Degradation Of Client Proteins Research Articles

Abstract 1619: Hsp90 inhibitor drug conjugates (HDCs): Construct design and preliminary evaluation

Abstract Background: Most cytotoxic agents are often broadly active but non-selective, and have the disadvantage of high toxicity due to collateral damage to normal tissues. Drugs that target specific protein drivers of cancer cell growth are more tumor selective, yet often lead to tumor resistance via point mutations in their target or activation of alternative signaling pathways. Targeted delivery strategies, such as Antibody-Drug Conjugates (ADCs), offer a solution to these limitations by delivering potent anti-cancer payloads more directly to tumors. Hsp90 is a chaperone protein required by many cancer cells to maintain the stability and function of numerous proteins that drive cancer cell growth, survival, and metastasis. Small molecule inhibitors of Hsp90 such as ganetespib, PU-H71 and 17-AAG are found to be retained in tumors with half- time up to 65 hours in mouse xenografts. These properties are believed to be due to overexpression of an active form of Hsp90 in cancer cells as compared to normal tissues, and have recently been applied for tumor imaging in patients. Results: We have developed a small molecule drug conjugate platform technology using the unique properties of Hsp90 proteins and Hsp90 inhibitors. We have synthesized HDCs with various Hsp90 inhibitor scaffolds including resorcinol, purine, geldanamycin, etc. A cell-based client protein degradation assay is carried out to confirm the intracellular uptake and the Hsp90 binding of the conjugates. While different Hsp90 inhibitor scaffolds offer different DMPK and toxicology profiles, cellular uptake and Hsp90 binding are not greatly affected by the different chemical classes of Hsp90 inhibitors. Over 30 payloads have been conjugated with Hsp90 inhibitors so far. We have selected the payloads based on the hypothesis that HDC can improve the safety profile of a cytotoxic drug, expand the application of chemotherapeutic agents to different tumor types, combat drug resistance, and enable novel anticancer approach. Examples of payloads include topoisomerase inhibitors (camptothecin), microtubule modulators (taxanes), proteasome inhibitors (carfilzomib), CDK inhibitors (flavopiridol) and others. Unlike most other conjugate technologies, HDC does not require lengthy spacing between the anchor Hsp90 inhibitor and the payload. Linker cleavage mechanism is considered a key feature in the HDC design. We have been able to incorporate several linkers such as disulfide, hydrazone, peptide, carbamate, carboxylate, etc. into our HDC designs. Conclusion: In this HDC platform, we provide a method which can be applied to many well-studied mechanisms for modulating cancer pathways and stopping tumor cell growth. We have created a promising platform technology which can result in many anticancer agents in the near future. Citation Format: Weiwen Ying, Dinesh Chimmanamada, Junyi Zhang, Teresa Przewloka, Jun Jiang, Genliang Lu, Sami Osman, James Loch, Dharma Vutukuri, Shoujun Chen, Robert Stein, John Chu, David Proia, Pat Rao, Takayo Inoue, Luisa Shin Ogawa, Ritu Singh, Noriaki Tatsuta. Hsp90 inhibitor drug conjugates (HDCs): Construct design and preliminary evaluation. [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 1619. doi:10.1158/1538-7445.AM2014-1619

Abstract 2509: Hsp90-inhibitor drug conjugates (HDC): Novel tumor-selective drug delivery platform with unprecedented anticancer activity

