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PDF file, 66K, Quantification of proteins from Western blots in Supplementary Figure S7 by densitometry using ImageJ software.

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Supplementary Figure 2 PDF file - 91K, Speed of recovery of downstream targets in NCI-N87

Supplementary Table 1 PDF file - 69K, NVP-AUY922 IC50 concentrations and response to trastuzumab in HER2 amplified breast cancer cell lines

PDF file, 77K, Comparison of the effect of dacomitinib at 48 hrs and 5 days on cell cycle and apoptosis in BT-474 and SK-BR-3 lines.

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PDF file, 60K, Effect of dacomitinib on apoptosis in BT-474 and SK-BR-3 parental lines and lines resistant to trastuzumab and lapatinib.

PDF file, 68K, Quantification of proteins from Western blots in Figure 3 by densitometry using ImageJ software.

Supplementary Legends PDF file - 78K, Supplementary Figure Legends

PDF - 69K, Supplemental Figure Legend

The human EGF (HER) family of receptors has been pursued as therapeutic targets in breast cancer and other malignancies. Trastuzumab and lapatinib are standard treatments for HER2-amplified breast cancer, but a significant number of patients do not respond or develop resistance to these drugs. Here we evaluate the in vitro activity of dacomitinib (PF-00299804), an irreversible small molecule pan-HER inhibitor, in a large panel of human breast cancer cell lines with variable expression of the HER family receptors and ligands, and with variable sensitivity to trastuzumab and lapatinib. Forty-seven human breast cancer and immortalized breast epithelial lines representing the known molecular subgroups of breast cancer were treated with dacomitinib to determine IC50 values. HER2-amplified lines were far more likely to respond to dacomitinib than nonamplified lines (RR, 3.39; P < 0.0001). Furthermore, HER2 mRNA and protein expression were quantitatively associated with response. Dacomitinib reduced the phosphorylation of HER2, EGFR, HER4, AKT, and ERK in the majority of sensitive lines. Dacomitinib exerted its antiproliferative effect through a combined G0–G1 arrest and an induction of apoptosis. Dacomitinib inhibited growth in several HER2-amplified lines with de novo and acquired resistance to trastuzumab. Dacomitinib maintained a high activity in lines with acquired resistance to lapatinib. This study identifies HER2-amplified breast cancer lines as most sensitive to the antiproliferative effect of dacomitinib and provides a strong rationale for its clinical testing in HER2-amplified breast cancers resistant to trastuzumab and lapatinib. Mol Cancer Ther; 11(9); 1978–87. ©2012 AACR.

Heat shock protein 90 (HSP90) is involved in protein folding and functions as a chaperone for numerous client proteins, many of which are important in non–small cell lung cancer (NSCLC) pathogenesis. We sought to define preclinical effects of the HSP90 inhibitor NVP-AUY922 and identify predictors of response. We assessed in vitro effects of NVP-AUY922 on proliferation and protein expression in NSCLC cell lines. We evaluated gene expression changes induced by NVP-AUY922 exposure. Xenograft models were evaluated for tumor control and biological effects. NVP-AUY922 potently inhibited in vitro growth in all 41 NSCLC cell lines evaluated with IC50 < 100 nmol/L. IC100 (complete inhibition of proliferation) < 40 nmol/L was seen in 36 of 41 lines. Consistent gene expression changes after NVP-AUY922 exposure involved a wide range of cellular functions, including consistently decreased dihydrofolate reductase after exposure. NVP-AUY922 slowed growth of A549 (KRAS-mutant) xenografts and achieved tumor stability and decreased EGF receptor (EGFR) protein expression in H1975 xenografts, a model harboring a sensitizing and a resistance mutation for EGFR-tyrosine kinase inhibitors in the EGFR gene. These data will help inform the evaluation of correlative data from a recently completed phase II NSCLC trial and a planned phase IB trial of NVP-AUY922 in combination with pemetrexed in NSCLCs. Mol Cancer Ther; 12(6); 890–900. ©2013 AACR.

PDF - 769K, Heat maps from microarray analyses of gene expression changes seen in at least 13 of 16 experiments after exposure to NVP-AUY922

PDF file, 247K, The effects of dacomitinib on protein expression in lines resistant to trastuzumab and lapatinib.

