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. Author manuscript; available in PMC: 2018 Oct 15.
Published in final edited form as: Clin Cancer Res. 2017 Jul 5;23(20):6215–6226. doi: 10.1158/1078-0432.CCR-16-3151

Efficacy of Onalespib a Long-acting Second Generation HSP90 Inhibitor as a Single Agent and in Combination with Temozolomide against Malignant Gliomas

Alessandro Canella 1,2, Alessandra M Welker 3, Ji Young Yoo 2, Jihong Xu 1,2, Fazly S Abas 4, Divya Kesanakurti 1,2, Prabhakaran Nagarajan 1,2, Christine E Beattie 3, Erik P Sulman 5, Joseph Liu 2, Joy Gumin 6, Frederick F Lang 6, Metin N Gurcan 4, Balveen Kaur 2, Deepa Sampath 7,§, Vinay K Puduvalli 1,2,§
PMCID: PMC5986078  NIHMSID: NIHMS890752  PMID: 28679777

Abstract

Purpose

HSP90, a highly conserved molecular chaperone that regulates the function of several oncogenic client proteins is altered in glioblastoma. However, HSP90 inhibitors currently in clinical trials are short-acting, have unacceptable toxicities or are unable to cross the blood brain barrier (BBB). We examined the efficacy of onalespib, a potent, long-acting novel HSP90 inhibitor as a single agent and in combination with temozolomide (TMZ) against gliomas in vitro and in vivo.

Experimental Design

The effect of onalespib on HSP90, its client proteins, and on the biology of glioma cell lines and patient-derived glioma-initiating cells (GSC) was determined. Brain and plasma pharmacokinetics of onalespib and its ability to inhibit HSP90 in vivo was assessed in non-tumor bearing mice. Its efficacy as a single agent or in combination with TMZ was assessed in vitro and in vivo using zebrafish and patient derived GSC xenograft mouse glioma models.

Results

Onalespib mediated HSP90 inhibition depleted several survival-promoting client proteins such as EGFR, EGFRvIII and AKT, disrupted their downstream signaling and decreased the proliferation, migration, angiogenesis and survival of glioma cell lines and GSCs. Onalespib effectively crossed the BBB to inhibit HSP90 in vivo and extended survival as a single agent in zebrafish xenografts and in combination with TMZ in both zebrafish and GSC mouse xenografts.

Conclusions

Our results demonstrate the long-acting effects of onalespib against gliomas in vitro and in vivo which combined with its ability to cross the BBB support its development as a potential therapeutic agent in combination with TMZ against gliomas.

Keywords: HSP90, malignant glioma, onalespib, HSP90 inhibitors, temozolomide

Introduction

Glioblastomas (GBM) are malignant primary brain tumors characterized by notable resistance to conventional chemotherapy and radiation therapy resulting in tumor recurrence and an overall median survival of about 16–18 months despite aggressive therapy (1, 2). GBM exhibits a marked heterogeneity of genetic and epigenetic alterations which influences tumor resistance and clinical outcome (3, 4). In addition, the presence of chemotherapy and radiation-resistant glioma-initiating cells (GSC) within GBM further contributes to the heterogeneity and highly malignant nature of these tumors (5). Significantly, both inter- and intra-tumoral driver events converge on a few key pathways involving EGFR, AKT, and ERK indicating that these pathways are central to tumorigenesis in GBM (6, 7). However, targeting individual pathways in GBM has largely failed to yield the expected improvement in outcome (8) suggesting that strategies to target GBMs may require an alternative approach that simultaneously blocks multiple pathways critical to their growth and survival. One such strategy involves targeting mechanisms of protein processing that are highly active in tumors compared to normal cells and simultaneously disabling several key components of the signaling pathways essential for GBM growth (9).

Heat shock protein 90 (HSP90) is an evolutionarily highly conserved molecular chaperone which functions predominantly as a cytoplasmic protein responsible for ATP-dependent folding and activation of client proteins and is overexpressed in cancer at levels up to 2 to 10 fold higher than in normal cells (10). A large number of receptor and protein kinases and DNA damage repair/response proteins depend on HSP90 for their function (11–13). Tumor cells respond to chemo, radiation or targeted therapy by adaptively activating DNA repair pathways and by oncogenic reprograming that switches tumor dependence to alternative kinase pathways, by a process facilitated by HSP90 (13, 14). This suggests that targeting HSP90 could potentially overcome these resistance mechanisms and improve sensitivity to therapies (15, 16). Currently, several HSP90 inhibitors are being evaluated in trials against solid tumors and are in different stages of clinical development (17–19). They bind to the N-terminal ATPase-pocket, affecting the chaperone activity and leading to the depletion of HSP90 substrates by subsequent proteosomal degradation (11–13). Recent studies in several solid tumor models have shown sensitization to currently used chemotherapeutic agents when combined with new generation HSP90 inhibitors (20, 21). Onalespib, a potent second-generation HSP90 inhibitor differs from other HSP90 inhibitors in its longer duration of target inhibition (22) and its favorable toxicity profile in phase I studies in patients with refractory solid tumors (17). However, there is no significant preclinical data regarding the effects of onalespib against glioblastoma; in addition, the potential for chemosensitization of onalespib in combination with alkylating agents such as temozolomide (TMZ) has not been previously studied.