Abstract Background: Various strategies have been used to make chemotherapeutic compounds more tumor selective in order to increase their safety and efficacy, but they were met with only limited success. Antibody Drug Conjugates (ADCs) and folate receptor mediated drug delivery (FRDC) are the emerging concepts in the drug delivery field, but they are limited to high potent toxins and narrow sets of indications. We established a novel tumor-directed drug delivery platform technology based on the unique property of Hsp90 inhibitors. One prominent feature of Hsp90 inhibitors is their ability to be retained selectively in tumor cells for a prolonged period of time. The difference in residency time in tumor against plasma and normal tissues is 10-25 fold. Moreover, the chaperone protein Hsp90 is overexpressed 2-3 fold in tumor compared to normal tissues. Combining the overexpression Hsp90 and longer residency of its inhibitors in tumor, a strategy to link anticancer drugs to an Hsp90 inhibitor, where the attached “payload” is released in the tumor selectively for prolonged periods, was conceived. Results: Though the HDC concept is applicable to most small molecule anticancer drugs, we chose a common topoisomerase-1 inhibitor (SN-38) for proof-of-concept studies. More than 100 Hsp90 inhibitor/SN38 conjugates were synthesized; we chose STA-12-8666 as our lead molecule based on its in vitro and in vivo pharmacokinetic properties. The conjugate binds strongly to Hsp90 as determined by client protein degradation assays and has good stability in plasma of several species (human>rat>mouse). In xenograft tumor tissue distribution studies, we demonstrate that mice dosed once weekly with 50 mg/kg STA-12-8666 clear the parental HDC and its cleavage products, SN-38 and the Hsp90 inhibitor, from plasma and normal tissue within 24 hr. However, we detected 0.6 µM and 0.3 µM SN-38 in tumor at 24h and 48h, respectively. SN-38 was not detected in tumor at 24h or 48h with an equivalent dose of the SN-38 prodrug, irinotecan. Xenograft studies using multiple cell lines demonstrated superior efficacy for STA-12-8666 compared to irinotecan. For example, in MCF-7 breast cancer xenografts, STA-12-8666 produced 70% regression in tumor volume compared to moderate tumor growth inhibition by irinotecan with no regression. Similar results were observed in in colon, SCLC, ovarian, bladder, NSCLC, melanoma models. Preliminary toxicological evaluation of STA-12-8666 resulted in better or comparable toxicities to that of irinotecan. Conclusion: We designed a novel tumor-directed drug delivery platform by conjugating previously validated small molecule chemotherapeutics to HSP90 inhibitors. Our lead candidate STA-12-8666, has great tissue distribution profile, superior in vivo antitumor activity and better safety profile than the active control irinotecan, warranting its clinical evaluation. Citation Format: Dinesh Chimmanamada, David Proia, Takayo Inoue, Luisa Shin Ogawa, Suqin He, Manuel Sequeira, Donald Smith, John-Paul Jimenez, Chaohua Zhang, Jim Sang, Jaime Acquaviva, Masazumi Nagai, Yuan Liu, Josephine Ye, Dan Zhou, Andrew Sonderfan, Ritu Singh, Noriaki Tatsuta, Teresa Przewloka, Jun Jiang, Junyi Zhang, Weiwen Ying. Hsp90-inhibitor drug conjugates (HDC): Novel tumor-selective drug delivery platform with unprecedented anticancer activity. [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 2509. doi:10.1158/1538-7445.AM2014-2509

Antiproliferative effect of HSP90 inhibitor Y306zh against pancreatic cancer is mediated by interruption of AKT and MAPK signaling pathways.

Inhibition of HSP90 is a potential strategy to treat pancreatic cancer (PC), since many client proteins of HSP90 are found to be over-expressed and/or genetically mutated in PC. These client proteins directly participate in the modulation of proliferation and aggravation of PC. Our previous works led to Y306zh as a novel and potent HSP90 inhibitor. In this study we further investigated the mode of actions of Y306zh as an anti-PC agent. The binding affinity of Y306zh towards HSP90 was analyzed by fluorescence polarization assay. The anti-proliferative activities and molecular mechanism of Y306zh were investigated in human PC cell lines and Miapaca2 xenograft model. Y306zh binds tightly to the NH2-terminus of HSP90 with an IC50 of 85 nM, and this causes ATP incapable to attach to the same binding site, and disrupts HSP90-p23 association. Y306zh also induces the expression of HSP70, recruits the complex of CHIP, HSP70 and HSP90, and eventually results in the degradation of HSP90 client proteins (EGFR, AKT, C-RAF and CDK4) via proteasomal pathway. Amazingly, coincident with the molecular mechanism, Y306zh also inhibits the proliferation of PC cells through G2/M phase arrest, but appears to be totally insensitive to normal mammalian cells. Furthermore, Y306zh effectively inhibits tumor growth of Mia-paca2 xenograft mice without any obvious effect on body weight. Y306zh, a novel HSP90 inhibitor, interrupts ATP binding to HSP90 and disrupts HSP90-p23 interaction, and eventually inhibits the growth of PC cells.

