An update on the status of HSP90 inhibitors in cancer clinical trials

Abstract

The evolutionary conserved molecular chaperone heat shock protein 90 (HSP90) plays an indispensable role in tumorigenesis by stabilizing client oncoproteins. Although the functionality of HSP90 is tightly regulated, cancer cells exhibit a unique dependence on this chaperone, leading to its overexpression, which has been associated with poor prognosis in certain malignancies. While various strategies targeting heat shock proteins (HSPs) involved in carcinogenesis have been explored, only inhibition of HSP90 has consistently and effectively resulted in proteasomal degradation of its client proteins. To date, a total of 22 HSP90 inhibitors (HSP90i) have been tested in 186 cancer clinical trials, as reported by clinicaltrials.gov. Among these trials, 60 % have been completed, 10 % are currently active, and 30 % have been suspended, terminated, or withdrawn. HSP90 inhibitors (HSP90i) have been used as single agents or in combination with other drugs for the treatment of various cancer types in clinical trials. Notably, improved clinical outcomes have been observed when HSP90i are used in combination therapies, as they exhibit a synergistic antitumor effect. However, as single agents, HSP90i have shown limited clinical activity due to drug-related toxicity or therapy resistance. Recently, active trials conducted in Japan evaluating TAS-116 (pimitespib) have demonstrated promising results with low toxicity as monotherapy and in combination with the immune checkpoint inhibitor nivolumab. Exploratory biomarker analyses performed in various trials have demonstrated target engagement that suggests the potential for identifying patient populations that may respond favorably to the therapy. In this review, we discuss the advances made in the past 5 years regarding HSP90i and their implications in anticancer therapeutics. Our focus lies in evaluating drug efficacy, prognosis forecast, pharmacodynamic biomarkers, and clinical outcomes reported in published trials. Through this comprehensive review, we aim to shed light on the progress and potential of HSP90i as promising therapeutic agents in cancer treatment.

Introduction

Cancer therapeutics have been predominantly focused on genetic aberrations. However, the discovery of geldanamycin (GA) as a heat shock protein 90 inhibitor (HSP90i) shifted the paradigm toward possibly targeting this and other chaperones, in turn uncovering the reliance of multiple cancers on the proteostasis network. While HSP90 is an evolutionarily conserved house-keeping gene, the amplified expression of HSPs and heat shock factor-1 (HSF-1), a transcriptional regulator of many HSPs, in various human cancers is associated with poor prognosis, tumor invasion, metastasis, and treatment resistance.1, 2, 3 The rapid induction of the HSF-1-HSP90 axis in response to cellular stress, post-translational modifications such as acetylation, phosphorylation and S-nitrosylation, and ectopic localization of HSP90 distinguishes HSP90 in cancer cells versus normal cells.1, 4, 5 Structurally, HSP90 exists as a homodimer with each protomer comprising an N-terminal nucleotide-binding domain (NTD) with ATPase activity, followed by a charged linker which provides dynamic conformational flexibility, followed by the client protein binding middle domain (MD), and a carboxy-terminal domain (CTD) for dimerization.6, 7, 8, 9 HSP90 has four functional paralogs in mammalian cells, HSP90α (HSP90AA1) and HSP90β (HSP90AB1) are localized to the cytosol and nucleus, GRP94 is found in the endoplasmic reticulum and TNF receptor-associated protein-1 (TRAP1) is localized to mitochondria.10, 11, 12, 13 The expression of HSP90 cytosolic isoforms has been implicated in various cancers, including lung cancer,¹⁴ hepatocellular carcinoma,¹⁵ colorectal cancer,¹⁶ etc. Recently, Liu et al.¹⁷ demonstrated that plasma HSP90AA1 predicted the risk of breast cancer onset and distant metastasis. The fold change in mRNA levels of HSP90AA1, HSP90AB1, HSP90B1, and TRAP1 in various cancer tissues are shown in Figure 1. The data were generated from a web server for gene expression profiling and interactive analysis http://gepia.cancer-pku.cn/.¹⁸ It is interesting to note that the expression of HSP90AA1 is highest in cervical squamous cell carcinoma and endocervical adenocarcinoma whereas, in lung adenocarcinoma tumor tissue and lung squamous cell carcinoma HSP90AB1 and TRAP1 have the highest expression respectively (Figure 1).

Fig. 1.

The gene expression profile of HSP90 homologs.

The heat map showing the gene expression profiling interactive analysis of HSP90 homologs expression in various cancer types- BRCA Breast invasive carcinoma, CESC- Cervical squamous cell carcinoma and endocervical adenocarcinoma, ESCA- Esophageal carcinoma, LAML- Acute Myeloid Leukemia, LUAD- Lung adenocarcinoma, LUSC- Lung squamous cell carcinoma, PRAD- Prostate adenocarcinoma, SARC- Sarcoma, SKCM- Skin Cutaneous Melanoma, TGCT- Testicular Germ Cell Tumors, UCEC- Uterine Corpus Endometrial Carcinoma UCS- Uterine Carcinosarcoma. Each individual block represents the fold change of the tumor tissue in expression of HSP90 genes from the normal tissue.

HSP90 buffers cell stress by stabilizing a wide array of client proteins whose functional stability promotes the hallmarks of cancer.¹⁹ HSP90i alters the HSP90-client protein complex interaction and causes proteasomal degradation of these proteins (Figure 2). There are over 400 client proteins of HSP90 that can be broadly categorized as follows20, 21, 22, 23:

• (1) Protein kinases such as the SRC family kinases (SRC, LCK, YES, and FYN), receptor tyrosine kinases (HER2, epidermal growth factor receptor [EGFR], IGF1R, and FLT3), serine/threonine kinases (RAF-1, AKT, and cyclin-dependent kinase 4 [CDK4]), cell cycle G2 checkpoint kinases (WEE1, MYT1, POLO-1), and mutant fusion kinases (BCR-ABL and NPM-ALK).

• (2) Steroid hormone receptors (glucocorticoid, androgen, estrogen, and progesterone receptors).

• (3) Transcription factors (p53, HSF-1, and hypoxia‐inducible factor‐1 [HIF‐1]).

• (4) Telomerase (hTERT).

• (5) Chromatin remodeling factors.

Fig. 2.

Role of HSP90i in stabilizing oncogenic client proteins and promoting hallmarks of cancer.

HSP90 facilitates the stabilization of immature oncogenic client protein which in turn promotes hallmarks of cancer while HSP90i attenuate client folding and cause proteasomal degradation of client proteins.

Many of these clients may be oncogenic in different scenarios and can often contribute to various cancer phenotypes. If one or multiple clients are indispensable for survival, cancer cells become “addicted” to the HSP90 machinery for their maintenance and stability.24, 25

The ability of HSP90 to stabilize complex circuitries in abnormal or upregulated signaling pathways present in most cancers makes it an attractive therapeutic target. The clinical success of HSP90i, which has entered into clinical trials, has been impeded by dose-limiting toxicities (DLTs) and adverse events (AEs).3, 26

In this review, we highlight the discovery and development timeline of HSP90i, the current status of HSP90i in the context of malignancies and the efficacy of HSP90i currently in clinical trials, the role of certain biomarkers as valuable pharmacodynamic (PD) tools in HSP90i clinical trials, and the limitations of HSP90i in clinical trials and the need for further development of HSP90i as anticancer therapeutics.