In this study, we characterized the anti-tumor activity of onalespib in glioma cell lines and glioma initiating cells (GSC) and provide evidence that its use in combination with TMZ causes a synergistic anti-proliferative effect in malignant glioma. The study also examines the ability of the drug to cross the blood brain barrier (BBB) and to inhibit in vivo tumor progression in combination with TMZ.

Methods

Cell lines and reagents

The human glioma cell lines, LN229 and A172, were obtained from ATCC (Manassas, VA). U251HF glioma cells were kindly provided by Dr. W. K. Alfred Yung (M. D. Anderson Cancer Center, Houston, TX); U251HF-Luc were generated by stably transfecting a firefly luciferase gene under a CMV promoter into U251HF cells and maintained as described with the addition of Geneticin (G418–800μg/ml) for U251HF-Luc cells (23). GSC2, GSC20, GSC11, GSC23 and GSC811 patient-derived glioma stem cell lines (24) were kindly provided by Dr. Frederick Lang (M. D. Anderson Cancer Center, Houston, TX) and cultured as neurospheres passaged every 5–7 days in serum-free DMEM/F12 medium containing B-27 Supplement (Life Technologies, Carlsbad, CA), bFGF (FGF2 Gold BioTechnology, St. Luis, MO) and EGF (Gold Bio Technology). Cell lines were authenticated at the University of Arizona Genetics Core (http://uagc.arl.arizona.edu/services/cell-line-authentication-human). Onalespib (AT13387) was purchased from Medkoo Biosciences (Morrisville, NC).

Cytotoxicity assays. WST-1

Glioblastoma cells were seeded into 96 well plates and exposed to vehicle (PBS), onalespib, TMZ or a combination of onalespib and TMZ for 24–96 h. Glioma and GSC lines were exposed to a 0.1 – 0.8 μM onalespib for varying times. Cell viability was measured using the WST-1 assay as per manufacturer’s instructions (Roche, Indianapolis, IN). Annexin PI: LN229, U251HF and A172 cells were exposed to increasing concentrations of onalespib following which annexin assays were conducted by incubating cells with annexin V–fluorescein isothiocyanate and propidium iodide (BD Biosciences) for 15 minutes and accumulating the fluorescence of at least 10,000 cells on a BD Biosciences FACSCalibur flow cytometer to determine the percentage of apoptotic and viable cells.

Colony forming assays

LN229, U251HF and A172 cell lines were treated with onalespib or vehicle (PBS) for 24 h and seeded at 1000 cells/well in triplicate. After 7–10 days colonies were fixed, stained with crystal violet and counted using Gelcount (Oxford Optronix, Oxford, UK). The Compusyn software (ComboSyn, Paramus, NJ) was used to determine the combinatorial index (CI).

Immunoblotting

Immunoblotting was performed with antibodies against HSP90, HSP70, p-AKT (Ser 308), AKT, pERK1/2 (Thr 202/Tyr 204), ERK 1/2, pS6 (Ser 240/244), S6, GAPDH (Cell Signaling Technology, Danvers, MA) pEGFR, EGFR, and anti-mouse and anti-rabbit IgG-HRP as secondary antibodies (GE Healthcare, Piscataway, NJ).

In vitro migration and angiogenesis assays

LN229, U251HF and A172 cells were seeded in triplicate on 6-well dishes. Upon reaching 80% confluence, they were exposed to vehicle or onalespib for 24 h after which a scratch was made and images taken at various time points using an AxioScope A1 (Zeiss, Thornwood, NY). The degree of migration was determined using the Image J software (https://imagej.nih.gov/ij/). In vitro angiogenesis assays were performed as described (25) and the formation of capillary-like structures were captured as images using a Nikon microscope attached to a CCD camera.

Intracranial Glioma xenografts

Animal experiments were conducted under a protocol approved by the Institutional Animal Safety Committee in compliance with the Humane Care and Use of Laboratory Animals Policy. Nude mouse intracranial glioma xenografts were generated by implanting U251HF-Luc cells (106 cells/5 μl) into the forebrain as described (26), after which luciferin solution (100mg/kg) was injected and tumor growth was determined by measuring the bioluminescence emitted using a ZFOV-24 zoom lens-installed IVIS Lumina Series III Pre-clinical In Vivo Imaging System (Perkin Elmer, American Fork, UT).

Confocal microscopy

Glioma cells isolated from normal brain or GBM xenografts were seeded at 4 × 105 on chamber slides, fixed in ethanol and blocked with 1%(w/v) BSA before being incubated with HSP90 antibody (Cell Signaling Technology, Danvers, MA), and subsequently incubated with fluorochrome (Cy3) tagged anti rabbit secondary antibodies. Nuclei were stained with DAPI, following which slides were washed and optical sections collected using an Olympus FV1000 confocal microscope equipped with three lasers and 100X oil immersion objective. Image processing was conducted using Olympus Fluoview software.

