Hsp90 Inhibitor Research Articles

Expanded ROS Generation and Hypoxia Reversal: Excipient-free Self-assembled Nanotheranostics for Enhanced Cancer Photodynamic Immunotherapy.

The efficacy of photodynamic therapy (PDT)-related cancer therapies is significantly restricted by two irreconcilable obstacles, i.e., low reactive oxygen species (ROS) generation capability and hypoxia which constrains the immune response. Herein, we developed a self-assembled clinical photosensitizer indocyanine green (ICG) and the HSP90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) nanoparticles (ISDN) without any excipient. We discovered that the hydrophobic interaction forces between ICG and 17-DMAG promote the photostability of ICG and its intersystem crossing process (ISC), thereby improving the ROS quantum yield from 0.112 to 0.46. Augmented ROS generation enhances PDT efficacy and further enhances immunogenic cell death (ICD) effects. 17-DMAG inhibits the HSP90/HIF-1α axis to dramatically reverse the immunosuppressive tumor microenvironment caused by PDT-aggravated hypoxia. In a mouse model of pancreatic cancer, ISDN markedly improve cytotoxic T lymphocyte infiltration and MHC I and MHC II activation, demonstrating the superior ICD effects in situ tumor and the powerful systematic antitumor immunity generation, eventually achieving vigorous antitumor and recurrence resistance. This study proposes an unsophisticated and versatile strategy to significantly improve PDT efficacy for enhancing systemic antitumor immunity and potentially extending it to multiple cancers. This article is protected by copyright. All rights reserved.

Heat Shock Protein HSP27 and the Status of the Glutathione System in Dexamethasone-Induced Apoptosis of Jurkat Tumor Cells.

We studied the effect of the HSP27 inhibitor, 5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl)-isoxazole, at a final concentration of 0.1 μM and/or the apoptosis inducer dexamethasone at a final concentration of 10 μM on the content of hydroxyl radical, reduced and oxidized glutathione, HSP27, activity of glutathione reductase, glutathione peroxidase, caspase-3, and the number of Annexin+ Jurkat tumor cells. The involvement of HSP27 in apoptosis of Jurkat tumor cells was demonstrated. Simultaneous exposure to the HSP27 inhibitor and dexamethasone resulted in an increase in the level of HSP27 against the background of developing oxidative stress (increase in the concentration of hydroxyl radicals and changes in the state of the glutathione system).

GSH-Responsive Liposomes with Heat Shock Protein Regulatory Ability for Efficient Photodynamic/Photothermal Combined Therapy of Tumors.

Phototherapy, represented by photodynamic therapy (PDT) and photothermal therapy (PTT), has great potential in tumor treatment. However, the presence of antioxidant glutathione (GSH) and the heat shock proteins (HSPs) expression caused by high temperature can weaken the effects of PDT and PTT. Here, a multifunctional nanocomplex BT&GA@CL is constructed to realize enhanced synergistic PDT/PTT. Cinnamaldehyde liposomes (CLs) formed by cinnamaldehyde dimer self-assembly were loaded with in gambogic acid (GA) and an aggregation-induced emission molecule BT to obtain BT&GA@CL. As a drug carrier, CL can consume glutathione (GSH) and release drugs responsively. The released BT aggregates can simultaneously act as both a photothermal agent and photosensitizer to achieve PDT and PTT under 660 nm laser irradiation. Specifically, GA as an HSP90 inhibitor can attenuate PTT-induced HSP90 protein expression, thereby weakening the tolerance of tumor cells to high temperatures and enhancing PTT. Such a multifunctional nanocomplex simultaneously modulates the content of GSH and HSP90 in tumor cells, thus enhancing both PDT and PTT, ultimately achieving the goal of efficient combined tumor suppression.