Supplementary Figure 3 PDF file - 58K, Activation of HSP70 in both N87-Parental and N87-TRC cell lines

PDF file, 75K, Dacomitinib response is marginally inversely correlated with relative HER3 mRNA levels.

PDF file, 50K, Effect of dacomitinib on cell cycle in BT-474 and SK-BR-3 parental lines and lines resistant to trastuzumab and lapatinib.

PDF file, 45K, Dacomitinib response is associated with the total HER2 protein levels in HER2-amplified cell lines.

PDF file, 60K, Dacomitinib response is associated with relative HER2 mRNA levels.

PDF - 47B, A tabular demonstration of the efficacy and tolerability of NVP-AUY922 in 32 mice carrying subcutaneous A549 or H1975 xenograft tumors randomized

Hepatocellular carcinoma (HCC) is the fifth most common malignancy and is the third leading cause of cancer death worldwide. Recently, the multitargeted kinase inhibitor sorafenib was shown to be the first systemic agent to improve survival in advanced HCC. Unlike other malignancies such as breast cancer, in which molecular subtypes have been clearly defined (i.e., luminal, HER2 amplified, basal, etc.) and tied to effective molecular therapeutics (hormone blockade and trastuzumab, respectively), in HCC this translational link does not exist. Molecular profiling studies of human HCC have identified unique molecular subtypes of the disease. We hypothesized that a panel of human HCC cell lines would maintain molecular characteristics of the clinical disease and could then be used as a model for novel therapeutics. Twenty human HCC cell lines were collected and RNA was analyzed using the Agilent microarray platform. Profiles from the cell lines in vitro recapitulate previously described subgroups from clinical material. Next, we evaluated whether molecular subgroup would have predictive value for response to the Src/Abl inhibitor dasatinib. The results demonstrate that sensitivity to dasatinib was associated with a progenitor subtype. Dasatinib was effective at inducing cell cycle arrest and apoptosis in “progenitor-like” cell lines but not in resistant lines. Conclusion: These findings suggest that cell line models maintain the molecular background of HCC and that subtype may be important for selecting patients for response to novel therapies. In addition, it highlights a potential role for Src family signaling in this progenitor subtype of HCC. (HEPATOLOGY 2013)

HSP90 enables the activation of many client proteins of which the most clinically validated is HER2. NVP-AUY922, a potent HSP90 inhibitor, is currently in phase II clinical trials. To explore its potential clinical use in HER2-amplified breast and gastric cancers, we evaluated the effect of AUY922 alone and in combination with trastuzumab in both trastuzumab-sensitive and -resistant models. A panel of 16 human gastric and 45 breast cancer cell lines, including 16 HER2-amplified (3 and 13, respectively) cells, was treated with AUY922 over various concentrations. In both breast and gastric cancer, we used cell lines and xenograft models with conditioned trastuzumab-resistance to investigate the efficacy of AUY922 alongside trastuzumab. Effects of this combination on downstream markers were analyzed via Western blot analysis. AUY922 exhibited potent antiproliferative activity in the low nanomolar range (

Introduction Estrogen receptor (ER) signaling and its interaction with epidermal growth factor receptor (EGFR) is a potential therapeutic target in non–small-cell lung cancer (NSCLC). To explore cross-communication between ER and EGFR, we have correlated ER pathway gene and protein expression profiles and examined effects of antiestrogens with or without EGFR inhibitors in preclinical models of human NSCLC. Methods We evaluated 54 NSCLC cell lines for growth inhibition with EGFR inhibitors, antiestrogen treatment, or the combination. Each line was evaluated for baseline ER pathway protein expression. The majority were also evaluated for baseline ER pathway gene expression. Human NSCLC xenografts were evaluated for effects of inhibition of each pathway, either individually, or in combination. Results The specific antiestrogen fulvestrant has modest single agent activity in vitro, but in many lines, fulvestrant adds to effects of EGFR inhibitors, including synergy in the EGFR-mutant, erlotinib-resistant H1975 line. ERα, ERβ, progesterone receptor-A, progesterone receptor-B, and aromatase proteins are expressed in all lines to varying degrees, with trends toward lower aromatase in more sensitive cell lines. Sensitivity to fulvestrant correlates with greater baseline ERα gene expression. Tumor stability is achieved in human tumor xenografts with either fulvestrant or EGFR inhibitors, but tumors regress significantly when both pathways are inhibited. Conclusions These data provide a rationale for further investigation of the antitumor activity of combined therapy with antiestrogen and anti-EGFR agents in the clinic. Future work should also evaluate dual ER and EGFR inhibition in the setting of secondary resistance to EGFR inhibition.

