HSP90 Inhibitor 17-DMAG Research Articles

Potential role of Hsp90 in rat islet function under the condition of high glucose.

The preservation of pancreatic β-cell function is a key point in the treatment of type 2 diabetes mellitus. There is substantial evidence demonstrating that heat-shock protein 90 (Hsp90) is needed for the stabilization and correct folding of client proteins and plays important roles in various biological processes. Here, we revealed the important role of Hsp90 in β-cell function. Islets from male Sprague-Dawley rats were isolated to be used for further RT-PCR, Western blot, and insulin secretion test ex vivo in response to different stimuli. Our results revealed that Hsp90 expression was significantly decreased in isolated rat islets exposed to high glucose, which was involved in glucokinase activation and glucose metabolism, not calcium signaling. Two kinds of Hsp90 inhibitors 17-DMAG and CCT018159 markedly enhanced glucose-stimulated insulin secretion from rat islets, along with increased expressions of genes closely related to β-cell function. These data indicate that Hsp90 may be involved in high glucose-induced islet function adaptation.

Role of Monocytes and Pro-Inflammatory Th17 Cells Expressing Multi-Drug Resistance Protein Type 1 in the Pathogenesis of Graft-Versus-Host Disease

Graft-versus-host disease (GvHD) is a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). The pathophysiology of GvHD is complex and remains poorly understood. However, it has been demonstrated that Th17 cells play an important role in the development and progression of acute and chronic GvHD. Furthermore, monocytes have an essential function in the induction of Th17 cells. It is known that the distribution of the three monocyte subtypes (classical (CD14++ CD16-), non-classical (CD14+ CD16++) and intermediate (CD14++ CD16+) monocytes) changes significantly in the peripheral blood of patients during GvHD: the level of classical monocytes is decreased, whereas the percentages of intermediate and non-classical monocytes are elevated. Additionally, there is first evidence that intermediate monocytes are expanded during rheumatoid arthritis and promote the expansion of Th17 cells. Recently it could be demonstrated that pro-inflammatory Th17 cells are restricted to a novel subset of CCR6+ CXCR3hi CCR4lo CCR10- CD161+ cells that stably express P-glycoprotein/multi-drug resistance protein type 1 (MDR1) - the so-called Th17.1 cells. These MDR1+ Th17.1 cells are enriched and activated in the gut of patients with Crohn's disease and are resistant to glucocorticoid-mediated T cell suppression. Thus, this subtype of Th17 cells might play an important role in steroid-resistant inflammatory diseases as well as steroid-refractory GvHD.Our previously performed study could demonstrate that monocytes isolated from patients with acute or chronic GvHD grade I-IV (n=13) induced significant higher percentages of IL-17-producing Th17 cells compared to monocytes from healthy donors (n=32) or patients without GvHD after HCT (n=21) (p<0.001). To further investigate the role of monocytes in the pathogenesis of GvHD the influence of classical, non-classical and intermediate monocytes on the induction of pro-inflammatory MDR1+ CCR6+ CXCR3hi CCR4lo CCR10- CD161+ Th17.1 cells was investigated in the present study. Therefore, these monocyte subtypes were isolated from peripheral blood mononuclear cells (PBMCs) by magnetic cell isolation technology and were co-cultured with isolated CD4+ T cells. The obtained results could show that intermediate and non-classical monocytes induced higher percentages of pro-inflammatory MDR1+ CCR6+ CXCR3hi CCR4lo CCR10- CD161+ Th17.1 cells compared to classical monocytes or whole monocyte population. Additionally, the influence of glucocorticoids used for the treatment of patients with GvHD and the Hsp90 inhibitor 17-Dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) was examined on the development of MDR1 expressing Th17.1 cells in vitro. Therefore, monocytes were co-cultured with CD4+ T cells in the presence and absence of prednisolone, dexamethasone and 17-DMAG. First data demonstrated that treatment of these co-cultures with glucocorticoids resulted in elevated levels of induced pro-inflammatory MDR1+ Th17.1 cells. In contrast, addition of the Hsp90 inhibitor 17-DMAG to the co-cultures led to decreased levels of these pro-inflammatory Th17 cells compared to the untreated control.In conclusion, our findings suggest that intermediate and non-classical monocytes trigger the induction of pro-inflammatory MDR1+ CCR6+ CXCR3hi CCR4lo CCR10- CD161+ Th17.1 cells and might therefore play a major role in the pathogenesis of GvHD. While glucocorticoids seem to promote the development of MDR1 expressing Th17.1 cells, the Hsp90 inhibitor 17-DMAG acts anti-inflammatory leading to decreased levels of induced pro-inflammatory Th17 cells. Thus, the chaperone Hsp90 could be an important regulator in monocyte-induced development of pro-inflammatory MDR1+ Th17.1 cells and might therefore be a therapeutic target for GvHD. DisclosuresNo relevant conflicts of interest to declare.

HSP90 inhibition blocks ERBB3 and RET phosphorylation in myxoid/round cell liposarcoma and causes massive cell death in vitro and in vivo.