A novel combination therapy targets sonic hedgehog signaling by the dual inhibition of HMG-CoA reductase and HSP90 in rats with non-alcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH) is characterized by liver inflammation, fat accumulation, and collagen deposition. Due to the limited availability of effective treatments, there is a pressing need to develop innovative strategies. Given the complex nature of the disease, employing combination approaches is essential. Hedgehog signaling has been recognized as potentially promoting NASH, and cholesterol can influence this signaling by modifying the conformation of PTCH1 and SMO activity. HSP90 plays a role in the stability of SMO and GLI proteins. We revealed significant positive correlations between Hedgehog signaling proteins (Shh, SMO, GLI1, and GLI2) and both cholesterol and HSP90 levels. Herein, we investigated the novel combination of the cholesterol-lowering agent lovastatin and the HSP90 inhibitor PU-H71 in vitro and in vivo. The combination demonstrated a synergy score of 15.09 and an MSA score of 22.85, as estimated by the ZIP synergy model based on growth inhibition rates in HepG2 cells. In a NASH rat model induced by thioacetamide and a high-fat diet, this combination therapy extended survival, improved liver function and histology, and enhanced antioxidant defense. Additionally, the combination exhibited anti-inflammatory and anti-fibrotic potential by influencing the levels of TNF-α, TGF-β, TIMP-1, and PDGF-BB. This effect was evident in the suppression of the Col1a1 gene expression and the levels of hydroxyproline and α-SMA. These favorable outcomes may be attributed to the combination's potential to inhibit key Hedgehog signaling molecules. In conclusion, exploring the applicability of this combination contributes to a more comprehensive understanding and improved management of NASH and other fibrotic disorders.

A pipeline for senolytics.

There is intense interest in identifying compounds that selectively kill senescent cells, termed senolytics, for ameliorating age-related comorbidities. However, screening for senolytic compounds currently relies on primary cells or cell lines where senescence is induced in vitro. Given the complexity of senescent cells across tissues and diseases, this approach may not target the senescent cells that develop under specific conditions in vivo. In this issue of the JCI, Lee et al. describe a pipeline for high-throughput drug screening of senolytic compounds where senescence was induced in vivo and identify the HSP90 inhibitor XL888 as a candidate senolytic to treat idiopathic pulmonary fibrosis.

Taxonomy of Candida parapsilosis complex isolated from neonates and the role of Hsp90 inhibitors to enhanced the antifungal activity of micafungin.

Species from Candida parapsilosis complex are frequently found in neonatal candidemia. The antifungal agents to treat this infection are limited and the occurrence of low in vitro susceptibility to echinocandins such as micafungin has been observed. In this context, the chaperone Hsp90 could be a target to reduce resistance. Thus, the objective of this research was to identify isolates from the C. parapsilosis complex and verify the action of Hsp90 inhibitors associated with micafungin. The fungal identification was based on genetic sequencing and mass spectrometry. Minimal inhibitory concentrations were determined by broth microdilution method according to Clinical Laboratory and Standards Institute. The evaluation of the interaction between micafungin with Hsp90 inhibitors was realized using the checkerboard methodology. According to the polyphasic taxonomy, C. parapsilosis sensu stricto was the most frequently identified, followed by C. orthopsilosis and C. metapsilosis, and one isolate of Lodderomyces elongisporus was identified by genetic sequencing. The Hsp90 inhibitor geladanamycin associated with micafungin showed a synergic effect in 31.25% of the isolates, a better result was observed with radicicol, which shows synergic effect in 56.25% tested yeasts. The results obtained demonstrate that blocking Hsp90 could be effective to reduce antifungal resistance to echinocandins.

Targeted delivery of HSP90 inhibitors for efficient therapy of CD44-positive acute myeloid leukemia and solid tumor-colon cancer

Heat shock protein 90 (HSP90) is overexpressed in numerous cancers, promotes the maturation of numerous oncoproteins and facilitates cancer cell growth. Certain HSP90 inhibitors have entered clinical trials. Although less than satisfactory clinical effects or insurmountable toxicity have compelled these trials to be terminated or postponed, these results of preclinical and clinical studies demonstrated that the prospects of targeting therapeutic strategies involving HSP90 inhibitors deserve enough attention. Nanoparticulate-based drug delivery systems have been generally supposed as one of the most promising formulations especially for targeting strategies. However, so far, no active targeting nano-formulations have succeeded in clinical translation, mainly due to complicated preparation, complex formulations leading to difficult industrialization, incomplete biocompatibility or nontoxicity. In this study, HSP90 and CD44-targeted A6 peptide functionalized biomimetic nanoparticles (A6-NP) was designed and various degrees of A6-modification on nanoparticles were fabricated to evaluate targeting ability and anticancer efficiency. With no excipients, the hydrophobic HSP90 inhibitor G2111 and A6-conjugated human serum albumin could self-assemble into nanoparticles with a uniform particle size of approximately 200 nm, easy fabrication, well biocompatibility and avoidance of hepatotoxicity. Besides, G2111 encapsulated in A6-NP was only released less than 5% in 12 h, which may avoid off-target cell toxicity before entering into cancer cells. A6 peptide modification could significantly enhance uptake within a short time. Moreover, A6-NP continues to exert the broad anticancer spectrum of Hsp90 inhibitors and displays remarkable targeting ability and anticancer efficacy both in hematological malignancies and solid tumors (with colon tumors as the model cancer) both in vitro and in vivo. Overall, A6-NP, as a simple, biomimetic and active dual-targeting (CD44 and HSP90) nanomedicine, displays high potential for clinical translation.

