Heat Shock Protein 90 Inhibitor Research Articles

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

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. This phase Ib trial administered onalespib with paclitaxel in patients with advanced TNBC to assess safety and establish a recommended phase II dose (RP2D). 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). 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. 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. 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. Onalespib and paclitaxel in treating patients with advanced TNBC https://clinicaltrials.gov/ct2/show/NCT02474173.

Timosaponin AIII promotes non-small-cell lung cancer ferroptosis through targeting and facilitating HSP90 mediated GPX4 ubiquitination and degradation.

Timosaponin AIII (Tim-AIII), a steroid saponin, exhibits strong anticancer activity in a variety of cancers, especially breast cancer and liver cancer. However, the underlying mechanism of the effects of Tim-AIII-mediated anti-lung cancer effects remain obscure. In this study, we showed that Tim-AIII suppressed cell proliferation and migration, induced G2/M phase arrest and ultimately triggered cell death of non-small cell lung cancer (NSCLC) cell lines accompanied by the release of reactive oxygen species (ROS) and iron accumulation, malondialdehyde (MDA) production, and glutathione (GSH) depletion. Interestingly, we found that Tim-AIII-mediated cell death was reversed by ferroptosis inhibitor ferrostatin-1 (Fer-1). Meanwhile, the heat shock protein 90 (HSP90) was predicted and verified as the direct binding target of Tim-AIII by SwissTargetPrediction (STP) and surface plasmon resonance (SPR) assay. Further study showed that Tim-AIII promoted HSP90 expression and Tim-AIII induced cell death was blocked by the HSP90 inhibitor tanespimycin, indicating that HSP90 was the main target of Tim-AIII to further trigger intracellular events. Mechanical analysis revealed that the Tim-AIII-HSP90 complex further targeted and degraded glutathione peroxidase 4 (GPX4), and promoted the ubiquitination of GPX4, as shown by an immunoprecipitation, degradation and in vitro ubiquitination assay. In addition, Tim-AIII inhibited cell proliferation, induced cell death, led to ROS and iron accumulation, MDA production, GSH depletion, as well as GPX4 ubiquitination and degradation, were markedly abrogated when HSP90 was knockdown by HSP90-shRNA transfection. Importantly, Tim-AIII also showed a strong capacity of preventing tumor growth by promoting ferroptosis in a subcutaneous xenograft tumor model, whether C57BL/6J or BALB/c-nu/nu nude mice. Together, HSP90 was identified as a new target of Tim-AIII. Tim-AIII, by binding and forming a complex with HSP90, further targeted and degraded GPX4, ultimately induced ferroptosis in NSCLC. These findings provided solid evidence that Tim-AIII can serve as a potential candidate for NSCLC treatment.

Remarkable anti-breast cancer activity and molecular docking studies of ferrocene tethered pyrimidobenzothiazoles and pyrimidobenzimidazoles

Two new series of structurally diverse ferrocene tethered pyrimidobenzothiazoles and pyrimidobenzimidazoles were rationally designed, synthesized and evaluated for their in vitro anticancer activity against human breast cancer cell lines. Among the synthesized series, five molecules displayed significantly higher anticancer activity against breast carcinoma MCF-7 and MDA-MB-231 cell lines (GI50 0.018–0.022 µM) as compared to the standard drug doxorubicin (GI50 0.018 µM). Furthermore, most of the synthesized compounds were found to exhibit significant antioxidant activity in DPPH, SOD and FRAP assays as compared to standard ascorbic acid and trolox. Moreover, some of the compounds were found to be potent anti-angiogenic agents with angiogenic score ranging 0.8–1.4 in in-ovo chick allantoic membrane (CAM) assay. The in-silico molecular docking analysis ascertained the mode of action of target compounds via inhibition of heat shock protein90 (Hsp90).

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.

HSP70 inhibitors upregulate prostaglandin E1-induced synthesis of interleukin-6 in osteoblasts.