Classification of HSP90i based on the target sites

HSP90i, in general, can be broadly classified into three types according to their target sites in the HSP90 machinery: (1) those targeting the NTD ATP-binding pocket,20, 26 (2) those targeting co-chaperone interactions,27, 28, 29 or (3) and those targeting the CTD and MD.³⁰ Of these, NTD ATPase binders have dominated clinical trials, with over 100 trials to date. However, none have received approval from the FDA. Marcu et al., via truncation studies discovered a second cryptic ATP-binding pocket in the HSP90 CTD and confirmed that naturally derived coumarins such as novobiocin bind to this site.³¹

NTD-ATP binding HSP90i

GA based HSP90i

GA was the first naturally derived HSP90i, purified in 1970 from Streptomyces hygroscopicus var. geldanus.³² It was initially misidentified as a novel kinase inhibitor, however, in 1994, Whitesell and Neckers reported that GA did not bind directly to v-src proteins; rather, it binds to HSP90 in the HSP90-v-src heteroprotein complex, thereby affecting src stabilization and its kinase activity.³³ GA was a first-generation ansamycin analog with a benzoquinone ring which was found to bind to the NTD ATP binding pocket of HSP90. Its binding blocked ATP binding and resulted in a conformational change that led to the degradation of HSP90 client proteins via the ubiquitin-proteasome pathway. Although GA looked like an attractive candidate for advancement into clinical trials, preclinical studies showed that the quinone moiety induced severe hepatotoxicity in vivo.³⁴ Apart from this, low solubility in water also proved to be a major pharmacological drawback for further in vivo GA. To overcome these issues, the parent structure of GA was modified at the C-17 position to create an allyl amino derivative called 17-N-allylamino-17-demethoxygeldanamycin (17-AAG).34, 35, 36 In 1999, the first-in-class HSP90i 17-AAG (tanespimycin) entered phase I clinical trial with the maximum tolerated dose (MTD) in patients of 295–450 mg/m².37, 38, 39, 40, 41 The reported side-effects still included mild hepatotoxicity associated with the drug vehicle DMSO.⁴² To date, in a total of 38 clinical trials, 17-AAG has been administered either alone or in combination with various drugs.⁴³ 17-AAG provided therapeutic benefit to patients when used in combination; however, due to its low water solubility and hepatotoxicity, Bristol-Myers Squibb halted the clinical development of 17-AAG.⁴⁴

To overcome the major challenge of solubility and bioavailability associated with 17-AAG, Kosan Biosciences developed a second-generation, semi-synthetic GA derivative called 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG; alvespimycin).20, 45 17-DMAG is more soluble in aqueous media, has better bioavailability, and has reduced metabolism compared to 17-AAG. Moreover, it can be administered both intravenously (i.v.) and orally. 17-DMAG has been used as a single agent or in combination in seven clinical trials against various cancer types, but most of them have been phase I.⁴⁶ In spite of showing promising results against refractory HER2+ metastatic breast cancer³⁷ and myelogenous leukemia,⁴⁷ 17-DMAG exhibited higher toxicity when compared to 17-AAG, and for this reason, Kosan suspended further clinical development of 17-DMAG.⁴⁸

Another oil-in-water nano emulsion derivative of 17-AAG; CNF1010 was developed by Biogen Idec (Conforma Therapeutics).⁴⁹ However, Biogen had to suspend further development on CNF1010 due to nonsignificant clinical response and grade-3 hepatotoxicity in 2 phase I trials in advanced solid tumors and ZAP-70 positive B-cell chronic lymphocytic leukemia.50, 51 Abraxis BioScience developed an albumin-bound nanometer-sized form of 17-AAG, ABI-010, to facilitate efficient uptake of the drug in tumor cells. It entered the phase I trial as a single agent or in combination with ABI-007 (Abraxane) in patients with advanced nonhematologic malignancies in 2009, but the trial was suspended in 2018.⁵²

In another approach to overcome the poor aqueous solubility of 17-AAG, a highly soluble hydroquinone hydrochloride IPI-504 (retaspimycin hydrochloride) was developed by Infinity Pharmaceuticals, Inc.⁴⁸ In a phase I trial in advanced non-small cell lung cancer (NSCLC), an overall response rate (ORR) of 26 % was observed in patients treated with IPI-504 and docetaxel.⁵³ Although IPI-504 showed modest clinical activity in an ERBB2-positive breast cancer trial, it did not meet the criteria for study expansion.⁵⁴ A phase III (RING) trial of IPI-504 in patients with refractory gastrointestinal stromal tumors (GIST) was terminated by Infinity due higher mortality rate in the first 46 patients enrolled in the study.⁵⁵ Infinity Pharmaceuticals also discontinued the development of IPI-493, an oral formulation of 17-AAG it codeveloped alongside IPI-504. Although it entered the phase I trial for advanced malignancies, the study was terminated due to superior drug exposure of IPI-504 over IPI-493.⁵⁶

Radicicol based HSP90i

A second naturally derived antifungal macrocyclic antibiotic is radicicol (RDC). It was first isolated from Monosporium bonorden in 1953 and was later found to bind to the HSP90 NTD ATP binding site with a greater affinity than GA.57, 58, 59 While it exhibited similar antitumor activity as GA in vitro, in vivo studies with RDC did not yield promising results due to its rapid metabolism of inactive metabolites.⁶⁰ Consequently, clinical application of RDC was unsuccessful. However, metabolically stable RDC analogs presented a new pharmacophore (a resorcinol that was utilized for the development of new HSP90i such as Ganetespib [STA-9090], NVP-AUY922 [VER-52269], HSP990, and AT13387 for cancer treatment).

To improve potency and physical and chemical properties, purine (PU) scaffolds were utilized to design new NTD inhibitors. PU3 was the first PU-scaffold-based synthetic HSP90i,⁶¹ while BIIB021 (Biogen Idec) was the first PU-based HSP90i to enter clinical trials.62, 63 Currently, there are no active clinical trials for BIIB021. The commercial production of PU-H71 is done by Samus Therapeutics which entered in Phase I/II clinical trials.⁶⁴

Targeting co-chaperones interactions

Certain naturally derived small molecules and peptides bind to the interaction sites on the co-chaperones of HSP90 and inhibit protein–protein interactions (PPIs), thereby disrupting the association of the client proteins with the Hsp90 machinery.²⁸ Recently discovered SEW84 binds to AHA1, a potential oncogene, and the main activator of ATPase of Hsp90; inhibited both wild-type and mutated variants of the androgen receptor (AR), implicated in prostate cancer in in vitro studies.27, 29 Several disruptors of HSP90 and cell cycle protein cdc37 such as celastrol A, DCZ3112, and FW-04-806 have been reported.²⁷ HSP90 is also known to stabilize HER-2, a member of ErbB family of receptor tyrosine kinases. Nefilnavir and Emodin have been identified as HSP90–HER-2 PPI disruptors.²⁷ Other known potential targets of PPIs like HSP90–HOP, HSP90–p23, HSP90–HIF-1α implicated in cell cycle progression, angiogenesis, and metastasis developmental processes of tumor cells have also been investigated. Although the efficacies of these PPIs have been proved in various in vitro and preclinical studies, they are still under the drug development process.

HSP90 C-terminal domain (CTD) and MD inhibitors

NTD inhibitors induce a heat shock response (HSR) which subsequently upregulates HSP27, HSP40, and HSP70, imparting cytoprotection to cancer cells and increasing resistance to HSP90 inhibition. Marcu et al., via truncation studies, discovered a second ATP-binding pocket in the CTD of HSP90 and confirmed that naturally derived coumarin antibiotics, such as novobiocin, bind to an ATP site in the CTD.³¹ The novobiocin structure was used to synthesize compound A4 and its analogs.³⁰ The most potent novobiocin compound made was KU-174 which demonstrated its efficacy in various cancer cell lines by breaking down the client proteins without inducing the HSR. Currently, there is no FDA-approved C-terminal inhibitor.⁶⁵

Sansalvamide A is an isolated cyclic pentapeptide that binds to the mid-domain N-terminal fragment of HSP90 and interrupts C-terminal binding co-chaperones and client proteins. Three compounds derived from Sansalvamide A H-10, H-15, and LY-15 were investigated in preclinical studies as HSP90 inhibitors.66, 67, 68 Another MD HSP90 inhibitor is Diptoindonesin G which degrades client protein by binding to the MD of HSP90 without inducing a HSR.69, 70 Targeting and developing MD and CTD inhibitors may be beneficial as they circumvent the HSR,⁷¹ but currently, no MD or CTD inhibitors have entered into clinical trials.⁶⁵

Current status of HSP90i clinical trials

From 2017 to 2023, 10 HSP90i have been evaluated as single agents or in combination in clinical trials for various cancer types. Currently, there are 18 ongoing clinical trials of HSP90i, and 17 trials have been completed. Another seven trials were suspended or withdrawn for various reasons (Figure 3). Here, we discuss in detail the current status of HSP90i in clinical trials since 2017⁷² (https://clinicaltrials.gov).

Fig. 3.

Overview of HSP90i in clinical trials, 2017-2023.