Cancer Cell-Mimicking Prussian Blue Nanoplatform for Synergistic Mild Photothermal/Chemotherapy via Heat Shock Protein Inhibition.

Combined mild-temperature photothermal/chemotherapy has emerged as a highly promising modality for tumor therapy. However, its therapeutic efficacy is drastically compromised by the heat-induced overexpression of heat shock proteins (HSPs) by the cells, which resist heat stress and apoptosis. The purpose of this study was to downregulate HSPs and enhance the mild-temperature photothermal/chemotherapy effect. In detail, the colon cancer cell membrane (CT26M)-camouflaged HSP90 inhibitor ganetespib and the chemotherapeutic agent doxorubicin (DOX)-coloaded hollow mesoporous Prussian blue (HMPB) nanoplatform (named PGDM) were designed for synergistic mild photothermal/chemotherapy via HSP inhibition. In addition to being a photothermal agent with a high efficiency of photothermal conversion (24.13%), HMPB offers a hollow hole that can be filled with drugs. Concurrently, the cancer cell membrane camouflaging enhances tumor accumulation through a homologous targeting mechanism and gives the nanoplatform the potential to evade the immune system. When exposed to NIR radiation, HMPB's photothermal action (44 °C) not only causes tumor cells to undergo apoptosis but also causes ganetespib to be released on demand. This inhibits the formation of HSP90, which enhances the mild photothermal/chemotherapy effect. The results confirmed that the combined treatment regimen of mild photothermal therapy (PTT) and chemotherapy showed a better therapeutic efficacy than the individual treatment methods. Therefore, this multimodal nanoparticle can advance the development of drugs for the treatment of malignancies, such as colon cancer, and has prospects for clinical application.

Quantitative proteomic analysis reveals unique Hsp90 cycle-dependent client interactions.

Hsp90 is an abundant and essential molecular chaperone that mediates the folding and activation of client proteins in a nucleotide-dependent cycle. Hsp90 inhibition directly or indirectly impacts the function of 10-15% of all proteins due to degradation of client proteins or indirect downstream effects. Due to its role in chaperoning oncogenic proteins, Hsp90 is an important drug target. However, compounds that occupy the ATP-binding pocket and broadly inhibit function have not achieved widespread use due to negative effects. More selective inhibitors are needed; however, it is unclear how to achieve selective inhibition. We conducted a quantitative proteomic analysis of soluble proteins in yeast strains expressing wild-type Hsp90 or mutants that disrupt different steps in the client folding pathway. Out of 2,482 proteins in our sample set (approximately 38% of yeast proteins), we observed statistically significant changes in abundance of 350 (14%) of those proteins (log2 fold change ≥1.5). Of these, 257/350 (∼73%) with the strongest differences in abundance were previously connected to Hsp90 function. Principal component analysis of the entire dataset revealed that the effects of the mutants could be separated into three primary clusters. As evidence that Hsp90 mutants affect different pools of clients, simultaneous co-expression of two mutants in different clusters restored wild-type growth. Our data suggests that the ability of Hsp90 to sample a wide range of conformations allows the chaperone to mediate folding of a broad array of clients and that disruption of conformational flexibility results in client defects dependent on those states.

Hsp90 inhibition leads to an increase in surface expression of multiple immunological receptors in cancer cells.