Myxoid sarcoma (MLS) is one of the most common types of malignant soft tissue tumors. MLS is characterized by the FUS-DDIT3 or EWSR1-DDIT3 fusion oncogenes that encode abnormal transcription factors. The receptor tyrosine kinase (RTK) encoding RET was previously identified as a putative downstream target gene to FUS-DDIT3 and here we show that cultured MLS cells expressed phosphorylated RET together with its ligand Persephin. Treatment with RET specific kinase inhibitor Vandetanib failed to reduce RET phosphorylation and inhibit cell growth, suggesting that other RTKs may phosphorylate RET. A screening pointed out EGFR and ERBB3 as the strongest expressed phosphorylated RTKs in MLS cells. We show that ERBB3 formed nuclear and cytoplasmic complexes with RET and both RTKs were previously reported to form complexes with EGFR. The formation of RTK hetero complexes could explain the observed Vandetanib resistence in MLS. EGFR and ERBB3 are clients of HSP90 that help complex formation and RTK activation. Treatment of cultured MLS cells with HSP90 inhibitor 17-DMAG, caused loss of RET and ERBB3 phosphorylation and lead to rapid cell death. Treatment of MLS xenograft carrying Nude mice resulted in massive necrosis, rupture of capillaries and hemorrhages in tumor tissues. We conclude that complex formation between RET and other RTKs may cause RTK inhibitor resistance. HSP90 inhibitors can overcome this resistance and are thus promising drugs for treatment of MLS/RCLS.

Organ‐specific efficacy of HSP90 inhibitor in multiple‐organ metastasis model of chemorefractory small cell lung cancer

Small-cell lung cancer (SCLC) accounts for nearly 15% of lung cancer cases and exhibits aggressive clinical behavior characterized by rapid growth and metastatic spread to multiple organs. About 70% of patients with SCLC present with extensive disease and distant metastases at diagnosis. HSP90 is a 90-kDa molecular chaperone whose association is required for the stability and function of its numerous "client proteins." Here, we assessed the therapeutic potential of the HSP90 inhibitor 17-DMAG in SCLC. Notably, 17-DMAG hindered the viability of human SCLC cell lines-regardless of their chemosensitivity-via the decreased expression of client proteins, including the proto-oncogene c-Raf (also known as RAF1). In an in vivo imaging model of SCLC multiple-organ metastasis with the human SCLC cell line SBC-5, treatment with 17-DMAG remarkably inhibited the formation of metastatic sites in the liver, but was ineffective in hindering the progression of bone lesions. The latter was likely the result of activation of osteoclasts. IGF-1, which is supposed to be rich in bone environment, preserved c-Raf expression and maintained viability of SBC-5 cells treated with 17-DMAG. Furthermore, the combined use of a bisphosphonate with 17-DMAG significantly attenuated the progression of metastases in both the liver and the bone. These findings suggest that therapeutic effects of HSP90 inhibitors may be organ-specific and should be carefully monitored in SCLC clinical trials.

Hsp90 at the Hub of Metabolic Homeostasis in Malignant B Cells

Abstract Reprogramming of tumor cell metabolism contributes to disease aggressiveness and chemoresistance, but how this process is regulated on the molecular levels is unclear. Hsp90 regulates diverse cellular processes through its interaction with co-chaperones and client proteins. Although basally expressed, Hsp90 is almost universally overexpressed in malignant cells due to increased levels of internal (oxidants, genomic instability, unfolded proteins) and external (hypoxia, nutrients limitation, drugs) stressors. Indeed, Hsp90 is starting to be acknowledged also for its role as an integrator of nutrient sensing, tumor bioenergetics and metabolic stress response (Chae et al, Cancer Cell, 2012). Here we report that active cytosolic Hsp90 binds and stabilizes enzymes regulating central metabolic processes in DLBCL, and provides an actionable target for this disease. Despite its overexpression, not all DLBCL are equally responsive to Hsp90 inhibitors. Using a panel of 10 DLBCL cell lines we found that those with higher glycolytic activity were more likely to respond to the Hsp90 inhibitors 17-DMAG and PU-H71. Global metabolomics profiling (GC-MS and UPLC-MS) of Hsp90-dependent (OCI-Ly7, OCI-Ly1, SU-DHL6, OCI-Ly10) vs. Hsp90-independent (Karpas422) cells identified “purine metabolism” (p=0.000027) and “pyrimidine metabolism” (p=0.0017) as upregulated, whereas “TCA cycle” was downregulated (p=0.000027). We validated these findings by short-term (6 h) exposure of OCI-Ly1 and OCI-Ly7 cells with non-cytotoxic doses of PU-H71 and found a highly reduced oxygen consumption rate (OCR) as well as glycolytic rate (ECAR). This was accompanied by a sharp decrease in glucose consumption and lactate production and by only a slight reduction in glutamine consumption. Unexpectedly, the drop in glycolysis and respiration in OCI-Ly1 and OCI-Ly7 did not lead to a significant decrease either in ATP or reducing power, suggesting that actually both the production and the utilization of ATP and redox equivalents are shut down when Hsp90 activity (an ATP-hydrolyzing enzyme) is inhibited. To identify the molecular basis for these differences, we characterized the active Hsp90-associated proteome in DLBCL by using chemical affinity purification followed by MS in cytosolic fractions of two Hsp90-dependent DLBCL cell lines (OCI-Ly1 and OCI-Ly7) in duplicates. This method pulls down only active Hsp90 multichaperone complexes (Moulick et al, Nat Chem Biol, 2011). Pathway enrichment analysis identified “cellular metabolism” as a significant process actively chaperoned by Hsp90 (ratio of enrichment 4.35, p=8.22E-15). Specifically, “nucleotides” and “carbohydrates” metabolism were the two most represented pathways, suggesting their critical role in Hsp90-dependent DLBCL cells. To functionally validate these findings, we integrated the proteomics analysis with serum metabolomics obtained from 10 OCI-Ly7-xenografted mice treated with PU-H71 vs vehicle (75 mg/m2 for 24 h (n=5)). Bioinformatic analysis revealed that PU-H71-treated mice had significant changes in 122 metabolites including lower levels of xanthine, hypoxanthine, adenosine and inosine, suggesting an alteration of the purines metabolism. IMPDH1/2 is the enzyme catalyzing the first committed and rate-limiting step of de novo guanine nucleotide biosynthesis. To determine whether IMPDH stability depends on Hsp90 activity, we treated a panel of 6 DLBCL cell lines (including OCI-Ly7) with their GI50 for PU-H71 or 17-DMAG for up to 24 h and found a time-dependent decrease in IMPDH2 protein levels. Finally, SCID mice were xenografted with OCI-Ly1 and SU-DHL6 and treated with vehicle, PU-H71, mycophenolate mofetil (MMF, the pro-drug of the IMPDH inhibitor mycophenolic acid) and the combination of PU-H71 and MMF. We found that the combination of drugs exhibited a greater antitumor effect than each drug alone (p=0.002 for SU-DHL6 and P&lt;0.001 for OCI-Ly1, T-test day 10). There were no toxic effects. Altogether, our work supports a role for Hsp90 as a necessary component in a subset of DLBCLs to build-up the metabolic features that allow these tumor cells to meet the requirements for their unrestrained growth and proliferation. Disclosures No relevant conflicts of interest to declare.

