Heat Shock Protein 90 Inhibitor Research Articles

HSP90 inhibitor (NVP-AUY922) enhances the anti-cancer effect of BCL-2 inhibitor (ABT-737) in small cell lung cancer expressing BCL-2

Small cell lung cancer (SCLC) cannot be efficiently controlled using existing chemotherapy and radiotherapy approaches, indicating the need for new therapeutic strategies. Although ABT-737, a B-cell lymphoma-2 (BCL-2) inhibitor, exerts anticancer effects against BCL-2-expressing SCLC, monotherapy with ABT-737 is associated with limited clinical activity because of the development of resistance and toxicity. Here, we examined whether combination therapy with ABT-737 and heat shock protein 90 (HSP90) inhibitor NVP-AUY922 exerted synergistic anticancer effects on SCLC. We found that the combination of ABT-737 and NVP-AUY922 synergistically induced the apoptosis of BCL-2-expressing SCLC cells. NVP-AUY922 downregulated the expression of AKT and ERK, which activate MCL-1 to induce resistance against ABT-737. The synergistic effect was also partly due to blocking NF-κB activation, which induces anti-apoptosis protein expressions. However, interestingly, targeting BCL-2 and MCL-1 or BCL2 and NF-κB did not induce the cytotoxicity. In conclusion, our study showed that combination of BCL2 inhibitor with HSP90 inhibitor increased activity in in vitro and in vivo study in only BCL-2 expressing SCLC compared to either single BCL2 inhibitor or HSP inhibitor. The enhanced activity might be led by blocking several apoptotic pathways simultaneously rather than a specific pathway.

Therapeutic potency of heat-shock protein-90 pharmacological inhibitors in the treatment of gastrointestinal cancer, current status and perspectives.

Heat-shock protein-90 (HSP90) chaperone machinery is critical to the folding, stability and activity of several client proteins including many responsible for tumour initiation, progression and metastasis. Overexpression of HSP90 is correlated with poor prognosis of GI cancer. Pharmacological inhibitors of HSP90 suppress tumorigenic effects of HSP90 by suppressing angiogenesis, survival, metastasis and drug resistance in GI cancer. This review summarizes the role of HSP90 inhibitors in the treatment of GI cancer. We have presented different antitumour mechanisms of HSP90 inhibitors in cancer treatment. Suppression of HSP90 signalling via specific and novel pharmacological inhibitors is a potentially novel therapeutic approach for patients with GI cancer for a better understanding and hence a better management of this disease.

Suppression by HSP90 inhibitors of BMP‑4‑stimulated osteoprotegerin synthesis in osteoblasts: Attenuation of p70 S6 kinase.

Heat shock protein 90 (HSP90) is an ATP‑dependent ubiquitous molecular chaperon which is important in cell homeostasis. The authors previously demonstrated that bone morphogenetic protein (BMP)‑4 stimulates osteoprotegerin (OPG) production in osteoblast‑like MC3T3‑E1 cells, and that p70 S6 kinase positively regulates the OPG synthesis by BMP‑4. The present study investigated the involvement of HSP90 in the BMP‑4‑stimulated OPG synthesis and the mechanism in MC3T3‑E1 cells. HSP90 inhibitors, 17‑allylamino‑17demethoxy‑geldanamycin (17‑AAG), 17‑dimethylamino‑ethylamino‑17‑demethoxy‑geldanamycin (17‑DMAG) and geldanamycin significantly suppressed the BMP‑4‑stimulated OPG release. Geldanamycin markedly reduced the BMP‑4‑induced mRNA expression of OPG. 17‑AAG and 17‑DMAG significantly attenuated the phosphorylation of p70 S6 kinase induced by BMP‑4 without affecting the BMP‑4‑induced phosphorylation of mothers against decapentaplegic homolog 1/5. The results suggest that HSP90 inhibitors suppress the BMP‑4‑stimulated OPG synthesis in osteoblasts, and that their suppressive effects are exerted through downregulating p70 S6 kinase.

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.

