concomitant effects on multiple oncogenic signaling pathways, cell cycle regulation, and DNA damage repair capacity to promote apoptosis. Combinations of ganetespib and low-dose ionizing radiation enhanced the radiosensitivity of HCT 116 cells and resulted in superior cytotoxic activity over either treatment alone. In vivo, the single-agent activity of ganetespib was relatively modest, suppressing HCT 116 xenograft tumor growth by approximately half. However, ganetespib significantly potentiated the antitumor efficacy of the 5-Fluorouracil (5-FU) prodrug capecitabine in HCT 116 xenografts, causing tumor regressions in a model that is intrinsically resistant to fluoropyrimidine therapy. This demonstration of combinatorial benefit afforded by an HSP90 inhibitor to a standard CRC adjuvant regimen provides an attractive new framework for the potential application of ganetespib as an investigational agent in this disease.Electronic supplementary materialThe online version of this article (doi:10.1007/s10637-014-0095-4) contains supplementary material, which is available to authorized users.

Heat shock protein 90 (HSP90) inhibition in squamous cell carcinoma of the head and neck (SCCHN): An in vitro analysis with a focus on p16 status.

2552 Background: Despite advances in therapy, the overall 5 year survival for SCCHN is close to 50%. Cisplatin and radiation therapy remain the current standard for treating locally advanced SCCHN. Novel treatment approaches are urgently needed, especially in HPV negative disease. We examined the expression of HSP90 in HPV positive and negative SCCHN and investigated if the HSP90 inhibitor ganetespib can sensitize mutant p53 SCCHN to cisplatin and radiation therapy in vitro. Methods: Phase 1 trials of ganetespib at 150 mg/m2 have shown the maximum clinically achievable concentration (CAC) in patients to be ~166 nM. We treated p53 deficient or mutant p53 SCCHN cells (SCC25 and Detroit 568, CAL27and FADU, respectively) with ganetespib at 10-50 nM alone and in combination with cisplatin or radiation therapy and assessed survival. We measured HSP90, HSP70 and p53 protein expression levels in SCCHN cell lines by immunoblotting and analyzed HSP90 protein levels in p16 positive and p16 negative SCCHN tumor samples by immunohistochemistry. Results: Ganetespib at CAC was significantly more cytotoxic in mutant p53 compared to p53 deficient SCCHN cells (p &lt; 0.05). When combined with CAC doses of cisplatin or radiation therapy, ganetespib displayed strong sensitizing activity (p &lt; 0.05 and p &lt; 0.01 respectively). Ganetespib treatment upregulated HSP70 and HSP90 expression while decreasing p53 expression in the mutant cell lines. HSP90 expression was significantly higher in p16 negative than p16 positive SCCHN tumor samples (79.5% vs. 51%, ANOVA p = 0.016). Conclusions: Our results demonstrate a potential efficacy of ganetespib as a single agent and in combination with cisplatin and radiation therapy in SCCHN. HSP90 expression is upregulated in p16 negative SCCHN. Further exploration of the role of HSP90 in SCCHN, and utility of HSP90 inhibitors, is warranted.

Abstract 1038: Inhibition of mTOR enhances the activity of HSP90 inhibitors in part through cessation of heat shock protein synthesis.

Abstract 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 &amp;gt;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 &amp;gt;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

Antiangiogenic activity of the HSP90 inhibitor ganetespib in pancreatic cancer models

Heat shock protein 90 (HSP90) has a central role in the regulation of transcriptional factors that modulate angiogenesis and growth. Ganetespib (G) is a functional inhibitor of HSP90 being studied in a broad range of clinical trials. We tested the hypothesis that functional inhibition of HSP90 by G can inhibit angiogenesis and growth in vitro and in vivo models of pancreatic cancer. PANC‐1, and HPAC cell lines were treated with vehicle or G (50nM) for 24 h and lysates were analyzed by Western blot. Egg CAM and matrigel plug assays were performed to quantify the effects of G on angiogenesis. Efficacy of G (100mg/kg) was assessed in mice bearing HPAC and ASPC‐1 xenograft. Western blot analyses demonstrated a significant reduction in intracellular HIF‐1á and VEGF protein levels in PANC‐1 and HPAC cells treated with G. Results from ELISA assays showed that G reduced VEGF secretion in the culture medium from both pancreatic lines. G treatment reduced angiogenesis compared to vehicle in all three models. Animals with human pancreatic tumor xenografts treated with G had significant tumor growth delay and inhibition of angiogenesis. The preclinical data demonstrates that G can inhibit pancreatic cancer growth and angiogenesis, suggesting that targeting HSP90 is a rational new approach to pancreatic cancer therapy to be explored in clinical trials.