Interleukin-6 (IL-6) is a pro-inflammatory and bone-resorptive cytokine that also regulates bone formation. We previously showed that prostaglandin E1 (PGE1) induces the synthesis of IL-6 by activating p44/p42 mitogen-activated protein kinase (MAPK), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and p38 MAPK in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether heat shock protein 70 (HSP70), a molecular chaperone that coordinates protein folding and homeostasis, affects PGE1-stimulated IL-6 synthesis in MC3T3-E1 cells through the MAPK activation. The osteoblast-like MC3T3-E1 cells were treated with HSP70 inhibitors-VER-155008 and YM-08-, PD98059, SB203580 or SP600125 and then stimulated with PGE1. IL-6 synthesis was evaluated using an IL-6 enzyme-linked immunosorbent assay kit. IL-6 mRNA expression was measured by real-time RT-PCR. The phosphorylation of p38 MAPK was evaluated by Western blotting. We found that VER-155008, an HSP70 inhibitor, enhanced the PGE1-stimulated IL-6 release and IL-6 mRNA expression. YM-08, another HSP70 inhibitor, also enhanced PGE1-stimulated IL-6 release. PD98059, a p44/p42 MAPK inhibitor, and SP600125, a SAPK/JNK inhibitor, upregulated PGE1-stimulated IL-6 release. On the other hand, SB203580, a p38 MAPK inhibitor, suppressed PGE1-stimulated IL-6 release. YM-08 stimulated the PGE1-induced phosphorylation of p38 MAPK. SB203580 suppressed the amplification by YM-08 of the PGE1-stimulated IL-6 release. Our results suggest that HSP70 inhibitors upregulate the PGE1-stimulated IL-6 synthesis through p38 MAPK in osteoblasts and therefore affect bone remodeling.

Heat shock protein 90 inhibitors induce cell differentiation via the ubiquitin-dependent aurora kinase A degradation in a MPLW515L mouse model of primary myelofibrosis.

Primary myelofibrosis (PMF) is characterized by immature megakaryocytic hyperplasia, splenomegaly, extramedullary hematopoiesis and bone marrow fibrosis. Our preclinical study had demonstrated that aurora kinase A (AURKA) inhibitor MLN8237 reduced the mutation burden of PMF by inducing differentiation of immature megakaryocytes. However, it only slightly alleviated splenomegaly, reduced tissue fibrosis, and normalized megakaryocytes in PMF patients of the preliminary clinical study. So enhancing therapeutic efficacy of PMF is needed. In this study, we found that AURKA directly interacted with heat shock protein 90(HSP90) and HSP90 inhibitors promoted the ubiquitin-dependent AURKAdegradation. We demonstrated that HSP90 inhibitors 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), normalized peripheral blood counts, improved splenomegaly, attenuated extramedullary hematopoiesis, decreased tissue fibrosis and reduced mutant burden in a MPLW515L mouse model of PMF. Importantly, both 17-AAG and 17-DMAG treatment at effective doses in vivo did not influence on hematopoiesis in healthy mice. Collectively, the study demonstrates that HSP90 inhibitors induce cell differentiation via the ubiquitin-dependent AURKA and also are safe and effective for the treatment of a MPLW515L mouse model of PMF, which may provide a new strategy for PMF therapy. Further, we demonstrate that combined therapy shows superior activity in acute megakaryocytic leukemia mouse model than single therapy.

354 Heat Shock Protein 90 Inhibitor Attenuates Inflammation Induced by 12-O-tetradecanoylphorbol-13-acetate in Primary Human Keratinocytes and Mice

Heat shock protein 90 (HSP90) is a molecular chaperone that folds and promotes the activity of a wide range of client proteins, including key signaling proinflammatory molecules involved in aberrant inflammation. RGRN-305 is a novel HSP90 inhibitor that has been reported to exhibit antiinflammatory properties in autoimmune skin diseases. Here, the antiinflammatory effects of RGRN-305 were investigated in vitro and in a mouse model in vivo. In primary human keratinocytes stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA; inducer of inflammation), the mRNA expression of the proinflammatory cytokines TNFα, IL-1β, IL-6 and IL-8 were significantly reduced by preincubation with RGRN-305, when measured by quantitative real-time PCR.