HSP90: a promising broad-spectrum antiviral drug target

The emergence of antiviral drug-resistant mutants is the most important issue in current antiviral therapy. As obligate parasites, viruses require host factors for efficient replication. An ideal therapeutic target to prevent drug-resistance development is represented by host factors that are crucial for the viral life cycle. Recent studies have indicated that heat shock protein 90 (HSP90) is a crucial host factor that is required by many viruses for multiple phases of their life cycle including viral entry, nuclear import, transcription, and replication. In this review, we summarize the most recent advances regarding HSP90 function, mechanisms of action, and molecular pathways that are associated with viral infection, and provide a comprehensive understanding of the role of HSP90 in the immune response and exosome-mediated viral transmission. In addition, several HSP90 inhibitors have entered clinical trials for specific cancers that are associated with viral infection, which further implies a crucial role for HSP90 in the malignant transformation of virus-infected cells; as such, HSP90 inhibitors exhibit excellent therapeutic potential. Finally, we describe the challenge of developing HSP90 inhibitors as anti-viral drugs.

Hyponatremia and V2 vasopressin receptor upregulation: a result of HSP90 inhibition

PurposeSmall-molecule inhibitors of heat-shock protein 90 (HSP90) have been under development as chemotherapeutic agents. The adverse events reported from early clinical trials included hyponatremia. Given the limited number of patients enrolled, the number of hyponatremia incidents was remarkable and repeatedly, the event was judged as severe. Inappropriate V2 vasopressin receptor stimulation is an established cause of hyponatremia. We explored the hypothesis that HSP90 inhibition produces hypersensitivity to vasopressin by upregulating V2-receptors.MethodsExperiments were carried out in cell culture using HEK293 cells with heterologous expression of the human V2-receptor and HELA cells with an endogenous V2-receptor complement. We tested the effect of HSP90 inhibition by three structurally unrelated compounds (alvespimycin, luminespib, radicicol) and asserted its specificity in cells depleted of cytosolic HSP90 (by RNA interference). Assays encompassed surface V2-receptor density and vasopressin-stimulated formation of cyclic AMP (cAMP).ResultsThe results demonstrate a twofold increase in cell-surface receptor density following pre-incubation with each of the HSP90 inhibitors. The effect had a concentration-dependence consistent with the individual potencies to inhibit HSP90. Similarly, depletion of cytosolic HSP90 increased surface-receptor density and at the same time, reduced the inhibitor effect. Upregulated V2-receptors were fully functional; hence, in culture treated with an HSP90 inhibitor, addition of vasopressin resulted in higher levels of cAMP than in controls.ConclusionSince formation of cAMP is the first signalling step in raising water permeability of the collecting duct epithelia, we suggest that V2-receptor upregulation generates hypersensitivity to vasopressin linking HSP90 inhibition to the development of hyponatremia.

A phase I trial of ganetespib in combination with paclitaxel and trastuzumab in patients with human epidermal growth factor receptor-2 (HER2)-positive metastatic breast cancer

BackgroundTargeted therapies in HER2-positive metastatic breast cancer significantly improve outcomes but efficacy is limited by therapeutic resistance. HER2 is an acutely sensitive Heat Shock Protein 90 (HSP90) client and HSP90 inhibition can overcome trastuzumab resistance. Preclinical data suggest that HSP90 inhibition is synergistic with taxanes with the potential for significant clinical activity. We therefore tested ganetespib, a HSP90 inhibitor, in combination with paclitaxel and trastuzumab in patients with trastuzumab-refractory HER2-positive metastatic breast cancer.MethodsIn this phase I dose-escalation study, patients with trastuzumab-resistant HER2-positive metastatic breast cancer received weekly trastuzumab (2 mg/kg) and paclitaxel (80 mg/m2) on days 1, 8, 15, and 22 of a 28-day cycle with escalating doses of ganetespib (100 mg/m2, 150 mg/m2, and a third cohort of 125 mg/m2 if needed) on days 1, 8, and 15. Therapy was continued until disease progression or toxicity. The primary objective was to establish the safety and maximum tolerated dose and/or recommended phase II dose (RP2D) of this therapy. The secondary objectives included evaluation of the effects of ganetespib on the pharmacokinetics of paclitaxel, and to make a preliminary assessment of the efficacy of the combination therapy.ResultsDose escalation was completed for the two main cohorts without any observed dose-limiting toxicities. Nine patients received treatment. The median prior lines of anti-HER2 therapy numbered three (range 2–4), including prior pertuzumab in 9/9 patients and ado-trastuzumab emtansine (T-DM1) in 8/9 patients. The most common grade 1/2 adverse events (AEs) were diarrhea, fatigue, anemia, and rash. There were no grade 4 AEs related to ganetespib. The overall response rate was 22% (2/9 patients had partial response) and stable disease was seen in 56% (5/9 patients). The clinical benefit rate was 44% (4/9 patients). The median progression-free survival was 20 weeks (range 8–55).ConclusionThe RP2D of ganetespib is 150 mg/m2 in combination with weekly paclitaxel plus trastuzumab. The combination was safe and well tolerated. Despite prior taxanes, pertuzumab, and T-DM1, clinical activity of this triplet regimen in this heavily pretreated cohort is promising and warrants further study in HER2-positive metastatic breast cancer.Trial registrationClinicalTrials.gov NCT02060253. Registered 30 January 2014.

