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Purpose: ALK-activating mutations are identified in approximately 10% of newly diagnosed neuroblastomas and ALK amplifications in a further 1%-2% of cases. Lorlatinib, a third-generation anaplastic lymphoma kinase (ALK) inhibitor, will soon be given alongside induction chemotherapy for children with ALK-aberrant neuroblastoma. However, resistance to single-agent treatment has been reported and therapies that improve the response duration are urgently required. We studied the preclinical combination of lorlatinib with chemotherapy, or with the MDM2 inhibitor, idasanutlin, as recent data have suggested that ALK inhibitor resistance can be overcome through activation of the p53-MDM2 pathway., Experimental Design: We compared different ALK inhibitors in preclinical models prior to evaluating lorlatinib in combination with chemotherapy or idasanutlin. We developed a triple chemotherapy (CAV: cyclophosphamide, doxorubicin, and vincristine) in vivo dosing schedule and applied this to both neuroblastoma genetically engineered mouse models (GEMM) and patient-derived xenografts (PDX)., Results: Lorlatinib in combination with chemotherapy was synergistic in immunocompetent neuroblastoma GEMM. Significant growth inhibition in response to lorlatinib was only observed in the ALK-amplified PDX model with high ALK expression. In this PDX, lorlatinib combined with idasanutlin resulted in complete tumor regression and significantly delayed tumor regrowth., Conclusions: In our preclinical neuroblastoma models, high ALK expression was associated with lorlatinib response alone or in combination with either chemotherapy or idasanutlin. The synergy between MDM2 and ALK inhibition warrants further evaluation of this combination as a potential clinical approach for children with neuroblastoma., (©2023 The Authors; Published by the American Association for Cancer Research.)

Purpose: Most ALK -positive lung cancers will develop ALK-independent resistance after treatment with next-generation ALK inhibitors. MET amplification has been described in patients progressing on ALK inhibitors, but frequency of this event has not been comprehensively assessed., Experimental Design: We performed FISH and/or next-generation sequencing on 207 posttreatment tissue ( n = 101) or plasma ( n = 106) specimens from patients with ALK-positive lung cancer to detect MET genetic alterations. We evaluated ALK inhibitor sensitivity in cell lines with MET alterations and assessed antitumor activity of ALK/MET blockade in ALK-positive cell lines and 2 patients with MET-driven resistance., Results: MET amplification was detected in 15% of tumor biopsies from patients relapsing on next-generation ALK inhibitors, including 12% and 22% of biopsies from patients progressing on second-generation inhibitors or lorlatinib, respectively. Patients treated with a second-generation ALK inhibitor in the first-line setting were more likely to develop MET amplification than those who had received next-generation ALK inhibitors after crizotinib ( P = 0.019). Two tumor specimens harbored an identical ST7-MET rearrangement, one of which had concurrent MET amplification. Expressing ST7-MET in the sensitive H3122 ALK-positive cell line induced resistance to ALK inhibitors that was reversed with dual ALK/MET inhibition. MET inhibition resensitized a patient-derived cell line harboring both ST7-MET and MET amplification to ALK inhibitors. Two patients with ALK-positive lung cancer and acquired MET alterations achieved rapid responses to ALK/MET combination therapy., Conclusions: Treatment with next-generation ALK inhibitors, particularly in the first-line setting, may lead to MET-driven resistance. Patients with acquired MET alterations may derive clinical benefit from therapies that target both ALK and MET., (©2020 American Association for Cancer Research.)

Purpose: Lorlatinib is a third-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor with proven efficacy in patients with ALK-rearranged lung cancer previously treated with first- and second-generation ALK inhibitors. Beside compound mutations in the ALK kinase domain, other resistance mechanisms driving lorlatinib resistance remain unknown. We aimed to characterize the mechanisms of resistance to lorlatinib occurring in patients with ALK -rearranged lung cancer and design new therapeutic strategies in this setting., Experimental Design: Resistance mechanisms were investigated in 5 patients resistant to lorlatinib. Longitudinal tumor biopsies were studied using high-throughput next-generation sequencing. Patient-derived models were developed to characterize the acquired resistance mechanisms, and Ba/F3 cell mutants were generated to study the effect of novel ALK compound mutations. Drug combinatory strategies were evaluated in vitro and in vivo to overcome lorlatinib resistance., Results: Diverse biological mechanisms leading to lorlatinib resistance were identified. Epithelial-mesenchymal transition (EMT) mediated resistance in two patient-derived cell lines and was susceptible to dual SRC and ALK inhibition. We characterized three ALK kinase domain compound mutations occurring in patients, L1196M/D1203N, F1174L/G1202R, and C1156Y/G1269A, with differential susceptibility to ALK inhibition by lorlatinib. We identified a novel bypass mechanism of resistance caused by NF2 loss-of-function mutations, conferring sensitivity to treatment with mTOR inhibitors., Conclusions: This study shows that mechanisms of resistance to lorlatinib are diverse and complex, requiring new therapeutic strategies to tailor treatment upon disease progression., (©2019 American Association for Cancer Research.)