The human EGF (HER) family of receptors has been pursued as therapeutic targets in breast cancer and other malignancies. Trastuzumab and lapatinib are standard treatments for HER2-amplified breast cancer, but a significant number of patients do not respond or develop resistance to these drugs. Here we evaluate the in vitro activity of dacomitinib (PF-00299804), an irreversible small molecule pan-HER inhibitor, in a large panel of human breast cancer cell lines with variable expression of the HER family receptors and ligands, and with variable sensitivity to trastuzumab and lapatinib. Forty-seven human breast cancer and immortalized breast epithelial lines representing the known molecular subgroups of breast cancer were treated with dacomitinib to determine IC50 values. HER2-amplified lines were far more likely to respond to dacomitinib than nonamplified lines (RR, 3.39; P < 0.0001). Furthermore, HER2 mRNA and protein expression were quantitatively associated with response. Dacomitinib reduced the phosphorylation of HER2, EGFR, HER4, AKT, and ERK in the majority of sensitive lines. Dacomitinib exerted its antiproliferative effect through a combined G0–G1 arrest and an induction of apoptosis. Dacomitinib inhibited growth in several HER2-amplified lines with de novo and acquired resistance to trastuzumab. Dacomitinib maintained a high activity in lines with acquired resistance to lapatinib. This study identifies HER2-amplified breast cancer lines as most sensitive to the antiproliferative effect of dacomitinib and provides a strong rationale for its clinical testing in HER2-amplified breast cancers resistant to trastuzumab and lapatinib. Mol Cancer Ther; 11(9); 1978–87. ©2012 AACR.

Purpose: HER2 amplification occurs in 18% to 27% of gastric and gastroesophageal junction cancers. Lapatinib, a potent ATP-competitive inhibitor simultaneously inhibits both EGFR and HER2. To explore the role of HER family biology in upper gastrointestinal cancers, we evaluated the effect of lapatinib, erlotinib, and trastuzumab in a panel of molecularly characterized human upper gastrointestinal cancer cell lines and xenografts. Experimental Design: EGFR and HER2 protein expression were determined in a panel of 14 human upper gastrointestinal cancer cell lines and HER2 status was assessed by fluorescent in situ hybridization. Dose-response curves were generated to determine sensitivity to lapatinib, erlotinib, and trastuzumab. In HER2-amplified cells, the combination of trastuzumab and lapatinib was evaluated using the median effects principal. The efficacy of lapatinib, trastuzumab, or the combination was examined in HER2-amplified xenograft models. Results: Lapatinib had concentration-dependent antiproliferative activity across the panel with the greatest effects in HER2-amplified cells. There was no association between EGFR protein expression and sensitivity to any of the HER-targeted agents. Cell cycle analysis revealed that lapatinib induced G1 arrest in sensitive lines and phosphorylated AKT and phosphorylated ERK were decreased in response to lapatinib as well. The combination of lapatinib and trastuzumab was highly synergistic in inhibiting cell growth with a combination index of Conclusion: Together, these data suggest that lapatinib has activity in HER2-amplified upper gastrointestinal cancer and supports the ongoing clinical investigation of lapatinib in patients with HER2-amplified disease. Clin Cancer Res; 16(5); 1509–19

PD-0332991 is an inhibitor of cyclin-dependent kinases (CDK) 4 and 6, and was evaluated to determine its anti-proliferative effects in 25 renal cell carcinoma (RCC) cell lines.Half-maximal inhibitory concentrations (IC50) of PD-0332991 were determined with cell line proliferation assays, as were its effects on the cell cycle, apoptosis, and retinoblastoma (RB) phosphorylation. Molecular markers for response prediction, including p16, p15, cyclin D1 (CCND1), cyclin E1 (CCNE1), E2F transcription factor 1 (E2F1), RB, CDK4 and CDK6, were studied using array comparative genomic hybridization (CGH) and gene expression.IC50 values for PD-0332991 ranged from 25.0 nM to 700 nM, and the agent demonstrated G0/G1 cell-cycle arrest, induction of late apoptosis, and blockade of RB phosphorylation. Through genotype and expression data p16, p15 and E2F1 were identified as having significant association between loss and sensitivity to PD-0332991: p16 (p=0.021), p15 (p=0.047), and E2F1 (p=0.041).PD-0332991 has antiproliferative activity in RCC cell lines, and molecular markers predict for sensitivity to this agent.