HSP90 inhibitor 17-AAG plays an important role in JAK3/STAT5 signaling pathways in HTLV-1 infection cell line HUT-102

目的通过体外实验探索成人T细胞白血病（ATL）细胞内JAK/STAT信号通路的异常持续激活与HSP90蛋白的相关性。方法使用不同浓度17-烯丙胺-17-脱甲氧格尔德霉素（17-AAG）作用于HTLV-1感染ATL细胞株HUT-102，CCK8法检测细胞增殖，流式细胞术检测细胞凋亡，RT-PCR检测JAK3基因mRNA表达，Western blot检测JAK3、STAT5、p-STAT5、HSP90蛋白表达。结果HUT-102细胞株内HSP90蛋白表达量显著高于正常人外周血单个核细胞（P<0.05）。17-AAG能显著抑制HUT-102细胞增殖，17-AAG的浓度在0~2 000 nmol/L时，细胞存活率随着给药浓度的提高逐渐下降，17-AAG浓度在2 000~8 000 nmol/L范围时，细胞存活率差异无统计学意义（P>0.05）。细胞凋亡实验中，未刺激组细胞凋亡率为（9.23±0.67）%，刺激组凋亡率随着17-AAG浓度增加而显著增加，浓度2 000 nmol/L时凋亡率达到（87.82±2.46）%；但在不同浓度（0、250、500、1 000 nmol/L）抑制剂刺激HUT-102细胞24 h后，JAK3 mRNA的表达无明显变化。结论HTLV感染的ATL细胞株HUT-102细胞中，JAK3激酶作为HSP90的客体蛋白被异常活化，导致其下游信号蛋白p-STAT5异常持续性激活。17-AAG能显著下调JAK3、p-STAT5蛋白表达，抑制HUT-102细胞增殖、诱导凋亡。

Heat shock protein 90 contributes to cutaneous vasodilation through activating nitric oxide synthase in young male adults exercising in the heat.

While the mechanisms underlying the control of cutaneous vasodilation have been extensively studied, there remains a lack of understanding of the different factors that may modulate cutaneous perfusion during an exercise-induced heat stress. We evaluated the hypothesis that heat shock protein 90 (HSP90) contributes to the heat loss response of cutaneous vasodilation via the activation of nitric oxide synthase (NOS) during exercise in the heat. In 11 young males (25 ± 5 yr), cutaneous vascular conductance (CVC) was measured at four forearm skin sites that were continuously treated with 1) lactated Ringer solution (control), 2) NOS inhibition with 10 mM NG-nitro-l-arginine methyl ester (l-NAME), 3) HSP90 inhibition with 178 μM geldanamycin, or 4) a combination of 10 mM l-NAME and 178 μM geldanamycin. Participants rested in a moderate heat stress (35°C) condition for 70 min. Thereafter, they performed a 50-min bout of moderate-intensity cycling (~52% V̇o2peak) followed by a 30-min recovery period. We showed that NOS inhibition attenuated CVC (~40-50%) relative to the control site during pre- and postexercise rest in the heat (P ≤ 0.05); however, no effect of HSP90 inhibition was observed (P > 0.05). During exercise, we observed an attenuation of CVC with the separate inhibition of NOS (~40-50%) and HSP90 (~15-20%) compared with control (both P ≤ 0.05). However, the effect of HSP90 inhibition was absent in the presence of the coinhibition of NOS (P > 0.05). We show that HSP90 contributes to cutaneous vasodilation in young men exposed to the heat albeit during exercise only. We also show that the HSP90 contribution is due to NOS-dependent mechanisms.NEW & NOTEWORTHY We show that heat shock protein 90 functionally contributes to the heat loss response of cutaneous vasodilation during exercise in the heat, and this response is mediated through the activation of nitric oxide synthase. Therefore, interventions that may activate heat shock protein 90 may facilitate an increase in heat dissipation through an augmentation of cutaneous perfusion. In turn, this may attenuate or reduce the increase in core temperature and therefore the level of heat strain.

