17-AAG Induces Endoplasmic Reticulum Stress-mediated Apoptosis in Breast Cancer Cells, Possibly Through PERK/eIF2α Up-regulation.

BACKGROUND/AIM: Breast cancer is the most predominant type of cancer affecting women worldwide and the current therapeutic treatment for breast cancer patients is not adequately effective. This study aimed to investigate the mechanism of 17-AAG, a heat shock protein (HSP90) inhibitor, as a treatment for inducing breast cancer cell apoptosis. MATERIALS AND METHODS: The pharmacology network was employed to examine the correlation of 17-AAG with the gene expression profiles of breast cancer, obtained by Gene Expression Profiling Interactive Analysis (GEPIA). MTT and flow cytometry were utilized to investigate cell proliferation and cell apoptosis, respectively. Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay and western blot analysis were employed to examine the correlation between cellular oxidant levels and protein expression. Immunofluorescence staining was utilized to confirm the protein localization and assess DNA damage. RESULTS: The pharmacological network analysis revealed that HSP90 serves as the common target connecting 17-AAG and breast cancer genes. Treatment with 17-AAG significantly increased cell apoptosis. Moreover, the treatment resulted in up-regulation of cellular oxidant levels and PERK/eIF2α expression. In line with these, protein localization after treatment revealed an increase in DNA damage, correlating with higher ER stress levels. Furthermore, GEPIA demonstrated that PERK and eIF2α expression were significantly higher in breast invasive carcinoma compared to other tumor types. CONCLUSION: HSP90 emerges as a potential target for inducing apoptosis in breast cancer cells by disrupting protein homeostasis in the endoplasmic reticulum, possibly through PERK/eIF2α up-regulation. 17-AAG, an HSP90 inhibitor, may therefore potentially hold an alternative therapeutic strategy for breast cancer treatment.

Cardiovascular safety of pimitespib in patients with advanced solid tumors: An open-label, nonrandomized, phase 1 study.

BACKGROUND: Pimitespib (TAS-116), a first-in-class, oral, selective heat-shock protein 90 inhibitor, is approved as fourth-line treatment for gastrointestinal stromal tumors in Japan. This phase 1 study evaluated the cardiac safety of pimitespib. METHODS: In this open-label, nonrandomized, multicenter study, Japanese patients (aged ≥20 years) with refractory, advanced solid tumors received placebo on day -1, then pimitespib 160 mg daily on days 1-5 of the cardiac safety evaluation period. Electrocardiograms were conducted at baseline, and on days -2, -1, 1, and 5; and blood samples were collected on days 1 and 5. Patients then received once-daily pimitespib for 5 days every 3 weeks. The primary end point was the time-matched difference in QT interval corrected for heart rate using the Fridericia correction (QTcF) between pimitespib and placebo. Pharmacokinetics, safety, and preliminary efficacy were also assessed. RESULTS: Of the 22 patients in the cardiac safety-evaluable population, no clinically relevant QTc prolongation was observed; the upper bound of the one-sided 95% confidence interval for the time-matched difference in change from baseline in QTcF was <20 msec at all time points on days 1 and 5. Pimitespib pharmacokinetic parameters were consistent with previous data, and the time-matched difference in change from baseline in QTcF showed no marked increase as plasma concentrations increased. The safety profile was acceptable; 40% of patients experienced grade 3 or greater adverse drug reactions, mostly diarrhea (20%). The median progression-free survival was 3.1 months. CONCLUSIONS: In Japanese patients with refractory, advanced solid tumors, pimitespib was not associated with clinically relevant QTc prolongation, and there were no cardiovascular safety concerns. PLAIN LANGUAGE SUMMARY: Pimitespib is a new anticancer drug that is being used to treat cancer in the stomach or intestines (gastrointestinal stromal tumors). This study demonstrated that pimitespib had no marked effect on heart rhythm or negative effects on the heart or blood vessels and had promising anticancer effects in Japanese patients with advanced solid tumors who were unable to tolerate or benefit from standard treatment.

The small-molecule drug homoharringtonine targets HSF1 to suppress pancreatic cancer progression.