Purpose: The first-generation ALK tyrosine kinase inhibitor (TKI) crizotinib is a standard therapy for patients with ALK-rearranged non-small cell lung cancer (NSCLC). Several next-generation ALK-TKIs have entered the clinic and have shown promising activity in crizotinib-resistant patients. As patients still relapse even on these next-generation ALK-TKIs, we examined mechanisms of resistance to the next-generation ALK-TKI alectinib and potential strategies to overcome this resistance., Experimental Design: We established a cell line model of alectinib resistance, and analyzed a resistant tumor specimen from a patient who had relapsed on alectinib. We developed Ba/F3 models harboring alectinib-resistant ALK mutations and evaluated the potency of other next-generation ALK-TKIs in these models. We tested the antitumor activity of the next-generation ALK-TKI ceritinib in the patient with acquired resistance to alectinib. To elucidate structure-activity relationships of ALK mutations, we performed computational thermodynamic simulation with MP-CAFEE., Results: We identified a novel V1180L gatekeeper mutation from the cell line model and a second novel I1171T mutation from the patient who developed resistance to alectinib. Both ALK mutations conferred resistance to alectinib as well as to crizotinib, but were sensitive to ceritinib and other next-generation ALK-TKIs. Treatment of the patient with ceritinib led to a marked response. Thermodynamics simulation suggests that both mutations lead to distinct structural alterations that decrease the binding affinity with alectinib., Conclusions: We have identified two novel ALK mutations arising after alectinib exposure that are sensitive to other next-generation ALK-TKIs. The ability of ceritinib to overcome alectinib-resistance mutations suggests a potential role for sequential therapy with multiple next-generation ALK-TKIs., (©2014 American Association for Cancer Research.)

Purpose: Heat shock protein 90 (HSP90) is required for the proper folding, function, and stability of various client proteins, two of which (KIT and PDGFRα) are critical in the pathogenesis and progression of gastrointestinal stromal tumors (GIST). This phase I study investigated the safety and maximum tolerated dose (MTD) of retaspimycin hydrochloride (IPI-504), a novel potent and selective HSP90 inhibitor, in patients with metastatic and/or unresectable GIST or other soft-tissue sarcomas (STS)., Experimental Design: IPI-504 was administered intravenously at doses ranging from 90 to 500 mg/m(2) twice weekly for 2 weeks on/1 week off. Safety, pharmacokinetic, and pharmacodynamic profiles were determined. Response was assessed by Response Evaluation Criteria for Solid Tumors (RECIST) 1.0 and optionally via 18-fluorodeoxyglucose positron emission tomography (18-FDG-PET) imaging., Results: Fifty-four patients received IPI-504; 37 with GIST and 17 with other STS. The MTD was 400 mg/m(2) twice weekly for 2 weeks on/1 week off. Common related adverse events were fatigue (59%), headache (44%), and nausea (43%). Exposure to IPI-504, 17-AAG, and 17-AG increased with IPI-504 dose. Stable disease (SD) was observed in 70% (26 of 37) of patients with GIST and 59% (10 of 17) of patients with STS. There was one confirmed partial response (PR) in a patient with GIST and one PR in a patient with liposarcoma. Metabolic partial responses occurred in 11 of 29 (38%) patients with GIST., Conclusions: In this study of advanced GIST or other STS, IPI-504 was generally well-tolerated with some evidence of antitumor activity, serving as a clinical proof-of-concept that HSP90 inhibition remains a promising strategy.

Purpose: We describe the anticancer activity of ganetespib, a novel non-geldanamycin heat shock protein 90 (HSP90) inhibitor, in non-small cell lung cancer (NSCLC) models., Experimental Design: The activity of ganetespib was compared with that of the geldanamycin 17-AAG in biochemical assays, cell lines, and xenografts, and evaluated in an ERBB2 YVMA-driven mouse lung adenocarcinoma model., Results: Ganetespib blocked the ability of HSP90 to bind to biotinylated geldanamycin and disrupted the association of HSP90 with its cochaperone, p23, more potently than 17-AAG. In genomically defined NSCLC cell lines, ganetespib caused depletion of receptor tyrosine kinases, extinguishing of downstream signaling, inhibition of proliferation and induction of apoptosis with IC(50) values ranging 2 to 30 nmol/L, substantially lower than those required for 17-AAG (20-3,500 nmol/L). Ganetespib was also approximately 20-fold more potent in isogenic Ba/F3 pro-B cells rendered IL-3 independent by expression of EGFR and ERBB2 mutants. In mice bearing NCI-H1975 (EGFR L858R/T790M) xenografts, ganetespib was rapidly eliminated from plasma and normal tissues but was maintained in tumor with t(1/2) 58.3 hours, supporting once-weekly dosing experiments, in which ganetespib produced greater tumor growth inhibition than 17-AAG. However, after a single dose, reexpression of mutant EGFR occurred by 72 hours, correlating with reversal of antiproliferative and proapoptotic effects. Consecutive day dosing resulted in xenograft regressions, accompanied by more sustained pharmacodynamic effects. Ganetespib also showed activity against mouse lung adenocarcinomas driven by oncogenic ERBB2 YVMA., Conclusions: Ganetespib has greater potency than 17-AAG and potential efficacy against several NSCLC subsets, including those harboring EGFR or ERBB2 mutation., (©2012 AACR.)