Ganetespib induces G2/M cell cycle arrest and apoptosis in gastric cancer cells through targeting of receptor tyrosine kinase signaling.

Heat shock protein 90 (HSP90) regulates several important cellular processes via its repertoire of 'client proteins'. These client proteins have been found to play fundamental roles in signal transduction, cell proliferation, cell cycle progression and survival, as well as other features of malignant cells, such as invasion, tumor angiogenesis and metastasis. Thus, HSP90 is an emerging target for cancer therapy. To this end, we evaluated ganetespib (STA-9090), a novel and potent HSP90 inhibitor, for its activity in gastric cancer cell lines. Ganetespib significantly inhibited the proliferation of AGS and N87 human gastric cancer cell lines and potently induced G2/M cell cycle arrest and apoptosis. Upregulation of cleaved poly(ADP-ribose) polymerase (c-PARP), c-caspase-3, c-caspase-8 and c-caspase-9 and suppression of gastric cancer-associated HSP90 client proteins, including ErbB2, Erk, Akt, mTOR, GSK3 and Src, were observed in ganetespib-treated cells. These findings demonstrate that the ganetespib-induced mechanism of cell growth inhibition involves the activation of death receptor and mitochondrial pathways and the inhibition of receptor tyrosine kinase signaling pathways. Our study implicates ganetespib as a potential strategy for gastric cancer treatment, which warrants further preclinical and clinical investigation.

Abstract 1330: HSP90 inhibitor has a possibility to overcome imatinib resistance in gastrointestinal stromal tumors

Abstract [Background] Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the digestive tract and are reported to harbor gain-of-function mutations in the KIT gene, which contribute to the development of sporadic GISTs. This knowledge has facilitated the development of targeted therapies with tyrosine kinase inhibitors and the revolutionary chemotherapeutic drug imatinib mesylate (IM). In clinical trials, the disease control rate was nearly 85%, and corresponding 2-year overall survival rates ranged from 70-80%, indicating markedly improved patient outcomes compared with anecdotal data in the pre-IM era. Despite its effectiveness, half of GISTs treated with IM develop resistance within 2 years, largely due to the accumulation of additional kinase domain mutations accompanied by concomitant re-activation of the KIT tyrosine kinase, even in the presence of IM. Heat shock protein 90 (HSP90) is one of chaperon molecules required for the proper folding, function, and stability of various client proteins such as KIT. The aim of this study is to clarify the efficacy of HSP90 inhibitor against IM resistant GIST. [Material and Method] We used the established human GIST cell line GIST-T1, and two IM-resistant cell lines (GIST-T1R8, GIST-T1R9), which had additional kinase domain mutations accompanied by concomitant re-activation of the KIT tyrosine kinase same as clinical samples, by exposure to IM. These resistant cell lines exhibited imatinib IC50 values (&amp;gt;10 μM) that were &amp;gt;1000-fold higher than the parental cell line. We investigated the cytotoxicity and signaling inhibition by HSP90 inhibitor using by the WST-8 assay, caspase3/7 apoptosis assay and western blotting. Immunobolts for KIT and for KIT-depending signaling pathways were observed at 12 hours of treatment. Caspase 3/7 apoptosis assay was measured after 24h. [Result] HSP90 inhibitor showed growth inhibition not only for GIST T1 but also GIST-T1R8 and GIST-T1R9. Apoptosis induction of these cell lines were also confirmed by the exposure to HSP90 inhibitor in a dose-dependent manner. To find the mechanism of apoptosis induced by HSP90 inhibitor, we investigated the signaling pathway depending on KIT. HSP90 inhibitor showed inhibition of phosphorylated-KIT, a client for HSP90, in both IM sensitive and resistant GIST cell lines. And, it also inhibited downstream of KIT signaling, e.g. p-ERK and p-AKT. [Conclusions]HSP90 inhibitor showed the anti-tumor efficacy for imatinib resistant GIST in vitro. It might have a possibility to apply for clinical use. Citation Format: Yurina Saito, Tsuyoshi Takahashi, Satoshi Serada, Minoru Fujimoto, Koji Tanaka, Yasuhiro Miyazaki, Tomoki Makino, Yukinori Kurokawa, Makoto Yamasaki, Kiyokazu Nakajima, Shuji Takiguchi, Masaki Mori, Yuichiro Doki, Tetsuji Naka. HSP90 inhibitor has a possibility to overcome imatinib resistance in gastrointestinal stromal tumors [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 1330. doi:10.1158/1538-7445.AM2017-1330