Abstract 1698: Identification of drugs with eribulin combinatorial activity that kill both eribulin-sensitive and eribulin-insensitive tumor cells

Abstract Eribulin mesylate, a nontaxane microtubule dynamics inhibitor with a mechanism distinct from most other antitubulin therapeutics, is clinically used in the United States for treatment of certain patients with locally advanced or metastatic breast cancer. Eribulin has broad anticancer activity in a wide variety of preclinical cancer models; as a result, numerous clinical trials are ongoing to investigate efficacy in other non-breast tumor types. We have examined eribulin sensitivity in a panel of 25 human cancer cell lines representing a variety of tumor types albeit with a preponderance of breast and lung cancer cell lines. As expected, the cell lines vary in sensitivity to eribulin at clinically relevant concentrations. To identify combination drugs capable of increasing anticancer effects in patients already responsive to eribulin, as well as inducing de novo anticancer effects in non-responders, we performed a combinatorial high throughput screen to identify drugs that combine with eribulin to selectively kill tumor cells. Among other observations, we found that inhibitors of ErbB1/ErbB2 (lapatinib, BIBW-2992, erlotinib), MEK (E6201, trametinib), PI3K (BKM-120), mTOR (AZD 8055, everolimus), and PI3K/mTOR (BEZ 235), and a BCL2 family antagonist (ABT-263) show combinatorial activity with eribulin. In addition, antagonistic pairings with other agents, such as topoisomerase I inhibitors (topotecan hydrochloride), an HSP-90 inhibitor (17-DMAG), and gemcitabine and cytarabine, were also identified. In summary, the preclinical studies described here have identified several combination drugs that have the potential to either augment or antagonize eribulin's anticancer activity. Further elucidation of the mechanisms responsible for such interactions may be important for identifying valuable therapeutic partners for eribulin. Citation Format: Toshimitsu Uenaka, Richard Rickles, Yasuhiro Funahashi, Ping Zhu, Jill M. Grenier, Janine Steiger, Nanding Zhao, Bruce A. Littlefield, Junji Matsui, Kenichi Nomoto. Identification of drugs with eribulin combinatorial activity that kill both eribulin-sensitive and eribulin-insensitive tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1698. doi:10.1158/1538-7445.AM2014-1698

Combined Pharmacological Induction of Hsp70 Suppresses Prion Protein Neurotoxicity in Drosophila

Prion diseases are rare and aggressive neurodegenerative disorders caused by the accumulation of misfolded, toxic conformations of the prion protein (PrP). Therapeutic strategies directed at reducing the levels of PrP offer the best chance of delaying or halting disease progression. The challenge, though, is to define pharmacologic targets that result in reduced PrP levels. We previously reported that expression of wild type hamster PrP in flies induces progressive locomotor dysfunction and accumulation of pathogenic PrP conformations, while co-expression of human Hsp70 delayed these changes. To validate the therapeutic potential of Hsp70, we treated flies with drugs known to induce Hsp70 expression, including the Hsp90 inhibitor 17-DMAG and the glucocorticoid dexamethasone. Although the individual treatment with these compounds produced no significant benefits, their combination significantly increased the level of inducible Hsp70, decreased the level of total PrP, reduced the accumulation of pathogenic PrP conformers, and improved locomotor activity. Thus, the combined action of two pharmacological activators of Hsp70 with distinct targets results in sustained high levels of inducible Hsp70 with improved behavioral output. These findings can have important therapeutic applications for the devastating prion diseases and other related proteinopathies.

Chronic treatment with 17-DMAG improves balance and coordination in a new mouse model of Machado-Joseph disease.

Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disease currently with no treatment. We describe a novel mouse model of MJD which expresses mutant human ataxin-3 at near endogenous levels and manifests MJD-like motor symptoms that appear gradually and progress over time. CMVMJD135 mice show ataxin-3 intranuclear inclusions in the CNS and neurodegenerative changes in key disease regions, such as the pontine and dentate nuclei. Hsp90 inhibition has shown promising outcomes in some neurodegenerative diseases, but nothing is known about its effects in MJD. Chronic treatment of CMVMJD mice with Hsp90 inhibitor 17-DMAG resulted in a delay in the progression of their motor coordination deficits and, at 22 and 24 weeks of age, was able to rescue the uncoordination phenotype to wild-type levels; in parallel, a reduction in neuropathology was observed in treated animals. We observed limited induction of heat-shock proteins with treatment, but found evidence that 17-DMAG may be acting through autophagy, as LC3-II (both at mRNA and protein levels) and beclin-1 were induced in the brain of treated animals. This resulted in decreased levels of the mutant ataxin-3 and reduced intranuclear aggregation of this protein. Our data validate this novel mouse model as a relevant tool for the study of MJD pathogenesis and for pre-clinical studies, and show that Hsp90 inhibition is a promising therapeutic strategy for MJD.