Many kinases and hormone receptors, important for cancer cell proliferation and survival, bind to and are dependent on the Hsp90 cycle for their folding and maturation. This provides the rationale for the development of small-molecule ATP competitors that, inhibiting Hsp90 function, lead to degradation of the "client" proteins. After continual efforts to improve the pharmacologic properties and the tolerability of these molecules, several Hsp90 inhibitors have exhibited activity in both preclinical models and in the clinical setting. As is the case with many other targeted agents, patient selection seems to be the major limitation to the success of these compounds. ERBB2-positive patients with breast cancer are exquisitely sensitive to Hsp90 inhibition. This is because ERBB2 is indispensable for growth and survival of this subtype of cancer, and at the same time ERBB2 is a client protein strictly dependent on Hsp90 for its maturation and stability. Extensive preclinical work identifying other ERBB-like client proteins will likely lead to the ability to enhance selection of appropriate patients for enrollment in more rational clinical trials. Hsp90 inhibition has also been reported to synergize with other therapeutic agents. Several ongoing studies testing different combinations of Hsp90 inhibitors with other targeted agents will confirm whether Hsp90 inhibition can potentiate the efficacy of targeted therapy and/or prevent the emergence of drug resistance., (©2012 AACR.)

Purpose: Targeting Hsp90 has significant potential as a treatment for prostate cancer, but prototypical agents such as 17-allylamino-17 demethoxygeldanamycin (17-AAG) have been ineffective in clinical trials. Recently, a phase I study aimed at defining a biologically active dose reported the first response to an Hsp90 inhibitor in a patient with prostate cancer, which supports the development of new generation compounds for this disease., Experimental Design: The biological actions of two new synthetic Hsp90 inhibitors, NVP-AUY922 and NVP-HSP990, were evaluated in the prostate cancer cell lines PC-3, LNCaP, and VCaP and in an ex vivo culture model of human prostate cancer., Results: In cell lines, both NVP-AUY922 and NVP-HSP990 showed greater potency than 17-AAG with regard to modulation of Hsp90 client proteins, inhibition of proliferation, and induction of apoptotic cell death. In prostate tumors obtained from radical prostatectomy that were cultured ex vivo, treatment with 500 nmol/L of NVP-AUY922, NVP-HSP990, or 17-AAG caused equivalent target modulation, determined by the pharmacodynamic marker Hsp70, but only NVP-AUY922 and NVP-HSP990 showed antiproliferative and proapoptotic activity., Conclusions: This study provides some of the first evidence that new generation Hsp90 inhibitors are capable of achieving biologic responses in human prostate tumors, with both NVP-AUY922 and NVP-HSP990 showing potent on-target efficacy. Importantly, the ex vivo culture technique has provided information on Hsp90 inhibitor action not previously observed in cell lines or animal models. This approach, therefore, has the potential to enable more rational selection of therapeutic agents and biomarkers of response for clinical trials., (©2012 AACR.)

Purpose: Protein kinase CK2 promotes multiple myeloma cell growth by regulating critical signaling pathways. CK2 also modulates proper HSP90-dependent client protein folding and maturation by phosphorylating its co-chaperone CDC37. Because the endoplasmic reticulum (ER) stress/unfolded protein response (UPR) is central in myeloma pathogenesis, we tested the hypothesis that the CK2/CDC37/HSP90 axis could be involved in UPR in myeloma cells., Experimental Design: We analyzed CK2 activity upon ER stress, the effects of its inactivation on the UPR pathways and on ER stress-induced apoptosis. The consequences of CK2 plus HSP90 inhibition on myeloma cell growth in vitro and in vivo and CK2 regulation of HSP90-triggered UPR were determined., Results: CK2 partly localized to the ER and ER stress triggered its kinase activity. CK2 inhibition reduced the levels of the ER stress sensors IRE1α and BIP/GRP78, increased phosphorylation of PERK and EIF2α, and enhanced ER stress-induced apoptosis. Simultaneous inactivation of CK2 and HSP90 resulted in a synergic anti-myeloma effect (combination index = 0.291) and in much stronger alterations of the UPR pathways as compared with the single inhibition of the two molecules. Cytotoxicity from HSP90 and CK2 targeting was present in a myeloma microenvironment model, on plasma cells from patients with myeloma and in an in vivo mouse xenograft model. Mechanistically, CK2 inhibition led to a reduction of IRE1α/HSP90/CDC37 complexes in multiple myeloma cells., Conclusions: Our results place CK2 as a novel regulator of the ER stress/UPR cascades and HSP90 function in myeloma cells and offer the groundwork to design novel combination treatments for this disease., (©2012 AACR.)

Purpose: To determine whether optical imaging can be used for in vivo therapy response monitoring as an alternative to radionuclide techniques. For this, we evaluated the known Her2 response to 17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride (17-DMAG) treatment, an Hsp90 inhibitor., Experimental Design: After in vitro 17-DMAG treatment response evaluation of MCF7 parental cells and 2 HER2-transfected clones (clone A medium, B high Her2 expression), we established human breast cancer xenografts in nude mice (only parental and clone B) for in vivo evaluation. Mice received 120 mg/kg of 17-DMAG in 4 doses at 12-hour intervals intraperitonially (n = 14) or PBS as carrier control (n = 9). Optical images were obtained both pretreatment (day 0) and posttreatment (day 3, 6, and 9), always 5 hours postinjection of 500 pmol of anti-Her2 Affibody-AlexaFluor680 via tail vein (with preinjection background subtraction). Days 3 and 9 in vivo optical imaging signal was further correlated with ex vivo Her2 levels by Western blot after sacrifice., Results: Her2 expression decreased with 17-DMAG dose in vitro. In vivo optical imaging signal was reduced by 22.5% in clone B (P = 0.003) and by 9% in MCF7 parental tumors (P = 0.23) 3 days after 17-DMAG treatment; optical imaging signal recovered in both tumor types at days 6 to 9. In the carrier group, no signal reduction was observed. Pearson correlation of in vivo optical imaging signal with ex vivo Her2 levels ranged from 0.73 to 0.89., Conclusions: Optical imaging with an affibody can be used to noninvasively monitor changes in Her2 expression in vivo as a response to treatment with an Hsp90 inhibitor, with results similar to response measurements in positron emission tomography imaging studies., (©2012 AACR.)