Abstract 2207: HSP90 inhibition leads to a differential proteomics profile in non small cell lung cancer cell lines

Abstract Background: The cancer chaperone, Heat Shock Protein 90 (HSP90), is known for its ability to regulate the stability of different oncogenic proteins. Thus, its overexpression has been related to unfavorable prognosis in some types of tumors. EGFR and EML4-ALK, two of the most important drivers in non-small cell lung cancer (NSCLC), are HSP90 clients and extremely depend on it. As a consequence, this chaperone is especially important in NSCLC hence HSP90 inhibition shows a lot of possibilities to future treatments in this tumor type. Nevertheless, to obtain a successful clinical development, will be essential supporting evidence of inhibitory efficacy in several molecularly defined subgroups of NSCLC. Methods: NSCLC cell lines carrying different gene mutations, whose direct (HCC827: EGFR mutated and H3122: EML4-ALK rearrangement) and indirect (A549: KRAS mutated) relationship with HSP90 has been reported, were used. In these cell lines, along with H1781 (EGFR, KRAS, ALK wild type) as control, the activity of the chaperone studied was interrupted. To this end, pharmacological inhibition of HSP90 was achieved through geldanamycin and resorcinol derivates. First, western blotting was carried out to confirm the effect of this inhibition. Later, to identify a proteomic profile associated with HSP90 inhibition, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of selected cell lines were performed. Results: The expression of the oncogenic HSP90 client proteins studied was decreased by the inhibitors in the NSCLC cell lines. The oncoproteins drivers EGFR and EML4-ALK showed a strong dependence on HSP90 observed through a high sensitivity of the cell lines HCC827 and H3122 to the inhibition. Therefore, these cell lines were selected with the purpose of identify a proteomic signature linked to HSP90 inhibition by 2D-PAGE. The untreated EGFR positive cell line presented 104 protein spots significantly up-regulated, whereas 80 spots were down-regulated compared to inhibited cell line. Meanwhile, the cell line harboring the EML4-ALK translocation showed 16 spots up-regulated and 5 down-regulated in the untreated versus inhibited cell line. In addition HSP70 overexpression, compared to unprocessed cell lines, was detected after treatments. This feedback, previously reported, confirmed HSP90 inhibition in the two cell lines studied. Conclusions: The evidence of treatment response, in the cell lines studied, was showed through oncogenic client proteins reduction as well as HSP70 induction. The proteomic profiles identified, of HSP90 inhibited and untreated tumor cells, revealed several deregulated pathways involved in the tumorigenesis. Citation Format: Angela Marrugal, Irene Ferrer, Maria Dolores Pastor, Alvaro Quintanal, Antonio Lucena-Cacace, Amancio Carnero, Luis Paz-Ares, Sonia Molina-Pinelo. HSP90 inhibition leads to a differential proteomics profile in non small cell lung cancer cell lines [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 2207. doi:10.1158/1538-7445.AM2017-2207