Pivotal Role Of Heat Shock Proteins In Monocyte and Endothelial Cell Pathology In Sickle Cell Disease

BackgroundIt is well established that sickle cell disease (SCD) manifests global perturbations of hemostasis. Vaso-occlusion, inflammation and coagulopathy all likely contribute to the protean complications of SCD. Central to both inflammation and coagulation is the monocyte. These cells can be profoundly pro-inflammatory and can express tissue factor on their surface and thus influence both inflammation and coagulation. Monocytosis is common in SCD, as is steady state monocyte activation. Exaggerated monocytic response to stimulus may also contribute to the severity of acute events. Thus, agents that regulate monocyte function are potentially of significant relevance in SCD.To this end, we have discovered that the heat shock proteins (HSPs) are potential master regulators of these cells. We previously demonstrated that inhibition of one such HSP, HSP90, could completely ablate the profound and hyper-responsive monocytic inflammatory release upon lipopolysacchiride (LPS) challenge. Inhibition of HSP90 also blocked LPS induced NFk-B translocation to the nucleus. Thus, these results suggested a potent role for HSP90 in LPS-induced monocyte based inflammation. However, the role of HSP90 is cytokine-induced monocyte activation was speculative. The role of HSP90 in monocyte tissue factor expression, or reactive oxygen species (ROS) generation remained unknown. ObjectivesWe sought to determine the role of HSP90 in regulating the pro-inflammatory, pro-coagulant, and ROS generating potential in monocytes. We then wanted to establish a potential role for HSP90 inhibition. MethodsPeripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll density separation. Flow cytometry was employed to measure the LPS- or cytokine-induced monocyte tissue factor expression and ROS generation. Monocyte ROS generation was detected using L012 based chemiluminescence or visualized in individual cells using flow cytometry with CELLROX. Inflammatory cytokines and tissue factor gene expression was evaluated using quantitative real-time polymerase chain reaction (qRT-PCR). NFkB translocation to the nucleus was detected via cell fractionation followed by western blotting or indirect immunofluorescence. PBMCs or THP-1 cells were pre-treated with the HSP90 inhibitors 17-DMAG or AUY922 prior to assay. Monocyte- induced endothelial cell permeability was measured using endothelial cell-substrate impedance sensing (ECIS). Unless otherwise stated p<0.05. ResultsAt baseline, PBMCs from patients with SCD demonstrated elevated monocyte ROS generation and tissue factor expression than those from healthy controls. Inhibition of significantly reduced these measures of steady state monocyte activation. HSP90 inhibition also inhibited both LPS and cytokine induced tissue mRNA accumulation and subsequent cell surface expression of tissue factor in monocytes. Cytokine- induced ROS generation was significantly interrupted in monocytes upon inhibition of HSP90. A panel of monocyte pro-inflammatory genes could be inhibited with AUY922, whereas the anti-inflammatory cytokine, IL-10, was induced upon HSP90 inhibition with AUY922. Mechanistically, we also noted a profound translocation of NFkB to the monocyte nucleus upon cytokine stimulation. Consistent with the effects on tissue factor expression, pro-inflammatory potential, and ROS generation, this translocation could be completely ablated with HSP90 inhibition. Importantly, HSP90 inhibitors significantly attenuated the LPS-activated monocyte -induced lung microvascular endothelial permeability. ConclusionOur data suggest that Hsp90 inhibitors significantly reduced the both pro-inflammatory and pro-coagulatory potential of PBMCs from patients with SCD. These results thus position HSP90 as a potential master regulator of hemostasis, endothelial cell permeability, and thus suggest that HSP90 is an attractive therapeutic target in patients with SCD. Disclosures:Kutlar:Celgene Corporation: Research Funding. Meiler:Celgene Corporation: Research Funding.

Synergistic Effect Of Nifurtimox and Inhibition Of Hsp70/Hsp90 In Treatment Of Neuroblastoma