201P - Exploratory Analysis of Circulating Free Dna to Identify Biomarkers Predictive of Outcome in Galaxy-1, a Large Randomized Phase Iib Study of Ganetespib in Combination with Docetaxel Versus Docetaxel Alone in Advanced Nsclc (Nct01348126)

ABSTRACT Aim: NSCLC is comprised of several distinct molecular subtypes driven by somatic oncogenic mutations, and several of these (eg, EML4-ALK, BRAF, KRAS, KIT) confer dependence on Hsp90. Inhibition of Hsp90 leads to client protein degradation and tumor apoptosis. In the clinic, the Hsp90 inhibitor ganetespib (G) has single agent activity in various molecularly defined cancers (eg, EML4-ALK, mut KRAS, HER2 +, mut BRAF). Circulating free DNA (cfDNA), present at low levels in plasma, allows detection of somatic mutations by deep sequencing. The aim of this work was to determine the mutational spectrum in cfDNA from patients (pts) enrolled in the GALAXY-1 trial to uncover plasma-borne somatic mutations predictive of clinical outcome to G-based therapy. Methods: Ultra-deep sequencing was performed using plasma samples isolated from whole blood of adenocarcinoma pts enrolled in GALAXY-1. CfDNA was isolated from 1ml of plasma collected at baseline using Qiagen kits and quantified by AQ real-time PCR. As only 3 of 105 samples (3%) met the desired cut-off of 1.7ng/µl cfDNA (10ng template) for direct sequencing, samples were concentrated by SpeedVac®; 36 samples (34%) met the requirement of 10ng template DNA, while the remaining samples had Results: cfDNA targeted sequence analysis of the first 105 pts identified somatic mutations in several genes commonly mutated in NSCLC (eg, EGFR, PIK3CA, MET), as well as germline mutations and single nucleotide variants (SNVs) in genes not commonly seen in NSCLC (eg, KIT). Preliminary results suggest that PTEN and KIT mutations may be predictive of response to G + D. Analysis of cfDNA from remaining samples, including paired samples collected during cycle 1 and at end-of-treatment, is ongoing. Conclusions: Ultra-deep re-sequencing of somatic mutations in circulating cfDNA represents a new approach to exploring tumor heterogeneity and biomarkers of treatment outcome. The early analysis of cfDNA in GALAXY-1 identified G sensitive subgroups. Final results will be presented at the meeting. Disclosure: D. Fennell: Advisory board; I. El-Hariry: I am an employee of Synta Pharmaceuticals. All other authors have declared no conflicts of interest.

Preclinical antitumor activity of SST0116CL1: a novel heat shock protein 90 inhibitor.

4-Amino substituted resorcino-isoxazole (SST0116CL1) (property of Sigma-Tau Research Switzerland S.A.) is a potent, second generation, small-molecule heat shock protein 90 inhibitor (Hsp90i). SST0116CL1 binds to the ATP binding pocket of Hsp90, and interferes with Hsp90 chaperone function thus resulting in client protein degradation and tumor growth inhibition. The aim of the study was to assess SST0116CL1 in various solid and haematological tumors. The antitumor properties of SST0116CL1 were assessed using in vitro cell proliferation and client protein degradation assays and in vivo different tumor xenograft models. Pharmacokinetic (PK) data were also generated in tumor-bearing mice to gain an understanding of optimal dosing schedules and regimens. SST0116CL1 was shown to inhibit recombinant Hsp90α and to induce the destabilization of different client proteins, often overexpressed and constitutively activated in different types of hematological or solid human tumors. In preclinical in vivo studies, it was revealed to induce antitumor effects in murine models of leukemia and of gastric and ovarian carcinoma. A modulation of PD biomarkers in terms of downregulation of Hsp90 client proteins in tumor-bearing mice was found. SST0116CL1 is a new clinical candidate for cancer therapy. The antitumor property of SST0116CL1, likely due to direct inhibition of the Hsp90 enzymatic activity, may prove to be a critical attribute as the compound enters phase I clinical trials.

89Zr-trastuzumab and 89Zr-bevacizumab PET to evaluate the effect of the HSP90 inhibitor NVP-AUY922 in metastatic breast cancer patients.

HSP90 chaperones have key client proteins that are involved in all hallmarks of breast cancer growth and progression. The primary aim of this clinical trial was to evaluate the feasibility of using (89)Zr-trastuzumab PET (for HER2-positive breast cancer) or (89)Zr-bevacizumab PET [for estrogen receptor (ER)-positive breast cancer] to determine in vivo degradation of client proteins caused by the novel HSP90 inhibitor NVP-AUY922. Of note, 70 mg/m(2) NVP-AUY922 was administered intravenously in a weekly schedule to patients with advanced HER2 or ER-positive breast cancer. Biomarker analysis consisted of serial PET imaging with 2[18F]fluoro-2-deoxy-D-glucose (FDG), (89)Zr-trastuzumab, or (89)Zr-bevacizumab. Response evaluation was performed according to RECIST1.0. FDG, (89)Zr-trastuzumab, and (89)Zr-bevacizumab distributions were scored visually and quantitatively by calculating the maximum standardized uptake values (SUVmax). In blood samples, serial HSP70 levels, extracellular form of HER2 (HER2-ECD), and pharmacokinetic and pharmacodynamic parameters were measured. Sixteen patients (ten HER2-positive and six ER-positive tumors) were included. One partial response was observed; seven patients showed stable disease. SUVmax change in individual tumor lesions on baseline versus 3 weeks (89)Zr-trastuzumab PET was heterogeneous and related to size change on CT after 8 weeks treatment (r(2) = 0.69; P = 0.006). Tumor response on (89)Zr-bevacizumab PET and FDG-PET was not correlated with CT response. NVP-AUY922 showed proof-of-concept clinical response in HER2-amplified metastatic breast cancer. Early change on (89)Zr-trastuzumab PET was positively associated with change in size of individual lesions assessed by CT.