AT13387 (Onalespib)

AT13387 is a synthetic NTD ATP binding site competitor that is orally bioavailable and long-acting.73, 74, 75 In tumor xenografts, the half-life of AT13387 is up to 65 h, thus demonstrating extended PD effects.⁷⁶ Recently, Spiegelberg et al.⁷⁷ showed that when combined with radiotherapy, onalespib exerted synergistic anticancer effects in vitro and in vivo. AT13387 has been given as therapy in a total of 13 clinical trials. Among these, 4 trials are still active/recruiting, 3 are terminated/withdrawn, and 6 are completed.⁷⁸ A phase I/II trial of onalespib in combination with abiraterone acetate (AA) and prednisone or prednisolone in patients with castration-resistant prostate cancer (CRPC), showed that the MTD was either 220 mg/m² (regimen 1; once weekly) or 120 mg/m² (regimen 2; twice weekly). DLTs were grade 3 diarrhea related to onalespib at 260 mg/m² or 160 mg/m². In regimen 1, the median progression-free survival (PFS) and overall survival (OS) were 77 days and 10.6 months, respectively, while in regimen 2, median PFS and OS were 84 days and 8.9 months, respectively.⁷⁹ Overall, this study suggested that onalespib, in combination with AA/prednisolone, demonstrated some biological effects but insufficient clinical activity in CRPC patients. Therefore, further exploration of this combination was not pursued.

Do et al.⁸⁰ presented a rational combination of onalespib and AT7519, a pan-CDK inhibitor, after in vitro investigation in a colorectal cancer cell line HCT-116. Onalespib induced HSP70 expression, which was suppressed in a dose-dependent manner by AT7519. AT7519 inhibited not only CDK9 but also HSP70 via CDK9-mediated phosphorylation of RNA Pol II. Downstream modulation of client proteins such as HER-2 and pAKT was also observed in these studies. Based on these results, a phase I study of onalespib in combination with AT7519 was done in patients with advanced solid tumors to determine the safety and tolerability of the combination. The MTD was 80 mg/m² and 1 DLTs were reported at this dose with grade 3 increase in cardiac troponins and oral mucositis. Exactly 69.5 % of the patients progressed during the first two cycles, but two patients with palate adenocarcinoma and Sertoli–Leydig tumor achieved a partial response (PR). A colorectal and an endometrial cancer patient had stable disease (SD) as the best response.⁸⁰ This study concluded that the combination of onalespib and AT7519 was tolerable with promising preliminary activity.

A combination of onalespib with erlotinib, an EGFR tyrosine kinase inhibitor (EGFR-TKI), was investigated in phase I/II trial in EGFR-mutant (focused on EGFR exon 20 Insertion [EGFR^ex20ins]) NSCLC. Eleven patients were treated, and 2 grade 3 DLTs occurred at 150 mg/m², while no DLTs were reported at 120 mg/m². Therefore, the recommended phase II dose (RP2D) was 120 mg/m². In 10 evaluable EGFR^ex20ins patients, no responses were observed, and the median PFS was 5.4 months with this combination therapy. Overlapping toxicities of erlotinib and onalespib limited the tolerability of this combination, and due to limited clinical activity, the trial was closed.⁸¹

Combination of onalespib (given i.v.) and the PARP inhibitor olaparib (given as per os) was also investigated in patients with unresectable solid tumor or recurrent ovarian, fallopian tube, primary peritoneal, or triple-negative breast cancer (TNBC). This phase I study revealed that dose levels (DLs) of olaparib 300 mg twice a day (b.i.d.) onalespib 40 mg/m² and olaparib 200 mg b.i.d. onalespib 80 mg/m² were safe without DLTs. There was no objective response, but the combination of olaparib and onalespib was feasible, and it prolonged SD ≥24 weeks in 32 % of evaluable patients with this combination therapy. However, due to limited clinical efficacy of onalespib in different cancer trials its further development has been discontinued.⁸²

Combination therapy of AT13387 with BRAF/MEK inhibitor in patients with BRAF-^mut solid tumors demonstrated modest clinical activity with a PR of 16 %, SD of 66.7 %, and a disease control rate of 16.7 % in a pretreated population. The MTD established for i.v. AT13387 was 260 mg/m² once weekly.⁸³

AUY922 (Luminespib)

AUY922 is a third-generation HSP90i that binds with high affinity to the HSP90 NTD ATP pocket, causing proteasomal degradation of oncogenic client proteins. AUY922 inhibited cell proliferation⁸⁴ and upregulated HSP72⁸⁵ in various human tumor cell lines. It was developed by the pharmaceutical company Vernalis in collaboration with the Institute of Cancer Research, London, and was later licensed to Novartis.⁸⁶ AUY922 entered phase I trial in 2007 and has been evaluated in 27 clinical trials to date. As per the information available on clinicaltrials.gov.,⁸⁷ out of 27 trials, 7 were terminated/withdrawn, 19 were completed, while the status of 1 trial remains unknown. Currently, there are no active clinical trials of AUY922. The first-in-human, phase I dose-escalation study of AUY922, conducted in patients with advanced solid tumors, recommended 70 mg/m² as a tolerable dose.⁸⁸ In 2018, the final results of 2 phase II trials of AUY922 in NSCLC, funded by Novartis, were published.⁸⁹ Patients with advanced NSCLC were stratified based on tumor molecular etiologies (ALK-rearranged, EGFR-mutant, KRAS-mutant and wild-type) that received more than two lines of prior chemotherapy and patients in the fifth stratum i.e., EGFR cohort received less than two lines of prior therapy (EGFR < 2). All the eligible patients received AUY922 at 70 mg/m² as monotherapy in 21-day treatment cycles. No drug-related AEs were reported. AUY922 showed clinical activity in patients with EGFR mutations and ALK rearrangement, whereas no clinical benefit was observed in patients with KRAS mutation, even though, preclinical evidence suggested that KRAS-mutant models were sensitive to HSP90.⁸⁹

Another phase II study evaluated the activity of AUY922 in NSCLC patients with EGFR^ins20.⁹⁰ AYU922 was well tolerated and showed clinical activity, as 38 % of patients had either PR or SD ≥3 months. Although the study treatment was prematurely stopped when luminespib was discontinued by the company, the trial concluded that AYU922 could be an active therapy for EGFR^ins20 NSCLC as it exceeded the predetermined target rate of effectiveness.⁹⁰

HS-196 and HS-201

Osada and colleagues⁹¹ linked a near-infrared fluorescing molecule to an analog of human HSP90i-SNX-5422, and designated this imaging probe as HS-196, which selectively and competitively binds to upregulated HSP90 in tumor cells. The near-infrared-tethered HS-196 noninvasively detects cancer cells in vivo. Exactly 1 × 10⁶ cells of 4 prostate cancer cell lines: AR-negative/irresponsive PC-3 and DU145, and AR-positive/responsive 22Rv1 and LNCaP were subcutaneously injected in SCID-beige mice. When tumor size reached 8–10 mm in diameter, HS-196 (10 nmol/injection) was administered via the tail vein. The imaging results of tumor xenografts demonstrated greater HS-196 fluorescence intensity within the aggressive/moderately aggressive cell lines PC3 and DU145, when compared to LNCaP. Among the two AR-positive prostate cancer cell lines, greater HS-196 fluorescence intensity was observed in 22Rv1 tumors as it has clinically aggressive, therapy-resistant disease features. These preclinical data suggest that HS-196 selectively identifies aggressive prostate cancers. In a phase I human study on prostate cancer patients who would be undergoing radical prostatectomies, clinical-grade HS-196 produced by Albany Molecular Research Inc. was systemically administered 36 h prior to the surgery. HS-196 was well tolerated with no toxic effects. HS-196 was detected in malignant nodules within prostatectomy specimens suggesting its utility for imaging deeper tissues.⁹¹

A theragnostic version of the HSP90-binding drug HS-201 was developed by replacing the near IR molecule with the photosensitizing molecule Verteporfin.⁹² HS-201 accumulation may allow visualization of tumors within the body, and verteporfin could facilitate photodynamic therapy of tumors. A phase I study of HS-201, for detection of solid malignancies, opened in July 2020, but was terminated 2 years later due to loss of funding.⁹³

Gamitrinib

Gamitrinib is a first-in-class mitochondrial matrix inhibitor that links GA HSP-90 inhibitor 17-AAG to triphenyl phosphonium which is an efficient mitochondrial import carrier⁹⁴ based mitochondrial matrix targeted HSP90i with potential anticancer activity. Gamitrinib was developed at the Wistar Institute to target and inhibit the activity of the HSP90 paralog TRAP1, which is specifically localized in the mitochondrial matrix. In susceptible tumor cells, gamitrinib caused mitochondrial dysfunction, loss of membrane potential, and membrane rupture, which initiates apoptosis.⁹⁵ The Fox Chase Cancer Center and the Wistar Institute announced the opening of a phase I clinical trial of gamitrinib in patients with advanced cancer.⁹⁶ However, due to limited drug supply, the trial was suspended in 2023.⁹⁷

PU-H71 (Zelavespib)