Heat shock protein 90 (Hsp90) is a molecular chaperone important for maintaining protein homeostasis (proteostasis) in the cell. Hsp90 inhibitors are being explored as cancer therapeutics because of their ability to disrupt proteostasis. Inhibiting Hsp90 increases surface density of the immunological receptor Major Histocompatibility Complex 1 (MHC1). Here we show that this increase occurs across multiple cancer cell lines and with both cytosol-specific and pan-Hsp90 inhibitors. We demonstrate that Hsp90 inhibition also alters surface expression of both IFNGR and PD-L1, two additional immunological receptors that play a significant role in anti-tumour or anti-immune activity in the tumour microenvironment. Hsp90 also negatively regulates IFN-γ activity in cancer cells, suggesting it has a unique role in mediating the immune system's response to cancer. Our data suggests a strong link between Hsp90 activity and the pathways that govern anti-tumour immunity. This highlights the potential for the use of an Hsp90 inhibitor in combination with another currently available cancer treatment, immune checkpoint blockade therapy, which works to prevent immune evasion of cancer cells. Combination checkpoint inhibitor therapy and the use of an Hsp90 inhibitor may potentiate the therapeutic benefits of both treatments and improve prognosis for cancer patients.

The protein phosphatase-2A subunit PR130 is involved in the formation of cytotoxic protein aggregates in pancreatic ductal adenocarcinoma cells

As a major source of cellular serine and threonine phosphatase activity, protein phosphatase-2A (PP2A) modulates signaling pathways in health and disease. PP2A complexes consist of catalytic, scaffolding, and B-type subunits. Seventeen PP2A B-type subunits direct PP2A complexes to selected substrates. It is ill-defined how PP2A B-type subunits determine the growth and drug responsiveness of tumor cells. Pancreatic ductal adenocarcinoma (PDAC) is a disease with poor prognosis. We analyzed the responses of murine and human mesenchymal and epithelial PDAC cells to the specific PP2A inhibitor phendione. We assessed protein levels by immunoblot and proteomics and cell fate by flow cytometry, confocal microscopy, and genetic manipulation. We show that murine mesenchymal PDAC cells express significantly higher levels of the PP2A B-type subunit PR130 than epithelial PDAC cells. This overexpression of PR130 is associated with a dependency of such metastasis-prone cells on the catalytic activity of PP2A. Phendione induces apoptosis and an accumulation of cytotoxic protein aggregates in murine mesenchymal and human PDAC cells. These processes occur independently of the frequently mutated tumor suppressor p53. Proteomic analyses reveal that phendione upregulates the chaperone HSP70 in mesenchymal PDAC cells. Inhibition of HSP70 promotes phendione-induced apoptosis and phendione promotes a proteasomal degradation of PR130. Genetic elimination of PR130 sensitizes murine and human PDAC cells to phendione-induced apoptosis and protein aggregate formation. These data suggest that the PP2A-PR130 complex dephosphorylates and thereby prevents the aggregation of proteins in tumor cells.Graphical

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.

Customized Enhancement of Thermal Sensitivity of Tumors at Different Subcutaneous Depths by Multichannel Lanthanide Nanocomposites.

The photothermal therapeutic effect on tumors located at different subcutaneous depths varies due to the attenuation of light by tissue. Here, based on the wavelength-dependent optical attenuation properties of tissues, the tumor depth is assessed using a multichannel lanthanide nanocomposite. A zeolitic imidazolate framework (ZIF-8)-coated nanocomposite is able to deliver high amounts of the hydrophilic heat shock protein 90 inhibitor epigallocatechin gallate through a hydrogen-bonding network formed by the encapsulated highly polarized polyoxometalate guest. It is superior to both bare and PEGylated ZIF-8 for drug delivery. With the assessment of tumor depth and accumulated amount of nanocomposite by fluorescence, an irradiation prescription can be customized to release sufficient HSP90 inhibitor and generate heat for sensitized photothermal treatment of tumors, which not only ensured therapeutic efficacy but also minimized damage to the surrounding tissues.