Heat shock factor 1 (HSF1), an essential transcription factor for stress response, is exploited by various tumors to facilitate their initiation, progression, invasion, and migration. Amplification of HSF1 is widely regarded as an indicator in predicting cancer severity, the likelihood of treatment failure and reduced patient survival. Notably, HSF1 is markedly amplified in 40% of pancreatic cancer (PC), which typically have limited treatment options. HSF1 has been proven to be a promising therapeutic target for multiple cancers. However, a direct small molecule HSF1 inhibitor with sufficient bioactivity and reliable safety has not been developed clinically. In this study, we successfully established a high-throughput screening system utilizing luciferase reporter assay specifically designed for HSF1, which leads to the discovery of a potent small molecule inhibitor targeting HSF1. Homoharringtonine (HHT) selectively inhibited PC cell viability with high HSF1 expression and induced a markedly stronger tumor regression effect in the subcutaneous xenograft model than the comparator drug KRIBB11, known for its direct action on HSF1. Moreover, HHT shows promise in countering the resistance encountered with HSP90 inhibitors, which have been observed to increase heat shock response intensity in clinical trials. Mechanistically, HHT directly bound to HSF1, suppressing its expression and thereby inhibiting transcription of HSF1 target genes. In conclusion, our work presents a preclinical discovery and validation for HHT as a HSF1 inhibitor for PC treatment.

FLT3 and IRAK4 Inhibitor Emavusertib in Combination with BH3-Mimetics in the Treatment of Acute Myeloid Leukemia.

Targeting the FLT3 receptor and the IL-1R associated kinase 4 as well as the anti-apoptotic proteins MCL1 and BCL2 may be a promising novel approach in the treatment of acute myeloid leukemia (AML). The FLT3 and IRAK4 inhibitor emavusertib (CA4948), the MCL1 inhibitor S63845, the BCL2 inhibitor venetoclax, and the HSP90 inhibitor PU-H71 were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells in vitro. AML cells represented all major morphologic and molecular subtypes, including FLT3-ITD and NPM1 mutant AML cell lines and a variety of patient-derived AML cells. Emavusertib in combination with MCL1 inhibitor S63845 or BCL2 inhibitor venetoclax induced cell cycle arrest and apoptosis in MOLM-13 cells. In primary AML cells, the response to emavusertib was associated with the presence of the FLT3 gene mutation with an allelic ratio >0.5 and the presence of NPM1 gene mutations. S63845 was effective in all tested AML cell lines and primary AML samples. Blast cell percentage was positively associated with the response to CA4948, S63845, and venetoclax, with elevated susceptibility of primary AML with blast cell fraction >80%. Biomarkers of the response to venetoclax included the blast cell percentage and bone marrow infiltration rate, as well as the expression levels of CD11b, CD64, and CD117. Elevated susceptibility to CA4948 combination treatments with S63845 or PU-H71 was associated with FLT3-mutated AML and CD34 < 30%. The combination of CA4948 and BH3-mimetics may be effective in the treatment in FLT3-mutated AML with differential target specificity for MCL1 and BCL2 inhibitors. Moreover, the combination of CA4948 and PU-H71 may be a candidate combination treatment in FLT3-mutated AML.

The HSP90 inhibitor HVH-2930 exhibits potent efficacy against trastuzumab-resistant HER2-positive breast cancer.

Rationale: Resistance to targeted therapies like trastuzumab remains a critical challenge for HER2-positive breast cancer patients. Despite the progress of several N-terminal HSP90 inhibitors in clinical trials, none have achieved approval for clinical use, primarily due to issues such as induction of the heat shock response (HSR), off-target effects, and unfavorable toxicity profiles. We sought to examine the effects of HVH-2930, a novel C-terminal HSP90 inhibitor, in overcoming trastuzumab resistance. Methods: The effect of HVH-2930 on trastuzumab-sensitive and -resistant cell lines in vitro was evaluated in terms of cell viability, expression of HSP90 client proteins, and impact on cancer stem cells. An in vivo model with trastuzumab-resistant JIMT-1 cells was used to examine the efficacy and toxicity of HVH-2930. Results: HVH-2930 was rationally designed to fit into the ATP-binding pocket interface cavity of the hHSP90 homodimer in the C-terminal domain of HSP90, stabilizing its open conformation and hindering ATP binding. HVH-2930 induces apoptosis without inducing the HSR but by specifically suppressing the HER2 signaling pathway. This occurs with the downregulation of HER2/p95HER2 and disruption of HER2 family member heterodimerization. Attenuation of cancer stem cell (CSC)-like properties was associated with the downregulation of stemness factors such as ALDH1, CD44, Nanog and Oct4. Furthermore, HVH-2930 administration inhibited angiogenesis and tumor growth in trastuzumab-resistant xenograft mice. A synergistic effect was observed when combining HVH-2930 and paclitaxel in JIMT-1 xenografts. Conclusion: Our findings highlight the potent efficacy of HVH-2930 in overcoming trastuzumab resistance in HER2-positive breast cancer. Further investigation is warranted to fully establish its therapeutic potential.