FW-04-806 inhibits proliferation and induces apoptosis in human breast cancer cells by binding to N-terminus of Hsp90 and disrupting Hsp90-Cdc37 complex formation.

BackgroundHeat shock protein 90 (Hsp90) is a promising therapeutic target and inhibition of Hsp90 will presumably result in suppression of multiple signaling pathways. FW-04-806, a bis-oxazolyl macrolide compound extracted from China-native Streptomyces FIM-04-806, was reported to be identical in structure to the polyketide Conglobatin.MethodsWe adopted the methods of chemproteomics, computational docking, immunoprecipitation, siRNA gene knock down, Quantitative Real-time PCR and xenograft models on the research of FW-04-806 antitumor mechanism, through the HER2-overexpressing breast cancer SKBR3 and HER2-underexpressing breast cancer MCF-7 cell line.ResultsWe have verified the direct binding of FW-04-806 to the N-terminal domain of Hsp90 and found that FW-04-806 inhibits Hsp90/cell division cycle protein 37 (Cdc37) chaperone/co-chaperone interactions, but does not affect ATP-binding capability of Hsp90, thereby leading to the degradation of multiple Hsp90 client proteins via the proteasome pathway. In breast cancer cell lines, FW-04-806 inhibits cell proliferation, caused G2/M cell cycle arrest, induced apoptosis, and downregulated Hsp90 client proteins HER2, Akt, Raf-1 and their phosphorylated forms (p-HER2, p-Akt) in a dose and time-dependent manner. Importantly, FW-04-806 displays a better anti-tumor effect in HER2-overexpressed SKBR3 tumor xenograft model than in HER2-underexpressed MCF-7 model. The result is consistent with cell proliferation assay and in vitro apoptosis assay applied for SKBR-3 and MCF-7. Furthermore, FW-04-806 has a favorable toxicity profile.ConclusionsAs a novel Hsp90 inhibitor, FW-04-806 binds to the N-terminal of Hsp90 and inhibits Hsp90/Cdc37 interaction, resulting in the disassociation of Hsp90/Cdc37/client complexes and the degradation of Hsp90 client proteins. FW-04-806 displays promising antitumor activity against breast cancer cells both in vitro and in vivo, especially for HER2-overexpressed breast cancer cells.

Novologues containing a benzamide side chain manifest anti-proliferative activity against two breast cancer cell lines

Hsp90 represents a promising target for the development of both anti-cancer and neuroprotective agents. Structure–activity relationship studies on novobiocin and novobiocin analogues, led to the development of KU-32 and recently, KU-596, as lead compounds for the potential treatment of neurodegenerative diseases. Similar to KU-32, we have demonstrated that upon replacement of the acetamide side chain present in KU-32 with a benzamide, this neuroprotective agent was transformed into a scaffold that manifests anti-proliferative activity. To assess structure–activity relationships for this new scaffold, a library of benzamide-containing novologues was prepared and evaluated against two breast cancer cell lines. Compound 14a manifested the most potent anti-proliferative activity from these studies and induced Hsp90-dependent client protein degradation in a concentration-dependent manner.