Up-regulation of HSP90α in HDM-induced asthma causes pyroptosis of airway epithelial cells by activating the cGAS-STING-ER stress pathway

Heat Shock protein 90 α (HSP90α), an main subtype of chaperone protein HSP90, involves important biological functions such as DNA damage repair, protein modification, innate immunity. However, the potential role of HSP90α in asthma occurrence and development is still unclear. This study aimed to elucidate the underlying mechanism of HSP90α in asthma by focusing on the cGAS-STING-Endoplasmic Reticulum stress pathway in inflammatory airway epithelial cell death (i.e., pyroptosis; inflammatory cell death). To accomplish that, we modeled allergen exposure in C57/6BL mice and bronchial epithelial cells with house dust mite. Protein technologies and immunofluorescence utilized to study the expression of HSP90α, activation of cGAS-STING pathway and pyroptosis. The effect of inhibitors on HDM-exposed mice detected by histological techniques and examination of bronchoalveolar lavage fluid. Results showed that HSP90α promotes asthma inflammation via pyroptosis and activation of the cGAS-STING-ER stress pathway. Treatment with the HSP90 inhibitor tanespimycin (17-AAG) significantly relieved airway inflammation and abrogated the effect of HSP90α on pyroptosis and cGAS-STING-ER stress in vitro and in vivo models of HDM. Further data indicated that up-regulation of HSP90α stabilized STING through interaction, which increased localization of STING on the ER. Activation of STING triggered ER stress and leaded to pyroptosis-related airway inflammation. The finding showed the potential role of pyroptosis caused by dysregulation of HSP90α on airway epithelial cells in allergic inflammation, suggested that targeting HSP90α in airway epithelial cells might prove to be a potential additional treatment strategy for asthma.

Hippocampal HDAC6 promotes POCD by regulating NLRP3-induced microglia pyroptosis via HSP90/HSP70 in aged mice

BackgroundPostoperative Cognitive Dysfunction (POCD) has attracted increased attention, but its precise mechanism remains to be explored. This study aimed to figure out whether HDAC6 could regulate NLRP3-induced pyroptosis by modulating the functions of HSP70 and HSP90 in microglia to participate in postoperative cognitive dysfunction in aged mice. MethodsAnimal models of postoperative cognitive dysfunction in aged mice were established by splenectomy under sevoflurane anesthesia. Morris water maze was used to examine the cognitive function and motor ability. Sixteen-months-old C57BL/6 male mice were randomly divided into six groups: control group (C group), sham surgery group (SA group), splenectomy group (S group), splenectomy + HDAC6 inhibitor ACY-1215 group (ACY group), splenectomy + HDAC6 inhibitor ACY-1215 + HSP70 inhibitor Apoptozole group (AP group), splenectomy + solvent control group (SC group). The serum and hippocampus of mice were taken after mice were executed. The protein levels of HDAC6, HSP90, HSP70, NLRP3, GSDMD-N, cleaved-Caspase-1 (P20), IL-1β were detected by western blotting. Serum IL-1β, IL-6 and S100β were measured using ELISA assay, and cell localization of HDAC6 was detected by immunofluorescence. In vitro experiments, BV2 cells were used to validate whether this mechanism worked in microglia. The protein levels of HDAC6, HSP90, HSP70, NLRP3, GSDMD-N, P20, IL-1β were detected by western blotting and the content of IL-1β in the supernatant was measured using ELISA assay. The degree of acetylation of HSP90, the interaction of HSP70, HSP90 and NLRP3 were analyzed by coimmunoprecipitation assay. ResultsSplenectomy under sevoflurane anesthesia in aged mice could prolong the escape latency, reduce the number of crossing platforms, increase the expression of HDAC6 and activate the NLRP3 inflammasome to induce pyroptosis in hippocampus microglia. Using ACY-1215 could reduce the activation of NLRP3 inflammasome, the pyroptosis of microglia and the degree of spatial memory impairment. Apoptozole could inhibit the binding of HSP70 to NLRP3, reduce the degradation of NLRP3 and reverse the protective effect of HDAC6 inhibitors. The results acquired in vitro experiments closely resembled those in vivo, LPS stimulation led to the pyroptosis of BV2 microglia cells and the release of IL-1β due to the activation of the NLRP3 inflammasome, ACY-1215 showed the anti-inflammatory effect and Apoptozole exerted the opposite effect. ConclusionsOur findings suggest that hippocampal HDAC6 promotes POCD by regulating NLRP3-induced microglia pyroptosis via HSP90/HSP70 in aged mice.