Gene expression markers in peripheral blood and outcome in patients with platinum-resistant ovarian cancer: A study of the European GANNET53 consortium.

Disease progression is a major problem in ovarian cancer. There are very few treatment options for patients with platinum-resistant ovarian cancer (PROC), and therefore, these patients have a particularly poor prognosis. The aim of the present study was to identify markers for monitoring the response of 123 PROC patients enrolled in the Phase I/II GANNET53 clinical trial, which evaluated the efficacy of Ganetespib in combination with standard chemotherapy versus standard chemotherapy alone. In total, 474 blood samples were collected, comprising baseline samples taken before the first administration of the study drugs and serial samples taken during treatment until further disease progression (PD). After microfluidic enrichment, 27 gene transcripts were analyzed using quantitative polymerase chain reaction and their utility for disease monitoring was evaluated. At baseline, ERCC1 was associated with an increased risk of PD (hazard ratio [HR] 1.75, 95% confidence interval [CI]: 1.20-2.55; p = 0.005), while baseline CDH1 and ESR1 may have a risk-reducing effect (CDH1 HR 0.66, 95% CI: 0.46-0.96; p = 0.024; ESR1 HR 0.58, 95% CI: 0.39-0.86; p = 0.002). ERCC1 was observed significantly more often (72.7% vs. 53.9%; p = 0.032) and ESR1 significantly less frequently (59.1% vs. 78.3%; p = 0.018) in blood samples taken at radiologically confirmed PD than at controlled disease. At any time during treatment, ERCC1-presence and ESR1-absence were associated with short PFS and with higher odds of PD within 6 months (odds ratio 12.77, 95% CI: 4.08-39.97; p < 0.001). Our study demonstrates the clinical relevance of ESR1 and ERCC1 and may encourage the analysis of liquid biopsy samples for the management of PROC patients.

Intervention treatment reducing cellular senescence inhibits tubulointerstitial fibrosis in diabetic mice following acute kidney injury.

Senescence of kidney tubules leads to tubulointerstitial fibrosis (TIF). Proximal tubular epithelial cells undergo stress-induced senescence during diabetes and episodes of acute kidney injury (AKI), and combining these injuries promotes the progression of diabetic kidney disease (DKD). Since TIF is crucial to progression of DKD, we examined the therapeutic potential of targeting senescence with a senolytic drug (HSP90 inhibitor) and/or a senostatic drug (ASK1 inhibitor) in a model of TIF in which AKI is superimposed on diabetes. After 8 weeks of streptozotocin-induced diabetes, mice underwent bilateral clamping of renal pedicles to induce mild AKI, followed by 28 days of reperfusion. Groups of mice (n=10-12) received either vehicle, HSP90 inhibitor (alvespimycin), ASK1 inhibitor (GS-444217), or both treatments. Vehicle-treated mice displayed tubular injury at day 3 and extensive tubular cell senescence at day 10, which remained unresolved at day 28. Markers of senescence (Cdkn1a and Cdkn2a), inflammation (Cd68, Tnf, and Ccl2), and TIF (Col1a1, Col4a3, α-Sma/Acta2, and Tgfb1) were elevated at day 28, coinciding with renal function impairment. Treatment with alvespimycin alone reduced kidney senescence and levels of Col1a1, Acta2, Tgfb1, and Cd68; however, further treatment with GS-444217 also reduced Col4a3, Tnf, Ccl2, and renal function impairment. Senolytic therapy can inhibit TIF during DKD, but its effectiveness can be improved by follow-up treatment with a senostatic inhibitor, which has important implications for treating progressive DKD.