PU-H71 was developed by Chiosis and colleagues at the Memorial Sloan Kettering Cancer Center (New York, NY, USA) as reported in the online newsletter of Memorial Sloan Kettering Cancer center.⁹⁸ The presence of an iodide functional group in its chemical structure facilitated the further insertion of radionuclide I-124 to produce the imaging agent ¹²⁴I-PU-H71. The iodine I-124 moiety is visualized by positron emission tomography (PET). The physical half-life of this agent is 4.02 days thereby allowing multiple assessments.⁶³

In 2017, clinical results of the first-in-human study of PU-H71 were published. PU-H71 was administered i.v. on days 1 and 8 of 21-day cycles in patients with refractory solid tumors. The goal of this study was to determine the safety, tolerability, and MTD of PU-H71.⁹⁹ The starting dose of PU-H71 in the trial was 10 mg/m²/day, and it was safely escalated to 470 mg/m² without the occurrence of DLT; however, the MTD was not established. The best response of SD >2 cycles was observed in 35 % of evaluable patients. The PD assessments of HSP90 client proteins in biopsy samples from patients treated at the MTD were planned, but due to the discontinuation of study drug supply by Samus Therapeutics Inc., the trial was closed prematurely in the dose-escalation phase.¹⁰⁰

In 2020, first-in-human trial results of PET imaging of cancer patients using ¹²⁴I-PUH71 were published. The data demonstrated the safety and feasibility of noninvasive in vivo detection of tumors by ¹²⁴I-PU-H71.¹⁰¹ Intravenously (201 + 12 MBq) administered ¹²⁴I-PU-H71 was detected in tumors of different cancer types (breast, lymphoma, neuroblastoma, genitourinary, gynecologic, sarcoma and pancreas), and the lesions were confirmed by the most recent CT, MRI, and/or 2[¹⁸F] fluoro-2-deoxy-D-glucose PET/CT scans before enrollment. ¹²⁴I-PUH71 was retained in the tumors for several days, but it was rapidly cleared from healthy tissues and blood.¹⁰¹ In another phase Ib trial, safety and tolerability of PU-H71 plus nanoparticle albumin bound nab-paclitaxel in HER2-negative metastatic breast cancer and the utility of PU-PET as a noninvasive predictive biomarker were demonstrated.¹⁰² The MTD of PU-H71 was established at 300 mg/m² plus (nab)-paclitaxel 260 mg/m² administered every 3 weeks. At the time of data cut-off, the clinical benefit rate was 42 % while ORR was 17 %. Time to progression was correlated to PU-H71 SUV_max (standardized uptake volume). The combination of PU-H71 and nab-paclitaxel was well tolerated and showed clinical activity, warranting further studies of this combination therapy, especially in TNBC.¹⁰²

SNX-5422

An orally bioavailable novel HSP90i, SNX-5422 was developed by Esanex Pharmaceuticals and has been safely tested in multiple phase I studies against various cancer types.¹⁰³ SNX-5422 is a prodrug that metabolizes to its active form SNX-2112, which accumulates readily in tumors.104, 105 Recently, a phase I study of SNX-5422 in combination with carboplatin and paclitaxel followed by SNX-5422 maintenance monotherapy in patients with advanced lung cancers reported that the MTD of SNX-5422 was 100 mg/m² in combination.¹⁰⁶ Exactly 48 % of patients experienced grade 3 or higher AEs, such as diarrhea, nausea, and neutropenia. ORR was 33 %, with 33 % PR, 56 % SD, and 11 % disease progression. The patients on maintenance monotherapy of SNX-5422 for 12 months and 7 months with HER2-^mutant NSCLC or KRAS-^mutant NSCLC, respectively, had a PR before progression. No activity was observed in SCLCs due to a small cohort.¹⁰⁶ The lack of a control group as well as uniform biomarker testing were major limitations of the study. Subsequent development of the drug was discontinued as the company went out of business.

STA-9090 (Ganetespib)

STA-9090 is a second-generation, resorcinol derivative that binds to the NTD ATP pocket of HSP90.107, 108 Developed by Synta Pharmaceuticals, this potent HSP90i arrests cell proliferation and induces apoptosis in various human cancer cell lines such as KIT-dependent malignant mast cell lines, MET-dependent osteosarcoma cell lines, Wilms tumor 1-dependent myeloid leukemias and JAK/STAT signaling dependent malignant hematologic cells.¹⁰⁹ It was also reported to significantly reduce tumor size in mouse xenograft models.¹¹⁰ To date, STA-9090 has been administered as a therapy in a total of 38 clinical trials, of which 24 were completed, 11 were terminated/withdrawn, 2 trials have unknown status, and 1 is still active/recruiting.¹¹¹ In a phase I dose-escalation study of ganetespib in combination with paclitaxel and trastuzumab in patients with HER2-positive metastatic breast cancer, the recommended phase II dose (RP2D) established was 150 mg/m^2. The combination of ganetespib with paclitaxel plus trastuzumab was well tolerated and the most common grade1/2 AEs were diarrhea, fatigue, anemia, and rash. The ORR was 22 %, SD was observed in 56 % of the patients, and the clinical benefit rate was 44 %.¹¹²

A second single-arm phase I study evaluated the combination of zaltrap (ziv) aflibercept, an antiangiogenic drug, with ganetespib in patients with advanced carcinomas and sarcomas. The starting DL 1 of ganetespib and ziv-aflibercept was 100 mg/m² (i.v.) and 4 mg/kg i.v., respectively. However, the dose of ganetespib was reduced to 100 mg/m² i.v. and ziv-aflibercept to 3 mg/kg i.v.¹¹³ Five patients were treated with this combination and three of four evaluable patients exhibited SD. Patients experienced multiple AEs, with gastrointestinal toxicities being the most common. Two deaths on trial were attributed to the treatment.¹¹³ The trial was terminated before the MTD was established, but the limited data suggested that at this dose, the combination was too toxic. In 2018, results from a phase II trial of ganetespib in metastatic uveal melanoma were published, which demonstrated that though ganetespib at 150 mg/m² twice weekly showed modest clinical benefit but was poorly tolerated and was associated with significant but manageable, gastrointestinal toxicity.¹¹⁴

Ganetespib has been administered as a single agent in EGFR-mutated, KRAS-mutated, and ALK-rearranged NSCLC.¹¹⁵ In the GALAXY-I trial, the addition of ganetespib to docetaxel improved OS and PFS versus docetaxel alone.¹¹⁶ These findings led to the GALAXY-2 study, which was the first phase III study in patients with NSCLC that compared ganetespib in combination with docetaxel versus docetaxel alone. The patients received 150 mg/m² ganetespib i.v. and docetaxel at 75 mg/m² i.v. in both treatment arms. The addition of ganetespib to docetaxel did not improve efficacy, and there were higher treatment-related AEs i.e., 82.5 % in the combination arm versus 77.2 % in the docetaxel arm. Likewise, the incidence of serious AEs in the combination arm was 41 % as compared to 31 % in docetaxel alone. Median PFS was 4.2 months in the combination arm and 4.3 months in the docetaxel arm. The ORR was 13.7 % in the combination arm compared to 16 % in docetaxel arm.¹¹⁷

Although, ganestespib in combination with docetaxel did not produce effective clinical outcomes in the trial described above, a dose escalation study of ganetespib in patients with pleural mesothelioma (MESO-2 trial) supported further investigation of ganetespib combination therapy for malignant pleural mesothelioma (MPM) in a large randomized controlled trial. Ganetespib at 200 mg/m² was safe in combination with pemetrexed and platinum chemotherapy, and it showed promising antitumor activity.¹¹⁸ The study concluded that the response rates for ganetespib (52 %) in combination with pemetrexed and platinum chemotherapy were better than the other novel agents in MPM trials.

Ganetespib demonstrated potent in vitro cytotoxicity in gastric cancer cell lines and significantly inhibited the growth of xenograft gastric tumors in vivo as a single agent or in combination with cisplatin.¹¹⁹ Based on the preclinical evidence, a phase II clinical trial of ganetespib in patients with refractory advanced esophagogastric cancer was conducted, and the results of this trial were published in 2020.¹²⁰ Patients received ganestespib 200 mg/m² i.v. and the most common drug-related AEs were diarrhea, fatigue, elevated ALKP, and elevated AST. One patient developed a grade 5 AE (non-neutropenic septic shock). Out of 26 evaluable patients, 1 patient achieved a complete response, 1 patient achieved SD, and 2 patients achieved minor responses. Median PFS and OS were 61 days and 94 days, respectively.¹²⁰ This phase II study was limited due to a lack of biomarker selection and insufficient therapeutic response of ganetespib as a single agent therefore, the trial was terminated early. With the closure of Synta Pharmaceuticals, ganetespib is no longer available for clinical use.