Abstract 2977: Novel HSP90 inhibitors with senolytic activity for a targeted therapy in a hormone-induced breast cancer senescence model

Abstract Background: The research into senescent cells, particularly their accumulation and contribution to aging and age-related diseases, has gained significant momentum in recent years. This interest is primarily driven by findings in mouse models, where the excessive buildup of senescent cells is linked to various age-related pathologies. Consequently, there has been a concerted effort in the scientific community to identify and develop senolytics. A notable development in this field is the identification of senolytics within the class of HSP90 inhibitors. These inhibitors, initially investigated for their potential in cancer therapy, have been repurposed due to their high selectivity for senescent cells. Our research has led to new compounds with mild inhibitory activity against HSP90α in response to these challenges. These compounds stand out for their lack of toxic side effects and their demonstrated senolytic activity across multiple cellular models. Objective: The primary objective of this research is to identify and characterize innovative HSP90 inhibitors that exhibit senolytic properties, specifically in the context of hormone-driven cancers. Methods and Results: Utilizing a structure-based virtual screening technique, we identified several compounds displaying strong affinity and predicted inhibitory activity against the target protein, HSP90α. Among these, two inhibitors designated K4 and K5, demonstrated notable senolytic activity in vitro without inducing cytotoxic effects in non-senescent cells. These compounds were tested for their efficacy in senolytic activity and displayed half-maximal inhibitory concentrations (IC50) of 155 nM and 111 nM, respectively. The experimental model for this study was the MCF7 cell line, which was treated with 4-Hydroxytamoxifen (Tam, 10uM for 96 hours) to induce a state of senescence. Subsequent growth curve analyses and beta-galactosidase assays established that Tam treatment effectively halted cell proliferation and induced a senescence state in about 40% of the cell population compared to the ethanol control. When treated with K4 and K5 (10 uM for an additional 96 hours), a significant reduction in the senescent cell population was observed, with K5 showing a particularly pronounced increase in the mortality rate of these cells (up to 2.5-fold compared to the DMSO control). Western blot analysis supported these results, suggesting the effectiveness of K5 combined with Tam in inhibiting proliferation and inducing cell death in MCF7 cells. Conclusion: Our findings propose a novel therapeutic approach for hormone-dependent breast cancer, combining the dual roles of tamoxifen and HSP90 inhibitors to target senescent cancer cells. This strategy could reduce tumor recurrence and contribute to the advancement of cancer treatment methods. Citation Format: Luca Cis, Michela Gottardi Zamperla, Veronica Barbi, Pietro Cigala Fulgosi, Sara De Martino, Aurora Aiello, Simona Nanni, Chiara Cencioni, Davide Pirolli, Paola Tabarelli De Fatis, Giovanni Battista Ivaldi, Marco Malavolta, Christian Steinkühler, Maria Cristina De Rosa, Antonella Farsetti, Sandra Atlante, Carlo Gaetano. Novel HSP90 inhibitors with senolytic activity for a targeted therapy in a hormone-induced breast cancer senescence model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2977.

Abstract 1082: Exploiting lurbinectedin-driven nucleolar relocalization of EWS-FLI1 to develop novel combination therapies for Ewing sarcoma

Abstract Background: Ewing sarcoma (ES) is a malignant bone tumor characterized by the oncogenic fusion protein EWS-FLI1. EWS-FLI1 acts as an aberrant transcription factor, inducing and silencing genes involved with increasing cell proliferation and survival. We have previously shown that lurbinectedin, a trabectedin analog, effectively inactivates the transcriptional activity of EWS-FLI1 by redistributing it within the nucleus to the nucleolus. Interestingly, this process activates a gene signature like that of the DNA damage response (DDR) to UV light, which was also shown to induce nucleolar translocation of WT Ewing sarcoma breakpoint 1 (EWSR1). However, the mechanism governing the nucleolar translocation of EWS-FLI1 induced by these drugs has yet to be elucidated. In addition, novel approaches to exploit this relocalization mechanism as a means to broaden the therapeutic window have yet to be established. Methods: We used immunoblotting, RT-qPCR, viability assays, cell fractionation, confocal microscopy, proximity ligation assays (PLA), mass spectrometry, genomics, and immunoprecipitation to investigate the mechanism of action of trabectedin/lurbinectedin-induced nucleolar relocalization of EWS-FLI1. Results: Through interrogation of the pathways triggered by trabectedin/lurbinectedin, we found that this mechanism is in part mediated by HSP70. Viability assays also revealed profound synergy between HSP70 inhibitors (HSP70is) and lurbinectedin. Immunoprecipitated EWS-FLI1 subjected to mass spectrometry revealed an association of EWS-FLI1 and several HSP70 isoforms after trabectedin treatment which was confirmed with PLA. Trabectedin/lurbinectedin treatment also revealed an upregulation of EWS-FLI1-suppressed targets and suppression of induced targets. Confocal microscopy showed that EWS-FLI1 “trapping” in the nucleolus was potentiated by combination treatment with HSP70is. Conclusion: Our results underscore the synergistic effect of combining HSP70is with trabectedin or lurbinectedin treatment to attenuate several aspects of ES tumorigenesis in vitro. In vivo experiments with patient-derived xenografts are underway to corroborate the synergy of HSP70is and trabectedin/lurbinectedin. Together, our data highlights the potential for a novel “EWS-FLI1 nucleolar trap” as a means to inhibit and sustain EWS-FLI1 repression with this class of compounds. Citation Format: Sridhar M. Veluvolu, Raphael D. Lopez, Jenna M. Gedminas, Elissa A. Boguslawski, Elizabeth R. Wilson, Benjamin P. Caiello, Maureen E. Murphy, Patrick J. Grohar. Exploiting lurbinectedin-driven nucleolar relocalization of EWS-FLI1 to develop novel combination therapies for Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1082.