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

Single-walled carbon nanotubes (SWCNTs) inhibit heat shock protein 90 (HSP90) signaling in human lung fibroblasts and keratinocytes

Single-walled carbon nanotubes (SWCNTs) are carbon-based nanomaterials that possess immense industrial potential. Despite accumulating evidence that exposure to SWCNTs might be toxic to humans, our understanding of the mechanisms for cellular toxicity of SWCNTs remain limited. Here, we demonstrated that acute exposure of short (1–3μm) and regular-length (5–30μm) pristine, carboxylated or hydroxylated SWCNTs inhibited cell proliferation in human somatic and human stem cells in a cell type-dependent manner. The toxicity of regular-length pristine SWCNT was most evidenced in NP69>CYT00086>MCF-10A>MRC-5>HaCaT > HEK-293T>HepG2. In contrast, the short pristine SWCNTs were relatively less toxic in most of the cells being tested, except for NP69 which is more sensitive to short pristine SWCNTs as compared to regular-length pristine SWCNTs. Interestingly, carboxylation and hydroxylation of regular-length SWCNTs, but not the short SWCNTs, significantly reduced the cytotoxicity. Exposure of SWCNTs also induced caspase 3 and 9 activities, mitochondrial membrane depolarization, and significant apoptosis and necrosis in MRC-5 embryonic lung fibroblasts. In contrast, SWCNTs inhibited the proliferation of HaCaT human keratinocytes without inducing cell death. Further analyses by gene expression profiling and Connectivity Map analysis showed that SWCNTs induced a gene expression signature characteristic of heat shock protein 90 (HSP90) inhibition in MRC-5 cells, suggesting that SWCNTs may inhibit the HSP90 signaling pathway. Indeed, exposure of MRC-5 cells to SWCNTs results in a dose-dependent decrease in HSP90 client proteins (AKT, CDK4 and BCL2) and a concomitant increase in HSP70 expression. In addition, SWCNTs also significantly inhibited HSP90-dependent protein refolding. Finally, we showed that ectopic expression of HSP90, but not HSP40 or HSP70, completely abrogated the cytotoxic effects of SWCNTs, suggesting that SWCNT-induced cellular toxicity is HSP90 dependent. In summary, our findings suggest that the toxic effects of SWCNTs are mediated through inhibition of HSP90 in human lung fibroblasts and keratinocytes.

Epigenetic effects of inhibition of heat shock protein 90 (HSP90) in human pancreatic and colon cancer

Silencing of tumor suppressor and DNA repair genes through methylation plays a role in cancer development, growth and response to therapy in colorectal and pancreatic cancers. Heat shock protein 90 (HSP90) regulates transcription of DNA methyltransferase enzymes (DNMT). In addition, DNMTs are client proteins of HSP90. The aim of this study is to evaluate the effects of HSP90 inhibition on DNA methylation in colorectal and pancreatic cancer cell lines. Our data shows that inhibition of HSP90 using ganetespib resulted in downregulation of mRNA and protein expression of DNMT1, DNMT3A, and DNMT3B in HT-29 and MIA PaCa-2 cell lines. This in turn was associated with a drop in the fraction of methylated cytosine residues and re-expression of silenced genes including MLH-1, P16 and SPARC. These effects were validated in HT-29 tumors implanted subcutaneously in mice following in vivo administration of ganetespib. This work demonstrates the effectiveness of ganetespib, an HSP90 inhibitor in modulating DNA methylation through downregulation of DNMT expression.