Neuroblastoma is the most common solid cancer in childhood with high relapse and mortality rates. Furthermore, high risk neuroblastoma is often accompanied by an infaust prognosis. The 5-nitrofuran nifurtimox, usually used in the treatment of Chagas disease, showed cytotoxic effects against neuroblastoma in vitro and in experimental therapy, which is presumably due to the formation of oxidative stress. Inducing oxidative stress is a well investigated and suitable strategy in the treatment of malignant diseases in vitro but often encounters difficulties in clinical administration. Thus, nifurtimox as a well-established drug represents a promising new approach in treating neuroblastoma. Combining the induction of reactive oxygen species by application of nifurtimox with a blockade of the cells' own stress response might even increase the cytotoxic effects. The chaperones heat shock protein 70 and 90 (Hsp70/Hsp90) are responsible for refolding or degrading damaged proteins, especially after stress situations such as heat or oxidative stress. Therefore, the roles of Hsp70 and Hsp90 were investigated in more detail.The commercially available human neuroblastoma cell lines IMR-32, LA-N-1 and the cell line LS, which has been established in the children's hospital Tuebingen, were exposed to increasing doses of nifurtimox (0.070 mM to 0.348 mM) and incubated for 1, 2 or 3 days. It could be observed that cell viability of all cell lines was significantly and dose-dependently reduced (p<0.01) after nifurtimox treatment. An average reduction of cell viability by 50% was achieved after 24h incubation with 0.348 mM nifurtimox (LS and IMR-32). The assumption that nifurtimox induces the formation of reactive oxygen species could be confirmed. The amount of intracellular reactive oxygen species was significantly increased (p<0.05) in a dose-dependent manner in all cell lines after 24h. Furthermore, expression levels of heat shock proteins Hsp70 and Hsp90 were investigated. Western blot analysis revealed increased intracellular expression levels for both heat shock proteins after 24h nifurtimox treatment. Concluding that Hsp70 and Hsp90 have important roles in tumor cell survival, it was decided to specifically inhibit Hsp90. For this purpose, the neuroblastoma cell lines were treated with the geldanamycin analog 17-Dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG). After inhibition of Hsp90 cells were additionally incubated with the previously used dosages of nifurtimox. A significant higher reduction of the cell viability (p<0.001) could be observed for all neuroblastoma cell lines compared to the application of nifurtimox or 17-DMAG alone.In conclusion, nifurtimox increases oxidative stress in neuroblastoma cell lines leading to significantly decreased cell viability. The specific inhibition of Hsp90 additionally intensifies this effect. The findings suggest that the combined administration of nifurtimox and the specific Hsp90 inhibitor 17-DMAG leads to a synergistic and favorable effect in the treatment of neuroblastoma. More importantly, being an approved medication and well investigated in a wide variety of clinical trials, nifurtimox and 17-DMAG are easy accessible and create a promising new approach not only in neuroblastoma treatment. Disclosures:No relevant conflicts of interest to declare.

Abstract C74: Stabilization of mutant BRCA1 confers PARP inhibitor and platinum resistance.

Abstract The BRCA1 gene is commonly mutated in hereditary breast and ovarian cancers. The BRCA1 protein has multiple domains that mediate protein interactions; BRCA1 gene mutations may produce truncated proteins that lose the ability to interact with associated proteins. Additionally, mutations in the BRCT domain of BRCA1 create protein folding defects that result in protease-mediated degradation. Cells that contain dysfunctional BRCA1 proteins are hypersensitive to DNA damaging agents. In particular, BRCA1-deficient cell lines are exquisitely sensitive to poly(ADP-ribose) polymerase (PARP) inhibitor treatment. Despite initial responses of BRCA1 mutant cancers to PARP inhibitor treatment, acquired resistance develops. To study PARP inhibitor resistance, specifically in the setting of BRCA1 C-terminal truncating mutations, we cultured the triple negative breast cancer cell line MDA-MB-436 (5396+1G&amp;gt;A) in the presence of the PARP inhibitor rucaparib. Drug resistant clones emerged approximately 2-4 months after initial exposure. Clones were highly resistant to rucaparib, and cross-resistant to olaparib, as well as cisplatin. No changes in the mutant BRCA1 DNA or mRNA sequences were detected and the PARP enzyme was efficiently inhibited by rucaparib. The mutant BRCA1 protein was undetectable in MDA-MB-436 parent cells, but was abundant in resistant clones by western blot. HSP90 co-immunoprecipitated with the stabilized mutant BRCA1 protein and treatment of resistant cells with the HSP90 inhibitor 17-DMAG reduced mutant BRCA1 protein levels and restored their sensitivity to PARP inhibition. The stabilized mutant BRCA1 protein also interacted with PALB2-BRCA2-RAD51, was essential for RAD51 focus formation and PARP inhibitor resistance. Resistant cells also acquired a TP53BP1 mutation that facilitated DNA end resection in the absence of a BRCA1 protein capable of binding CtIP. Finally, concomitant increased mutant BRCA1 and decreased 53BP1 protein expression occurs in clinical samples of BRCA1-mutated recurrent ovarian carcinomas that have developed resistance to platinum. These results provide evidence for a novel mechanism by which BRCA1-mutant tumors acquire anti-cancer therapy resistance. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C74. Citation Format: Neil Johnson, Shawn F. Johnson, Yifan Wang, Andrea Bernhardy, Wei Yao, Young-Eun Choi, Marzia Capelletti, Kristopher Sarosiek, Lisa Moreau, Dipanjan Chowdhury, Joyce Liu, Alan D'Andrea, Alexander Miron, Anneka Wickramanayake, Maria Harrell, Elizabeth M. Swisher, Geoffrey I. Shapiro. Stabilization of mutant BRCA1 confers PARP inhibitor and platinum resistance. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C74.

Inhibition of Hsp90 and 70 sensitizes melanoma cells to hyperthermia using ferromagnetic particles with a low Curie temperature

Heat shock protein (Hsp) 90 is a key regulator of various oncogene products and cell-signaling molecules, while Hsp70 protects against heat-induced apoptosis. We previously described a system in which hyperthermia was produced using thermosensitive ferromagnetic particles (FMPs) with a Curie temperature (T c) of 43 °C to mediate automatic temperature control, and demonstrated its antitumor effect in a mouse melanoma model. In the present study, the antitumor effects of combining Hsp90 inhibitor (17DMAG) and Hsp70 inhibitor (quercetin) with FMP-mediated hyperthermia were examined. Expressions of Hsp90/70 and Akt were evaluated using Western blotting in vitro. In an in vivo study, melanoma cells were subcutaneously injected into the backs of C57BL/6 mice. FMPs were then injected into the resultant tumors, and the mice were divided into groups treated with quercetin and/or 17DMAG with/without hyperthermia. When exposed to a magnetic field, the temperature of tissues containing FMPs increased and stabilized at the T c. The TUNEL method was used to determine whether hyperthermia induced apoptosis within tumors. In the group pretreated with hyperthermia + quercetin + 17DMAG, Akt expression was reduced in vitro, the incidence of apoptosis within tumors was greater, and tumor growth was significantly suppressed 20 days after FMP injection in vivo, compared with other treatment groups. The survival rates among tumor-bearing mice observed for a period of 40 days were significantly higher in the hyperthermia + quercetin + 17DMAG group. Combining Hsp90/70 inhibition with hyperthermia appears to increase their antitumor effects. Thus, the combination of FMP-mediated, self-regulating hyperthermia with Hsp90/70 inhibition has important implications for cancer treatment.