Celastrol Induces Apoptosis in Gefitinib-Resistant Non-Small Cell Lung Cancer Cells via Caspases-Dependent Pathways and Hsp90 Client Protein Degradation

Celastrol, a triterpene extracted from the Chinese herb Tripterygium wilfordii, has been shown to have multiple bioactivities. Although among these activities, its anti-cancer effects have attracted the most attention, the effect of celastrol on gefitinib-resistant non-small cell lung cancer (NSCLC) cells is not clearly known. Here, we examined the potency of celastrol in three different NSCLC cell lines. We explored its treatment mechanism in two gefitinib-resistant NSCLC cell lines (H1650 and H1975). Our data demonstrated that celastrol exerted its apoptotic effect in a dose- and time-dependent manner. Also, the mitochondria membrane potential was gradually lost and the ratio of Bax/Bcl-2 increased after the treatment of celastrol, both of which are indicators of mitochondria membrane integrity. Although the caspases were activated, the treatment with pan-caspase inhibitor could partially inhibit the level of apoptosis. Moreover, the protein level of Hsp90 client proteins, EGFR and AKT, was measured. Interestingly, both client proteins were remarkably down-regulated after the treatment of celastrol. Taken together, our data showed that celastrol may be developed as a promising agent for treating gefitinib-resistant NSCLCs by inducing apoptosis through caspase-dependent pathways and Hsp90 client protein degradation.

Cruentaren A Binds F1F0 ATP Synthase To Modulate the Hsp90 Protein Folding Machinery

The molecular chaperone Hsp90 requires the assistance of immunophilins, co-chaperones, and partner proteins for the conformational maturation of client proteins. Hsp90 inhibition represents a promising anticancer strategy due to the dependence of numerous oncogenic signaling pathways upon Hsp90 function. Historically, small molecules have been designed to inhibit ATPase activity at the Hsp90 N-terminus; however, these molecules also induce the pro-survival heat shock response (HSR). Therefore, inhibitors that exhibit alternative mechanisms of action that do not elicit the HSR are actively sought. Small molecules that disrupt Hsp90-co-chaperone interactions can destabilize the Hsp90 complex without induction of the HSR, which leads to inhibition of cell proliferation. In this article, selective inhibition of F1F0 ATP synthase by cruentaren A was shown to disrupt the Hsp90-F1F0 ATP synthase interaction and result in client protein degradation without induction of the HSR.

Abstract IA36: Treatment strategies to overcome EGFR TKI resistance.

Abstract IA36: Treatment strategies to overcome EGFR TKI resistance.

WTAP is a novel oncogenic protein in acute myeloid leukemia

WTAP is a novel oncogenic protein in acute myeloid leukemia

Triazole Containing Novobiocin and Biphenyl Amides as Hsp90 C-Terminal Inhibitors.

Hsp90 C-terminal inhibitors are advantageous for the development of new cancer chemotherapeutics due to their ability to segregate client protein degradation from induction of the prosurvival heat shock response, which is a major detriment associated with Hsp90 N-terminal inhibitors under clinical investigation. Based upon prior SAR trends, a 1,2,3-triazole side chain was placed in lieu of the aryl side chain and attached to both the coumarin and biphenyl scaffold. Antiproliferative studies against SKBr3 and MCF-7 breast cancer cell lines demonstrated these triazole-containing compounds to exhibit improved activity. These compounds were shown to manifest Hsp90 inhibitory activity through Western blot analysis and represent a new scaffold upon which more potent inhibitors can be pursued.

Sulphoxythiocarbamates modify cysteine residues in HSP90 causing degradation of client proteins and inhibition of cancer cell proliferation

Background:Heat shock protein 90 (HSP90) has a key role in the maintenance of the cellular proteostasis. However, HSP90 is also involved in stabilisation of oncogenic client proteins and facilitates oncogene addiction and cancer cell survival. The development of HSP90 inhibitors for cancer treatment is an area of growing interest as such agents can affect multiple pathways that are linked to all hallmarks of cancer. This study aimed to test the hypothesis that targeting cysteine residues of HSP90 will lead to degradation of client proteins and inhibition of cancer cell proliferation.Methods:Combining chemical synthesis, biological evaluation, and structure–activity relationship analysis, we identified a new class of HSP90 inhibitors. Click chemistry and protease-mass spectrometry established the sites of modification of the chaperone.Results:The mildly electrophilic sulphoxythiocarbamate alkyne (STCA) selectively targets cysteine residues of HSP90, forming stable thiocarbamate adducts. Without interfering with the ATP-binding ability of the chaperone, STCA destabilises the client proteins RAF1, HER2, CDK1, CHK1, and mutant p53, and decreases proliferation of breast cancer cells. Addition of a phenyl or a tert-butyl group in tandem with the benzyl substituent at nitrogen increased the potency. A new compound, S-4, was identified as the most robust HSP90 inhibitor within a series of 19 derivatives.Conclusion:By virtue of their cysteine reactivity, sulphoxythiocarbamates target HSP90, causing destabilisation of its client oncoproteins and inhibiting cell proliferation.