Phase Ib study of HSP90 inhibitor, onalespib (AT13387), in combination with paclitaxel in patients with advanced triple-negative breast cancer.

Background: Heat shock protein 90 (HSP90) is a molecular chaperone required for stabilization of client proteins over-activated in triple-negative breast cancer (TNBC). Over-expression of HSP90 client proteins has been implicated in paclitaxel resistance. Onalespib (AT13387) is a potent inhibitor of HSP90 that could improve paclitaxel efficacy when administered in combination. Design: This phase Ib trial administered onalespib with paclitaxel in patients with advanced TNBC to assess safety and establish a recommended phase II dose (RP2D). Objectives: The primary objectives were determining the dose-limiting toxicities and maximum tolerated dose of combination therapy. Secondary objectives included pharmacokinetic (PK) analysis and determination of overall response rate (ORR), duration of response (DOR), and progression-free survival (PFS). Methods: Patients with advanced TNBC were treated with standard dose intravenous paclitaxel in combination with intravenous onalespib at doses ranging from 120 to 260 mg/m2 administered on days 1, 8, and 15 of a 28-day cycle using a standard 3 + 3 design. A total of 15 patients were enrolled to dose expansion cohort at RP2D to confirm safety profile. Results: Thirty-one patients were enrolled in the study, of which over 90% had received prior taxane therapy. Paclitaxel was given for metastatic disease in 23% of patients. Adverse events (AEs) included anemia (grade 3: 20%), lymphopenia (grade 3: 17%), and neutropenia (grade 3: 33%, grade 4: 4%). The most frequent grade ⩾3 non-hematologic AE was diarrhea (7%). The established RP2D was 260 mg/m2 onalespib when given with paclitaxel at 80 mg/m2. PK analysis revealed a modest drug interaction profile for onalespib in the combination regimen. ORR was 20%. Three patients achieved complete responses, all of whom had received prior taxane therapy. Median DOR was 5.6 months; median PFS was 2.9 months. Conclusion: Combination treatment with onalespib and paclitaxel had an acceptable toxicity profile and RP2D was determined to be 260 mg/m2 of onalespib. Combination therapy showed antitumor activity in patients with advanced TNBC. Trial registration: Onalespib and paclitaxel in treating patients with advanced TNBC https://clinicaltrials.gov/ct2/show/NCT02474173.

Phase 1b study of HSP90 inhibitor called onalespib in combination with paclitaxel in patients with advanced triple-negative breast cancer This Phase 1b study demonstrated that treatment with a combination of onalespib and paclitaxel was reasonably well tolerated by most patients. Onalespib at 260 mg/m² given intravenously on days 1, 8 and 15 on 28-day cycles in combination with standard dose and schedule of paclitaxel was established as the recommended phase 2 dose for further clinical development. Despite minor drug-drug interactions between these 2 agents, onalespib did not alter paclitaxel exposure and paclitaxel did not affect exposure to onalespib. While onalespib with paclitaxel combination therapy did not yield durable objective responses or prolonged progression-free survival, there were several patients with long-lasting benefit from this combination including patients who previously experienced progression on taxane therapy.

Enhanced Therapeutic Effects of 177Lu-DOTA-M5A in Combination with Heat Shock Protein 90 Inhibitor Onalespib in Colorectal Cancer Xenografts.

Carcinoembryonic antigen (CEA) has emerged as an attractive target for theranostic applications in colorectal cancers (CRCs). In the present study, the humanized anti-CEA antibody hT84.66-M5A (M5A) was labeled with 177Lu for potential CRC therapy. Moreover, the novel combination of 177Lu-DOTA-M5A with the heat shock protein 90 inhibitor onalespib, suggested to mediate radiosensitizing properties, was assessed in vivo for the first time. M5A antibody uptake and therapeutic effects, alone or in combination with onalespib, were assessed in human CRC xenografts and visualized using SPECT/CT imaging. Although both 177Lu-DOTA-M5A and onalespib monotherapies effectively reduced tumor growth rates, the combination therapy demonstrated the most substantial impact, achieving a fourfold reduction in tumor growth compared to the control group. Median survival increased by 33% compared to 177Lu-DOTA-M5A alone, and tripled compared to control and onalespib groups. Importantly, combination therapy yielded comparable or superior effects to the double dose of 177Lu-DOTA-M5A monotherapy. 177Lu-DOTA-M5A increased apoptotic cell levels, indicating its potential to induce tumor cell death. These findings show promise for 177Lu-DOTA-M5A as a CRC therapeutic agent, and its combination with onalespib could significantly enhance treatment efficacy. Further in vivo studies are warranted to validate these findings fully and explore the treatment's potential for clinical use.