Calycosin ameliorates spinal cord injury by targeting Hsp90 to inhibit oxidative stress and apoptosis of nerve cells

BackgroundZhenbao pill is effective in protecting against spinal cord injury (SCI). We attempt to explore the characteristics of calycosin (a main monomer of Zhenbao pill) in SCI and its relative mechanism. MethodsThe target of calycosin was screened using pharmacological network analysis. The SCI cell model was constructed using hydrogen peroxide (H2O2), and the animal model was developed by compressing spinal cord with a vascular clamp. Flow cytometry was conducted to test reactive oxygen species (ROS) levels and cell apoptosis. Detection of malondialdehyde (MDA) activity and Superoxide dismutase (SOD) activity were performed using relative kits. Heat shock protein 90 (HSP90) was examined using western blot and quantitative real-time PCR. Motor function tests were carried out. The hematoxylin-eosin and Nissl staining were conducted. ResultsIn SCI models, ROS, MDA, and cell apoptosis were elevated, SOD and HSP90 levels were restrained, while calycosin addition reversed the above results. Besides, calycosin application or HSP90 overexpression enhanced phosphorylation of protein kinase B (Akt) but weakened that of apoptosis signal-regulating kinase 1 (ASK1) and p38, while HSP90 inhibitor 17-AAG treatment restrained the above results. Meanwhile, the injection of calycosin improved the motor function in SCI model rats. Furthermore, the pathologic results also clarified the positive effect of calycosin on SCI. ConclusionHSP90 was lowly expressed in SCI models. Calycosin alleviated SCI by promoting HSP90 up-regulation and inhibiting oxidative stress and apoptosis of nerve cells.

HSP90β chaperoning SMURF1-mediated LATS proteasomal degradation in the regulation of bone formation

Heat shock protein 90 (HSP90) molecular chaperone is responsible for the stabilization and biological activity of a diverse set of client proteins. We have previously demonstrated that inhibition of HSP90 by 17-Demethoxy-17-allyaminogeldanmycin (17-AAG) not only reverses the glucocorticoid-induced bone loss but also enhances the basal level of bone mass in mice. Here, we investigate the potential mechanism underlying HSP90-associated osteoblast differentiation and bone formation. Knockdown of HSP90β but not HSP90α or inhibition of HSP90 by 17-AAG or NVP-BEP800 negates the protein levels of large tumor suppressor (LATS), the core kinases of Hippo signaling, resulting in the inactivation of LATS and activation of Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), in the enhancement of osteoblastic differentiation. In contrast, genetic ablation of Lats1 in mesenchymal stem cells is sufficient to abolish the HSP90 inhibition-induced osteoblastic differentiation and bone formation. Mechanistically, HSP90β but not HSP90α chaperones and prevents the SMAD specific E3 ubiquitin protein ligase 1 (SMURF1)-mediated and ubiquitination-dependent LATS protein proteasomal degradation, whereas 17-AAG abolishes these effects of HSP90β. Thus, these results uncover the HSP90β chaperoning SMURF1-mediated LATS protein proteasomal degradation and the subsequent YAP/TAZ activation as a hitherto uncharacterized mechanism controlling osteoblastic differentiation and bone formation.

Mechanism of HSP90 Inhibitor in the Treatment of DSS-induced Colitis in Mice by Inhibiting MAPK Pathway and Synergistic Effect of Compound Sophorae Decoction.