XL888

An orally bioavailable, small-molecule inhibitor of HSP90, XL-888, developed by Exelixis,¹²¹ is currently in phase I trial for melanoma and gastrointestinal adenocarcinomas. A phase I dose escalation clinical trial of vemurafenib in combination with XL888 was conducted in patients with advanced BRAF^V600-mutant melanoma to determine MTD, safety, and efficacy of the combination.¹²² Twenty-one patients with advanced BRAF^{V600E/K-mutant} melanoma were enrolled for the study in 4 dose-level cohorts of XL888: 30 mg/kg per os biweekly (b.i.w.). Three patients with 45 mg/kg b.i.w., 3 patients with 90 mg/kg b.i.w., 9 patients with 135/kg mg b.i.w., and 6 patients with (960 mg/kg b.i.d.) standard dose of vemurafenibwere administered in each cohort. Grade 3 DLTs, including diarrhea and pancreatitis, were observed at DL 135 mg/kg therefore, 90 mg b.i.w was established as MTD. Objective responses were observed in 15 out of 20 evaluable patients with 3 CR and 12 PR. However, there was no definite correlation between the level of response and the XL888 dose. Two patients achieved a pathologic CR following resection of all residual diseases. This study suggested further evaluation of XL888 in combination with BRAF–MEK inhibitor, a current standard-of-care treatment for advanced BRAF^V600-mutant melanoma.¹²²

A meeting abstract published in JCO 2020 reported the results of the dose escalation study of a phase Ib trial of XL888 and pembrolizumab combination in advanced gastrointestinal adenocarcinomas.¹²³ The XL888 and pembrolizumab combination had an acceptable safety profile, and the established recommended phase II dose (RP2D) of XL888 was 90 mg orally twice weekly combined with pembrolizumab 200 mg (i.v.) every 3 weeks. The most common treatment-related toxicities included 1 grade 3 autoimmune hepatitis, 2 grade 2 retinopathy, 1 grade 2 nausea, 1 grade 2 constipation, and 3 grade 2 diarrhea. Pretreatment and on-treatment peripheral blood specimens were collected for immunologic correlative studies, which are underway.¹²³

TAS-116 (pimitespib)

TAS-116 (pimitespib) is an oral HSP90i discovered and developed by Taiho Pharmaceutical Company, Japan.¹²⁴ TAS-116 selectively inhibits cytosolic HSP90 isoforms α and β and not the endoplasmic reticulum and mitochondrial paralogs (GRP94 and TRAP1).¹²⁵ It displays anticancer activity by destabilizing HSP90 client proteins such as KIT, PDGFRA, HER2, and EGFR, which have been implicated in the growth and survival of different cancer types.¹²⁶ According to clinicaltrials.gov, there are 4 trials of TAS-116, 2 recruiting, 1 complete, and 1 upcoming for TAS-116 plus palbociclib in breast and Rb-null cancer which has not yet started recruiting patients.¹²⁷ A first in-human phase I trial of TAS-116 in solid tumors was conducted in Japan and the UK. The results determined 107.5 mg/m²/day for everyday, and 210.7 mg/m²/day for every other day as MTD. Around 160 mg/m² was selected as the MTD for the expansion phase. Grade 3 night blindness, visual impairment, and an increase in AST/ALT/γ-glutamyl transferase were observed at a dose of 150.5 mg/m². Two patients with NSCLC and one patient with GIST had a PR.¹²⁸ The trial supported further development of TAS-116 as it had an acceptable safety profile with antitumor activity. The efficacy and safety of TAS-116 were also evaluated in patients with metastatic or unresectable GIST refractory to imatinib, sunitinib and regorafenib where TAS-116 demonstrated clinical benefit including a PFS of 4.4 months irrespective of KIT or PDGFRA mutations, while OS was 11.5 months. Although 20 % of patients experienced eye disorders, the treatments were not discontinued.¹²⁹ Tsuge and his group showed that TAS-116 exerted antitumor activity in vivo and in vitro by inhibiting IL2-STAT5 signaling and thus reducing regulatory T-cells (Tregs).¹³⁰ They also reported that a combination of an immune checkpoint inhibitor (nivolumab) with TAS-116 had superior antitumor activity when compared to TAS-116 monotherapy. Based on these data, a phase Ib trial of this combination therapy was conducted for colorectal cancer and other solid tumors to assess its safety and efficacy.¹³¹ TAS-116 monotherapy was given orally once daily at 80–160 mg dose for 2 weeks, followed by in combination with nivolumab (3 mg/kg i.v. every 2 weeks). The MTD of TAS-116 in combination with nivolumab was 160 mg, and no DLTs were observed. Overall ORR was 14 % in the total patient population; 16 % in microsatellite stability (MSS) colorectal patients without prior anti-PD1/PDL1 therapy, 0 % in patients with liver metastasis, and 13 % in patients with lung metastasis. The overall median PFS and OS were 3.0 months and 10.1 months, respectively, in the total patient population and 3.2 months and 13.5 months, respectively, in MSS colorectal cancer patients without prior anti–PD-1/PD-L1 treatment.¹³¹ While the study was limited by patient population, the combination of TAS-116 and nivolumab was found to be safe and to exhibit antitumor activity. A randomized phase III trial evaluated the efficacy and safety of TAS-116 in patients with advanced GIST refractory to TKI therapy.¹³² Consistent with the phase II studies, TAS-116 was safe, and the most common AEs observed were diarrhea and decreased appetite. The median PFS with TAS-116 was 2.8 months versus 1.4 months in patients receiving a placebo. Based on these findings, the Japanese FDA approved TAS-116 in GIST patients refractory to TKI therapy.¹³³ However, the study was limited to only Japanese patients; to evaluate long term safety data for clinical practice additional patients are required.¹³² The clinical trials of HSP90 inhibitors are summarized in Table 1.

Table 1.

Summary of HSP90 inhibitors in recent cancer clinical trials.