HSP90 Inhibitor PU-H71 in Combination with BH3-Mimetics in the Treatment of Acute Myeloid Leukemia.

Targeting the molecular chaperone HSP90 and the anti-apoptotic proteins MCL1 and BCL2 may be a promising novel approach in the treatment of acute myeloid leukemia (AML). The HSP90 inhibitor PU-H71, MCL1 inhibitor S63845, and BCL2 inhibitor venetoclax were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells. AML cells represented all major morphologic and molecular subtypes including FLT3-ITD and TP53 mutant AML cell lines and a variety of patient-derived AML cells. Results: PU-H71 and combination treatments with MCL1 inhibitor S63845 or BCL2 inhibitor venetoclax induced cell cycle arrest and apoptosis in susceptible AML cell lines and primary AML. The majority of the primary AML samples were responsive to PU-H71 in combination with BH3 mimetics. Elevated susceptibility to PU-H71 and S63845 was associated with FLT3 mutated AML with CD34 < 20%. Elevated susceptibility to PU-H71 and venetoclax was associated with primary AML with CD117 > 80% and CD11b < 45%. The combination of HSP90 inhibitor PU-H71 and MCL1 inhibitor S63845 may be a candidate treatment for FLT3-mutated AML with moderate CD34 positivity while the combination of HSP90 inhibitor PU-H71 and BCL2 inhibitor venetoclax may be more effective in the treatment of primitive AML with high CD117 and low CD11b positivity.

Cytokine-Induced Killer Cells in Combination with Heat Shock Protein 90 Inhibitors Functioning via the Fas/FasL Axis Provides Rationale for a Potential Clinical Benefit in Burkitt's lymphoma.

Constant efforts are being made to develop methods for improving cancer immunotherapy, including cytokine-induced killer (CIK) cell therapy. Numerous heat shock protein (HSP) 90 inhibitors have been assessed for antitumor efficacy in preclinical and clinical trials, highlighting their individual prospects for targeted cancer therapy. Therefore, we tested the compatibility of CIK cells with HSP90 inhibitors using Burkitt's lymphoma (BL) cells. Our analysis revealed that CIK cytotoxicity in BL cells was augmented in combination with independent HSP90 inhibitors 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin) and ganetespib. Interestingly, CIK cell cytotoxicity did not diminish after blocking with NKG2D (natural killer group 2, member D), which is a prerequisite for their activation. Subsequent analyses revealed that the increased expression of Fas on the surface of BL cells, which induces caspase 3/7-dependent apoptosis, may account for this effect. Thus, we provide evidence that CIK cells, either alone or in combination with HSP90 inhibitors, target BL cells via the Fas-FasL axis rather than the NKG2D pathway. In the context of clinical relevance, we also found that high expression of HSP90 family genes (HSP90AA1, HSP90AB1, and HSP90B1) was significantly associated with the reduced overall survival of BL patients. In addition to HSP90, genes belonging to the Hsp40, Hsp70, and Hsp110 families have also been found to be clinically significant for BL survival. Taken together, the combinatorial therapy of CIK cells with HSP90 inhibitors has the potential to provide clinical benefits to patients with BL.

A Coumarin-Imidazothiadiazole Derivative, SP11 Abrogates Tumor Growth by Targeting HSP90 and Its Client Proteins.