The mechanism of Heat Shock Protein 90 (HSP90) in Ulcerative Colitis (UC) has been studied, and mitogenic-activated protein kinases (MAPK) also contribute to the pathogenesis of UC. However, the effect of the HSP90/MAPK pathway in UC is still unclear. Therefore, the mainstay of this research is to explore the mechanism of action of this pathway in UC. Compound sophorae decoction (CSD), as a Chinese herbal decoction, can synergistically affect the above process. This study aimed to uncover the synergistic effects of HSP90 inhibitors regulating the MAPK pathway for treating DSS-induced colitis in mice and the synergistic effects of CSD. This experiment used oral administration of standard diets containing 3% dextran sodium sulfate (DSS) to establish an experimental colitis model in mice. The model was treated with HSP90 inhibitor, CSD, or dexamethasone. Mouse feces, mobility, body weight, colon length, and colon histopathology scores were recorded daily to assess the degree of colitis inflammation. Expression levels of HSP90 and MAPK pathway-related genes and proteins were evaluated by Western blot and qPCR. The evaluation of intestinal mucosal permeability was measured by enzyme-linked immunosorbent assay (ELISA), which could detect the protein level of D-Amino Acid Oxidase (DAO) and D-lactic acid (D-LA). The same went for downstream molecules AFT-2, p53, and apoptosis-related proteins BAX, BCL-2, Caspase3, and survivin in the MAPK pathway. Immunohistochemical measured p-38, p-JNK, and p-ERK expressions. JAM-A and claudin-1 connexin were tested by immunofluorescence staining. The TUNEL method was for measuring the apoptosis rate of colonic epithelial cells. CBA kit determined the level of inflammatory factors of colons. HSP90 inhibitor can improve the degree of pathological damage in the colon of mice treated with DSS, increase the mice's weight and the length of the colon, and significantly reduce the disease activity index (DAI) score. Intraperitoneal injection of HSP90 inhibitor can reduce the expression of MAPK pathway markers P38, JNK, ERK, and their phosphorylation and decrease the content of AFT-2 and p53, which is downstream of the MAPK pathway. In addition, treatment of the HSP90 inhibitor up-regulated the expression of anti-apoptotic proteins BCL-2 and survivin, as well as down-regulated apoptotic protein caspase3, BAX in the colon of mice with colitis. Lower levels of inflammatory factors such as IL-6, MCP-1, IFN-γ, TNF, IL-12p70, and increased IL-10 were observed after HSP90 inhibitor therapy. Furthermore, the combination treatment of CSD can enhance the effect of the single HSP90 inhibitor treatment and play a synergistic effect. These data suggest that an HSP90 inhibitor is available to treat UC by inhibiting the MAPK signaling pathway. This axis can restore the intestinal mucosa barrier's function by reducing intestinal mucosa's permeability and inhibiting apoptosis of intestinal epithelial cells. The specific mechanism is that HSP90 inhibitor can reduce the pathological damage and inflammation levels of colitis mice, and reduce the apoptosis rate of colonic epithelial cells and the mucosal permeability, thereby restoring the mucosal barrier function. During this process, CSD works synergistically to improve the therapeutic effect of the HSP90 inhibitor.

RF07 | PSUN356 Synergistic Combination of HSP90 and PI3K inhibitors in Adrenocortical Cancer