Discovery of hybrid Hsp90 inhibitors and their anti-neoplastic effects against gefitinib-resistant non-small cell lung cancer (NSCLC)

Heat shock protein 90 (Hsp90) represents an attractive cancer therapeutic target due to its role in the stabilization and maturation of many oncogenic proteins. We have designed a series of hybrid Hsp90 inhibitors by connecting the resorcinol ring of VER-49009 (2) and the trimethoxyphenyl ring of PU3 (3) using structure-based approach. Subsequent testing established that compound 1f inhibited gefitinib-resistant H1975 cell proliferation, brought about the degradation of Hsp90 client proteins including EGFR, Met, Her2 and Akt and induced the expression of Hsp70. The design, synthesis, and evaluation of 1f are described herein.

Ganetespib blocks HIF-1 activity and inhibits tumor growth, vascularization, stem cell maintenance, invasion, and metastasis in orthotopic mouse models of triple-negative breast cancer

Targeted therapy against triple-negative breast cancers, which lack expression of the estrogen, progesterone, and HER2 receptors, is not available and the overall response to cytotoxic chemotherapy is poor. One of the molecular hallmarks of triple-negative breast cancers is increased expression of genes that are transcriptionally activated by hypoxia-inducible factors (HIFs), which are implicated in many critical aspects of cancer progression including metabolism, angiogenesis, invasion, metastasis, and stem cell maintenance. Ganetespib is a second-generation inhibitor of heat shock protein 90 (HSP90), a molecular chaperone that is essential for the stability and function of multiple client proteins in cancer cells including HIF-1α. In this study, human MDA-MB-231 and MDA-MB-435 triple-negative breast cancer cells were injected into the mammary fat pad of immunodeficient mice that received weekly intravenous injections of ganetespib or vehicle following the development of palpable tumors. Ganetespib treatment markedly impaired primary tumor growth and vascularization, and eliminated local tissue invasion and distant metastasis to regional lymph nodes and lungs. Ganetespib treatment also significantly reduced the number of Aldefluor-positive cancer stem cells in the primary tumor. Primary tumors of ganetespib-treated mice had significantly reduced levels of HIF-1α (but not HIF-2α) protein and of HIF-1 target gene mRNAs encoding proteins that play key roles in angiogenesis, metabolism, invasion, and metastasis, thereby providing a molecular basis for observed effects of the drug on the growth and metastasis of triple-negative breast cancer. Triple-negative breast cancers (TNBCs) respond poorly to available chemotherapy. TNBCs overexpress genes regulated by hypoxia-inducible factors (HIFs). Ganetespib induces degradation of HSP90 client proteins, including HIF-1α. Ganetespib inhibited TNBC orthotopic tumor growth, invasion, and metastasis. Ganetespib inhibited expression of HIF-1 target genes involved in TNBC progression.

Butein Disrupts Hsp90's Molecular Chaperoning Function and Exhibits Anti-proliferative Effects Against Drug-resistant Cancer Cells

Hsp90 shows great promise as a therapeutic target due to its potential to disable multiple signaling pathways simultaneously. In this study, we discovered that a natural product, butein moderately inhibited the growth of drug-resistant cancer cells (A2780cis and H1975), and brought about the degradation of oncogenic Hsp90 client proteins. The study demonstrated that butein would be a therapeutic lead to circumvent drug-resistance in cancer chemotherapy. The structure-based screening, synthesis, and biological evaluation of butein are described herein.

Antitumor Activities Of An Oral Selective HSP90α/β Inhibitor, TAS-116, In Combination With Bortezomib In Multiple Myeloma