Immunohistochemistry and high resolution image analysis

Formalin-fixed, paraffin-embedded tissue from normal brain tissue or GBM xenografts were cut into 15 μm thick coronal sections for immunohistochemical staining. Sections were deparaffinized in xylene, dehydrated in ethanol and rinsed with distilled water before incubation with antibodies against HSP90 (Cell Signaling Technology, Danvers, MA) or HSP70 (Enzo Life Science, Farmingdale, NY) which were visualized using the a DAB staining kit (Roche-Ventana, Tucson, AZ) according to the manufacturer’s instructions followed by counterstaining with hematoxylin. High resolution images were acquired and analyzed as detailed in supplemental methods.

Pharmacokinetics of onalespib

ICR wild type mice were injected with a single intravenous tail vein bolus dose of onalespib (30 mg/kg) following which brain tissue and plasma were harvested at various time points after injection (5 min, 10 min, 20 min, 30 min, 1 hr, 2 hr, 4 hr, 6 hr, 8 hr and 24 h), lysed and analyzed by LC-MS-MS (TSQ Quantum Discovery Max mass spectrometer, Thermo Scientific, Waltham, MA) in the Ohio State University Pharmacoanalytic Shared Resource.

Zebra fish in vivo studies

U251HF-GFP cells, grown to 70–80% confluence were transplanted (25–50 cells/animal) in the vicinity of the midbrain-hindbrain boundary of 36 hours post-fertilization tricaine-anesthetized zebrafish embryos (Sigma-Aldrich, St Louis, MO) and larvae screened at 24 h post-transplantation to determine implantation (27). Five days after transplantation (5 dpt) animals (24 animals/group) were randomized to vehicle (DMSO), onalespib at 0.5 μM, temozolomide (TMZ) at 10 μM or a combination of onalespib and TMZ in fish water for 5 days (from 5–10 dpt). After 5 days of treatment, tumor burden was assessed by imaging live fish on an Andor spinning disc confocal microscope as described previously (27).

Mouse GSC xenograft in vivo studies

Human patient derived GS811 neurospheres were dissociated and stereotactically implanted (0.8 × 106 cells in 5μl) into the right frontal lobe of 7–8-week-old female athymic nude mice (Charles River, Wilmington, MA). Fifteen days post implantation, mice were randomized to vehicle (15% Captosil, San Diego, CA) or treatment cohorts of temozolomide (4mg/kg), onalespib (30mg/kg) or a combination with temozolomide and onalespib. Temozolomide was administered by oral gavage daily for 5 consecutive days and onalespib by tail vein injection twice (day 16 and 19). Animals were observed daily and euthanized when they showed signs of morbidity (hunched posture and weight loss).

Statistics

All in vitro experiments were conducted at least three times independently and expressed as mean ± standard error (SEM). Comparisons between groups in vitro and in vivo were performed using two-tailed t-tests, and comparisons between multiple groups were performed using 1-way analysis of variance (ANOVA). In vivo experiments with zebrafish and mouse were analyzed for survival with the Kaplan Meier method and the significance evaluated by ANOVA.

Results

HSP90 is expressed in the cytoplasm of glioma xenograft cells

HSP90 is known to be overexpressed in several solid and hematological malignancies (10). To assess the expression levels of HSP90 in glioma versus non-tumor tissue, U251HF-luc cells were implanted into the forebrain of nude mice to generate bioluminescent intracranial glioma xenografts (Fig. 1A). Hematoxylin-eosin (H & E) staining (Fig. 1B, upper panels) was used to classify tissue sections as non-tumor (NTB) or tumor bearing (GBM) areas. Both non-tumor and glioma-containing sections were assessed for HSP90 expression by immunocytochemistry and immunofluorescence. HSP90 was robustly expressed in tumor tissue compared to non-tumor brain (Fig. 1B lower panel). Additionally, using confocal microscopy, HSP90 expression was seen to be predominantly cytoplasmic in location in the tumor cells (Fig. 1C). These results confirm the in vivo cytoplasmic overexpression of HSP90 in human glioma xenografts compared with adjacent non-tumor mouse brain.

Fig. 1. Expression of HSP90 in normal or glioma tissue in vivo and consequence of HSP90 inhibition on HSP70 in glioma cell lines.

Fig. 1

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A) Nude mice were intracranially implanted with 106 U251HF-Luc cells in the right frontal lobe and tumor growth was determined by measuring bioluminescence intensity 4 weeks after the tumor implantation (n=4). B) Non-tumor brain (NTB) and tumor (GBM) tissues sections from one of the intracranial xenograft containing mouse were assessed with H&E (upper panel) and evaluated for HSP90 expression by immunocytochemistry (lower panel). C) Normal and tumor tissue sections were prepared as above and were labeled with DAPI (nuclear staining) and HSP90 to determine the cellular localization of HSP90 confocal microscopy. D) HSP90 and HSP70 protein expression (upper panel) were evaluated by immunoblotting after treatment with onalespib 0.1, 0.2 and 0.4μM for 48h on LN229, U251HF and A172 cell lines; GAPDH was used as loading control (lower panel). Immunoblots are representative of three independent experiments and DMSO was used as vehicle control.

HSP90 inhibition by onalespib inhibits proliferation, survival, colony formation and migration in glioma cell lines

Onalespib inhibits HSP90 activity by blocking its chaperone function (28) which is associated with a reciprocal increase in the levels of HSP70, which appears to be a class effect of all HSP90 inhibitors and has been used as a biomarker of HSP90 inhibition (13). Exposure of LN229, U251HF and A172 cells to increasing concentrations of onalespib resulted in HSP90 inhibition as measured by the characteristic increase in HSP70 levels in cells in a dose-dependent manner (Fig. 1D).