Heat shock protein 90 (HSP90) is involved in protein folding and functions as a chaperone for numerous client proteins, many of which are important in non–small cell lung cancer (NSCLC) pathogenesis. We sought to define preclinical effects of the HSP90 inhibitor NVP-AUY922 and identify predictors of response. We assessed in vitro effects of NVP-AUY922 on proliferation and protein expression in NSCLC cell lines. We evaluated gene expression changes induced by NVP-AUY922 exposure. Xenograft models were evaluated for tumor control and biological effects. NVP-AUY922 potently inhibited in vitro growth in all 41 NSCLC cell lines evaluated with IC50 < 100 nmol/L. IC100 (complete inhibition of proliferation) < 40 nmol/L was seen in 36 of 41 lines. Consistent gene expression changes after NVP-AUY922 exposure involved a wide range of cellular functions, including consistently decreased dihydrofolate reductase after exposure. NVP-AUY922 slowed growth of A549 (KRAS-mutant) xenografts and achieved tumor stability and decreased EGF receptor (EGFR) protein expression in H1975 xenografts, a model harboring a sensitizing and a resistance mutation for EGFR-tyrosine kinase inhibitors in the EGFR gene. These data will help inform the evaluation of correlative data from a recently completed phase II NSCLC trial and a planned phase IB trial of NVP-AUY922 in combination with pemetrexed in NSCLCs. Mol Cancer Ther; 12(6); 890–900. ©2013 AACR.

Background: Heat Shock Protein 90 (HSP90) enables the activation of a large number of client proteins of which the most clinically validated is HER2. NVP-AUY922 is a a highly potent, non-geldamycin HSP90 inhibitor that has shown preliminary activity in HER2 positive metastatic breast cancer. To explore its potential clinical utility specifically in HER2 amplified gastric and breast cancers, we evaluated the effect of AUY922 alone and in combination with trastuzumab in both trastuzumab-sensitive and resistant in vitro and in vivo models. Methods: A panel of 16 human gastric and 45 breast cancer cell lines, with 3 and 13 HER2-amplified lines respectively, were exposed in vitro to AUY922 over various concentrations to generate dose response curves. In both breast and gastric cancer, we used cell lines and xenograft models with conditioned trastuzumab-resistance to investigate the efficacy of AUY922 alongside trastuzumab. In all in vivo experiments, trastuzumab was dosed at 10 mg/kg (IP; twice/week) and AUY922 at 50 mg/kg, (IP; 5 days on, 2 days off). Effects of this combination on downstream markers (HER2, AKT, ERK) were analyzed via western blot. Results: AUY922 was found to have potent anti-proliferative activity in the low nanomolar range (< 40 nM) for 59 of the 61 gastric and breast cancer cell lines tested. Additionally, in both gastric and breast cancer, HER2-amplified cells expressed greater sensitivity to the compound when compared to the HER2-negative cells. In conditioned trastuzumab-resistant cell line models, AUY922 continued to have potent activity and exhibited a synergistic effect with trastuzumab. The in vitro combination also induced greater decreases in HER2, induced a G0/G1 cell cycle arrest, and increased the rates of apoptosis. In a conditioned trastuzumab-resistant gastric cancer in vivo model (N87-Res), the combination of AUY922 and trastuzumab showed greater anti-tumor efficacy than either drug alone. Conclusions: These data suggest that AUY922 in combination with trastuzumab has unique efficacy in trastuzumab-resistant models. The combination of HSP90 inhibitors and trastuzumab represents a novel approach to the treatment of HER2 amplified cancers. Clinical trials combining AUY922 and trastuzumab in HER2 amplified breast and gastric cancers are currently ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3668. doi:1538-7445.AM2012-3668