Heat-shock protein 90 (HSP90) is a molecular chaperone considered to be attractive therapeutic target in many cancers, including multiple myeloma (MM), since HSP90 inhibition triggers downregulation /degradation of numerous client proteins to trigger apoptosis. In this study, we examined the anti-MM activities of a novel and orally bioavailable HSP90α/β inhibitor TAS-116 (TAIHO PHARMACEUTICAL CO., LTD., Japan) in MM. TAS-116 triggered downregulation of known client proteins associated with significant growth inhibition in MM cell lines and patient MM, without cytotoxicity in normal peripheral blood mononuclear cells. Importantly, TAS-116 showed cytotoxicity even in 17-allylamino-17-demethoxygeldanamycin (17-AAG)-resistant acute B cell leukemia cell line NALM6. TAS-116 induced cell cycle arrest, associated with downregulation of cell cycle regulatory proteins; followed by apoptosis, evidenced by increased annexin V-positive staining, as well as cleavage of caspases and PARP. Since the bone marrow (BM) microenvironment plays a crucial role in MM pathogenesis including drug resistance, we further examined the effect of TAS-116 in the presence of BM stromal cells (BMSCs). TAS-116 induced significant cytotoxicity in MM cells even in the presence of BMSCs, associated with downregulation of phospho-Akt and phospho-ERK. In addition, neither exogenous IL-6 nor IGF-1 was able to protect against TAS-116. Importantly, TAS-116 did not induce cytotoxicity in BMSCs. Our previous studies have shown that combined HSP90 inhibitor and proteasome inhibitor treatment induces synergistic MM cell death; therefore we similarly examined whether TAS-116 enhances bortezomib-induced cytotoxicity. TAS-116 enhanced bortezomib-induced cytotoxicity in MM.1S and RPMI-8226 cells, associated with decreased phospho-Akt, phospho-RelA (p65), phospho-IkBa, and phospho-IKKα/β; as well as increased CHOP and PARP cleavage. These results suggest that TAS-116 blocks bortezomib-induced canonical NF-κB pathway and augments bortezomib-induced endoplasmic reticulum (ER) stress. Finally, we examined the in vivo efficacy of TAS-116 in combination with bortezomib using a xenograft murine model of human MM. Although both monotherapy of TAS-116 and bortezomib were effective, TAS-116 in combination with bortezomib significantly enhanced growth inhibition (80%, p<0.05) and prolonged overall survival (29 vs 57 days, p<0.001). Immunohistochemical analysis of harvested human MM confirmed a significant increase in cleaved caspase-3- and TUNEL-positive cells in TAS-116 animals. These treatments were well tolerated, and no significant body weight loss of the animals was observed. Taken together, our studies show that the novel and selective HSP90α/β inhibitor TAS-116 blocks MM cell growth both in vitro and in vivo, providing the preclinical framework for clinical evaluation of this agent to improve patient outcome in MM. Disclosures: Hideshima: Acetylon Pharmaceuticals: Consultancy. Ohkubo:TAIHO PHARMACEUTICAL Co.,LTD: Employment. Richardson:Millenium: Consultancy; Celgene: Consultancy; Johnson & Johnson: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Munshi:Celgene: Consultancy; Novartis: Consultancy; Millennium: Consultancy. Utsugi:TAIHO PHARMACEUTICAL CO.,LTD: Employment. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.

Abstract C01: Systematic blueprinting of genetic vulnerabilities using ultra-complex shRNA libraries identifies genes synergizing with proteasome inhibition or blockade of IRE1 in multiple myeloma cells

Abstract Multiple myeloma (MM) remains a devastating incurable disease. Because of their secretory nature, MM cells rely on proper endoplasmic reticulum (ER) function. The unfolded protein response (UPR) oversees the folding capacity of the ER and adjusts it according to need. Paraprotein production by MM cells imposes an ER burden that provides an opportunity for therapeutic intervention. High ER function exceeds the buffering capacity of the cell for protein degradation, rendering MM cells exquisitely sensitive to proteasome inhibitors. While successful, standard-of-care proteasome inhibitors, like bortezomib, remain palliative, as patients ultimately become refractory, suggesting the selection of genetic escape routes. ER and proteasome functions are intimately connected. Proteasomal degradation of unfolded ER client proteins is accomplished after dislocation and ubiquitination of unfolded proteins from the ER. Therefore, genetic escape routes can exist within the ubiquitin-proteasome system and the UPR. The most conserved branch of the UPR, overseen by the sensor kinase IRE1 and its downstream effector, XBP1, has been a primary focus of the MM research community in recent years. In healthy cells IRE1/XBP1 allow adaptation to rises in ER function, while in MM cells IRE1/XBP1 are thought to confer a survival advantage. For these reasons, we hypothesized that modulation of the UPR, alone or in combination with proteasome inhibitors, while therapeutically beneficial, might also provide the driving force for acquired drug resistance. Therefore, a single agent is unlikely to lead to long-term remission. Here we show that the therapeutic benefit of blocking the proteasome or IRE1 in MM cells can be enhanced when combined with additional therapies targeting synthetic-lethal interactions, and that these can be systematically identified using an shRNA screening method developed by our groups. Employing an ultra-complex shRNA library, we performed pooled shRNA screens in RPMI-8226 and MM1-S MM cells and identified genes whose diminished function impact the cell's response to proteasome or IRE1 inhibition. Importantly, we show that unlike proteasome inhibition, blocking IRE1 function does not lead to substantial MM cell death, strongly suggesting that IRE1 alone is not a fate determinant. Instead, we posit IRE1 is a handicap that might be therapeutically exploited through the identification of synthetic-lethal combinations. We applied the same rationale to proteasome inhibition and found several synthetic-lethal pairs. Some of these genes can be targeted pharmacologically, which allowed us to query the susceptibility of a panel of MM cells to combinations of bortezomib and several drug-like molecules, including novel Hsp70 inhibitors. Our results inform which pathways may be selectively blocked to shut down potential escape routes that may lead to drug resistance. Notably, retrospective analyses on publicly available gene expression datasets of MM patients indicate that several of the genes we found on our screens are predictors of poor survival. Taken together, our preliminary results indicate our screening platform is a powerful tool for the discovery of (i) novel therapy targets, (ii) synthetic-lethal pairs that can be exploited in potential combination therapies, and (iii) diagnostics biomarkers. Currently, we continue to validate our results in additional cell lines prior to verification in primary patient samples and in an orthotopic xenograft model developed by our groups (DAA and PW, unpublished). Citation Format: Diego Acosta-Alvear, Martin Kampmann, Michael C. Bassik, Crystal P. Lee, Min Y. Cho, Blake T. Aftab, Xiaokai Li, Jason E. Gestwicki, Marc A. Shuman, Jonathan S. Weissman, Peter Walter. Systematic blueprinting of genetic vulnerabilities using ultra-complex shRNA libraries identifies genes synergizing with proteasome inhibition or blockade of IRE1 in multiple myeloma cells. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C01.