Gliomas are characterized by enhanced proliferation, survival, and cellular migration which contribute to their poor prognosis (29–31). We hence examined the effects of onalespib on these characteristics in LN229, U251HF and A172 cells and found a dose- and time-dependent decrease in proliferation as measured by the WST assay in all cell lines tested, with an IC50 ≤0.25 μM and with maximal inhibition seen at 72 h (Fig. 2A). Inhibition of growth was paralleled by a dose-dependent activation of caspase-3 and caspase-7 (Supp. Fig. 1A) and increase in the percentage of annexin positive cells (Fig. 2B) in response to onalespib. Correspondingly, colony forming assays demonstrated that exposure of LN229, U251 and A172 cells to 0.4 μM onalespib for 24 h significantly inhibited the proliferative capacity of glioma cell lines (Fig. 2C). These results show a broad and sustained in vitro antitumor activity by onalespib in several different glioma cell lines,

Fig. 2. Onalespib exposure inhibits the proliferation, survival and migration of glioma cells.

Fig. 2

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A) LN229, U251HF and A172 cells were exposed to vehicle control (DMSO) or increasing concentrations of AT13397 (μM) for 72 h, following which proliferation was measured using the WST1 assay. B) LN229, U251HF and A172 cells were exposed to onalespib as indicated (μM) for 48h following which the percentage of annexin V positive cells were measured. C) LN229, U251HF and A172 cells were exposed to 0.4μM onalespib flowing which the drug was washed out and assayed for colony formation after 10 days. ***p<0.001 (two-tailed t-test). D) LN229, U251HF and A172 cells were exposed to 0.4μM onalespib for 24 h, the conditioned medium (CM) was then added to endothelial cells plated in a matrigel and assayed for microtubule formation as a measure of angiogenesis in vitro. **p<0.01***p<0.001 (two-tailed t-test) (E). LN229, U251HF and A172 cells were exposed to 0.4μM onalespib for 24 h before being assayed for cell migration using the wound healing assay. The upper panel shows representative results obtained in U251HF cells and the graph below quantitates the results obtained in all 3 cell lines ***p<0.001, ****p<0.0001 (two-tailed t-test). Each of the above experiments was conducted three independent times in triplicate and DMSO was used as control.

GBMs are also characterized by neoangiogenesis and invasion, which is a hallmark of these tumors and is associated with poorer prognosis (31). Quantitation of the microtubules formed by endothelial cells after exposure to media conditioned (CM) by glioma cells was used as a surrogate of tumor mediated angiogenesis (25, 31) and showed that exposure to 0.4 μM onalespib for 24 h strongly inhibited the number of microtubules formed in response to CM in LN229, U251HF and A172 cells (Fig 2D). Further, when we examined the effect of onalespib on glioma cell migration in vitro using a wound healing assay, U251HF, LN229 and A172 cells treated with 0.4 μM onalespib showed reduced ability to migrate compared to untreated controls (Fig 2E and Supp. Fig. 1B). Taken together, these data provided the evidence that onalespib-induced HSP90 inhibition also inhibits the proliferation, migration, angiogenic potential and survival of glioma cells.

Onalespib inhibits the EGFR-AKT-ERK-S6 signaling network in glioma cell lines

Recent evidence indicated that the cell death response to HSP90 inhibitors does not fully correlate with degradation of individual oncogenic clients but instead may depend on the inhibition of entire signaling networks driven by key client proteins (32). In this context, amplification and mutations of EGFR (e.g. EGFRvIII), leading to its constitutive activation, is a signature abnormality in GBM that facilitates cell survival and chemo-resistance by activating the AKT, ERK and S6 signaling (33, 34). Hence, we examined the impact of onalespib on the EFGR signaling pathway in gliomas.

Exposure of U251HF, LN229 and A172 cells to onalespib for 48 h resulted in a robust dose-dependent decrease in the levels of EGFR and p-EGFR and its downstream signaling intermediaries, phospho- and total AKT (also a HSP90 client), phospho- and total ERK1/2 and phospho- and total S6 (Fig. 3A). Of particular note, HSP90 inhibition was equally effective at depleting mutant EGFRvIII and its downstream intermediates (Fig. 3B). In a parallel time-course experiment, exposure to 0.4 μM onalespib resulted in a time-dependent decline in the HSP90 client EGFR, with accompanying losses in p-EGFR as well as an inhibition of their downstream signaling intermediaries phospho- and total AKT, phospho- and total ERK1/2 and phospho- and total S6 by 24 h in each cell line tested (Fig. 3C). These results point to a profound impact on the entire EGFR signaling pathway in glioma cells upon onalespib treatment.

Fig. 3.