Purpose: KIT activity is crucial for gastrointestinal stromal tumors (GIST). Imatinib (IMA) and sunitinib (SUN) are very effective KIT-inhibitors in patients with advanced GIST but have no curative potential. We evaluated the efficacy of the novel HSP90 inhibitor IPI-493 alone, or in combination with IMA or SUN in GIST xenografts with KIT mutations., Experimental Design: Nude mice (n = 98) were grafted bilaterally with human GIST carrying KIT exon 11 (GIST-PSW), KIT exon 9 (GIST-BOE), or double, KIT imatinib-sensitive exon 11 and imatinib-resistant exon 17 mutations (GIST-48). Mice were divided into six treatment groups and dosed orally for 15 days as follows: (i) control group, sterile water; (ii) IMA alone; (iii) SUN alone; (iv) IPI-493 alone; (v) IPI-493+IMA; and (vi) IPI-493+SUN., Results: Treatment with IPI-493 resulted in tumor growth stabilization, variable proliferation arrest, induction of apoptosis and necrosis, and downregulation of KIT and its signaling cascade, especially in the GIST-BOE model. Significant reduction of vessel density was observed with IPI-493 treatment, and was equal to SUN treatment in GIST-PSW and GIST-BOE xenografts. IPI-493 treatment effects were enhanced in combination with TKIs, especially with IPI-493+SUN. In our hands, IPI-493 showed dose-dependent liver damages., Conclusions: When administered as a single agent in a xenograft model, the HSP90 inhibitor IPI-493 has consistent antitumor activity and induces KIT downregulation in GISTs with heterogeneous KIT mutations. IPI-493 synergizes with TKIs that are commonly used for the treatment of advanced or IMA-resistant GIST. The antitumor response of IPI-493 is particularly enhanced in combination with SUN., (©2011 AACR.)

Purpose: The underlying molecular mechanisms of thymic epithelial malignancies (TEMs) are poorly understood. Consequently, there is a lack of efficacious targeted therapies and patient prognosis remains dismal, particularly for advanced TEMs. We sought to investigate protumorigenic mechanism relevant to this understudied cancer., Experimental Design: Recently established cell lines derived from thymic epithelial tumors were used as a model system. The antitumor activity of specific heat shock protein 90 (Hsp90) inhibitors was investigated by an analysis of cell viability, cell cycle, and apoptosis using MTT-assays and flow cytometry. Western blotting was used to investigate the altered expression of Hsp90 clients. Pharmacological inhibitors against select Hsp90 clients, as well as RNAi, were employed to test the relevance of each client independently. Tissue microarray analysis was performed to match the in vitro findings with observations obtained from patient-derived samples., Results: Hsp90 inhibition significantly reduces cell viability of thymic carcinoma cells, induces cell cycle arrest and apoptosis, and blocks invasiveness. Hsp90 inhibition triggers the degradation of multiple oncogenic clients, for example insulin-like growth factor 1 receptor (IGF-1R), CDK4, and the inactivation of PI3K/Akt and RAF/Erk signaling. Mechanistically, the IGF/IGF-1R-signaling axis contributes to the establishment of the antiapoptotic phenotype of thymic cancer cells. Finally, IGF-1R is overexpressed in advanced TEMs., Conclusions: We have unraveled a novel protumorigenic mechanism in TEMs, namely Hsp90-capacitated overexpression of IGF-1R, which confers apoptosis evasion in malignant thymic epithelial cells. Our data indicate that Hsp90 inhibition, which simultaneously blocks multiple cancer hallmarks, represents a therapeutic strategy in TEMs that may merit evaluation in clinical trials., (©2011 AACR.)

Purpose: Prostate cancer responds initially to antiandrogen therapies; however, progression to castration-resistant disease frequently occurs. Therefore, there is an urgent need for novel therapeutic agents that can prevent the emergence of castrate-resistant prostate cancer (CRPC). HSP90 is a molecular chaperone involved in the stability of many client proteins including Akt and androgen receptor (AR). 17-Allylamino-17-demethoxy-geldanamycin (17-AAG) has been reported to inhibit tumor growth in various cancers; however, it induces tumor progression in the bone microenvironment., Methods: Cell growth, apoptosis, and AR transactivation were examined by crystal violet assay, flow cytometric, and luciferase assays, respectively. The consequence of HSP90 therapy in vivo was evaluated in LNCaP xenograft model. The consequence of PF-04928473 therapy on bone metastasis was studied using an osteoclastogenesis in vitro assay., Results: PF-04928473 inhibits cell growth in a panel of prostate cancer cells, induces cell-cycle arrest at sub-G(1), and leads to apoptosis and increased caspase-3 activity. These biological events were accompanied by decreased activation of Akt and Erk as well as decreased expression of Her2, and decreased AR expression and activation in vitro. In contrast to 17-AAG, PF-04928473 abrogates RANKL-induced osteoclast differentiation by affecting NF-κB activation and Src phosphorylation. Finally, PF-04929113 inhibited tumor growth and prolonged survival compared with controls. Surprisingly, PF-04929113 did not reduce serum prostate-specific antigen (PSA) levels in vivo; in parallel, these decrease in tumor volume., Conclusion: These data identify significant anticancer activity of PF-04929113 in CRPC but suggest that serum PSA may not prove useful as pharmacodynamic tool for this drug., (©2011 AACR.)