Abstract A48: Enhanced efficacy of cisplatin and 5-fluorouracil combination with AUY-922 in esophageal adenocarcinoma cells

Abstract Background: Esophageal Adenocarcinoma (EAC) continues to rise in incidence, with prognosis remaining poor despite advances in multimodality therapy. Several novel target agents are now being explored as an option for treating EAC. One potential target is heat shock protein 90 (Hsp90), a chaperone protein that is involved in many diverse biological processes including cell signaling, proliferation, and survival. Many of the client proteins are known oncoproteins that allow Hsp90 to stabilize cancer cell growth by supporting proliferation and preventing apoptosis. The isoform, Hsp90β, is constituently expressed, while Hsp90α is inducible during times of stress, with expression increased 2-10 fold in cancers. Our hypothesis is that Hsp90 inhibition, in combination with standard chemotherapeutic drugs, will cause EAC cancer cells to be more susceptible to apoptosis and reduce the rate of proliferation. Methods: EAC cell lines, OE19 and OE33, were used to evaluate the effects of Hsp90 inhibitor, AUY-922, in cancer cell growth and apoptosis. ELISA of WST-1 and BrdU were used to determine the effective dosage and assess proliferation. Pathway inhibition was evaluated by Western Blot of Hsp90α and Hsp70. OE19, OE33, and patient samples of EAC tumor and gastroesophageal reflux tissue were used to assess the gene expression of Hsp90 and several client protein pathways by reverse transcription polymerase chain reaction (RT-PCR). Results: The ED50 of AUY-922 was determined to be 30ηM. A combination of chemotherapeutic drugs, cisplatin and 5-fluorouracil (5-Fu), along with AUY-922 showed significantly decreased proliferation compared to untreated and single agent treated cell lines. Western blot demonstrated that Hsp90 was inhibited by AUY-922 treatment, by a decrease in expression of Hsp90α, and an increase in the expression of Hsp70. RT-PCR in the cell line treatment groups showed an impact on many client oncoproteins involved in cancer cell survival and Hsp90 was shown to be upregulated in tumor samples when compared to normal GERD samples. Conclusion: The use of Hsp90 inhibitor, AUY-922, leads to reduced Hsp90 pathway expression, resulting in a degradation of many Hsp90 client proteins involved in cancer genesis. Cell proliferation was decreased with AUY-922 treatment, with the greatest demonstrated effect when used in combination with cisplatin and 5-Fu. Therefore, Hsp90 inhibition may have an application in multimodal EAC chemotherapy. Citation Format: Christina L. Rotoloni, Jaclyn M. LaRosa, Michael J. Porter, Lori A. Kelly, Katherine Nega, Emily Wolfson, Yoshihiro Komastu, Juliann E. Kosovec, Pashtoon M. Kasi, Toshitaka Hoppo, Ali H. Zaidi, Blair A. Jobe. Enhanced efficacy of cisplatin and 5-fluorouracil combination with AUY-922 in esophageal adenocarcinoma cells. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A48.