Fig. 3

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Effect of the onalespib treatment on the EGFR pathway in LN229, U251HF and A172 cell lines. A) LN229, U251HF and A172 cells were exposed to 0.1, 0.2 and 0.4 μM onalespib for 48 h before being evaluated for the expression of EGFR, pEGFR, AKT, pAKT, ERK1/2, p-ERK1/2, S6, p-S6 by immunoblotting. GAPDH was assayed as a loading control. (B). U87 cells expressing mutant EGFRVIII were exposed to 0.1, 0.2 and 0.4μM onalespib before being assayed for the expression of EGFRvIII, HSP90, HSP70, AKT, p-AKT, ERK1/2, p-ERK1/2 and GAPDH. (C). LN229, U251HF and A172 cells were exposed to 0.4 μM onalespib for upto 24 h and assayed for the expression of EGFR, pEGFR, AKT, pAKT, ERK, p-ERK, S6, p-S6 and GAPDH by immunoblotting. Immunoblots are representative of three independent experiments with DMSO used as vehicle control.

Onalespib depletes pro-survival client proteins and stem cell markers in glioma stem cells (GSC)

Glioma stem cells (GSCs) represent a subpopulation of glioma cells postulated to contribute to treatment resistance and to have the ability to repopulate the tumor causing disease recurrence (35). GSC cell lines established from primary tumors recapitulate patient specific disease phenotypes and thus are valuable as preclinical models (36). Cell lines derived from GBMs (GSC11 and GSC23) when exposed to onalespib, exhibited a characteristic increase in HSP70 levels (Fig. 4A). Given the relevance of the EGFR signaling to GSC (37), we determined the effects of onalespib on the EGFR pathway in GSC cells. Exposure to onalespib for 48 h led to steep declines in the levels of total EGFR, pEGFR and an inhibition of its signaling via the AKT-ERK and S6 axis suggesting a potent effect against this key signaling pathway in GSCs (Fig. 4A). In parallel, inhibition of HSP90 in GSC11 and GSC23 cells was also associated with a dose-dependent decrease in the expression of the stem cell markers CD133 (Supp. Fig. 2A) and Nestin (Supp. Fig. 2B) with accompanying decreases in the levels of Olig2 which has been reported to be highly expressed in GSC (38) (Supp. Fig 2B).

Fig. 4.

Fig. 4

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Effect of onalespib on the HSP90, stem cell markers, oncogenic client proteins and survival of patient derived glioma initiating cells. (A) Patient-derived glioma initiating neurosphere cultures (GSC11 and GSC23) were exposed to onalespib (μM) or DMSO for 48h and immunoblotted for HSP90 and HSP70, EGFR, pEGFR, AKT, pAKT, ERK, p-ERK, S6, p-S6 and GAPDH (loading control) levels. Immunoblots are representative of three independent experiments. (B). Patient derived GSC lines were classified based on their molecular subtype into Mesenchymal (MES) or proneural (PN). (C) MES (GSC2 and GSC20) and PN (GSC11 and GSC23) GSC lines were exposed to increasing concentrations and times of onalespib before being assayed for viability using the WST-1 assay in all four GSC lines *p<0.05. Data represents the mean ± SEM of triplicate values from two independent experiments. (D) MES and PN GSC lines were exposed to increasing concentrations and times of onalespib before being assayed for phospho and total Stat3, GAPDH assayed as loading control. Immunoblot representative of three independent experiments.

Gene expression analysis has revealed that GBMs can be classified into classical, mesenchymal (MES) or proneural (PN) subtypes; of these, PN tumors exhibit the most favorable prognosis whereas MES subtypes exhibit are more resistant to treatment and have the most aggressive phenotype (3). To examine if there were differences in response of these subtypes to HSP90 inhibition, we tested the efficacy of onalespib against MES or PN GSCs (39). Exposure of MES (GSC2 and GSC20) and PN (GSC11 and GSC23) GSC lines (Fig. 4B) to onalespib showed a dose- and time-dependent decrease in survival (Fig. 4C) irrespective of molecular subtype. However, at the highest concentrations of onalespib both MES-GSC lines lost viability to 50% whereas the response of PN-GSCs was variable with loss of viability from 50% in GSC23 to over 90% in GSC11 cells.

While EGFR activation is necessary for the survival of both bulk tumor cells and GSCs, phosphorylation and activation of STAT3 appears to occur preferentially in GSCs and increases the tumorigenic potential of the GSC population (40). p-STAT3 was clearly evident in MES-GSC and less apparent in PN-GSCs; however exposure to onalespib for 48h inhibited p-STAT3 in both GSCs (Fig. 4D). Finally, exposure to onalespib markedly decreased the invasive ability of each of these cell lines when measured using the transwell migration assay (Supp. Fig. 2 C) Taken together, these results confirm the inhibitory effects of onalespib against glioma tumor initiating cells. They also support the likelihood that onalespib induced antitumor activity is not dependent to gene expression based subtypes of GBM.

Onalespib crosses the blood-brain barrier (BBB) and causes sustained inhibition of HSP90

A major requirement for agents targeting gliomas is their ability to cross the BBB and achieve target inhibition; the lack of transport of most chemotherapeutic and targeted agents across the BBB is a known reason for their lack of activity against gliomas (41). Hence, we assessed the ability of onalespib to cross the BBB in non-tumor bearing nude mice (with intact BBB) by intravenously injecting a single dose of onalespib at a dose of 30 mg/kg following which brain tissue and plasma samples were collected at various time points and analyzed by LC-MS/MS to determine onalespib concentration. As expected, the Cmax of onalespib was higher in plasma compared with brain tissue in the early time points after intravenous injection. However, levels of onalespib were found to be higher in brain tissue compared to the plasma at each time point analyzed from 2 h up to 24 h indicating the ability of the drug to cross the BBB and achieve higher than plasma levels (Fig. 5A).