TARGETING HEAT SHOCK PROTEIN 90 AND ITS DOWNSTREAM SIGNALING HUBS FOR THE TREATMENT OF MANTLE CELL LYMPHOMA

Introduction: Characterized by aberrant expression of cyclin D1, mantle cell lymphoma (MCL) is a distinct, especially aggressive, and highly heterogeneous mature B-cell lymphoma. Heat shock protein 90 (HSP90) has become an attractive therapeutic target in treating many cancers including blood cancers. Methods: In the current study, using cultured cell, xenograft mouse models and patients' peripheral blood, we sought to evaluate the effects of HSP90 inhibition and its downstream signaling hubs on mantle cell lymphoma. Results: In our study, we show that ganetespib, a clinically promising and actively investigated second-generation HSP90 inhibitor, dramatically reduced growth of mantle cell lymphoma cell lines in a dose-dependent manner. It induced G2/M cell-cycle arrest and apoptosis in Jeko-1 and Granta-519, and it significantly inhibited growth of xenograft tumors in vivo. Furthermore, a panel of signaling network proteins with a hub of HSP90 was screened for the effects of ganetespib and these pathways were studied mechanistically. Its downstream signaling hubs, common transcription factors (TFs), were identified and proved. We found inhibiting these TFs can reach similar inhibition effects as HSP90 and even decrease side effects of HSP90. Results of H&E staining, TUNEL assays, and immunohistochemistry staining of proliferation, apoptosis and other pathway relevant biomarkers in xenograft tumors revealed significant differences in tumors treated by ganetespib or by inhibiting these TFs. In addition, lymphoma cells from patients' samples from peripheral blood were tested by ganetespib or by inhibiting these TFs. Conclusions: In summary, our data suggest that targeting downstream TFs of HSP90 and HSP90 itself can be potentially used for the molecularly targeted therapy of MCL patients. Keywords: mantle cell lymphoma (MCL).

17-Allylamino-17-demethoxygeldanamycin-induced Changes in [18F]Fluorothymidine Uptake.

The proliferation status of tumor cells can be imaged using [18F]fluorothymidine ([18F]FLT), which is trapped by cell cycle-dependent thymidine kinase 1 (TK1). Targeting of heat shock protein 90 (HSP90) disrupts multiple client proteins, leading to heterogeneous cell-cycle phenotypes. To investigate whether [18F]FLT uptake reflects the growth arrest caused by various mechanisms of HSP90 inhibition, we used HCT116 cells that were arrested at G0/G1 phase, and Hep3B cells at G2/M phase, by the HSP90 inhibitor 17-AAG. In HCT116 cells, 17-AAG did not induce significant changes in [18F]FLT uptake despite the decreased expression of TK1, cyclin A, and cyclin B. The mRNA level of 5'3'-deoxynucleotidase 1 (NT5C), which antagonizes TK1 by de-phosphorylating [18F]FLT monophosphate, was also decreased in 17-AAG-treated HCT116 cells by 55.3±18.1% compared to vehicle-treated cells. In Hep3B cells, 17-AAG treatment did not induce TK1 expression, TK1 activity nor [18F]FLT uptake. Nucleoside transporter activity in the plasma membrane was unchanged in both cell lines. These results showed that a HSP90 inhibitor exerts multifaceted effects on [18F]FLT uptake before inducing cell death in a cell-cycle-independent manner. Therefore, the use of [18F]FLT-positron emission tomography for monitoring treatment by HSP90 inhibitor would be more appropriate when tumor cell death is induced after growth arrest.

Drug-Induced HSP90 Inhibition Alleviates Pain in Monoarthritic Rats and Alters the Expression of New Putative Pain Players at the DRG.

Purinergic receptors (P2XRs) have been widely associated with pain states mostly due to their involvement in neuron-glia communication. Interestingly, we have previously shown that satellite glial cells (SGC), surrounding dorsal root ganglia (DRG) neurons, become activated and proliferate during monoarthritis (MA) in the rat. Here, we demonstrate that P2X7R expression increases in ipsilateral DRG after 1week of disease, while P2X3R immunoreactivity decreases. We have also reported a significant induction of the activating transcriptional factor 3 (ATF3) in MA. In this study, we show that ATF3 knocked down in DRG cell cultures does not affect the expression of P2X7R, P2X3R, or glial fibrillary acidic protein (GFAP). We suggest that P2X7R negatively regulates P2X3R, which, however, is unlikely mediated by ATF3. Interestingly, we found that ATF3 knockdown in vitro induced significant decreases in the heat shock protein 90 (HSP90) expression. Thus, we evaluated in vivo the involvement of HSP90 in MA and demonstrated that the HSP90 messenger RNA levels increase in ipsilateral DRG of inflamed animals. We also show that HSP90 is mostly found in a cleaved form in this condition. Moreover, administration of a HSP90 inhibitor, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), attenuated MA-induced mechanical allodynia in the first hours. The drug also reversed the HSP90 upregulation and cleavage. 17-DMAG seemed to attenuate glial activation and neuronal sensitization (as inferred by downregulation of GFAP and P2X3R in ipsilateral DRG) which might correlate with the observed pain alleviation. Our data indicate a role of HSP90 in MA pathophysiology, but further investigation is necessary to clarify the underlying mechanisms.