Introduction: Activating mutations in the PIK3CA gene (encoding for the P110 catalytic subunit of PI3K) are found in more than 25% of human breast cancers [Saal LH Cancer Res 2005]. PF-04691502 is a potent orally-available ATP-competitive and reversible dual inhibitor of PI3K and mTOR. This study evaluates the anti-proliferative and cytotoxic activity of PF-04691502 across a large panel of breast cancer cell lines that recapitulate the clinical spectrum of the disease. Methods: Over 40 human breast cancer cell lines were exposed in vitro to PF-0469102 over 9 concentrations to generate dose-response curves. These assays were performed over a 6-day incubation period using direct cell counts. The ability of PF-04691502 to inhibit proliferation and induce cell death was calculated using IC50 and LD50 values, respectively. PI3K mutation status was available from public databases. In addition, baseline gene expression profiles were generated using the Agilent platform. A subset of breast cancer cell lines with varying levels of sensitivity to the compound were incubated with PF-04691502 concentrations ranging from 10 to 1000 nM, and processed for Western blotting. Results: Twenty-two cell lines had IC50 values < 100 nM, with a trend towards increased sensitivity to PF-04691502 in breast cancer cell lines harboring PIK3CA mutations, and in those belonging to the luminal subtype. Lethality (LD50 < 1 M) was observed in 10 out of 12 breast cancer cell lines harboring PI3KCA mutations, and was prevalent in cell lines of luminal subtype. Inhibition of the downstream target pAKT was observed. Conclusions: PF-04691502 has anti-cancer activity across a spectrum of breast cancer cell lines, and demonstrates lethality against a subset of cell lines. A first-in-human phase I trial of PF-04691502 is ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-70.

Abstract #5064 Background: Cell cycle dysregulation is a common molecular finding in malignancy and the cyclin-D kinases (CDK) represent an attractive target in this pathway. PD 0332991 (Pfizer Inc.) is an orally active potent and highly selective inhibitor of CDK 4 and CDK6 kinases with the ability to block pRb phosphorylation at serine 780 and 795 in the low nanomolar range. To identify predictors of response to PD 0332991, we determined the in vitro sensitivity to PD 0332991 of a large panel of molecularly characterized breast cancer cell lines, and then used baseline expression profiles to determine genes associated with response. Methods: 44 human breast cancer cell lines representing the known molecular subgroups of breast cancer (i.e. luminal, HER2, basal, etc) and 3 immortalized breast lines were treated in duplicate with PD 0332991 using two-fold dilutions over 12 concentrations. Dose response curves were generated using a cell count assay to calculate the IC50 across the panel. These data were then analyzed against baseline gene expression data (Agilent microarray) to identify genes associated with sensitivity and resistance to PD 0332991. ANOVA analysis identified genes associated with response and/or resistance to PD 0332991. Western blot analysis was performed analyzing the effects of PD 0332991 on pRb phosphorylation. Cell cycle analysis was performed using NIM-DAPI staining and flow-cytometry. In addition, combination studies were performed to analyze the interaction between PD 0332991 and tamoxifen and trastuzumab. These data were analyzed using Calcusyn software to generate a combination index (CI) to define the interaction as additive (CI=1), synergisitc (CI1). Results: Cell lines representing the luminal ER+ subtype (including HER2 amplified) were most sensitive to inhibition by PD 0332991 and non-luminal/basal (triple-negative) were most resistant. ANOVA analysis identified 563 differentially expressed genes between the sensitive and resistant groups. While, several of these genes are associated with breast cancer subtype, pRb was elevated and CDKN2A (p16) was decreased in the most sensitive lines. Cell lines treated with PD 0332991 showed clear G0/G1 arrest and a decrease in S-phase fraction in sensitive but not in resistant cell lines. In addition, Western blot showed that pRb phosphorylation is blocked in a time dependent manner. Finally, the combination of tamoxifen and PD 0332991 was strongly synergistic in three ER+ cell lines evaluated and appeared synergistic with trastuzumab as well. Conclusion: These studies suggest that a subgroup of breast cancers may be more likely to benefit from treatment with the PD 0332991 CDK4/6 inhibitor than others. This group represents ER+ luminal breast cancer and is associated with elevated pRb and low p16. The combination of PD 0332991 and tamoxifen and trastuzumab shows promising biologic activity in ER+ and HER2 amplified breast cancer cell lines, respectively. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5064.