Purpose: A phase I study to define toxicity and recommend a phase II dose of the HSP90 inhibitor alvespimycin (17-DMAG; 17-dimethylaminoethylamino-17-demethoxygeldanamycin). Secondary endpoints included evaluation of pharmacokinetic profile, tumor response, and definition of a biologically effective dose (BED)., Patients and Methods: Patients with advanced solid cancers were treated with weekly, intravenous (i.v.) 17-DMAG. An accelerated titration dose escalation design was used. The maximum tolerated dose (MTD) was the highest dose at which ≤ 1/6 patients experienced dose limiting toxicity (DLT). Dose de-escalation from the MTD was planned with mandatory, sequential tumor biopsies to determine a BED. Pharmacokinetic and pharmacodynamic assays were validated prior to patient accrual., Results: Twenty-five patients received 17-DMAG (range 2.5-106 mg/m(2)). At 106 mg/m(2) of 17-DMAG 2/4 patients experienced DLT, including one treatment-related death. No DLT occurred at 80 mg/m(2). Common adverse events were gastrointestinal, liver function changes, and ocular. Area under the curve and mean peak concentration increased proportionally with 17-DMAG doses 80 mg/m(2) or less. In peripheral blood mononuclear cells significant (P < 0.05) HSP72 induction was detected (≥ 20 mg/m(2)) and sustained for 96 hours (≥ 40 mg/m(2)). Plasma HSP72 levels were greatest in the two patients who experienced DLT. At 80 mg/m(2) client protein (CDK4, LCK) depletion was detected and tumor samples from 3 of 5 patients confirmed HSP90 inhibition. Clinical activity included complete response (castration refractory prostate cancer, CRPC 124 weeks), partial response (melanoma, 159 weeks), and stable disease (chondrosarcoma, CRPC, and renal cancer for 28, 59, and 76 weeks, respectively)., Conclusions: The recommended phase II dose of 17-DMAG is 80 mg/m(2) weekly i.v., (©2011 AACR.)

Purpose: This study aimed to characterize the preclinical activity of the first class of combinatorial, mitochondria-targeted, small molecule heat shock protein-90 (Hsp90) inhibitors, gamitrinibs, in models of hormone-refractory, drug-resistant, localized, and bone metastatic prostate cancer in vivo., Experimental Design: Mitochondrial permeability transition, apoptosis, and changes in metabolic activity were examined by time-lapse videomicroscopy, multiparametric flow cytometry, MTT, and analysis of isolated mitochondria. Drug-resistant prostate cancer cells were generated by chronic exposure of hormone-refractory PC3 cells to the Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG). The effect of gamitrinibs on s.c. or intratibial prostate cancer growth was studied in xenograft models. Bone metastatic tumor growth and bone parameters were quantified by micro-computed tomography imaging., Results: In the NCI 60-cell line screening, gamitrinibs were active against all tumor cell types tested, and efficiently killed metastatic, hormone-refractory, and multidrug-resistant prostate cancer cells characterized by overexpression of the ATP binding cassette transporter P-glycoprotein. Mechanistically, gamitrinibs, but not 17-AAG, induced acute mitochondrial dysfunction in prostate cancer cells with loss of organelle membrane potential, release of cytochrome c, and caspase activity, independently of proapoptotic Bcl-2 proteins Bax and Bak. Systemic administration of gamitrinibs to mice was well tolerated, and inhibited s.c. or bone metastatic prostate cancer growth in vivo., Conclusions: Gamitrinibs have preclinical activity and favorable safety in models of drug-resistant and bone metastatic prostate cancer in vivo., (©2010 AACR.)

Purpose: High-frequency microsatellite-instable (MSI-H) tumors account for approximately 15% of colorectal cancers. Therapeutic decisions for colorectal cancer are empirically based and currently do not emphasize molecular subclassification despite an increasing collection of gene expression information. Our objective was to identify low molecular weight compounds with preferential activity against MSI colorectal cancers using combined gene expression data sets., Experimental Design: Three expression/query signatures (discovery data set) characterizing MSI-H colorectal cancer were matched with information derived from changes induced in cell lines by 164 compounds using the systems biology tool "Connectivity Map." A series of sequential filtering and ranking algorithms were used to select the candidate compounds. Compounds were validated using two additional expression/query signatures (validation data set). Cytotoxic, cell cycle, and apoptosis effects of validated compounds were evaluated in a panel of cell lines., Results: Fourteen of the 164 compounds were validated as targeting MSI-H cell lines using the bioinformatics approach; rapamycin, LY-294002, 17-(allylamino)-17-demethoxygeldanamycin, and trichostatin A were the most robust candidate compounds. In vitro results showed that MSI-H cell lines due to hypermethylation of MLH1 are preferentially targeted by rapamycin (18.3 versus 4.4 mumol/L; P = 0.0824) and LY-294002 (15.02 versus 10.37 mumol/L; P = 0.0385) when compared with microsatellite-stable cells. Preferential activity was also observed in MSH2 and MSH6 mutant cells., Conclusion: Our study shows that the phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin pathway is of special relevance in mismatch repair-deficient colorectal cancer. In addition, we show that amalgamation of gene expression information across studies provides a robust approach for selection of potential therapies corresponding to specific groups of patients.

