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

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Pharmacokinetic/pharmacodynamic analysis of NVP-AUY922 in BT-474 tumor-bearing nude mice

A pharmacokinetic profile of NVP-AUY922 in BT-474 tumor xenografts, plasma, and organs (liver, heart, lung, and muscle) after administration of a single dose is shown. Female athymic mice bearing subcutaneous xenotransplants of the human ductal breast carcinoma BT-474 of approximately 250 mmreceived a single dose of 30 mg/kg of NVP-AUY922 (intravenous) at 0 hours. NVP-AUY922 concentrations were determined by HPLC/MS-MS analysis using an internal standard method. The limits of quantification for plasma and tissues were set to 4.0 ng/mL and 10 ng/g, respectively. Bars represent standard error of the mean (n = 4). Pharmacodynamic analysis in BT-474 xenografts following a single NVP-AUY922 dose. The amount of p23 associated with HSP90 was determined by immunoprecipitating HSP90 followed by immunoblotting for p23. The levels of AKT, phosphorylated AKT and β-tubulin were determined by immunoblotting. HPLC/MS-MS, high-pressure liquid chromatography/tandem mass spectrometry; HSP90, heat shock protein 90; IP, immunoprecipitation.<p><b>Copyright information:</b></p><p>Taken from "NVP-AUY922: a small molecule HSP90 inhibitor with potent antitumor activity in preclinical breast cancer models"</p><p>http://breast-cancer-research.com/content/10/2/R33</p><p>Breast Cancer Research : BCR 2008;10(2):R33-R33.</p><p>Published online 22 Apr 2008</p><p>PMCID:PMC2397535.</p><p></p

Analysis of the kinetics of HSP70 induction and ERBB2 degradation following a singe dose of NVP-AUY922

BT-474 tumor-bearing animals were administered 50 mg/kg NVP-AUY922 at 0 hours. The protein levels of HSP70 were determined by immunohistochemistry during the following week. The quantification of the protein levels of HSP70. ERBB2 protein levels were determined by immunohistochemistry and Western blot analysis. The lowest single dose of NVP-AUY922 which mediates HSP90-p23 dissociation and reduced AKT phosphorylation was determined by immunoprecipitation and Western blot analysis. HSP70, heat shock protein 70; HSP90, heat shock protein 90.<p><b>Copyright information:</b></p><p>Taken from "NVP-AUY922: a small molecule HSP90 inhibitor with potent antitumor activity in preclinical breast cancer models"</p><p>http://breast-cancer-research.com/content/10/2/R33</p><p>Breast Cancer Research : BCR 2008;10(2):R33-R33.</p><p>Published online 22 Apr 2008</p><p>PMCID:PMC2397535.</p><p></p

Protein expression analysis in a panel of human breast cancer cell lines and effect of NVP-AUY922 on the HSP90-p23 complex in BT-474 cells

The expression of HSP90 and specific proteins affected by HSP90 inhibition (Her3 [EGFR3], Her2 [ErbB2, EGFR2], phospho Her2 [pHer2], EGFR, AKT [PKB], phospho-AKT [pAkt], estrogen receptor-alpha [ERa], PI3K [p110α], PDK1, HSP70, Hsc70, Rb, pMEK1/2, pERK, Bax, Bcl-2, Bad, and Bcl-XL) was analyzed in seven human breast cancer cell lines (BT20, BT-474, MDA-MB-157 [MB-157], MDA-MB-231 [MB-231], MDA-MB-468 [MB-468], SKBr3, and MCF-7) by Western blot analysis. The ERBB2-overexpressing and estrogen receptor-positive cell line BT-474 was chosen for studies of the kinetics and concentration-dependent dissociation of HSP90-p23 complexes and client proteins in the presence of NVP-AUY922 or tanespimycin (17-AAG). The amount of p23 associated with HSP90 was determined by immunoprecipitating p23 followed by immunoblotting for HSP90. The levels of ERBB2, AKT, phosphorylated AKT and β-tubulin were determined by immunoblotting. DMSO, dimethyl sulfoxide; HSP90, heat shock protein 90.<p><b>Copyright information:</b></p><p>Taken from "NVP-AUY922: a small molecule HSP90 inhibitor with potent antitumor activity in preclinical breast cancer models"</p><p>http://breast-cancer-research.com/content/10/2/R33</p><p>Breast Cancer Research : BCR 2008;10(2):R33-R33.</p><p>Published online 22 Apr 2008</p><p>PMCID:PMC2397535.</p><p></p

Antitumor effect and tolerability of NVP-AUY922 in a BT-474 human breast cancer xenograft model

