Amrita J. Desai

PD 0332991, a selective cyclin D kinase 4/6 inhibitor, preferentially inhibits proliferation of luminal estrogen receptor-positive human breast cancer cell lines in vitro

IntroductionAlterations in cell cycle regulators have been implicated in human malignancies including breast cancer. PD 0332991 is an orally active, highly selective inhibitor of the cyclin D kinases (CDK)4 and CDK6 with ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. To identify predictors of response, we determined the in vitro sensitivity to PD 0332991 across a panel of molecularly characterized human breast cancer cell lines.MethodsForty-seven human breast cancer and immortalized cell lines representing the known molecular subgroups of breast cancer were treated with PD 0332991 to determine IC50 values. These data were analyzed against baseline gene expression data to identify genes associated with PD 0332991 response.ResultsCell lines representing luminal estrogen receptor-positive (ER+) subtype (including those that are HER2 amplified) were most sensitive to growth inhibition by PD 0332991 while nonluminal/basal subtypes were most resistant. Analysis of variance identified 450 differentially expressed genes between sensitive and resistant cells. pRb and cyclin D1 were elevated and CDKN2A (p16) was decreased in the most sensitive lines. Cell cycle analysis showed G0/G1 arrest in sensitive cell lines and Western blot analysis demonstrated that Rb phosphorylation is blocked in sensitive lines but not resistant lines. PD 0332991 was synergistic with tamoxifen and trastuzumab in ER+ and HER2-amplified cell lines, respectively. PD 0332991 enhanced sensitivity to tamoxifen in cell lines with conditioned resistance to ER blockade.ConclusionsThese studies suggest a role for CDK4/6 inhibition in some breast cancers and identify criteria for patient selection in clinical studies of PD 0332991.

Lapatinib, a dual EGFR and HER2 kinase inhibitor, selectively inhibits HER2-amplified human gastric cancer cells and is synergistic with trastuzumab in vitro and in vivo.

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 <1. The combination also induced greater decreases in AKT and ERK activation, G0-G1 cell cycle arrest, and increased rates of apoptosis. In vivo studies showed that the combination of lapatinib and trastuzumab had greater antitumor efficacy than either drug alone. 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

Inhibition of HSP90 with AUY922 Induces Synergy in HER2-Amplified Trastuzumab-Resistant Breast and Gastric Cancer

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 (<40 nmol/L) for 59 of 61 cell lines. In both histologies, HER2-amplified cells expressed greater sensitivity to AUY than HER2-negative cells. In conditioned trastuzumab-resistant models, AUY922 showed a synergistic effect with trastuzumab. In vitro, the combination induced greater decreases in HER2, a G2 cell-cycle arrest, and increased apoptosis. In a trastuzumab-resistant gastric cancer xenograft model, the combination of AUY922 and trastuzumab showed greater antitumor efficacy than either drug alone. These data suggest that AUY922 in combination with trastuzumab has unique efficacy in trastuzumab-resistant models. The combination of HSP90 inhibition and direct HER2 blockade represents a novel approach to the treatment of HER2-amplified cancers and clinical trials based on the above data are ongoing. Mol Cancer Ther; 12(4); 509–19. ©2013 AACR.

Dacomitinib (PF-00299804), an Irreversible Pan-HER Inhibitor, Inhibits Proliferation of HER2-Amplified Breast Cancer Cell Lines Resistant to Trastuzumab and Lapatinib

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.

Identification of common predictive markers of in vitro response to the MEK inhibitor selumetinib (AZD6244; ARRY-142886) in human breast cancer and non-small cell lung cancer cell lines