Purpose: Activating mutations in platelet-derived growth factor receptor-alpha (PDGFRA) have been reported in approximately 5% to 10% of patients with gastrointestinal stromal tumors (GIST). Imatinib efficiently inhibits the juxtamembrane PDGFRA mutations, whereas many tyrosine kinase domain activation loop PDGFRA mutations confer primary resistance to imatinib. In this study, we compared the efficacy of second-line tyrosine kinase inhibitors such as dasatinib, sorafenib, and nilotinib against two GIST-related PDGFRA mutants, PDGFRA(D842V) and PDGFRA(DeltaDIM842-844). In addition, we sought to investigate the inhibitory effect of the heat shock protein 90 inhibitor, IPI-504, on these mutants., Experimental Design: Primary imatinib-resistant tumor cells and cell lines expressing imatinib-resistant PDGFRA(D842V) or imatinib-sensitive PDGFRA(DeltaDIM842-844) mutants were treated with different concentrations of dasatinib, sorafenib, nilotinib, and IPI-504. The effect of treatment on proliferation, survival, and signaling was determined., Results: All inhibitors tested exhibited a high efficacy toward the PDGFRA(DeltaDIM842-844) mutant. In contrast, ex vivo and in vitro assays revealed that only dasatinib potently inhibited the PDGFRA(D842V) isoform with an IC(50) value of 62 nmol/L. Sorafenib and nilotinib were significantly less efficacious against this mutation, inhibiting the PDGFRA kinase activity at >1,000 and >5,000 nmol/L, and suppressing the proliferation of the cells expressing the PDGFRA(D842V) mutant with an IC(50) value of 239 and 1,310 nmol/L, respectively. IPI-504 treatment potently inhibited PDGFRA kinase activity by inducing the degradation of PDGFRA(D842V) and PDGFRA(DeltaDIM842-844) at 256 and 182 nmol/L, respectively., Conclusions: Treatment with dasatinib or the heat shock protein 90 inhibitor IPI-504 may provide a therapeutic alternative for GIST patients whose tumors carry the imatinib-resistant PDGFRA(D842V) mutant isoform.

Purpose: We hypothesized that targeting both Akt and heat shock protein (HSP) 90 would induce cytotoxic activity against multiple myeloma (MM) cells and target the bone marrow (BM) microenvironment to inhibit angiogenesis, osteoclast formation, as well as migration and adhesion of MM cells., Experimental Design: MM cell lines were incubated with perifosine (5 and 10 micromol/L) and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG; 50 and 100 nmol/L) alone and in combination., Results: The combination of Akt inhibitor perifosine and HSP90 inhibitor 17-DMAG was synergistic in inducing MM cell cytotoxicity, evidenced by inhibition of DNA synthesis and induction of apoptosis. In addition, perifosine and 17-DMAG almost completely inhibited osteoclast formation: perifosine interfered with both early and late stages of osteoclast progenitor development, whereas 17-DMAG targeted only early stages. We next showed that combined therapy overcomes tumor growth and resistance induced by BM stromal cells and endothelial cells as well as the proliferative effect of exogenous interleukin-6, insulin-like growth factor-I, and vascular endothelial growth factor. Moreover, the combination also induced apoptosis and growth inhibition in endothelial cells and inhibited angiogenesis. Finally, we showed that the two agents prevented migration of MM cells toward stromal-derived factor-1 and vascular endothelial growth factor, which are present in the BM milieu, and also prevented adhesion of MM cells to fibronectin., Conclusions: This study provides the preclinical framework for treatment protocols targeting both the Akt and HSP pathways in MM.

Purpose: To examine the immunohistochemical profile of heat shock protein 90 (Hsp90) in uveal melanoma and the cytotoxicity of an Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), in uveal melanoma cell lines., Experimental Design: Hsp90 expression was determined by immunohistochemistry in 44 paraffin-embedded sections of primary human uveal melanoma and in five uveal melanoma cell lines (92.1, OCM-1, MKT-BR, SP6.5, and UW-1). Sulforhodamine B-based proliferation assay was used to compare uveal melanoma cell growth with a range of concentrations of 17-AAG. Changes in cell migration, invasion, cell cycle fractions, and apoptotic activity were also evaluated. Expression of intracellular proteins was determined by Western blot analysis after 17-AAG exposure., Results: Immunohistochemical expression of Hsp90 was identified in 68% of the paraffin-embedded sections and significantly associated with largest tumor dimension (P = 0.03). 17-AAG significantly reduced the proliferation rates of uveal melanoma cell lines, with concentrations of 100 to 0.1 micromol/L. 17-AAG also significantly reduced the migratory and invasive capabilities of uveal melanoma cell lines. Cell cycle analysis showed that 17-AAG induced accumulations of cells in G(1). Caspase-3 protease activity analysis, a marker for apoptosis, showed a significant increase after drug exposure. The cytotoxic effect of 17-AAG was associated with decreased levels of phosphorylated Akt and cyclin-dependent kinase 4., Conclusions: The immunohistochemical expression of Hsp90 in uveal melanoma indicates worse prognosis. To the best of our knowledge, this is the first report showing the inhibitory effect on uveal melanoma cells using 17-AAG to target Hsp90. Therefore, Hsp90 may be used as a potential target for treatment of patients with uveal melanoma.