Despite several treatment options for blood cancer, mortality remains high due to relapse and the disease's aggressive nature. Elevated levels of HSP90, a molecular chaperone essential for protein folding, are associated with poor prognosis in leukemia and lymphoma. HSP90 as a target for chemotherapy has been met with limited success due to toxicity and induction of heat shock. This study tested the activity of an HSP90 inhibitor, SP11, against leukemic cells, mouse lymphoma allograft, and xenograft models. SP11 induced cytotoxicity in vitro in leukemic cell lines and induced cell death via apoptosis, with minimal effect on normal cells. SP11 induced cell death by altering the status of HSP90 client proteins both in vitro and in vivo. SP11 reduced the tumor burden in allograft and xenograft mouse models without apparent toxicity. The half-life of SP11 in the plasma was approximately 2 h. SP11 binding was observed at both the N-terminal and C-terminal domains of HSP90. C-terminal binding was more potent than N-terminal binding of HSP90 in silico and in vitro using isothermal calorimetry. SP11 bioavailability and minimal toxicity in vivo make it a potential candidate to be developed as a novel anticancer agent.

Molecular determinants of Hsp90 dependence of Src kinase revealed by deep mutational scanning.

Hsp90 is a molecular chaperone involved in the refolding and activation of numerous protein substrates referred to as clients. While the molecular determinants of Hsp90 client specificity are poorly understood and limited to a handful of client proteins, strong clients are thought to be destabilized and conformationally extended. Here, we measured the phosphotransferase activity of 3929 variants of the tyrosine kinase Src in both the presence and absence of an Hsp90 inhibitor. We identified 84 previously unknown functionally dependent client variants. Unexpectedly, many destabilized or extended variants were not functionally dependent on Hsp90. Instead, functionally dependent client variants were clustered in the αF pocket and ß1-ß2 strand regions of Src, which have yet to be described in driving Hsp90 dependence. Hsp90 dependence was also strongly correlated with kinase activity. We found that a combination of activation, global extension, and general conformational flexibility, primarily induced by variants at the αF pocket and ß1-ß2 strands, was necessary to render Src functionally dependent on Hsp90. Moreover, the degree of activation and flexibility required to transform Src into a functionally dependent client varied with variant location, suggesting that a combination of regulatory domain disengagement and catalytic domain flexibility are required for chaperone dependence. Thus, by studying the chaperone dependence of a massive number of variants, we highlight factors driving Hsp90 client specificity and propose a model of chaperone-kinase interactions.

A Boolean-based machine learning framework identifies predictive biomarkers of HSP90-targeted therapy response in prostate cancer.

Precision medicine has emerged as an important paradigm in oncology, driven by the significant heterogeneity of individual patients' tumour. A key prerequisite for effective implementation of precision oncology is the development of companion biomarkers that can predict response to anti-cancer therapies and guide patient selection for clinical trials and/or treatment. However, reliable predictive biomarkers are currently lacking for many anti-cancer therapies, hampering their clinical application. Here, we developed a novel machine learning-based framework to derive predictive multi-gene biomarker panels and associated expression signatures that accurately predict cancer drug sensitivity. We demonstrated the power of the approach by applying it to identify response biomarker panels for an Hsp90-based therapy in prostate cancer, using proteomic data profiled from prostate cancer patient-derived explants. Our approach employs a rational feature section strategy to maximise model performance, and innovatively utilizes Boolean algebra methods to derive specific expression signatures of the marker proteins. Given suitable data for model training, the approach is also applicable to other cancer drug agents in different tumour settings.

Native Size-Exclusion Chromatography-Based Mass Spectrometry Reveals New Components of the Early Heat Shock Protein 90 Inhibition Response Among Limited Global Changes.

The molecular chaperone heat shock protein 90 (HSP90) works in concert with co-chaperones to stabilize its client proteins, which include multiple drivers of oncogenesis and malignant progression. Pharmacologic inhibitors of HSP90 have been observed to exert a wide range of effects on the proteome, including depletion of client proteins, induction of heat shock proteins, dissociation of co-chaperones from HSP90, disruption of client protein signaling networks, and recruitment of the protein ubiquitylation and degradation machinery-suggesting widespread remodeling of cellular protein complexes. However, proteomics studies to date have focused on inhibitor-induced changes in total protein levels, often overlooking protein complex alterations. Here, we use size-exclusion chromatography in combination with mass spectrometry (SEC-MS) to characterize the early changes in native protein complexes following treatment with the HSP90 inhibitor tanespimycin (17-AAG) for 8 h in the HT29 colon adenocarcinoma cell line. After confirming the signature cellular response to HSP90 inhibition (e.g., induction of heat shock proteins, decreased total levels of client proteins), we were surprised to find only modest perturbations to the global distribution of protein elution profiles in inhibitor-treated HT29 cells at this relatively early time-point. Similarly, co-chaperones that co-eluted with HSP90 displayed no clear difference between control and treated conditions. However, two distinct analysis strategies identified multiple inhibitor-induced changes, including known and unknown components of the HSP90-dependent proteome. We validate two of these-the actin-binding protein Anillin and the mitochondrial isocitrate dehydrogenase 3 complex-as novel HSP90 inhibitor-modulated proteins. We present this dataset as a resource for the HSP90, proteostasis, and cancer communities (https://www.bioinformatics.babraham.ac.uk/shiny/HSP90/SEC-MS/), laying the groundwork for future mechanistic and therapeutic studies related to HSP90 pharmacology. Data are available via ProteomeXchange with identifier PXD033459.