Identifier	HSP90 Inhibitor/Combination drug	Cancer type	Phase	MTD	DLT	Clinical activity	Trial conclusion	Reference
NCT01685268	AT13387 + abiraterone acetate Prednisolone/ prednisone	CRPC	I/II	220 mg/m2 once weekly 120 mg/m2 twice weekly	Grade (Gr) 3 diarrhea at 260 mg/m2 or 160 mg/m2	Regimen 1 PFS = 77 days OS = 10.6 months Regimen 2 PFS = 84 days OS = 8.9 months	Not effective clinical activity	77
NCT02503709	AT13387 + AT7519	Advanced solid tumors	I	80 mg/m2	Gr-3 increase in cardiac troponins Gr- 3 oral mucositis	69.5% Patients progressed Partial response: 2 patients Stable disease: 2 patients	Combination therapy tolerable promising preliminary activity	78
NCT02535338	AT13387 + erlotinib	NSCLC	I/II	120 mg/m2	Two Gr-3 DLT at 150 mg/m2 No DLT at 120 mg/m2	No responses observed Median PFS = 5.4 months	Limited tolerability limited clinical activity	79
NCT02898207	AT13387 + Olaparib	Advanced solid tumors	I	40 mg and 80 mg	No DLTs observed at 40/80 mg dose of onalespib	Stable disease ≥24 in 32% patients	Combination therapy feasible limited clinical activity	80
NCT02097225	AT13387+ Dabrafenib and trametinib	BRAF-mut solid tumors	I	260 mg/m2 once weekly	N/A	Partial response: 16.7% Stable disease: 66.7% Disease control: 16.7 %	AT13387 in combination with BRAF/MEKi safe	81
NCT01124864	AUY922	NSCLC	II	70 mg/m2	N/A	ALK-PFS = 2.35; OS = 9.53 EGFR-mut. PFS = 2.56; OS = 8.44 EGFR<2-mut PFS = 3.68; OS = 14.52 KRAS-mut PFS = 1.38; OS = 4.68 wild-type PFS = 1.25; OS = 7.82	Manageable safety profile	88
NCT01854034	AUY922	NSCLC	II	70 mg/m2	N/A	ORR: 17%, median PFS = 2.9 months OS = 13 months PR/SD ≥3 months 38%	Well tolerated	87
NCT03333031	HS-196	Detection prostate cancer	I	7 mg	N/A	HS-196 detected in tumor	Well tolerated	89
NCT01393509	PU-H71	Advanced solid tumors	I	MTD (not established)	No DLTs till 470 mg/m2	Stable disease >2 cycles: 35% patients	Trial closed: discontinuation of drug supply	97
NCT01269593	PU-H71	PET imaging-various cancer types	I	N/A	N/A	Tumor tissue: 124I-PU-H71 retained Healthy tissue: cleared rapidly	Supports clinical development of PU-H71	99
NCT03166085	PU-H71 + nab-paclitaxel	PET imaging- HER2-ve m breast cancer	I	300 mg/m2	Cohort 1-1 patient had febrile neutropenia Cohort 2 no DLT	Clinical benefit rate: 42% Overall response rate: 17%. Time to progression: 18.6 weeks	Combination therapy well tolerated showed clinical activity	100
NCT01892046	SNX-5422 + carboplatin paclitaxel	Advanced lung cancers	I	100 mg/m2	No DLTs observed at 50/75 mg/m2 At 100 mg/m2- Elevated AST/ALT Diarrhea and nausea	ORR: 33% Partial response: 33% Stable disease: 56% Progressive disease: 11%	SNX-5422 maintenance monotherapy well tolerated suggests rationale for HSP90i combination therapy	104
NCT02060253	STA9090 + paclitaxel trastuzumab	HER2 positive metastatic breast cancer	I	MTD/R2PD- 150 mg/m2	No DLT observed	ORR: 22% Stable disease: 56% Clinical benefit rate: 44%	The combination therapy safe and well tolerated	110
NCT02192541	STA9090 + ziv-aflibercept	Advanced carcinoma and sarcoma	I	MTD (not established)	N/A	Stable disease: 3 patients	Combination was too toxic	111
NCT01200238	STA9090	mUveal melanoma	II	MTD not defined	N/A	Partial response: 1 patient Stable disease: 4 patients Progressive disease: 11 patients ORR: 5.9% Cohort 1 PFS = 1.6; OS = 8.5 months Cohort 2 PFS = 1.8; OS = 4.9	Ganetespib was poorly tolerated associated with gastrointestinal toxicity	112
NCT01798485	STA9090 + docetaxel	NSCLC	III	N/A	N/A	Ganetespib arm; PFS in = 4.2; OS = 10.9 months Docetaxel arm = 4.3 months; OS = 10.5 months	HSP90 inhibition in NSCLC will be unlikely studied further	115
NCT01590160	STA9090 + pemetrexed cisplatin	Malignant pleural mesothelioma (MPM)	Ib	200 mg/m2	Gr-3 nausea Gr-2 infusion related reaction	Partial response: 56% Disease control: 83% PFS = 6.3 months	Combination therapy safe should be investigated in larger studies	116
NCT01167114	STA9090	Esophagogastric cancer	II	200 mg/m2	N/A	Complete response: 1 patient Stable disease: 1 patient Minor response: 2 patients ORR: 4% PFS = 2 months OS = 3.1 months	Manageable toxicity a subset of EG patients could benefit from ganetespib	118
NCT01657591	XL888 + vemurafenib	BRAFV600-mutant melanoma	I	90 mg	Gr- 3 DLTs diarrhea and pancreatitis at 135 mg	Complete response: 3 patients Partial response: 12 patients Pathologic complete response: 2 patients ORR: 4% PFS = 2 months OS = 3.1 months	Suggests further evaluation of XL888 in combination to BRAF–MEK inhibitor	120
NCT03095781	XL888 + pembrolizumab	Advanced gastrointestinal adenocarcinoma	I	RP2D-90 mg	N/A	N/A	Study ongoing	121
NCT02965885	TAS-116	Solid tumors	I	107.5 mg/m2 q.d. 210.7 mg/m2 q.o.d. 160 mg/m2 Expansion phase	Gr-3 night blindness, visual impairment and, AST/ALT/γ-glutamyl transferase (γ-GTP) at 150.5 mg/m2	Partial response: 2 NSCLC patients; 1-GIST	Acceptable safety Antitumor activity	126
JapicCTI-163182	TAS-116	Metastatic or unresectable GIST	II	N/A	N/A	PFS = 4.4; OS = 11.5 Stable disease >6 weeks: 85% patients	Clinical activity observed Phase III studies underway	127
EPOC1704	TAS-116 + Nivolumab	Colorectal cancer Other solid tumors	Ib	160 mg	No DLTs observed	Overall ORR = 14% Median PFS = 3.0 months Median OS = 10.1 months	Combination therapy safe antitumor activity	129
JapicCTI-184094	TAS-116	Advanced GIST	III	N/A	N/A	Median PFS = 2.8 months	Acceptable safety Improved PFS and cross-over OS versus placebo	130

Abbreviations used: CRPC, castration-resistant prostate cancer; DLT, dose-limiting toxicities; EGFR, epidermal growth factor receptor; GIST, gastrointestinal stromal tumors; HSP90, heat shock protein 90; HSP90i, heat shock protein 90 inhibitor; MTD, maximum tolerated dose; NSCLC, non-small cell lung cancer; ORR, overall response rate; OS, overall survival; PET, positron emission tomography; PFS, progression-free survival; PR, partial response; QD, everyday; QOD, every other day; SD, stable disease.

PEN-866

PEN-866 is a miniaturized drug conjugate linked to SN-38, the active metabolite of irinotecan, which targets and binds to activated HSP90 in tumors.¹³⁴ Compared to SN-38 alone, PEN-866 preferentially targets and is retained in the tumor due to its binding to HSP90. Inside the tumor cells the SN-38 moiety is released in a sustained manner and inhibits topoisomerase I, which results in DNA breaks, inhibition of DNA replication, and apoptosis.¹³⁵ A first in human phase I/IIa study of PEN-866 was conducted to assess safety and preliminary efficacy of PEN-866 in solid tumors.¹³⁴ Of 15 evaluable patients, 1 patient with squamous cell cancer of the anus had a PR, 7 patients had SD at 8 weeks, 4 patients had SD for 12–57 weeks. Phase I results concluded that PEN-866 was well tolerated with acceptable evidence of antitumor activity.¹³⁴ However, further clinical evaluation of PEN-866 has been suspended due to company-related business issues.

PD and pharmacogenomic assessment in HSP90i clinical trials

PD biomarkers are indicators of the effects of therapeutic interventions.¹³⁶ Focused PD biomarker measurements are critical for prognosis and provide a rationale for different drug combinations of targeted agents. In most recent HSP90i clinical trials, various PD markers such as imaging biomarker, molecular biomarkers, circulating tumor cells (CTCs), and immune subsets have proved invaluable to study target engagement and therapeutic outcomes.

Imaging biomarkers

¹²⁴IPU-H71 and HS-196 have been used as imaging agents for the noninvasive screening of human tumors.¹⁰²¹²⁴I- PU-H71 was the first-in-class investigational PET biomarker for tumor epichaperome detection. Epichaperomes reflect an integrated network of the HSP90 chaperome that facilitates tumor survival, has diagnostic implications, and serves as a potential target for drug interventions.¹⁰¹ Phase I clinical trial results showed enhanced PU-H71 retention in tumors by PET because of their increased reliance on the epichaperome network. On the contrary, normal tissues exhibited low PU-H71 tracer concentrations. In HER2-negative metastatic breast cancer, the tumor epichaperome expression was measured using [¹²⁴I] PU-H71 PET as a biomarker of response for PU-H71+Nab-Paclitaxel combination therapy. The PU-PET scans demonstrated that in epichaperome-positive tumors, the molar concentrations of PU-H71 were higher when compared to epichaperome-negative tumors. In TNBC patients, a significant correlation between SUV_max (standardized uptake volume) (PU-PET) and time to progression was observed.¹⁰² These observations suggest that PU-PET can be potentially used for targeting epichaperomes, and it can also be used as a predictive biomarker for PU-H71 combination therapy.

Another phase I study identified aggressive prostate cancer using HS-196 imaging agent.⁹¹ HS-196 was synthesized by linking near Infra-Red molecule to HSP90 targeting probe, which fluoresces at longer wavelengths (790 nm absorption/800 nm emission). Resected prostate tissues from the patients enrolled in this trial were analyzed by LI-COR Odyssey imager which detected fluorescence in malignant nodules within the tissue.⁹¹ The results of these phase I trials demonstrate potential noninvasive clinical usage of HSP90i imaging agents for a new diagnostic approach to detect tumors and understand in vivo drug distribution and responses in early-phase cancer trials.