Proteomic analysis of proteome and histone post-translational modifications in heat shock protein 90 inhibition-mediated bladder cancer therapeutics.

e16030 Background: Heat shock protein 90 (HSP90) is a chaperone for many proteins integral development of cancer, making inhibition of HSP90 an attractive strategy for cancer treatment. However, little is known about HSP90 inhibition-mediated bladder cancer therapy. Here, we present a quantitative proteomic study that evaluates alterations in protein expression and histone post-translational modifications (PTMs) in bladder cancer in response to HSP90 inhibition. Methods: We assessed the antitumor activity of five HSP90 inhibitors in 5637 bladder cancer cell line using MTS, cell viability, and caspase 3/7 assays. We then performed a proteomic analysis of cells before and after treatment with the HSP90 inhibitors and assessed for changes. Results: We show that the five HSP90 inhibitors (AUY922, ganetespib, SNX2112, AT13387, and CUDC305) potently inhibited the proliferation of bladder cancer 5637 cells. HSP90 inhibitors also had differential effects on cell survival between bladder cancer cells and uroepithelial cells. Our proteomic study quantified 518 twofold up-regulated and 811 twofold downregulated proteins common to both AUY922 and ganetespib treatment (P &lt; 0.05). The subsequent bioinformatic analysis revealed that those differentially expressed proteins were involved in chromatin modifications and cell death-associated pathways. Further, quantitative proteome studies identified 14 different types of PTMs with 93 marks on the core histones, including 34 novel histone marks of butyrylation, citrullination, 2-hydroxyisobutyrylation, propionylation, and succinylation in AUY922- and ganetespib-treated 5637 cells, suggesting that HSP90 inhibitors may exert multiple cytotoxic actions in bladder cancer cells by inhibiting HSP90 activity or altering the structure of chromatin. Conclusions: This study outlines the association between proteomic changes and histone PTMs in response to HSP90 inhibitor treatment in bladder cancer cells, and supports the notion that HSP90 inhibitors both as single agents and in combination might provide new therapeutic options for bladder cancer treatment and thus warrant further preclinical exploration.

Phase Ib study of heat shock protein 90 inhibitor, onalespib in combination with paclitaxel in patients with advanced, triple-negative breast cancer (NCT02474173).

TPS1127 Background: Heat shock protein 90 (HSP90) is a molecular chaperone which is necessary for proper folding and stabilization of proteins. Client proteins of HSP90 include many oncogenic proteins known to be over-activated in triple negative breast cancer such as AKT, EGFR, members of RAS/MAPK signaling pathway and androgen receptor. High expression of HSP90 in breast cancer has been associated with poor outcome. In addition, over-expression of HSP90 client proteins such as AKT and c-RAF has been implicated in paclitaxel resistance. Onalespib (AT13387) is a synthetic non-ansamycin small molecule that acts as an inhibitor of HSP90 by binding to the amino terminal of the protein and has dissociation constant (Kd) of 0.71 nM. Methods: Patients with inoperable or metastatic, triple negative or &lt; 10% hormone receptor positive breast cancer are treated with onalespib and paclitaxel on days 1, 8, 15 every 28 days. Paclitaxel is given at a standard dose of 80 mg/m2 while the dose of onalespib is gradually increased using standard 3+3 design (see table). In order to assess the effect of each drug on pharmacokinetics of the other drug, onalespib is given on day -7 prior to cycle 1 and skipped on day 1 of cycle 1 during which paclitaxel is administered alone. The primary objective of the study is to determine recommended phase II dose and assess the toxicity profile of the combination. The secondary objectives include pharmacokinetic of each agent. Overall response rate, response duration and progression-free survival will also be assessed. The study is currently enrolling patients to dose level 2. Clinical trial information: NCT02474173. [Table: see text]