Abstract Adrenocortical cancer (ACC) is a rare and aggressive cancer (0.5-2 cases/ million/ year) with a poor 5-year survival (<40%). Most patients inevitably succumb to the wide-spread metastasis because of the lack of effective systemic treatment options or to complications from uncontrolled hypercortisolism. Therefore, it is critically important to identify new effective treatments that can be readily used in a clinical trial. In this direction, we identified a potent synergistic combination of Heat Shock Protein 90 (HSP90) and PI3K inhibitors via quantitative high-throughput drug screening (qHTS) for ACC, using a pharmaceutical library comprised of ∼5000 drugs (FDA approved or investigational), and evaluation of drug synergy by a computerized drug combination matrix analysis. The synergistic efficacy of HSP90 inhibitor STA9090 and PI3K inhibitor, PIK75 was validated in vitro, in ACC cell lines H295R and SW13, by proliferation assay, which also determined the most efficacious doses of individual and combinatorial drugs. The drug combination significantly induced apoptosis and disintegrated the 3D-ACC spheroids as compared to the individual drugs, with a simultaneous upregulation of apoptotic protein markers (cleaved-Caspase3, cleaved-PARP), inhibition of the phosphorylation of PI3K pathway members (AKT1, mTOR, S6K, 4EB1 and GSK3α/β), and alteration in the G2/M phase of the cell cycle, in ACC cells. The synergetic effect of the drug combination was also effective in reducing the metastatic potential as revealed by invasion/migration assay, which was confirmed by the downregulation of the mesenchymal protein (Vimentin, N-cadherin, SNAIL and Zeb1) markers. Consistent with our data, observations from the gene expression data from The Cancer Genome Analysis (TCGA) & Gene Expression Omnibus (GEO) databases, revealed significant mRNA overexpression of HSP90AA1, HSP90AB1 HSP90B1 and its clients AKT2, CDK1, CDK4, NR3C1 indicating both HSP90 and PI3K/AKT pathways are highly activated in ACC cohorts. Overexpression and a positive correlation of mesenchymal markers TWIST1, ZEB1, VIM with HSP90AB1 and PI3KCA, in GEO and TCGA respectively, was consistent with the invasive potential of the ACC cells. In conclusion, our studies on the combination of HSP90 and PI3K inhibitors demonstrated a promising preliminary synergistic efficacy by inhibiting proliferation and epithelial-to-mesenchymal transition of ACC. Our findings can lead to development of an efficient mechanism-specific novel combination therapy that can be expedited to clinical trial in advanced ACC patients. Presentation: Saturday, June 11, 2022 1:12 p.m. - 1:17 p.m., Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

The Role of HSP90 Inhibitors in the Treatment of Cardiovascular Diseases.

Cardiovascular disease is the result of complicated pathophysiological processes in the tissues that make up the blood vessels and heart. Heat shock protein 90 (HSP90) can interact with 10% of the proteome and is the most widely studied molecular chaperone in recent years. HSP90 is extensively involved in the regulation of protein folding and intracellular protein stability, making HSP90 a hopeful target for the treatment of multiple cardiovascular diseases. Numerous client proteins of HSP90 have been identified in known cardiac disease pathways, including MAPK signaling, PI3K/AKT (PKB)/mTOR, and TNF-α signaling. Therefore, these pathways can be controlled by regulating HSP90. Among them, the activity of HSP90 can be regulated via numerous inhibitors. In this review, first, we will discuss the function of HSP90 and its role in pathological pathways. In addition, HSP90 plays a significant role in most cardiovascular diseases, including hypertension, pulmonary venous hypertension, atherosclerosis, and heart failure; next we will focus on this part. Finally, we will summarize the currently known HSP90 inhibitors and their potential in the treatment of heart disease.

Advances in research related to heat shock protein 90 and autoimmune dermatoses

Autoimmune dermatoses result from immune imbalances due to aberrant immune responses to self-antigens. Heat shock protein 90 (HSP90), as a member of a highly conserved family of stress proteins, plays an important role in inflammation and immune responses. It has been suggested that HSP90 is related to the occurrence and development of autoimmune dermatoses, but the exact mechanisms involved remain unclear. In this report, we review the evidence indicating a potential link between HSP90 and three common autoimmune dermatoses, bullous dermatoses, psoriasis and lupus erythematosus. In addition, the progress of research involving HSP90 inhibitors as potential therapeutic targets is assessed.