Purpose: The heat shock protein 90 (Hsp90) chaperone plays an important role in transformation by regulating the conformational maturation and stability of oncogenic kinases and transcription factors. Ansamycins, such as 17-(allylamino)-17-demethoxygeldanmycin (17-AAG), inhibit Hsp90 function; induce the degradation of Hsp90 client proteins such as HER2, and have shown activity in early clinical trials. However, the utility of these drugs has been limited by their hepatotoxicity, poor solubility, and poorly tolerated formulations., Experimental Design: We determined the pharmacodynamic and antitumor properties of a novel, synthetic Hsp90 inhibitor, SNX-2112, in cell culture and xenograft models of HER kinase-dependent cancers., Results: We show in a panel of tumor cell lines that SNX-2112 and its prodrug SNX-5542 are Hsp90 inhibitors with properties and potency similar to that of 17-AAG, including: degradation of HER2, mutant epidermal growth factor receptor, and other client proteins, inhibition of extracellular signal-regulated kinase and Akt activation, and induction of a Rb-dependent G(1) arrest with subsequent apoptosis. SNX-5542 can be administered to mice orally on a daily schedule. Following oral administration, SNX-5542 is rapidly converted to SNX-2112, which accumulates in tumors relative to normal tissues. A single dose of SNX-5542 causes HER2 degradation and inhibits its downstream signaling for up to 24 h, and daily dosing results in regression of HER2-dependent xenografts. SNX-5542 also shows greater activity than 17-AAG in a non-small cell lung cancer xenograft model expressing mutant EGFR., Conclusions: These results suggest that Hsp90 inhibition with SNX-2112 (delivered as a prodrug) may represent a promising therapeutic strategy for tumors whose growth and survival is dependent on Hsp90 clients.

Purpose: Inhibitors of heat-shock protein 90 (Hsp90) may interfere with oncogenic signaling pathways, including Erk, Akt, and hypoxia-inducible factor-1alpha (HIF-1alpha). Because insulin-like growth factor-I receptor (IGF-IR) and signal transducer and activator of transcription 3 (STAT3) signaling pathways are implicated in the progression of pancreatic cancer, we hypothesized that blocking Hsp90 with geldanamycin derivates [17-allylamino-geldanamycin (17-AAG), 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG)] would impair IGF-I- and interleukin-6-mediated signaling and thus reduce pancreatic tumor growth and angiogenesis in vivo., Experimental Design: Human pancreatic cancer cells (HPAF-II, L3.6pl) were used for experiments. Changes in signaling pathway activation upon Hsp90 blockade were investigated by Western blotting. Effects of Hsp90 inhibition (17-AAG) on vascular endothelial growth factor were determined by ELISA and real-time PCR. Effects of 17-DMAG (25 mg/kg; thrice a week; i.p.) on tumor growth and vascularization were investigated in a s.c. xenograft model and in an orthotopic model of pancreatic cancer., Results: 17-AAG inhibited IGF-IR signaling by down-regulating IGF-IRbeta and directly impairing IGF-IR phosphorylation. Hypoxia- and IL-6-mediated activation of HIF-1alpha or STAT3/STAT5 were substantially inhibited by 17-AAG. Moreover, a novel IL-6/STAT3/HIF-1alpha autocrine loop was effectively disrupted by Hsp90 blockade. In vivo, 17-DMAG significantly reduced s.c. tumor growth and diminished STAT3 phosphorylation and IGF-IRbeta expression in tumor tissues. In an orthotopic model, pancreatic tumor growth and vascularization were both significantly reduced upon Hsp90 inhibition, as reflected by final tumor weights and CD31 staining, respectively., Conclusions: Blocking Hsp90 disrupts IGF-I and IL-6-induced proangiogenic signaling cascades by targeting IGF-IR and STAT3 in pancreatic cancer, leading to significant growth-inhibitory effects. Therefore, we suggest that Hsp90 inhibitors could prove to be valuable in the treatment of pancreatic cancer.