Evaluating the HSP90 Inhibitor Ganetespib as a Radiosensitizing Agent in Breast Cancer Models In Vitro

• Experiments were conducted using the inflammatory breast cancer cell line SUM-149, and the triple negative cell line MDA-MB-231. The BT-474 (ER/PR positive, HER2 positive) and MCF-7 (ER/PR positive, HER2 negative) cell lines underwent XTT testing. • XTT Assays: • Cells were plated in 96 well plates, exposed to ganetespib in concentrations ranging from 50-250 nM for 24 hrs. Media was changed and cells were incubated for 72 hrs. XTT assays were then performed. • Clonogenic Assays: • Cells were plated on 6-well plates, ganetespib was added to the media at 10 or 50 nM and allowed to incubate 20 hours. Cells were then irradiated and incubated for 4 more hours. Media was changed, and 14 days later, plates were fixed, stained, and colonies counted. • Cell Cycle Analysis: • Cells were plated in flasks and treated with media or media + ganetespib at a concentration of 150 nM. The cells were incubated for 24 hours. They were then harvested, fixed in 70% ice cold ethanol, washed in 1xPBS, and re-suspended in 1 mL of PI solution containing DNAse-free RNAse. The cells were analyzed with a Beckman Coulter Flow Cytometer. • Western Blots: • Cells were treated with 100 nM ganetespib for 24 hours, harvested, then analyzed for expression of several marker proteins (HSP90, HSP70, AKT, p-AKT, pRb) Conclusions

Abstract 5601: Overcoming acquired resistance to BRAF inhibitors in melanoma with the HSP90 inhibitor ganetespib

Abstract Activating BRAF(V600E) kinase mutations occur in 50% of melanoma, 45% of papillary thyroid cancer and 10% of colon cancer. BRAF inhibitors have elicited significant clinical response in melanomas but acquired resistance frequently develops after initial responses. Therefore, consideration of new treatment strategies to block activity of mutant BRAF and deter therapeutic resistance is warranted. Heat shock protein 90 (HSP90) is a molecular chaperone required for the stability of hundreds of client proteins, including the RAF family of kinases, and has emerged as a highly relevant anticancer target. Ganetespib is a second generation, small molecule HSP90 inhibitor currently being evaluated in multiple clinical trials. To investigate the potential for ganetespib in the treatment of melanoma we evaluated its preclinical activity as monotherapy, and in combination with targeted agents, in cells sensitive or resistant to BRAF inhibitors. Ganetespib displayed potent anticancer activity in a large panel of BRAF(V600E) mutant melanoma, thyroid and colon cancer cell lines, with IC50 between 4 and 40 nM. Further analysis showed that ganetespib disrupted oncogenic signaling by inducing the destabilization of mutant BRAF and subsequent inactivation of MEK and ERK. Levels of wild-type BRAF were only slightly reduced in response to ganetespib treatment, implying that the stability of activated BRAF is more reliant on HSP90 and that ganetespib may selectively deplete oncogenic forms of BRAF. Notably, ganetespib treatment also led to depletion of CRAF, which is implicated in driving therapeutic resistance to BRAF inhibitors. In an effort to maximize antitumor activity, we combined low levels of ganetespib with BRAF or MEK inhibitors in vitro and observed increased cytotoxicity compared to single agent treatment. Both the BRAF and MEK inhibitors were cytostatic on their own, which may be due to continued PI3K/mTOR signaling. We subsequently assayed the combination of ganetespib with a PI3K/mTOR inhibitor and observed synergistic increases in cell death. Importantly, ganetespib was capable of blocking the feedback activation on MAPKK induced by a MEK inhibitor as well as the activation of ERK by PI3K/mTOR inhibitors. To determine if these results translated in vivo, similar combinations were performed in BRAF mutant human melanoma xenografts. Co-administration of ganetespib with either a BRAF or PI3K/mTOR inhibitor suppressed in vivo tumor growth to a greater extent compared to monotherapy. Finally, we show that ganetespib effectively blocked MAPK signaling and killed RPMI-7951 melanoma cells (IC50 = 4 nM), which are resistant to BRAF and MEK inhibitors due to the overexpression of COT. In conclusion, targeting HSP90 with ganetespib represents a promising approach for the treatment of BRAF(V600E)-driven cancers and may be especially relevant in the context of acquired resistance to BRAF inhibitors. 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 5601. doi:1538-7445.AM2012-5601