1 Cellular protein TTC4 and its cofactor HSP90 are pro-viral for bovine herpesvirus 1

Bovine Herpesvirus Type 1 (BoHV-1) infection causes infectious bovine rhinotracheitis and genital disease in cattle, with significant economic and welfare impacts. However, the role of cellular host factors during viral replication remains poorly characterised. A previously performed genome-wide CRISPR knockout screen identified pro- and antiviral host factors acting during BoHV-1 replication. Herein we validate a pro-viral role for a candidate from this screen: the cellular protein tetracopeptide repeat protein 4 (TTC4). We show that TTC4 transcript production is upregulated during BoHV-1 infection. Depletion of TTC4 protein impairs BoHV-1 protein production but does not reduce production of infectious virions, whereas overexpression of exogenous TTC4 results in a significant increase in production of infectious BoHV-1 virions. TTC4 itself is poorly characterized (especially in the context of virus infection), but is a known co-chaperone of heat shock protein 90 (HSP90). HSP90 has a well-characterized pro-viral role during the replication of diverse herpesviruses, and we therefore hypothesized that HSP90 is also pro-viral for BoHV-1. Drug-mediated inhibition of HSP90 using geldanamycin at sub-cytotoxic concentrations inhibited both BoHV-1 protein production and viral genome replication, indicating a pro-viral role for HSP90 during BoHV-1 infection. Our data demonstrates pro-viral roles for both TTC4 and HSP90 during BoHV-1 replication; possibly, interactions between these two proteins are required for optimal BoHV-1 replication, or the two proteins may have independent pro-viral roles.

Combination of AURKA inhibitor and HSP90 inhibitor to treat breast cancer with AURKA overexpression and TP53 mutations.

Breast cancer is the most common cancer among women worldwide. Researches show that Aurora kinase A (AURKA) is highly expressed in approximately 73% of breast cancer patients, which induces drug resistance in breast cancer patients and decreases the median survival time. AURKA regulates spindle assembly, centrosome maturation, and chromosome alignment. AURKA overexpression affects the occurrence and development of breast cancer. Besides AURKA overexpression, heat shock protein 90 (HSP90) maintains the survival and proliferation of tumor cells by stabilizing the structure of oncoproteins, including P53 mutants (mtP53). TP53 mutations accounted for approximately 13%, 40%, 80%, 33%, 71%, and 82% of luminal A, Luminal B, Luminal C, normal basal-like, HER2-amplified, and basal-like breast cancers, respectively. TP53 mutation can aggravate cell genome instability and enhance the invasion, migration, and resistance of cancer cell. This review describes the research status of AURKA and HSP90 in breast cancer, summarizes the structure, function, and the chaperone cycle of HSP90, elaborates the interrelation between HSP90, mtP53, P53, and AURKA, and proposes the combination of HSP90 inhibitor and AURKA inhibitor to treat breast cancer. Targeting AURKA and HSP90 to treat cancer with AURKA overexpression and TP53 mutations will help improve the specificity and efficiency of breast cancer treatment and solve the problem of drug resistance.

Targeting Heat-Shock Protein 90 in Cancer: An Update on Combination Therapy.

Heat-shock protein 90 (HSP90) is an important molecule chaperone associated with tumorigenesis and malignancy. HSP90 is involved in the folding and maturation of a wide range of oncogenic clients, including diverse kinases, transcription factors and oncogenic fusion proteins. Therefore, it could be argued that HSP90 facilitates the malignant behaviors of cancer cells, such as uncontrolled proliferation, chemo/radiotherapy resistance and immune evasion. The extensive associations between HSP90 and tumorigenesis indicate substantial therapeutic potential, and many HSP90 inhibitors have been developed. However, due to HSP90 inhibitor toxicity and limited efficiency, none have been approved for clinical use as single agents. Recent results suggest that combining HSP90 inhibitors with other anticancer therapies might be a more advisable strategy. This review illustrates the role of HSP90 in cancer biology and discusses the therapeutic value of Hsp90 inhibitors as complements to current anticancer therapies.