Fig. 5.

Fig. 5

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Pharmacokinetics and pharmacodynamics of onalespib in mouse brain tissues. A) 6 week old ICR mice were injected with a single dose of onalespib (30mg/kg by tail vein). Brain and plasma tissues were collected at 5 min,10 min, 20min, 30 min, 1h, 2h, 4h,8h and 24h (3 mice/time point) and subjected to LC-MS/MS to measure the levels of onalespib (nM) in brain and plasma tissues. B) Brain tissue sections were assayed for the levels of Hsp70 protein by IHC, high resolution images obtained by high resolution microscopy and converted into heat maps to quantitate the intensity of HSP70 staining across the tissue section, a representative image of which is shown. C) Mice were injected with onalespib (30mg/kg) or vehicle (PBS) and brain tissue collected at various time points. Whole brain tissue sections (1 section/mouse, 3 mice/time point) were prepared from onalespib treated mice at 0, 1, 2, 4, 8 and 24h and from vehicle-treated mice at 0 and 24h and subjected to HSP70 immunohistochemistry with DAB (3,3″-diaminobenzidine). Subimages (500 × 500 regions of interest) were obtained from each digitized tissue section images and the whole image mean brown pixel intensity of HSP70 signal was determined. The area x intensity of signal was measured as whole image mean brown pixel intensity and background x % light brown pixels and plotted against time as shown. Representative IHC for HSP70 from onalespib and vehicle treated mice are shown in the right panel.

In parallel, normal brain tissue sections obtained from mice intravenously injected with 30 mg/kg of onalespib or from untreated controls were evaluated for evidence of HSP90 inhibition by measuring the levels of HSP70 by immunohistochemistry (IHC). The IHC image was then scanned and digitized to enable an unbiased quantitation of the intensity as well as the percentage of DAB stained HSP70 positive light brown pixels within the 100 × 100 region of interest (ROI). This data was also converted into a color-coded population maps (heat maps) which were used to visually locate positive areas on whole-slide images (Fig. 5B). Our data indicate that onalespib exposure resulted in successful HSP90 inhibition within brain tissues in vivo; however, the HSP70 staining also demonstrated heterogeneity across tissue sections with some areas showing dense HSP70 pixels and others a variable intensity (Fig. 5B). Quantitation of the pixel intensity demonstrated the increase in the percentage and intensity of HSP70 pixels over time in onalespib treated mice in comparison to the vehicle treated mice (Fig. 5C).

Onalespib synergizes with temozolomide in vitro and in vivo

The standard of care first-line treatment for glioblastoma consists of radiation therapy with concurrent and adjuvant administration of temozolomide (TMZ), an alkylating agent (2). Resistance mechanisms that lead to tumor progression during alkylator therapy include the repair of TMZ-induced monofunctional alkylation and single strand DNA lesion by methyl-guanyl methyltransferase (MGMT) and repair of double stranded DNA breaks (DSB) by specific DNA repair enzymes in glioma cells and in GSCs (42). Consequently, therapeutic strategies that can potentiate the activity of TMZ are of high clinical relevance. To determine whether exposure to onalespib would sensitize glioma cells to TMZ, we treated U251HF, LN229 and A172 glioma cells with various concentrations of onalespib alone, TMZ alone or a combination of onalespib and TMZ (Supp. Fig 3A) and determined the combinatorial index (CI) of the treatments (43); the combination of onalespib and TMZ was synergistic in the U251 and LN229 cells with a CI of 0.11± 0.06 and 0.7± 0.1 respectively and was additive in the A172 cell line (CI=1.0 ± 0.16). The IC50 for the combination was achieved by 48 h of drug exposure in LN229 and U251 but between 48–72 h in A172 cells (Supp. Fig 3B). These results demonstrate that the combination of onalespib and TMZ is synergistic or additive in vitro in glioma cells.

To examine whether the combination of onalespib and TMZ was synergistic in vivo, we employed two distinct glioma xenograft models. The first was an orthotopic intracranial xenograft glioma model in zebrafish which we had utilized to demonstrate the efficacy of TMZ in reducing tumor burden and improving survival (27). To investigate whether HSP90 inhibition would potentiate the cytotoxic effects of TMZ, we transplanted U251HF cells expressing green fluorescent protein (U251HF-GFP) into the midbrain of zebrafish embryos to generate gliomas, which were evident at 5 days post-transplant (dpt) (Fig. 6A) and grew robustly by 10 days dpt. Zebrafish were randomized to the following four groups: 1% DMSO, TMZ (10 μM), onalespib (0.5 μM) or TMZ (10 μM) plus onalespib (0.5 μM), and treated between 5 and 10 dpt (Fig. 6A). Fish treated with DMSO showed an exponential increase in intracranial tumor growth during this period (Fig. 6A, top panel) whereas those exposed to TMZ or onalespib showed moderate reductions in tumor burden (Fig. 6A and 6B). However, fish exposed to the combination of onalespib and TMZ showed the greatest reduction in tumor burden (Fig. 6B). Correspondingly, onalespib in combination with TMZ significantly extended survival to a greater extent than each individual treatment alone indicating that the combination of onalespib with TMZ synergizes to reduce tumor growth and extends survival in this in-vivo vertebrate model of glioma (Fig 6C).