The Anticancer Drug AUY922 Generates a Proteomics Fingerprint That Is Highly Conserved among Structurally Diverse Hsp90 Inhibitors

AUY922 is a potent synthetic Hsp90 antagonist that is moving steadily through clinical trials against a small range of cancers. To identify protein markers that might measure the drug's effects, and to gain understanding of mechanisms by which AUY922 might inhibit the proliferation of leukemia cells, we characterized AUY922's impacts on the proteomes of cultured Jurkat cells. We describe a robust and readily assayed proteomics fingerprint that AUY922 shares with the flagship Hsp90 inhibitors 17-DMAG and radicicol. We also extend our proteomics findings, demonstrating that an unrelated antagonist of protein folding potentiates the antiproliferative effects of AUY922. Results provide a set of candidate biomarkers for responses to AUY922 in leukemia cells and suggest new modalities for enhancing AUY922's anticancer activities.

The Hsp90 Inhibitor 17-DMAG Increases SOCS3 and Regulates Cytokine Production, Migration and Cell Death in Chronic Lymphocytic Leukemia

Abstract 1362Epigenetic or transcriptional silencing of important tumor suppressors has been described to contribute to cell survival and tumorigenesis in chronic lymphocytic leukemia (CLL). We investigated gene silencing in CLL using gene expression microarray analysis, and found that over 2000 genes are repressed more than 2-fold in CLL B cells compared to normal B cells, including genes involved in cell signaling and survival (Jun/Fos signaling, ATF family, cyclin dependent kinases and suppressors of cytokine signaling). In particular, the suppressor of cytokine signaling protein 3 (SOCS3) was decreased 60-fold in CLL B cells compared to peripheral blood B cells from normal donors. Despite this profound effect, few therapeutic approaches have focused on reversing this gene silencing in CLL. NF-κB has been shown to transcriptionally silence gene expression in several types of cancer, and our group has previously identified a similar role for this transcription factor in CLL. In addition, we have previously shown that the Hsp90 inhibitor 17-DMAG targets NF-κB signaling to induce apoptosis in CLL through transcriptional down-regulation of oncogenes such as MCL1 and BCL2. However, our microarray analysis revealed that treatment with 17-DMAG also leads to the re-expression of nearly 200 genes silenced in CLL compared to normal B cells. One of the genes significantly silenced in CLL and re-expressed by 17-DMAG is SOCS3. This increase in SOCS3 was evident as early as 8 hours following treatment with 17-DMAG, and peaking at 16–24 hours just prior to 17-DMAG induced cell death (up-regulated 5.6 fold at 8 hours, 59.8 fold at 16 hours, and 25.7 fold at 24 hours). The magnitude of induction in CLL cells was significantly greater than SOCS3 induction in peripheral blood B cells from normal donors, which correlates with a decreased apoptotic response of normal B cells to 17-DMAG (25.7 fold up-regulated in CLL versus 3.5 fold in normal B cells). While little is known about its regulation or functional impact in CLL, SOCS3 has been shown to be methylated in solid tumors as well as myeloid leukemia, leading to aberrant cytokine production and cell survival. While it is known that promoter hypermethylation and subsequent gene silencing contributes to CLL disease progression, we found that there was no significant methylation of the SOCS3 promoter in CLL compared to normal B lymphocytes, indicating an alternative mechanism of SOCS3 silencing in CLL. In order to further characterize the downstream effect of SOCS3 regulation, we investigated the pathways known to be regulated by this protein, specifically IL-6 and CXCR4 signaling. We found that 17-DMAG prevents phosphorylation of STAT3 induced by IL-6 stimulation, which leads to decreased production of pro-survival cytokines including negative feedback by decreasing IL-6 itself. While STAT3 is a known Hsp90 client protein, the effect on phosphorylation of STAT3 was evident before a decrease in the total protein was observed, indicating a distinct effect on the signaling pathway independent of Hsp90’s role as a protein chaperone. SOCS3 has also been shown to prevent phosphorylation of focal adhesion kinase (FAK) and therefore block both integrin and CXCR4 signaling pathways. We found that 17-DMAG prevents constitutive phosphorylation of FAK in primary CLL cells, and subsequently reduces AKT phosphorylation following recombinant SDF-1 stimulation. In order to determine if 17-DMAG inhibits migration of CLL cells towards recombinant SDF-1 as well as the direct signaling through the CXCR4 receptor, we performed transwell migration assays and found that 17-DMAG significantly inhibits migration towards both recombinant SDF-1 and CXCL13 (migration towards CXCL12, 12.4% with Vehicle vs. 8.9% with 17-DMAG, p=0.0061, towards CXCL13, 12.4% with Vehicle vs. 6.1% with 17-DMAG, p<0.0001). Similar results were obtained by over-expression of SOCS3 in a CLL B cell line, suggesting that 17-DMAG inhibits migration through an increase in SOCS3. Based on these results, we suggest that 17-DMAG reverses gene silencing in CLL, and through re-expression SOCS3 inhibits the migration and signaling associated with SDF-1/CXCR4, an important factor in the tumor microenvironment that contributes to CLL cell survival. Therefore Hsp90 inhibitors represent a novel approach to target transcriptional silencing in CLL and other B cell lymphoproliferative disorders. Disclosures:No relevant conflicts of interest to declare.