Abstract 152: Exploring the impact of heat shock protein 90 on the antitumor efficacy and stemness in medulloblastoma cells

Abstract Background: Medulloblastoma (MB) is the most common children’s malignant brain tumor. Although MB can be treated with conventional chemotherapies, chemoresistance has been frequently developed and thereby hampers their therapeutic effectiveness. The preliminary reports of our research showed that some heat shock proteins have higher levels in MB cells. Among these heat shock proteins, HSP70 and HSP90 expressions are particularly high in MB CD133+ cancer stem cells (CSCs), which arouses our attention and interest. Numerous studies demonstrated that heat shock proteins are prominent in some chemotherapy-resistant cancer cells and CSCs. As known HSP90 and its client proteins are required for the maintenance of self-renewal/stemness and regulation of cell cycle, synergistic killing of cancer cells by simultaneous inhibition of HSP90 expression and activity may be a considerable good therapeutic strategy for treating MB. The purpose of this study is to verify the regulatory function of HSP90 in chemoresistance and stemness of MB. Materials and Methods: The levels of HSP90, its client proteins (P13K/pAKT, Erk, NFκB, and c-Myc), and stemness makers were detected using immunoblotting and immunoprecipitation after the treatment of HSP90 inhibitor (17-AAG) or knockdown of HSP90 by siRNA in MB and MB CSCs. The antitumor effects of HSPs inhibitors or knockdown of HSPs on these cells were evaluated by MTT assay, Annexin V/PI analysis and cell cycle analysis. Results: Our findings revealed elevated expression of HSP90 and its target client proteins in MB cells compared to normal astrocytes. Silencing or inhibiting HSP90 expression induced cell cycle arrest in MB cells. Notably, CD133+ MB CSCs exhibited higher HSP90 levels than parental MB cells. Inhibition of HSP90 led to reduced sphere formation and expression of stemness markers in CD133+ MB CSCs. Treatment with 17-AAG demonstrated antitumor activity in CD133+ MB CSCs, and a synergistic effect was observed when combining conventional chemotherapeutic agents with 17-AAG in MB cells Conclusion: These results illustrated that inhibition of HSP90 could eliminate CSCs, and thereby overcome resistance to chemotherapeutic agents in MB. Chemotherapies in combination with HSP90 blockade may be an emerging MB therapy in the future. Citation Format: Shu-Mei Chen, Hsieng-Yun Huang, Chien-Kai Wang, Yi-Yuan Yang, Sy-Jye Leu, Ying-Ying Li. Exploring the impact of heat shock protein 90 on the antitumor efficacy and stemness in medulloblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 152.