Design, synthesis and biological evaluation of a new class of Hsp90 inhibitors vibsanin C derivatives.

Hsp90, an ATP-dependent chaperone that is essential for a wide range of protein assembly and folding processes, has long been recognized as a potential target for cancer. Hsp90 has more recently been identified as having a significant pathogenic role in viral infection, neurodegenerative disease, and inflammation, therefore, the development of the agents to inhibit the chaperone could potentially treat such intractable diseases. Here, on the basis of primary structure-activity relationships and docking analysis, a series of novel vibsanin C analogues with an emphasis on the C18 position was first designed, synthesized and biologically evaluated. The most effective Hsp90 inhibitory activity among these analogues was demonstrated by 29 and 31, with IC50 values of 0.39 and 0.27 µM respectively. Direct interaction between Hsp90 and its inhibitors were further confirmed. Mechanism studies indicated that 29 promoted HL-60 cell apoptosis by mitochondrial-mediated apoptosis pathway. In addition, 29 suppressed tumor growth in the H22 tumor-bearing mice model and revealed low acute toxicity in mice (LD50 > 500 mg/kg), suggesting its potential for further investigations.

Heterocyclic Compounds as Hsp90 Inhibitors: A Perspective on Anticancer Applications.

Heat shock proteins (Hsps) have garnered special attention in cancer therapy as molecular chaperones with regulatory/mediatory effects on folding, maintenance/stability, maturation, and conformation of proteins as well as their effects on prevention of protein aggregation. Hsp90 ensures the stability of various client proteins needed for the growth of cells or the survival of tumor cells; therefore, they are overexpressed in tumor cells and play key roles in carcinogenesis. Accordingly, Hsp90 inhibitors are recognized as attractive therapeutic agents for investigations pertaining to tumor suppression. Natural Hsp90 inhibitors comprising geldanamycin (GM), reclaimed analogs of GM including 17-AAG and DMAG, and radicicol, a natural macrocyclic antifungal, are among the first potent Hsp90 inhibitors. Herein, recently synthesized heterocyclic compounds recognized as potent Hsp90 inhibitors are reviewed along with the anticancer effects of heterocyclic compounds, comprising purine, pyrazole, triazine, quinolines, coumarin, and isoxazoles molecules.

Discovery of Quinacrine as a Potent Topo II and Hsp90 Dual-Target Inhibitor, Repurposing for Cancer Therapy.

Topo II and Hsp90 are promising targets. In this study, we first verified the structural similarities between Topo IIα ATPase and Hsp90α N-ATPase. Subsequently, 720 compounds from the Food and Drug Administration (FDA) drug library and kinase library were screened using the malachite green phosphate combination with the Topo II-mediated DNA relaxation and MTT assays. Subsequently, the antimalarial drug quinacrine was found to be a potential dual-target inhibitor of Topo II and Hsp90. Mechanistic studies showed that quinacrine could specifically bind to the Topo IIα ATPase domain and inhibit the activity of Topo IIα ATPase without impacting DNA cleavage. Furthermore, our study revealed that quinacrine could bind Hsp90 N-ATPase and inhibit Hsp90 activity. Significantly, quinacrine has broad antiproliferation activity and remains sensitive to the multidrug-resistant cell line MCF-7/ADR and the atypical drug-resistant tumor cell line HL-60/MX2. Our study identified quinacrine as a potential dual-target inhibitor of Topo II and Hsp90, depending on the ATP-binding domain, positioning it as a hit compound for further structural modification.