Fig. 6.

Fig. 6

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Onalespib synergizes with TMZ to extend survival in orthotopic intracranial zebrafish glioma model. A. Zebrafish were treated with 1% DMSO, 10 μm TMZ, 0.5 μm onalespib or 10 μm TMZ plus 0.5 μm onalespib continuously from 5 to 10 days post-transplant (dpt). Tumor burden was determined at 5 dpt (before treatment) and 10 dpt (after 5 days of treatment). (B) Results were quantitated using MetaMorph software (n=24 animals/group. p value of the DMSO group at day 5 Vs day 10 < 0.0001; p value of the combination group at day 5 Vs day 10 < 0.0001. (C) Zebrafish treated as above were assessed for survival (n = 24 animals/group) p value of the onalespib+TMZ vs DMSO group < 0.0001. (D) GSC811 patient derived xenografts were randomized to treatment with vehicle, onalespib (30mg/kg), TMZ (4mg/kg), or a combination of onalespib and TMZ and followed for survival (n=10 animals/group; onalespib+ TMZ vs control p<0.5, log rank test; onalespib+ TMZ vs TMZ p<0.01, log rank test; onalespib+ TMZ vs onalespib p<0.01, log rank test).

Given that conventional cell lines do not fully represent human tumors, we further evaluated the efficacy of this combination in a more clinically relevant patient derived xenograft mouse model. GSC811 cells represent aggressive GSCs and were implanted intracranially into NOD/SCID mice. 15 days post implant, mice bearing GSC811 xenografts were randomized to vehicle, TMZ, onalespib or a combination of onalespib with TMZ treatment cohorts. While treatment with TMZ or onalespib alone did not extend survival compared to vehicle treated mice, the combination of onalespib and TMZ significantly improved survival (p<0.01, log rank test) in comparison to mice treated with vehicle or each single agent (Fig 6D).

Discussion

Increased expression and activity of HSP90 has been implicated in the malignant process in several human tumors including gliomas. Hsp90 inhibitors have been shown to induce a variety of biological effects against gliomas in preclinical evaluations. For instance, geldanamycin, a benzoquinone ansamycin antibiotic, or its derivatives 17-AAG or 1-DMAG, have been shown to inhibit glioma growth in vitro through their potent inhibition of HSP90 and consequent biological anti-tumor effects that included inhibition of survival, invasion and migration (30), and effects against glioma stem cells (9). Similar results have been seen with newer non-geldanamycin HSP90 inhibitors such as NVP-AUY922 and NVP-BEP800 (44) and IPI-504 (45). Of note, upregulation of HSP70 levels upon inhibition of HSP90 is considered a clinically identifiable class-effect of HSP90 inhibitors across tumor types and can serve as a marker of target inhibition in tumor tissue. This effect is mediated by HSF1, which is normally bound by HSP90 and maintained in an inactive state in the cytoplasm but which upon HSP90 inhibition translocates in its active trimeric form to the nucleus) and binds to HSP70 promoter, transcriptionally inducing this target gene resulting in increased HSP70 levels (46).

Although 80% of the kinome in the cell and 30% of transcription factors are strong or weak clients of HSP90 (11), strength of binding of client proteins to HSP90 alone does not appear to determine sensitivity to HSP90 inhibition. Rather, the degree to which the tumor cell depends on the HSP90 client signaling network and the extent to which the signaling networks are inhibited as a whole as opposed to individual oncogenic clients appear critical in determining the cellular response to HSP90 inhibitors (32). EGFR amplification occurs in ~40–50 % of GBMs and results in an increase in pro-survival and proliferative signals. In response to onalespib, there was rapid reduction of EGFR which was accompanied by an inhibition of the key downstream signaling intermediaries, AKT, ERK and S6 signaling indicating an onalespib-mediated shutdown of the signaling network activated by EGFR. Over half of the tumors that show EGFR amplification also express of EGFRvIII mutant, which is recognized as a strong driver of tumor progression in this subset of GBMs and correlates with poor prognosis (33). The potent inhibition of downstream signaling in EGFRvIII expressing cells demonstrates that onalespib can exhibit a profound effect on this pathway even in the setting of constitutive ligand-independent signaling.

Onalespib treatment was also effective against glioma stem-like cells which are considered the treatment-resistant subset of tumor initiating clones. Tumorigenicity is preferentially augmented in GSCs by the phosphorylation and activation of STAT3 whereas activation of EGFR signaling is key to survival in both GSCs and bulk tumor cells. The finding that onalespib exposure inhibited p-STAT3 and EGFR signaling to compromise survival in GSCs suggest that onalespib could be active against GBM regardless of EGFR mutation status or stem cell subtype. Further, the activity of onalespib was independent of GBM subtypes in patient-derived GSC lines that recapitulated the relatively favorable proneural or more aggressive mesenchymal signature. These findings taken in conjunction with the reduction of stem cell markers in glioma initiating cells and the observed inhibition of migration, angiogenesis and proliferation in glioma lines exposed to onalespib, suggest a potential for this agent to effectively target the diverse cell populations and pathways that drive heterogeneity and resistance in gliomas.