Serum Metabolomics Uncovers a New Therapeutic Target in Diffuse Large B Cell Lymphoma (DLBCL)

Abstract 1648DLBCL is a molecularly heterogeneous disease usually treated with chemoimmunotherapy ultimately curing ∼65% of pts. In order to improve therapy for these pts, the identification of broadly relevant therapeutic targets is critical. One such target is HSP90. Tumor cells are enriched for a fraction of HSP90 found in higher-order multi-chaperone complexes. Tumor-enriched HSP90 (teHSP90) displays higher affinity for HSP90 inhibitors than normal tissues, which contain latent, uncomplexed HSP90. Many client proteins are depleted upon exposure to teHSP90 inhibitors. PU-H71 is a highly teHSP90 selective inhibitor with an excellent toxicity profile currently being tested for DLBCL in clinical trials. Combination therapies involving teHSP90 inhibitors may synergize with drugs targeting client proteins of teHSP90 by more powerfully inhibit survival pathways. Cell replication involves substantial metabolic demands and PU-H71-induced degradation of enzymatic client proteins may affect critical metabolic pathways. We hypothesized that by identifying PU-H71-induced metabolic changes, metabolomics could point to potential new targets for combinatorial therapeutic intervention.We therefore analyzed the global metabolic consequence of teHSP90 inhibition in DLBCL by PU-H71. The metabolome was analyzed in the serum of LY7 DLBCL xenografted mice treated with 75 mg/m2 of PU-H71 for 24 h (n=5) or vehicle (n=5) by HPLC/MS. Bioinformatic analysis revealed significant changes in 122 metabolites in PUH-71- vs. vehicle-treated mice; including significantly lower levels of xanthine, hypoxanthine, adenosine, xanthosine monophosphate (XMP), depletion of the guanine nucleoside pool, together with higher levels of inosine and inosine monophosphate (IMP). These metabolic changes pointed towards possible PU-H71 mediated inhibition of inosine monophosphate dehydrogenase (IMPDH). IMPDH catalyzes the NAD-dependent oxidation of IMP to XMP, which is the committed step in de novo guanosine nucleotide biosynthesis. This reaction is particularly important to lymphocytes, which depend on IMPDH activity to generate the guanosine nucleotide levels needed to initiate a proliferative response to antigen. Increased IMPDH activity has also been observed in leukemia and lymphoma, mostly as consequence of up-regulation of the IMPDH2 isoform. In order to determine whether IMPDH1/2 stability depends on teHSP90, we treated a panel of 6 DLBCL cell lines (including LY7) with the mean GI50 of PU-H71 (1 μM) for up to 24 h and checked for IMPDH1/2 abundance. We found a time-dependent decrease in IMPDH2 protein levels. Similar results were obtained with the chemically unrelated HSP90 inhibitor 17-DMAG. To confirm that IMPDH2 binds to teHSP90, we took advantage of an affinity-based PUH-71 pull-down method we recently developed. In this assay PU-H71-beads preferentially bind to teHSP90 complexes pecipitating cancer-related client proteins. By using this assay, we determined that IMPDH2 was indeed a teHSP90 client in DLBCL cells. We also found that PU-H71 (and 17-DMAG) inhibited the activity of IMPDH in DLBCL cells by shortening its half-live (IMPDH t1/2from 2 h to 45 min). To determine whether the combination of teHSP90 inhibitors will synergize with IMPDH inhibitors in killing DLBCL, we treated a panel of 10 DLBCL cell lines with the combination of PU-H71 and two IMPDH inhibitors in clinical use, mycophenolic acid (MPA, an uncompetitive inhibitor) and ribavirin (RIB, a competitive inhibitor). We found that most cell lines showed synergistic killing effect when treated with the combination of drugs compared to each drug alone (determined by isobologram method). This prompted us to test the combination in vivo. SCID mice were xenografted with LY7 and SUDHL6 and once tumor developed, they were treated with vehicle, PU-H71, mycophenolate mofetil (MMF, the pro-drug of MPA), RIB and the combination of PU-H71 and MMF or RIB. We found that mice treated with the combination of drugs exhibited greater effect that each drug alone (p=0.002 for SU-DHL6 and p>0.001 for LY7 for PU-H71+MMF, and p=0.01 for SU-DHL6 and p=0.02 for LY7 for PU-H71+RIB, all T-test day 10). There were no toxic effects.In sum, our work uses serum metabolomics to provide new insights into the pharmacological targets of a particular HSP90 inhibitor, and unveiled a critical survival pathway in DLBCL that was harnessed to develop a rationally combined targeted therapy. Disclosures:No relevant conflicts of interest to declare.