Allosteric HSP70 inhibitors perturb mitochondrial proteostasis and overcome proteasome inhibitor resistance in multiple myeloma

Proteasome inhibitor (PI) resistance remains a central challenge in multiple myeloma. To identify pathways mediating resistance, we first mapped proteasome-associated genetic co-dependencies. We identified heat shock protein 70 (HSP70) chaperones as potential targets, consistent with proposed mechanisms of myeloma cells overcoming PI-induced stress. We therefore explored allosteric HSP70 inhibitors (JG compounds) as myeloma therapeutics. JG compounds exhibited increased efficacy against acquired and intrinsic PI-resistant myeloma models, unlike HSP90 inhibition. Shotgun and pulsed SILAC mass spectrometry demonstrated that JGs unexpectedly impact myeloma proteostasis by destabilizing the 55S mitoribosome. Our data suggest JGs have the most pronounced anti-myeloma effect not through inhibiting cytosolic HSP70 proteins but instead through mitochondrial-localized HSP70, HSPA9/mortalin. Analysis of myeloma patient data further supports strong effects of global proteostasis capacity, and particularly HSPA9 expression, on PI response. Our results characterize myeloma proteostasis networks under therapeutic pressure while motivating further investigation of HSPA9 as a specific vulnerability in PI-resistant disease.

FOXM1 regulates glycolysis and energy production in multiple myeloma

The transcription factor, forkhead box M1 (FOXM1), has been implicated in the natural history and outcome of newly diagnosed high-risk myeloma (HRMM) and relapsed/refractory myeloma (RRMM), but the mechanism with which FOXM1 promotes the growth of neoplastic plasma cells is poorly understood. Here we show that FOXM1 is a positive regulator of myeloma metabolism that greatly impacts the bioenergetic pathways of glycolysis and oxidative phosphorylation (OxPhos). Using FOXM1-deficient myeloma cells as principal experimental model system, we find that FOXM1 increases glucose uptake, lactate output, and oxygen consumption in myeloma. We demonstrate that the novel 1,1-diarylethylene small-compound FOXM1 inhibitor, NB73, suppresses myeloma in cell culture and human-in-mouse xenografts using a mechanism that includes enhanced proteasomal FOXM1 degradation. Consistent with the FOXM1-stabilizing chaperone function of heat shock protein 90 (HSP90), the HSP90 inhibitor, geldanamycin, collaborates with NB73 in slowing down myeloma. These findings define FOXM1 as a key driver of myeloma metabolism and underscore the feasibility of targeting FOXM1 for new approaches to myeloma therapy and prevention.

Therapeutic Response Monitoring with 89Zr-DFO-Pertuzumab in HER2-Positive and Trastuzumab-Resistant Breast Cancer Models.

Immuno-positron emission tomography (PET) has great potential to evaluate the target expression level and therapeutic response for targeted cancer therapy. Immuno-PET imaging with pertuzumab, due to specific recognition in different binding sites of HER2, could be useful for the determination of the therapeutic efficacy of HER2-targeted therapy, trastuzumab, and heat shock protein 90 (HSP90) inhibitor, in HER2-expressing breast cancer. The aim of this study is to evaluate the feasibility of monitoring therapeutic response with 89Zr-DFO-pertuzumab for the treatment of HER2-targeted therapeutics, trastuzumab, or the HSP90 inhibitor 17-DMAG, in trastuzumab-resistant JIMT-1 breast cancer models. We prepared an immuno-PET imaging agent using desferoxamine (DFO)-pertuzumab labeled with 89Zr and performed the biodistribution and PET imaging in breast cancer xenograft models for monitoring therapeutic response to HER2-targeted therapy. 89Zr-DFO-pertuzumab was successfully prepared and showed specific binding to HER2 in vitro and clearly visualized HER2 expressing JIMT-1 tumors. 89Zr-DFO-pertuzumab had prominent tumor uptake in HER2 expressing JIMT-1 tumors. JIMT-1 tumors showed trastuzumab-resistant and HSP90 inhibitor sensitive characterization. In immuno-PET imaging, isotype antibody-treated JIMT-1 tumors had similar uptake in trastuzumab-treated JIMT-1 tumors, but 17-DMAG-treated JIMT-1 tumors showed greatly reduced uptake compared to vehicle-treated tumors. Additionally, HER2 downregulation evaluated by immuno-PET imaging was verified by western blot analysis and immunofluorescence staining which resulted in a significant reduction in the tumor’s HER2 level in 17-DMAG-treated JIMT-1 tumors. 89Zr-DFO-pertuzumab immuno-PET may be clinically translated to select pertinent patients for HER2-targeted therapy and to monitor the therapeutic response in HER2-positive cancer patients under various HER2-targeted therapeutics treatments.