Although preclinical reports have shown broad activity of HSP90 inhibitors across various tumor types, single agent activity of HSP90 inhibitors against most malignancies including gliomas has been modest likely due to insufficient target inhibition, short duration of action or toxicities (47). Unlike other HSP90 inhibitors, Onalespib has a favorable pharmacokinetic and pharmacodynamic profile exerting a prolonged inhibition of HSP90 resulting in a sustained reduction in levels of its client oncoproteins and their downstream targets (22). This may be particularly relevant clinically where sustained inhibition of oncogenic client proteins (12) could result in a more pronounced clinical effect. Onalespib also has the unique advantage of being able to traverse the blood brain barrier. Although the brain Cmax was 4.7 fold lower than that in plasma, the overall brain exposure of onalespib was greater than of plasma. This was reflected with a consistently higher level of HSP70 staining (marker of HSP90 inhibition) in the non-tumor brain tissue of mice exposed to onalespib compared to vehicle. We also observed variations in the intensity of HSP70 signal (marker of HSP90 inhibition) across the IHC stained section which could be of relevance to the variability of the drug’s effects across various portions of the tumor, a finding that could be further examined in clinical specimens as a measure of heterogeneity in target and client protein inhibition across the tumor.

Single agent HSP90 inhibitor therapy has shown only modest activity in clinical studies suggesting that while these agents induce changes in a variety of cellular processes, they may not be able to induce a robust anticancer activity alone; instead, several studies suggest that they could potentially serve to prime cancer cells by depleting HSP90 client oncoproteins and sensitize them to cytotoxic signals from other anticancer agents (20, 48, 49). Previous studies of combining shorter acting HSP90 inhibitors with temozolomide, a monofunctional alkylating agent that is the most commonly used cytotoxic chemotherapeutic agent against GBM, did not reveal any significant additive or synergistic activity (9, 48). Our results however show that while both onalespib or TMZ could inhibit glioma growth individually in vivo, onalespib was able to potentiate the cytotoxic effects of TMZ in several glioma cells as well as improve survival in two independent glioma xenograft models, one generated using a glioma cell line in zebrafish and the second using a patient-derived GSC xenograft in mice suggesting a potential for efficacy of the combination in the treatment of bulk tumor as well as the GSC populations in GBM. We attribute this finding to the prolonged inhibition of HSP90 by onalespib and to its broad effects on key signaling pathways potentiating the effects of TMZ.

This study is the first to determine the preclinical activity of onalespib against gliomas and assess its potential as an anti-tumor agent both as a single agent and in combination with TMZ. In addition, onalespib was seen to cross the intact BBB and show target inhibition, which are key requirements for agents targeting infiltrative gliomas. The prolonged pharmacodynamic effect of onalespib differentiates it from other HSP90 inhibitors and its effects on diverse glioma cells and GSC suggest a potential for targeting glioma in a manner that can overcome tumor resistance; this was shown in its activity in combination with TMZ; these results provide a strong rationale for development of onalespib in combination with TMZ as a therapeutic approach against gliomas.

Supplementary Material

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Translational Relevance.

HSP90 is a molecular chaperone required for the function of multiple oncogenic hubs in glioblastoma (GBM). Onalespib, a novel and potent long-acting HSP90 inhibitor, depleted the expression of survival promoting client proteins such as EFGR (wild type and mutant) and AKT to suppress downstream signaling via ERK and S6 to inhibit of survival, proliferation, invasion and migration across glioma cell lines and patient-derived glioma-initiating cells (GSCs). Onalespib was brain penetrant with a favorable pharmacokinetic profile, and caused sustained HSP90 inhibition in brain tissues of mice. Further, onalespib in combination with temozolomide conferred an in vivo survival advantage in a zebrafish glioma xenograft model as well as in a patient-derived GSC xenograft mouse model compared to onalespib or temozolomide alone. These properties highlight key advantages of onalespib over non-brain permeable HSP90 inhibitors for treatment of malignant gliomas and its potential for combination with temozolomide.

Acknowledgments

Funding: This study was supported by NCI grant K24CA160777, the Salvino Family & Accenture Brain Cancer Research Fund, Lisa B. Landes Brain Cancer Fund, and the Roc on Research Fund for Neuro-oncology (VK Puduvalli); the Ohio State University Cancer Center Support grant NCI-CA16058 and the MD Anderson Brain Cancer SPORE P50 CA127001 (FF Lang and EP Sulman).

This project was supported by the Ohio State University Comprehensive Cancer Center core grant (NCIP30CA016058) through the Target Validation Shared Resource (TVSR) core facility (providing athymic nude mice), the Pharmacoanalytic Shared Resource and the Comparative Pathology and Mouse Phenotyping (assistance with tissue processing and immunohistochemistry) and NINDS P30NS045758 for the zebrafish studies.

Footnotes

Author contributions

AC, AW, PN, JYY, JX, FAS, JL and DK performed experiments. JG, ES and FL provided GSC lines and data. All the authors have contributed substantially to the design of experiments, and analysis and interpretation of data. AC, AW, DS and VKP wrote the manuscript. VKP approved the final manuscript.

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