BT-474 cells were inoculated subcutaneously in female nude mice carrying an estrogen-release pellet. When the tumors reached 100 to 200 mm, drug treatment was initiated. Each group consisted of eight animals. NVP-AUY922 was administered as a single injection, three times per week (3qw), or once per week (qw) at the indicated dose levels. Tumor volumes and body weights were measured three times per week. Each point represents the mean ± standard error of the mean (SEM). Arrows in right panels indicate NVP-AUY922 treatment days. Asterisks indicate statistical significance compared with vehicle-treated controls (< 0.05, one-way analysis of variance Dunnett). i.v., intravenous.<p><b>Copyright information:</b></p><p>Taken from "NVP-AUY922: a small molecule HSP90 inhibitor with potent antitumor activity in preclinical breast cancer models"</p><p>http://breast-cancer-research.com/content/10/2/R33</p><p>Breast Cancer Research : BCR 2008;10(2):R33-R33.</p><p>Published online 22 Apr 2008</p><p>PMCID:PMC2397535.</p><p></p

Calcium-activated chloride channel ANO1 promotes breast cancer progression by activating EGFR and CAMK signaling

The calcium-activated chloride channel anoctamin 1 (ANO1) is located within the 11q13 amplicon, one of the most frequently amplified chromosomal regions in human cancer, but its functional role in tumorigenesis has remained unclear. The 11q13 region is amplified in ̄15% of breast cancers. Whether ANO1 is amplified in breast tumors, the extent to which gene amplification contributes to ANO1 overexpression, and whether overexpression of ANO1 is important for tumor maintenance have remained unknown. We have found that ANO1 is amplified and highly expressed in breast cancer cell lines and primary tumors. Amplification of ANO1 correlatedwith disease grade and poor prognosis. Knockdown of ANO1 in ANO1-amplified breast cancer cell lines and other cancers bearing 11q13 amplification inhibited proliferation, induced apoptosis, and reduced tumorgrowth inestablishedcancer xenografts.Moreover,ANO1chloride channel activity was important for cell viability. Mechanistically, ANO1 knockdown or pharmacological inhibition of its chloride-channel activity reduced EGF receptor (EGFR) and calmodulin-dependent protein kinase II (CAMKII) signaling, which subsequently attenuated AKT, v-src sarcoma viral oncogene homolog (SRC), and extracellular signal-regulated kinase (ERK) activation in vitro and in vivo. Our results highlight the involvement of the ANO1 chloride channel in tumor progression and provide insights into oncogenic signaling in human cancerswith 11q13 amplification, thereby establishing ANO1 as a promising target for therapy in these highly prevalent tumor types

Identification of a novel NAMPT inhibitor by CRISPR/Cas9 chemogenomic profiling in mammalian cells

Chemogenomic profiling is a powerful and unbiased approach to elucidate targets and mechanism of bioactive compounds. Until recently, high-quality experiments of this nature have been limited to fungal systems due to lack of mammalian genome-wide deletion collections. Here we show that the CRISPR/Cas9 system enables the generation of such libraries and allows for the identification of targets and pathways mediating hypersensitivity and resistance relevant to the compound mechanism of action, using a novel NAMPT inhibitor as an example

Calcium-activated chloride channel ANO1 promotes breast cancer progression by activating EGFR and CAMK signaling

The calcium-activated chloride channel anoctamin 1 (ANO1) is located within the 11q13 amplicon, one of the most frequently amplified chromosomal regions in human cancer, but its functional role in tumorigenesis has remained unclear. The 11q13 region is amplified in ¯15% of breast cancers. Whether ANO1 is amplified in breast tumors, the extent to which gene amplification contributes to ANO1 overexpression, and whether overexpression of ANO1 is important for tumor maintenance have remained unknown. We have found that ANO1 is amplified and highly expressed in breast cancer cell lines and primary tumors. Amplification of ANO1 correlatedwith disease grade and poor prognosis. Knockdown of ANO1 in ANO1-amplified breast cancer cell lines and other cancers bearing 11q13 amplification inhibited proliferation, induced apoptosis, and reduced tumorgrowth inestablishedcancer xenografts.Moreover,ANO1chloride channel activity was important for cell viability. Mechanistically, ANO1 knockdown or pharmacological inhibition of its chloride-channel activity reduced EGF receptor (EGFR) and calmodulin-dependent protein kinase II (CAMKII) signaling, which subsequently attenuated AKT, v-src sarcoma viral oncogene homolog (SRC), and extracellular signal-regulated kinase (ERK) activation in vitro and in vivo. Our results highlight the involvement of the ANO1 chloride channel in tumor progression and provide insights into oncogenic signaling in human cancerswith 11q13 amplification, thereby establishing ANO1 as a promising target for therapy in these highly prevalent tumor types