NVP-AUY922: a small molecule HSP90 inhibitor with potent antitumor activity in preclinical breast cancer models

INTRODUCTION:Heat shock protein 90 (HSP90) is a key component of a multichaperone complex involved in the post-translational folding of a large number of client proteins, many of which play essential roles in tumorigenesis. HSP90 has emerged in recent years as a promising new target for anticancer therapies.METHODS:The concentrations of the HSP90 inhibitor NVP-AUY922 required to reduce cell numbers by 50% (GI50 values) were established in a panel of breast cancer cell lines and patient-derived human breast tumors. To investigate the properties of the compound in vivo, the pharmacokinetic profile, antitumor effect, and dose regimen were established in a BT-474 breast cancer xenograft model. The effect on HSP90-p23 complexes, client protein degradation, and heat shock response was investigated in cell culture and breast cancer xenografts by immunohistochemistry, Western blot analysis, and immunoprecipitation.RESULTS:We show that the novel small molecule HSP90 inhibitor NVP-AUY922 potently inhibits the proliferation of human breast cancer cell lines with GI50 values in the range of 3 to 126 nM. NVP-AUY922 induced proliferative inhibition concurrent with HSP70 upregulation and client protein depletion � hallmarks of HSP90 inhibition. Intravenous acute administration of NVP-AUY922 to athymic mice (30 mg/kg) bearing subcutaneous BT-474 breast tumors resulted in drug levels in excess of 1,000 times the cellular GI50 value for about 2 days. Significant growth inhibition and good tolerability were observed when the compound was administered once per week. Therapeutic effects were concordant with changes in pharmacodynamic markers, including HSP90-p23 dissociation, decreases in ERBB2 and P-AKT, and increased HSP70 protein levels.CONCLUSION:NVP-AUY922 is a potent small molecule HSP90 inhibitor showing significant activity against breast cancer cells in cellular and in vivo settings. On the basis of its mechanism of action, preclinical activity profile, tolerability, and pharmaceutical properties, the compound recently has entered clinical phase I breast cancer trials

Identification of piggyBac transposon insertions in malignant tumors

In the last decades, many important genes responsible for the genesis of various cancers have been discovered, their mutations precisely identified, and the pathways through which they act characterized. Nevertheless the wide range of possible genetic alterations, their functional relevance, their downstream targets or effector pathways represent a major hindrance to the therapeutical success. Genetic instability and heterogeneity in cancer vary between the different types and stages, tissues and individuals. While there has been considerable progress in understanding the impact of genetic mechanisms in tumorigenesis, large efforts must be made to develop experimental strategies to understand these cellular processes. The current challenge in the oncology field is to develop efficient tools to try to identify gene responsible for tumorigenesis processes to treat human cancers. Here we describe the use of piggyBac transposon system to identify driver genes in cancer. Transgenic mice carrying several transposon copies able to randomly hop around the genome (RosaPB/+; ATP1/+) were generated. ATP1 transposon can confer proliferative advantage by activating or repressing genes expression. To accelerate the disease onset, we added a sensitizing mutation (Arf-/-) and studied mice with the three genetic components. After aging of mice, tumors developed due to Arf-/- and PB insertional mutagenesis, and were harvested. To create a bank with a broad range of tumor models, harvested tumors were passaged and expanded in recipient mice. Interestingly, our ATP1 DNA copy numbers quantification often showed gain of ATP1 in tumors compared to normal tissue, suggesting PB insertional mutagenesis is favorable for tumors. Genes of interest were identified by deep sequencing of amplicons of DNA fragments that contain extremities of ATP1 sequences. Statistical analysis of transposon integration sites in tumor genomes identified Braf gene as target in almost all RosaPB/+; ATP1/+; Arf-/- tumors, suggesting a genetic cooperation between Arf deletion and Braf activation. Other genes are now evaluated. Here we have developed a powerful in vivo genomic tool allowing the identification of genes driving cancer

Hsp90 inhibitors: Clinical development and future opportunities in oncology therapy

The development of small molecular mass Hsp90 inhibitors has become an increasingly competitive field of research in recent years. Progress in preclinical and clinical research has provided increasing evidence that Hsp90 represents a promising molecular target for the treatment of cancer and other diseases. Although many challenges remain, recent clinical trial results for the most clinically advanced inhibitors indicate that clinical proof of concept in oncology therapy is achievable, and that Hsp90 inhibitors have the potential to contribute to the collection of anticancer treatments available to clinicians and patients in the near future

E2F7, a novel E2F featuring DP-independent repression of a subset of E2F-regulated genes

The E2F family of transcription factors play an essential role in the regulation of cell cycle progression. In a screen for E2F-regulated genes we identified a novel E2F family member, E2F7. Like the recently identified E2F-like proteins of Arabidopsis, E2F7 has two DNA binding domains and binds to the E2F DNA binding consensus site independently of DP co-factors. Consistent with being an E2F target gene, we found that the expression of E2F7 is cell cycle regulated. Ectopic expression of E2F7 results in suppression of E2F target genes and accumulation of cells in G(1). Furthermore, E2F7 associates with E2F-regulated promoters in vivo, and this association increases in S phase. Interestingly, however, E2F7 binds only a subset of E2F-dependent promoters in vivo, and in agreement with this, inhibition of E2F7 expression results in specific derepression of these promoters. Taken together, these data demonstrate that E2F7 is a unique repressor of a subset of E2F target genes whose products are required for cell cycle progression