S153: DECRYPTING THE ROLE OF HSP90Α AND Β ISOFORMS TO OVERCOME RESISTANCE IN BCR-ABL1 LEUKEMIA

Background: The BCR-ABL1 fusion protein, which results from the chromosomal translocation t(9;22)(q34;q11) is detected in over 95% of CML, 25% of adult BCP-ALL and 3-5% of childhood BCP-ALL patients. The development of tyrosine kinase inhibitors (TKi) has enabled targeted treatment, however development of resistance is common and the BCR-ABL1 leukemia subtype is still associated with poor prognosis. Among the chaperone proteins involved in maintaining the homeostasis of cancerous cells, heat shock protein 90 (HSP90) have been widely studied, due to its crucial involvement in stabilizing a variety of oncogenic proteins, including BCR-ABL1 and STAT3/5. Therefore, inhibition of HSP90 can be an effective alternative approach to target TKi resistant and BCR-ABL1T315I mutant expressing cells. In the past, over 17 HSP90 inhibitors (HSP90i) have been evaluated in clinical trials, however associated resistances (e.g. HSR) and dose limited toxicity have thus far precluded their clinical approval. Aims: In mammalian cells, there are two predominant cytosolic isoforms of HSP90, a stress-inducible HSP90α and a constitutively expressed HSP90β isoform. In the past, the distinct roles of the isoforms were studied by applying pan- and isoform-targeted HSP90i. We aimed to decrypt the role of cytosolic HSP90 isoforms using genetically edited models and to identify novel therapeutic vulnerabilities to target therapy refractory BCR-ABL1+ leukemia. Methods: To study the kinetic implications of the resistance mechanism associated with pharmacological targeting of HSP90, we generated a transient knockdown (Kd) and a stable CRISPR-Cas9 based knockout (KO) model of HSP90α/β in BCR-ABL1+ cells. The edited cells were analyzed in ex vivo functional assays (e.g. IF staining and WB) and their transplantation efficiency was determined in NSG mice. Global transcriptomic and proteomic profiling was performed in conjunction with high throughput drug screening (HTDS) to identify therapeutic vulnerabilities upon loss of HSP90α/β. Results: HSP90α/β-KO or -Kd did not affect the expression of other HSP90 paralogues (TRAP1 and GRP94). Only HSP90β-KO cells displayed hyperactive pro-survival HSR. Other reported HSP90 isoform-dependent clients, such as Survivin, CDK4 and CDK6 were also not affected upon KO or Kd of HSP90α/β. As HSP90 is involved in stabilizing, proper folding and subcellular localization of BCR-ABL1 protein, immunofluorescence imaging demonstrated two fold higher abundance of BCR-ABL1 foci (cytoplasmic/nucleocytoplasmic region) in HSP90α KO cells, with hyperactive BCR-ABL1 and downstream signaling (pBCR-ABL and pSTAT5a). When KO cells were transplanted into NSG mice (n=5 mice per arm) the engraftment of the HSP90α-KO cells was significantly reduced with a prolonged overall survival of the animals (19 days, p=0.0083) as compared to HSP90β-KO and control group. To find possible target (s) in order to circumvent resistance associated with the use HSP90i, we performed HTDS with HSP90α/β KO cells, which revealed distinct sensitivities toward certain classes of inhibitors. One of these classes was CDK7i, which was differentially active in HSP90α KO cells. These results can be explained by our global transcriptomic results, which revealed hyperactive androgen receptor signaling in HSP90α KO cells (GSEA), which is known to be a prognostic marker for CDK7 inhibition. Summary/Conclusion: In vivo targeting of HSP90α is a promising approach to target BCR-ABL1+ leukemia cells. Moreover, the combination with CDK7i may help to overcome the resistance associated with clinical use of pan HSP90i.