2[¹⁸F] fluoro-2-deoxy-D-glucose-PET scans in patients during early treatment with ganetespib in metastatic uveal melanoma showed a reduction in metabolic activity in 24 % of patients, implying a PD effect of HSP90 inhibition which did not correlate with clinical benefit.¹¹⁴

Molecular biomarkers

NTD inhibitors induce HSRs, which subsequently upregulate HSP27, HSP40, and HSP70, imparting cytoprotection to cancer cells and increasing resistance to HSP90 inhibition. Target engagement in HSP90i clinical trials was assessed by measuring the levels of various HSPs, such as HSP70 and HSP72, among others. In a phase I/II clinical trial of CRPC, onalespib upregulated HSP72 and moderately reduced the expression of AR and GR (glucocorticoid receptor). But no significant changes were observed in Ki-67(proliferation marker) and cleaved caspase3 (apoptotic biomarker).⁷⁹ In a trial of solid tumors, onalespib treatment increased HSP70 level in plasma and peripheral blood mononuclear cells (PBMCs) but, with the addition of AT7519 (pan-CDKi) HSP70 decreased.⁸⁰ In dose escalation trial of onalespib for patients with BRAF-^mut. solid tumors, the HSP70 levels in PBMCs increased in response to combination therapy; however, HSP70 levels did not correlate with onalespib dose.⁸² Expression of 45 different client proteins studied in patient biopsy samples of patients on treatment (dosing day 0 and day 8) revealed that overall protein expression was heterogenous. Higher BRAF expression was observed in patients who received high doses of trametinib, a MEK inhibitor drug and AT13387. The patients who achieved disease control also had high BRAF and MET expression but low ERBB3 and AKT1/AKT2 expression as compared to the patients who had rapid progression.⁸² In another phase I trial of solid tumors, TAS-116 administration induced HSP70 expression in normal PBMCs in a dose-dependent manner from 4.8 mg/m² to 107.5 mg/m².¹²⁸ In cancer, HSP90 plays an important role in angiogenesis by modulating VEGF signaling and stabilizing the HIF-1α complex.¹³⁷ In a phase I study of advanced carcinoma, administration of ganestespib with the antiangiogenic agent ziv-aflibercept was planned with the endpoint measurement being HIF-1α in tumor tissues of the expansion cohort after MTD was achieved. However, the trial was terminated because this therapy combination was too toxic.¹¹³ In the GALAXY-2 trial of ganestespib with docetaxel in NSCLC patients, plasma was collected for correlative studies.¹¹⁷ It was observed that once every 2-week dosing of ganetespib was not optimal for suppression of client oncoproteins. Lack of biomarker selection and inadequate tissue availability limited biomarker assessment in patients with advanced esophagogastric cancer treated with ganetespib. However, immunohistochemistry analysis showed that patients with 3+ HER2 positive tumors had minor responses.¹¹⁷ In BRAF^600-mut melanoma, proteome-level responses to the BRAF inhibitor vemurafenib and HSP90i XL-888 were heterogenous even in the responders. In this trial, LC-MRM mass spectrometry assays were performed with patient-derived PBMCs, which showed an increase in HSP70 expression in most of the patients. Several HSP90 client proteins were also analyzed in the pre (day 0) and on therapy (day 8) tumor biopsy samples. For example, ERBB3, the mediator of BRAF inhibitor resistance, BAD, Bcl-2, BIM, Bok, c-MET, and IRS1 all decreased in patients receiving vemurafenib + XL888 but not in patients that received vemurafenib alone. The RTK signaling pathway, the MAPK pathway, AKT signaling, β-catenin pathway, and negative apoptosis regulators also exhibited variable expression in responders. The other HSP90 client proteins implicated in BRAF inhibition, such as RAF-1 decreased and KRAS increased after combination therapy. The complex observations from this trial highlight the importance of ‘system level analysis of HSP90 client proteins which should be degraded for a robust clinical response.¹²² In a combination trial of TAS-116 with nivolumab, PDL1 expression was studied by immunohistochemistry, which showed that patients having a combined positive score of PD-L1 ≥1 had 27 % ORR, while patients with combined positive score <1 had 0 %.¹³¹

CTCs and immune subset analysis

In a phase I/II prostate cancer trial in which patients were treated with a combination of HSP90 Inhibitor onalespib and AA, CTC enumeration as well as level of AR in CTCs were evaluated as PD biomarkers.⁷⁹ The therapy transiently decreased CTCs and AR + CTCs in more than 30 % of patients on treatment, relative to baseline CTC values. In the dose escalation cohort of TAS-116 monotherapy,¹³¹ while the frequency of effector Tregs (eTregs) among the CD4+ T-cells decreased, the CD8+ T cells/eTregs ratio increased after therapy at any dose. However, in tumor-infiltrating lymphocytes, the frequency of eTregs among the CD4+ T-cells remained unchanged. At doses of 120 and 160 mg, the proportion of CTLA-4, a suppressive Treg functional marker, + eTregs among CD4+ T-cells and CTLA4+ eTregs among CD8+ T-cells significantly decreased, suggesting that TAS-116 inhibited Treg-mediated immunosuppression in tumor-infiltrating lymphocytes. After 6 weeks of combination therapy with TAS-116 and nivolumab, decreased eTregs among the CD4+ T-cells were observed, but this combination therapy increased the CD8+ T cell/eTreg ratio in PBMCs. It was concluded that this combination therapy enhanced antitumor activity via Treg reduction.¹³¹

Pharmacogenomics analysis

Measuring variations in spatially defined gene expression can be a useful tool for understanding target engagement and treatment response.¹³⁸ For instance, mutations detected by next-generation sequencing (NGS) corresponded with clinical outcomes in EGFR^ex20ins NSCLC and advanced solid tumors.⁸¹ NGS circulating tumor (ct)DNA assay detected EGFR^ex20ins and TP53 co-mutations at baseline in most patients; however, erlotinib + onalespib combination therapy failed to decrease ctDNA, a potential prognostic marker, which was consistent with lack of clinical response.⁸¹ Furthermore, patients with BRCA1/BRCA2-mutated ovarian cancers and acquired PARP inhibitor resistance as well as patients with RB-pathway alterations (e.g., biallelic RB1 loss) detected by targeted NGS, had SD ≥24 weeks and a patient with CCNE1 amplification had tumor shrinkage by 28 % in a phase I study of PARP inhibitor and HSP90i (olaparib + AT13387) for treatment of advanced solid tumors.⁸² For HSP90i-AUY922, NGS data showed its clinical activity as PR in patients with EGFR mutations, exon19 deletions, exon 20 insertions, and acquired T790 mutations.⁸⁹ In the first study of TAS-116 in patients with advanced GIST, NGS assay in ctDNA detected mutations in client proteins of HSP90: KIT, PDGFRA, and BRAF in 75 % of patients. These results suggest that TAS-116 was effective in patients with KIT and PDGFRA mutations. While SD >10 months was observed in patients with BRAF mutation (A728T), in the nonresponders an alternate signaling pathway or induction of HSP70 and HSP27 might have compromised TAS-116 activity.¹²⁹ Similarly, in a randomized double-blind phase III trial in GIST patients, TAS-116 improved PFS in patients with KIT mutations (exon 9 and exon 11; 13/14 or 17/18, respectively). However, due to a small number of patients, the authors were unable to evaluate the effect of TAS-116 in patients with PDGFRA mutations.¹³² In phase I/II trial of TAS-116 in combination with nivolumab, objective tumor responses were observed in MSS colorectal cancer patients regardless of RAS/BRAF mutational status, suggesting clinical activity of this therapy. Tumor mutation burden (TMB) was also analyzed in some patients and revealed that patients with TMB ≥10 mut/Mb (high) had a 25 % ORR, while patients with low TMB had a 13 % ORR.¹³¹

Somatic copy number alterations, global loss of heterozygosity, and total homozygous deletions were assessed in baseline tumor samples of patients enrolled in the MESO-02 trial.¹¹⁸ The results showed that patients with genomically unstable MPMs had a shorter time to progression. Furthermore, higher baseline loss of heterozygosity was not only associated with less reduction in total tumor burden but also with worse clinical outcomes. Although it was hypothesized that the response rate to ganetespib therapy could be higher in patients with lower somatic copy number alterations, this study could not establish any interaction between specific copy number alterations and sensitivity to HSP90 inhibition.¹¹⁸ The hotspot mutations analyzed by SNaPshot profiling in phase II advanced esophagogastric trial showed that a patient with KRAS G12D mutation had a complete response and another patient with EGFR amplification along with 2+ HER2 overexpression had progressive disease as the best response.¹²⁰ This suggests that certain molecular subsets of patients may benefit from HSP90i therapy. Single-cell RNA sequencing can reveal complex and rare cell populations and their temporal-spatial distribution within the tumor tissue, which might correspond to drug response and therapeutic outcomes. In the phase I prostate cancer study,⁹¹ single-cell RNA sequencing analyses of tumor tissue identified differential uptake of HS-196 by cells in the peripheral zone of tumors (HS-196+) compared to cells in the transition/central zone (HS-196 negative). The peripheral zone cells also expressed prostate tumor markers such as TMPRSS2, AMACR, ERG, AR, SYP, and ENO2, which are associated with therapeutic resistance and metastatic progression.⁹¹ The PDs and pharmacogenomic analysis are summarized in Table 2.