Selumetinib (AZD6244; ARRY-142886) is a tight-binding, uncompetitive inhibitor of mitogen-activated protein kinase kinases (MEK) 1 and 2 currently in clinical development. We evaluated the effects of selumetinib in 31 human breast cancer cell lines and 43 human non–small cell lung cancer (NSCLC) cell lines to identify characteristics correlating with in vitro sensitivity to MEK inhibition. IC50 <1 μmol/L (considered sensitive) was seen in 5 of 31 breast cancer cell lines and 15 of 43 NSCLC cell lines, with a correlation between sensitivity and raf mutations in breast cancer cell lines (P = 0.022) and ras mutations in NSCLC cell lines (P = 0.045). Evaluation of 27 of the NSCLC cell lines with Western blots showed no clear association between MEK and phosphoinositide 3-kinase pathway activation and sensitivity to MEK inhibition. Baseline gene expression profiles were generated for each cell line using Agilent gene expression arrays to identify additional predictive markers. Genes associated with differential sensitivity to selumetinib were seen in both histologies, including a small number of genes in which differential expression was common to both histologies. In total, these results suggest that clinical trials of selumetinib in breast cancer and NSCLC might select patients whose tumors harbor raf and ras mutations, respectively. Mol Cancer Ther; 9(7); 1985–94. ©2010 AACR.

The HSP90 inhibitor NVP-AUY922 potently inhibits non-small cell lung cancer growth

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.

Antiestrogen fulvestrant enhances the antiproliferative effects of epidermal growth factor receptor inhibitors in human non-small-cell lung cancer.

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. ER&agr;, ER&bgr;, 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 ER&agr; 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.

Molecular subtype and response to dasatinib, an Src/Abl small molecule kinase inhibitor, in hepatocellular carcinoma cell lines in vitro

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)

Panobinostat (LBH589), a pan-DAC inhibitor, induces cell death in ER+ and HER2 amplified cell lines in vitro and is synergistic in vivo with trastuzumab.

Abstract #4047 Background: Histone modifications have been investigated as causing altered gene expression resulting in activation of oncogenic pathways. Panobinostat (LBH589) is a potent pan-deacetylase (DAC) inhibitor in clinical development. Given the potentially broad biological effects of this class of agent, we assessed the in vitro effects of panobinostat on a large panel of molecularly profiled human breast cancer cell lines to better define the differential effects of panobinostat across subtypes and to help guide clinical development. In addition, preclinical studies evaluated the effects of panobinostat and the HER2-targeted antibody, trastuzumab.
 Methods: 43 human cell lines representing known molecular subgroups of breast cancer, and 3 immortalized breast lines were treated in duplicate with panobinostat using two-fold dilutions over 12 concentrations. Dose response curves were generated using a cell count assay to calculate IC 50 and LD 50 of panobinostat. Data were analyzed against baseline gene expression data (Agilent microarray) to identify genes associated with sensitivity (S) and resistance (R) to panobinostat. 12 cell lines (6 S; 6 R) were then treated with clinically relevant concentrations of panobinostat; RNA was isolated after 48 hrs. Agilent expression profiles compared pre-and post-treatment cell lines to identify differentially expressed genes (DEGs) that change in S and R cell lines. In vivo , nude mice bearing HER2+ and pathway-dependent BT-474 xenografts were treated with panobinostat alone and in combination with trastuzumab. Tumor growth inhibition and delay were measured.
 Results: In vitro panobinostat has potent anti-proliferative effects across all breast cancer subtypes with most IC 50 values 50 50nM (Chi-square 50 >1000nM), which suggests some differential activity in malignant cell lines versus those that are more “normal”. When gene expression profiles were analyzed pre-and post-treatment, a remarkable overlap was seen in number and specific genes changing after panobinostat exposure in both S and R lines. However, the degree of gene changes was greater in S lines than R lines. In vivo , panobinostat acted synergistically with trastuzumab to inhibit growth of the HER2 amplified breast cancer cell line, BT-474.
 Conclusion: These studies suggest that ER+ and HER2 amplified breast cancers may be more likely to respond to the pan-DAC inhibitor panobinostat. Profiling studies identified 218 DEGs that were significantly changed between S and R lines. These data support the ongoing development of panobinostat in breast cancer and provide rationale for patient selection. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4047.

Abstract LB-70: In vitro anti-cancer activity of PF-04691502, a potent dual inhibitor of phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) in a panel of human breast cancer cell lines

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 IC 50 and LD 50 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 IC 50 values 50 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.