Correction: Combination Therapy with HSP90 Inhibitor 17-DMAG Reconditions the Tumor Microenvironment to Improve Recruitment of Therapeutic T Cells

Correction: Combination Therapy with HSP90 Inhibitor 17-DMAG Reconditions the Tumor Microenvironment to Improve Recruitment of Therapeutic T Cells

Combination Therapy with HSP90 Inhibitor 17-DMAG Reconditions the Tumor Microenvironment to Improve Recruitment of Therapeutic T cells

Ineffective recognition of tumor cells by CD8+ T cells is a limitation of cancer immunotherapy. Therefore, treatment regimens that coordinately promote enhanced antitumor CD8+ T-cell activation, delivery, and target cell recognition should yield greater clinical benefit. Using an MCA205 sarcoma model, we show that in vitro treatment of tumor cells with the HSP90 inhibitor 17-DMAG results in the transient (proteasome-dependent) degradation of the HSP90 client protein EphA2 and the subsequent increased recognition of tumor cells by Type-1 anti-EphA2 CD8+ T cells. In vivo administration of 17-DMAG to tumor-bearing mice led to slowed tumor growth, enhanced/prolonged recognition of tumor cells by anti-EphA2 CD8+ T cells, reduced levels of myeloid-derived suppressor cells and regulatory T cells in the tumor microenvironment, and activation of tumor-associated vascular endothelial cells in association with elevated levels of Type-1 tumor-infiltrating lymphocytes. When combined with EphA2-specific active vaccination or the adoptive transfer of EphA2-specific CD8+ T cells, 17-DMAG cotreatment yielded a superior tumor therapeutic regimen that was capable of rendering animals free of disease. Taken together, our findings indicate that 17-DMAG functions as an immune adjuvant in the context of vaccines targeting EphA2.

FRMD4A Upregulation in Human Squamous Cell Carcinoma Promotes Tumor Growth and Metastasis and Is Associated with Poor Prognosis

New therapeutic strategies are needed to improve treatment of head and neck squamous cell carcinoma (HNSCC), an aggressive tumor with poor survival rates. FRMD4A is a human epidermal stem cell marker implicated previously in epithelial polarity that is upregulated in SCC cells. Here, we report that FRMD4A upregulation occurs in primary human HNSCCs where high expression levels correlate with increased risks of relapse. FRMD4A silencing decreased growth and metastasis of human SCC xenografts in skin and tongue, reduced SCC proliferation and intercellular adhesion, and stimulated caspase-3 activity and expression of terminal differentiation markers. Notably, FRMD4A attenuation caused nuclear accumulation of YAP, suggesting a potential role for FRMD4A in Hippo signaling. Treatment with the HSP90 inhibitor 17-DMAG or ligation of CD44 with hyaluronan caused nuclear depletion of FRMD4A, nuclear accumulation of YAP and reduced SCC growth and metastasis. Together, our findings suggest FRMD4A as a novel candidate therapeutic target in HNSCC based on the key role in metastatic growth we have identified.

AKT-aro and HER2-aro, models for de novo resistance to aromatase inhibitors; molecular characterization and inhibitor response studies

Aromatase inhibitors (AI) are currently the first line therapy for estrogen receptor (ER)-positive postmenopausal women. De novo AI resistance is when a patient intrinsically does not respond to an AI therapy as well as other targeted endocrine therapy. To characterize this type of resistance and to examine potential therapies for treatment, we have generated two cell models for de novo resistance. These models derive from MCF-7 cells that stably overexpress aromatase and Akt (AKT-aro) or HER2 (HER2-aro). Evaluation of these cell lines revealed that the activities of aromatase and ER were inhibited by AI and ICI 187280 (ICI) treatment, respectively; however, cell growth was resistant to therapy. Proliferation in the presence of the pure anti-estrogen ICI, indicates that these cells do not require ER for cell growth and distinguishes these cells from the acquired AI resistant cells. We further determined that the HSP90 inhibitor 17-DMAG suppressed the growth of the AI-resistant cell lines studied. Our analysis revealed 17-DMAG-mediated decreased expression of growth promoting signaling proteins. It was found that de novo AI resistant AKT-aro and HER2-aro cells could not be resensitized to letrozole or ICI by treatment with 17-DMAG. In summary, we have generated two cell lines which display the characteristics of de novo AI resistance. Together, these data indicate the possibility that HSP90 inhibitors may be a viable therapy for endocrine therapy resistance although additional clinical evaluation is needed.

Effect of inhibition of the Ubiquitin-Proteasome System and Hsp90 on growth and survival of Rhabdomyosarcoma cells in vitro

BackgroundThe ubiquitin-proteasome system (UPS) and the heat shock response (HSR) are two critical regulators of cell homeostasis, as their inhibition affects growth and survival of normal cells, as well as stress response and invasiveness of cancer cells. We evaluated the effects of the proteasome inhibitor Bortezomib and of 17-DMAG, a competitive inhibitor of Hsp90, in rhabdomyosarcoma (RMS) cells, and analyzed the efficacy of single-agent exposures with combination treatments.MethodsTo assess cytotoxicity induced by Bortezomib and 17-DMAG in RMS cells, viability was measured by MTT assay after 24, 48 and 72 hours. Western blotting and immunofluorescence analyses were carried out to elucidate the mechanisms of action. Apoptosis was measured by FACS with Annexin-V-FITC and Propidium Iodide.ResultsBortezomib and 17-DMAG, when combined at single low-toxic concentrations, enhanced growth inhibition of RMS cells, with signs of autophagy that included intensive cytoplasmic vacuolization and conversion of cytosolic LC3-I protein to its autophagosome-associated form. Treatment with lysosomal inhibitor chloroquine facilitates apoptosis, whereas stimulation of autophagy by rapamycin prevents LC3-I conversion and cell death, suggesting that autophagy is a resistance mechanism in RMS cells exposed to proteotoxic drugs. However, combination treatment also causes caspase-dependent apoptosis, PARP cleavage and Annexin V staining, as simultaneous inhibition of both UPS and HSR systems limits cytoprotective autophagy, exacerbating stress resulting from accumulation of misfolded proteins.ConclusionThe combination of proteasome inhibitor Bortezomib with Hsp90 inhibitor 17-DMAG, appears to have important therapeutic advantages in the treatment of RMS cells compared with single-agent exposure, because compensatory survival mechanisms that occur as side effects of treatment may be prevented.