Table 2.

PD/PGx evaluated in HSP90 inhibitors cancer clinical trials.

Identifier	HSP90 Inhibitor/Combination Drug	Cancer type	PD/PGx	PD-Outcome	Reference
NCT01685268	AT13387 + abiraterone acetate Prednisolone prednisone	CRPC	IHC in paired tumor biopsies for 1HSP72, 2AR, GR 3Ki-67 and Capase-3 4CTCs-Whole blood	1AT13387 upregulated HSP72 2AR and GR-decreased3 Ki-67, Caspase-3: No change 4Transient reduction of CTCs	77
NCT02503709	AT13387 + AT7519	Advanced solid tumors	HSP70 expression by Western Blot in PBMCs and ELISA in Plasma	AT13387 alone increased HSP70 in PBMCs and plasma AT7519 decreased HSP70	78
NCT02535338	AT13387 + erlotinib	NSCLC	NGS in ctDNA	Mutations detected Combination therapy failed to clear ctDNA	79
NCT02898207	AT13387 + Olaparib	Advanced solid tumors	NGS in tumor specimen	BRCA mut. HGSOC patients progressed; patient with biallelic RB1 loss-had stable disease ≥24 patient with CCNE1 amplification-tumor shrinkage by 28%	80
NCT02097225	AT13387+ Dabrafenib and trametinib	BRAF-mut solid tumors	Liquid chromatography in PBMCs for HSP70 IHC-other client proteins	HSP70 increased but did not correlate to AT13387 Expression of other client proteins heterogenous	81
NCT01124864	AUY922	NSCLC	NGS in tumor tissue	Partial response observed in patients with EGFR mutations, exon19 deletions, exon 20 insertions, and T790M mutations	87
NCT03333031	HS-196	Detection prostate cancer	1Fluorescence imaging of resected tissue 2scRNA-seq	1Fluorescence in malignant nodules confirmed presence of HS-196 2Identified differential uptake of HS-196 by peripheral epithelial and central zone cells	89
NCT01269593	PU-H71	PET imaging-various cancer types	PET-Scan	Retention of PU-H71 in tumors Low PU-H71tracer concentrations in normal tissues	99
NCT03166085	PU-H71 + Nab-paclitaxel	PET imaging- HER2-ve m Breast cancer	PET-Scan	Epichaperome-positive tumors-PU-H71 High concentration epichaperome-negative tumors-PU-H71 low concentration PU-PET SUVmax correlated to disease progression	100
NCT02192541	STA9090 + Ziv-aflibercept	Advanced carcinoma and Sarcoma	HIF-1α planned in tumor tissue	Trial was terminated	111
NCT01200238	STA9090	mUveal melanoma	FDG-PET scan	24% Reduction in metabolic activity	112
NCT01590160	STA9090 + Pemetrexed cisplatin	Malignant pleural mesothelioma (MPM)	Genomic variable in tumor tissues measured at baseline using Oncoscan FFPE assay kit	Total SCNA, LOH, and homozygous deletions measured. Genomically unstable MPMs-shorter progression time LOH associated with less TTB reduction, poor clinical outcome	116
NCT01167114	STA9090	Esophagogastric cancer	1IHC- tumor tissues for HER2 2 Hotspot mutations-SNaPshot profiling	13+ HER2 positive tumors-minor response 2Patient with KRAS G12D mutation-Complete response EGFR amplification + 2 + HER2 mut.: Progressive disease	118
NCT01657591	XL888 + vemurafenib	BRAFV600-mutant melanoma	1LC-MRM mass spectrometry-HSP70 2Tumor biopsy-other client proteins	1HSP70 in PBMCs increased 2Other client proteins- decreased	120
NCT02965885	TAS-116	Solid tumors	HSP70 in PBMCs using ELISA	TAS-116 induced HSP70 expression in PBMCs	126
JapicCTI-163182	TAS-116	Metastatic or unresectable GIST	NGS in ctDNA	Mutations in KIT, PDGFRA and BRAF detected TAS-116 was effective in patients with KIT and PDGFRA Stable disease >10 months in BRAF mut. patients	127
EPOC1704	TAS-116 + Nivolumab	Colorectal cancer Other solid tumors	1IHC-PDL1 2 Immune subset analysis in whole blood 3TMB-Oncomine tumor mutation load assay	1PDL1 expression correlated to ORR 2Decreased e-Tregs on CD4 T cells 2Increased CD8+ T cells/e-Tregs ratio 2At 120/160 mg TAS-116 significantly decreased Tregs in TILs 3High TMB 25% ORR, low TMB 13% ORR	129
JapicCTI-184094	TAS-116	Advanced GIST	NGS in whole blood	Mutations in KIT, PDGFRA detected TAS-116 improved PFS in patients with KIT mut. PDGFRA mut: not evaluated, less patient number	130

Abbreviations used: CRPC, castration-resistant prostate cancer; CTCs, circulating tumor cells; ctDNA, circulating tumor DNA; EGFR, epidermal growth factor receptor; e-Tregs, effector Tregs; HIF, hypoxia-inducible factor; HSP90, heat shock protein 90; IHC, immunohistochemistry; LOH, loss of heterozygosity; NGS, next-generation sequencing; NSCLC, non-small cell lung cancer; ORR, overall response rate; PBMCs, peripheral blood mononuclear cells; PD, pharmacodynamic; PET, positron emission tomography; PGx, pharmacogenomic; scRNA-seq, single-cell RNA sequencing; SUV, standardized uptake volume; TIL, tumor-infiltrating lymphocytes; TMB, tumor mutation burden.

Conclusions

Despite the long journey of HSP90i in clinical trials, no HSP90 inhibitor has been approved by the US FDA. The low effectiveness of HSP90i in clinical trials can be attributed to various factors, such as lack of appropriate biomarker selection, limited drug efficacy, toxicity profiles, and limited patient accrual. To maximize the clinical efficacy of HSP90i, precision delivery systems using nanocarriers could be employed to avoid systemic and organ toxicity. Furthermore, novel drug combinations must be explored to overcome therapy resistance. One potential approach is the development of chimeric drugs capable of simultaneously targeting various HSPs involved in cancer progression. The disruption of PPIs also offers an alternative strategy to selectively inhibit HSP90-co-chaperones. Identifying the potential sites of interaction with HSP90 machinery and developing inhibitors have appeared to be effective so far in various in vitro and preclinical studies against different cancer types. For example, MJC13 a compound targeting FK506 binding protein, FKBP52 an important regulator of AR, inhibited AR-dependent prostate cancer cell proliferation in vitro, and therefore could be exploited against prostate cancer therapeutics.¹³⁹ Similarly, P053, a competitive inhibitor of PP5 co-chaperone of HSP90, which regulates tumor initiation and apoptotic pathways,¹⁴⁰ and SEW84, inhibitor of Aha1-dependent Hsp90 activities²⁹ could be reckoned for therapeutical approaches against cancer treatment. Another approach involves combining immune checkpoint inhibitors with drugs targeting HSP90 and its paralogs. By selecting cancers driven by HSP90 client proteins, the therapeutic efficacy of any HSP90i drug combination can be improved. Identification of novel prognostic biomarkers relevant to HSP90 inhibition should be explored to optimize treatment strategies. Recent advances in NGS technology offer researchers the opportunity to discover novel mutations associated with clinical outcomes. Additionally, accurate diagnosis using NGS technology, could benefit clinicians to design effective treatment plans. Overall, this review highlights the importance of the mechanisms and challenges involved in the clinical application of HSP90 inhibitors. By addressing the limitations and exploring innovative approaches, we can enhance the potential of HSP90 inhibitors as valuable therapeutic agents in cancer treatment.

Declarations of interest

The authors have no conflict of interest to disclose.

Data availability

No data was used for the research described in the article.