Purpose: Constitutive signal transducer and activator of transcription 3 (STAT3) activity, observed in approximately 50% of acute myelogenous leukemia cases and associated with adverse treatment outcome, is down-regulated by arsenic trioxide (ATO). Heat shock protein (HSP) 90 is a molecular chaperone involved in signal transduction pathways. We hypothesized that HSP90 inhibitors will potentiate ATO effect on constitutive STAT3 activity and cell killing. One concern was that the effect of ATO and HSP90 inhibitors will result in up-regulation of HSP70, a protein known to inhibit apoptosis., Experimental Design: We have used a semimechanistic pharmacodynamic model to characterize concentration-effect relationships of ATO and HSP90 inhibitors on constitutive STAT3 activity, HSP70 expression, and cell death in a cell line model., Results: Pharmacodynamic interaction of ATO and three HSP90 inhibitors showed synergistic interactions in inhibiting constitutive STAT3 activity and inducing cell death, in spite of a concurrent synergistic up-regulation of HSP70., Conclusions: These preliminary results provide a basis for studying the combined role of ATO with HSP90 inhibitors in acute myelogenous leukemia with constitutive STAT3 activity.

Purpose: DNA-damaging agents, such as etoposide, while clinically useful in leukemia therapy, are limited by DNA repair pathways that are not well understood. 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG), an inhibitor of the molecular chaperone heat shock protein 90 (Hsp90), inhibits growth and induces apoptosis in FLT3(+) leukemia cells. In this study, we evaluated the effects of etoposide and 17-AAG in leukemia cells and the roles of Hsp90, FMS-like tyrosine kinase 3 (FLT3), checkpoint kinase 1 (Chk1), Rad51, and topoisomerase II in this inhibition., Experimental Design: The single and combined effects of 17-AAG and etoposide and the mechanism of these effects were evaluated. FLT3 and the DNA repair-related proteins, Chk1 and Rad51, were studied in small interfering RNA (siRNA)-induced cell growth inhibition experiments in human leukemia cells with wild-type or mutated FLT3., Results: We found that etoposide and the Hsp90/FLT3 inhibitor 17-AAG, had synergistic inhibitory effects on FLT3(+) MLL-fusion gene leukemia cells. Cells with an internal tandem duplication (ITD) FLT3 (Molm13 and MV4;11) were more sensitive to etoposide/17-AAG than leukemias with wild-type FLT3 (HPB-Null and RS4;11). A critical role for FLT3 was shown in experiments with FLT3 ligand and siRNA targeted to FLT3. An important role for topoisomerase II and the DNA repair-related proteins, Chk1 and Rad51, in the synergistic effects was suggested from the results., Conclusions: The repair of potentially lethal DNA damage by etoposide in leukemia cells is dependent on intact and functioning FLT3 especially leukemias with ITD-FLT3. These data suggest a rational therapeutic strategy for FLT3(+) leukemias that combines etoposide or other DNA-damaging agents with Hsp90/FLT3 inhibitors such as 17-AAG.

Purpose: The phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (mTOR) pathway and the heat shock protein family are up-regulated in multiple myeloma and are both regulators of the cyclin D/retinoblastoma pathway, a critical pathway in multiple myeloma. Inhibitors of mTOR and HSP90 protein have showed in vitro and in vivo single-agent activity in multiple myeloma. Our objective was to determine the effects of the mTOR inhibitor rapamycin and the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) on multiple myeloma cells., Experimental Design: Multiple myeloma cell lines were incubated with rapamycin (0.1-100 nmol/L) and 17-AAG (100-600 nmol/L) alone and in combination., Results: In this study, we showed that the combination of rapamycin and 17-AAG synergistically inhibited proliferation, induced apoptosis and cell cycle arrest, induced cleavage of poly(ADP-ribose) polymerase and caspase-8/caspase-9, and dysregulated signaling in the phosphatidylinositol 3-kinase/AKT/mTOR and cyclin D1/retinoblastoma pathways. In addition, we showed that both 17-AAG and rapamycin inhibited angiogenesis and osteoclast formation, indicating that these agents target not only multiple myeloma cells but also the bone marrow microenvironment., Conclusions: These studies provide the basis for potential clinical evaluation of this combination for multiple myeloma patients.

Purpose: Loss of p53 function impairs apoptosis induced by DNA-damaging agents used for cancer therapy. Here, we examined the effect of the heat shock protein 90 (HSP90) inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (DMAG) on doxorubicin-induced apoptosis in lymphoma. We aimed to establish the optimal schedule for administration of both drugs in combination and the molecular basis for their interaction., Experimental Design: Isogenic lymphoblastoid and nonisogenic lymphoma cell lines differing in p53 status were exposed to each drug or combination. Drug effects were examined using Annexin V, active caspase-3, cell cycle, and cytotoxicity assays. Synergy was evaluated by median effect/combination index. Protein expression and kinase inhibition provided insight into the molecular mechanisms of drug interaction., Results: Presence of mutant p53 conferred increased survival to single agents. Nevertheless, DMAG showed synergistic toxicity with doxorubicin independently of p53 status. Synergy required exposure to doxorubicin before DMAG. DMAG-mediated down-regulation of CHK1, a known HSP90 client, forced doxorubicin-treated cells into premature mitosis followed by apoptosis. A CHK1 inhibitor, SB-218078, reproduced the effect of DMAG. Administration of DMAG before doxorubicin resulted in G1-S arrest and protection from apoptosis, leading to additive or antagonistic interactions that were exacerbated by p53 mutation., Conclusions: Administration of DMAG to doxorubicin-primed cells induced premature mitosis and had a synergistic effect on apoptosis regardless of p53 status. These observations provide a rationale for prospective clinical trials and stress the need to consider schedule of exposure as a critical determinant of the overall response when DMAG is combined with chemotherapeutic agents for the treatment of patients with relapsed/refractory disease.