Abstract 4567: Dual targeting of HSP70 and AURKA improves treatment in neuroendocrine prostate cancer

Abstract Background: Neuroendocrine prostate cancer (NEPC) is the most aggressive type of prostate cancer with no effective treatments. Therefore, there is an urgent need to develop new treatment strategies. N-Myc plays a key role in driving NEPC progression, and Aurora kinase A (AURKA) prevents N-Myc degradation by interact with N-Myc and disallowing the intervention of ubiquitin ligases. The AURKA inhibitor Alisertib inhibits NEPC tumor growth by disrupting N-Myc signaling, but it is failed in a phase II clinical trial. In this study, we tested the synergy of novel HSP70 allosteric inhibitor JG231 and Alisertib in NEPC cells, organoids, and patient derived xenograft (PDX) models, which will provide new therapeutic strategies for the NEPC treatment. Methods: RT-PCR, western blotting, or immunohistochemistry (IHC) were used to determine the expression of N-Myc, neuroendocrine maker (NSE, SYP, CHGA, etc) in different prostate cancer cell lines and PDX tumors. After the knockdown of HSP70 and AURKA by siRNA transfection, cell proliferation and N-Myc expression were assessed by using cell viability assay, RT-PCR and Western blotting respectively. The co-immunoprecipitation assay was performed to detect the N-Myc ubiquitination. RNA-sequencing, gene set enrichment analysis was employed to determine the changes of JG231 on gene programs. The effects of JG231 and Alisertib on the proliferation were examined in NEPC cell lines and PDX organoids models. Results: We determined that PC3, CWR22Rv1, H660 cell lines, LuCaP49, and LuCaP93 PDX tumors had significantly increased the expression of N-Myc and neuroendocrine makers. Knockdown of HSP70 or using the HSP70 inhibitor, JG231, significantly inhibit the growth of H660 and CWR22Rv1 cells and leads to a synergistic effect with Alisertib (p&amp;lt;0.001). JG231 combined with Alisertib also inhibits growth and induces the death of LuCaP93 and H660 tumor derived organoids in a dose-dependent manner (p&amp;lt;0.001). Mechanistically, JG231 inhibits the protein expression of N-Myc through the ubiquitin-proteasome system and promote STUB1 to enter the nucleus to bind to N-Myc. Conclusions: JG231, a novel HSP70 inhibitor, can improves the treatment efficacy of AURKA inhibitor Alisertib against NEPC through the regulation of N-Myc protein homeostasis. Citation Format: Pengfei Xu, Joy C. Yang, Bo Chen, Shu Ning, Leyi Wang, Christopher Nip, Christopher P. Evans, Marc A. Dall'Era, Allen C. Gao, Jason Gestwicki, Chengfei Liu. Dual targeting of HSP70 and AURKA improves treatment in neuroendocrine prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4567.

Abstract 6205: Identify drugs for metastatic castration-resistance prostate cancer (mCRPC) patients with high glycolytic activity through in silico approach

Abstract Prostate cancer (PC) remains a prominent cause of cancer-related mortality among men in the United States. A significant factor contributing to the mortality of PC patients is the development of resistance to second-generation antiandrogen therapies, particularly androgen receptor-signaling inhibitors (ARSI), which is pronounced in patients with late-stage PC, metastatic castration-resistance prostate cancer (mCRPC). Therefore, there is an urgent need for novel therapeutics beyond AR signaling inhibition for patients with mCRPC. One of the key drivers of resistance to ARSI and a critical feature of CRPC is cancer metabolism reprogramming to elevated glycolysis. However, the current research on finding drugs to impede high-glycolytic tumor growth is hurdled by a resistance raised from metabolic plasticity. The aim of this research is to quickly repurpose existing drugs for inhibiting high-glycolytic mCRPC growth while proactively circumstancing resistance from metabolic plasticity through computational approaches. To do this, we imputed a large number of drug responses in patients with mCRPC using a computational algorithm (OncoPredict), identified drugs that were projected to show higher sensitivity in high-glycolytic tumors, and performed in vitro validation. Furthermore, to maximize drug efficacy, we identified biomarkers for drug candidates. By implementing this computational pipeline, we identified a number of drugs with various mechanisms of action that show higher efficacy in mCRPC with high glycolysis. Furthermore, we successfully validated the efficacy of these nominated drug candidates including an anti-parasitic agent, an HSP90 inhibitor, and a CDK inhibitor. These drug candidates exhibited superior tumor growth inhibition in in vitro models of high glycolysis-induced advanced PC. In conclusion, this study introduces an innovative in silico approach to unveil the existing drugs’ unknown potential for the treatment of patients with mCRPC. Our findings hold promise for development of effective therapeutics for combating this challenging disease. Citation Format: Mei-Chi Su, Adam M. Lee, R. Stephanie Huang. Identify drugs for metastatic castration-resistance prostate cancer (mCRPC) patients with high glycolytic activity through in silico approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6205.