An open-label, crossover study to compare different formulations and evaluate effect of food on pharmacokinetics of pimitespib in patients with advanced solid tumors.

This study compared the bioavailability of two pimitespib formulations (Formulations A and B), evaluated the food effect on Formulation A, and evaluated the safety and efficacy of multiple pimitespib doses in patients with solid tumors. This clinical, pharmacological multicenter study had two cohorts and periods. A single dose of Formulation A or B was administered in a crossover design to compare the pharmacokinetics in Cohort 1. In Cohort 2, the effects of fed vs fasting conditions were evaluated among those receiving Formulation A. Subsequently, multiple Formulation A doses were administered to all patients for safety and efficacy assessments. In Cohorts 1 and 2, 12 and 16 patients, respectively, were analyzed for pharmacokinetics. Thirty patients were analyzed for safety and efficacy. Maximum concentration (Cmax), area under the curve (AUC)last, and AUCinf geometric mean ratios for Formulations A and B (90% confidence interval [CI]) were 0.8078 (0.6569-0.9933), 0.7973 (0.6672-0.9529), and 0.8094 (0.6697-0.9782), respectively; 90% CIs were not within the bioequivalence range (0.80-1.25). In Cohort 2, mean Cmax, AUClast, and AUCinf were higher in fed vs fasting conditions. No safety concerns emerged with single or multiple administration. Overall response rate, disease control rate, and median progression-free survival were 0%, 33%, and 1.5 months, respectively. Four patients had stable disease ≥ 5 months. Bioequivalence of the two formulations was unconfirmed. Systemic exposure of Formulation A was approximately 20% less than Formulation B. A high-fat/calorie meal increased the relative pharmacokinetics and bioavailability of a single 160-mg dose. Trial Registration: JapicCTI-184191 (Japan Pharmaceutical Information Center) registered on November 5, 2018.

[Chloroquine Enhances BIIB021-induced Apoptosis in Chronic Myeloid Leukemia Cells Bearing T315I Mutation].

OBJECTIVE: To explore the combined pro-apoptosis effect of HSP90 inhibitor BIIB021 and chloroquine (CQ) in chronic myeloid leukemia (CML) cells bearing T315I mutation and its mechanism. METHODS: The p210-T315I cells were divided into 4 groups by different treatment: control, BIIB021, CQ, and BIIB021 + CQ. After treated with BIIB021 or/and CQ for 24 hours, Annexin V/PI binding assay was used to detect apoptosis rates of CML cells. DAPI staining was used to observe nuclear fragmentation, and Western blot was used to detect the expression of caspase 3, PARP (apoptosis related proteins) and p62, LC3-I/II (autophagy related proteins). P210-T315I cells were inoculated subcutaneously into mice and CML mouse models were established. The mice in treatment groups were injected with BIIB021 and/or CQ while mice in control group were treated with PBS and normal saline. The tumor volume of mice was measured every 4 days, and protein level of cleaved-caspase 3 and LC3-II in tumor tissue were detected by immunohistochemistry. RESULTS: The results showed that BIIB021 induced apoptosis of CML cells in a dose-dependent manner ( r=0.91). CQ could enhance the apoptosis-inducing effect of BIIB021. Flow cytometry analysis results showed that the apoptosis rate of p210-T315I cells in combination group was higher than that in BIIB021 or CQ only group (P<0.05). DAPI staining showed nuclear fragmentation in combination group could be observed more obviously. Western blot analysis showed that BIIB021 could induce LC3-I to convert to LC3-II and decrease p62 protein levels (P<0.05). Moreover, the combination group had higher expression of LC3-II, p62 (P<0.05), activated PARP and activated caspase 3 than BIIB021 only group (P<0.05). Besides, experiment in vivo showed the mean tumor volume in co-treatment group was lower than that in single drug group (P<0.01). Immunohistochemistry of tumor tissue also showed the protein level of cleaved-caspase 3 and LC3-II in combined group was higher than that in BIIB021 only group. CONCLUSION: HSP90 inhibitor BIIB021 induced significant apoptosis of CML cells bearing T315I both in vivo and in vitro. CQ can enhance this effect probably by autophagy inhibition.