Deciphering mechanisms of response and resistance in large-scale mouse cancer screens

Acquired resistance is a major limitation for the successful treatment of cancer patients. Although numerous efficacious cancer therapeutics have been developed in the past decades, resistance arises due to a variety of reasons including tumoral genetic alterations, or modulation of factors in the tumor environment. Understanding the mechanistic reasons for tumor relapse supports the identification of novel combination therapies that could lead to more durable responses. Here, we will review large-scale in vivo screens in pre-clinical cancer models that employed genetic and pharmacological agents toward elucidating acquired drug resistance and informing on beneficial combinations to be tested in clinical trials

Evaluation of the Hsp90 inhibitor NVP-AUY922 in multicellular tumour spheroids with respect to effects on growth and PET tracer uptake

BACKGROUND: Molecular targeting has become a prominent concept in cancer treatment and heat shock protein 90 (Hsp90) inhibitors are suggested as promising anticancer drugs. The Hsp90 complex is one of the chaperones that facilitate the refolding of unfolded or misfolded proteins and plays a role for key oncogenic proteins such as Her2, Raf-1, Akt/PKB, and mutant p53. NVP-AUY922 is a novel low-molecular Hsp90 inhibitor, currently under clinical development as an anticancer drug. Disruption of the Hsp90-client protein complexes leads to proteasome-mediated degradation of client proteins and cell death. The aim of the current study was to use a combination of the multicellular tumour spheroid (MTS) model and positron emission tomography (PET) to investigate the effects of NVP-AUY922 on tumour growth and its relation to PET tracer uptake for the selection of appropriate PET tracer. A further aim was to evaluate the concentration and time dependence in the relation between growth inhibition and PET tracer uptake as part of translational imaging activities. METHODS: MTS of two breast cancer cell lines (MCF-7 and BT474), one glioblastoma cell line (U87MG) and one colon carcinoma cell line (HCT116) were prepared. Initially, we investigated MTS growth pattern and (3)H-thymidine incorporation in MTS after continuous exposure to NVP-AUY922 in order to determine dose response. Then the short-term effect of the drug on the four PET tracers 2-[(18)F] fluoro-2-deoxyglucose (FDG), 3'-deoxy-3'-fluorothymidine (FLT), methionine and choline was correlated to the long-term effect (changes in growth pattern) to determine the adequate PET tracer with high predictability. Next, the growth inhibitory effect of different dose schedules was evaluated to determine the optimal dose and time. Finally, the effect of a 2-h exposure to the drug on growth pattern and FDG/FLT uptake was evaluated. RESULTS: A dose-dependent inhibition of growth and decrease of (3)H-thymidine uptake was observed with 100% growth cessation in the dose range 7-52 nM and 50% (3)H-thymidine reduction in the range of 10-23 nM, with the most pronounced effect on BT474 cells. The effect of the drug was best detected by FLT. The results suggested that a complete cessation of growth of the viable cell volume was achieved with about 50% inhibition of FLT uptake 3 days after continuous treatment. Significant growth inhibition was observed at all doses and all exposure time spans. Two-hour exposure to NVP-AUY922 generated a growth inhibition which persisted dose dependently up to 10 days. The uptake of FDG per viable tumour volume was reduced by just 25% with 300 nM treatment of the drug, whereas the FLT uptake decreased up to 75% in correlation with the growth inhibition and recovery. CONCLUSIONS: Our results indicate a prolonged action of NVP-AUY922 in this cell culture, FLT is a suitable tracer for the monitoring of the effect and a FLT PET study within 3 days after treatment can predict the treatment outcome in this model. If relevant in vivo, this information can be used for efficient planning of animal PET studies and later human PET trial

Preclinical Activity of the Novel Orally Bioavailable HSP90 Inhibitor NVP-HSP990 against Multiple Myeloma Cells

BACKGROUND: HSP90 inhibitors effectively reduce expression and activity levels of oncogenic survival proteins. However, their clinical anti-multiple myeloma (MM) activity has been found to be rather weak, spurring the exploration of combination therapies and development of compounds with improved physicochemical properties. MATERIALS AND METHODS: Preclinical effects of the novel orally bioavailable HSP90 inhibitor NVP-HSP990 on the viability, apoptosis and client protein levels of MM cells (established cell lines and clinical specimens) were tested alone and in combination with other drugs. RESULTS: NVP-HSP990 exerted profound activity against MM cells, with a molecular mode of action conforming well with its role as HSP90 inhibitor. Enhanced activity was most obvious in combination with melphalan. Combination with a phosphatidylinositol-3-kinase (PI3-kinase)/mammalian target of rapamycin (mTOR) inhibitor, rendered the HSP90 blockade-mediated stress response ineffective and considerably increased the anti-MM toxicity. CONCLUSION: Given the current interest in both HSP90 and PI3-kinase/mTOR as potential clinical targets, these observations could broaden the therapeutic utility of either class of inhibitor in MM