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OncoPanel Poster Aacr 2008

OncoPanel Poster Aacr 2008






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    OncoPanel Poster Aacr 2008 OncoPanel Poster Aacr 2008 Presentation Transcript

    • ASSESSING CANCER THERAPEUTIC AGENTS ACROSS A FIFTEEN HUMAN TUMOR CELL LINE PANEL Ovechkina, Y.Y., Nguyen, P.T.B., Keyser, R.F., Shively, R.D., Marcoe, K.F. and O’Day, C.O. MDS Pharma Services INTRODUCTION Lack of Correlation Between Cell Growth Rate and EC50 Cell Cycle Changes Precede Apoptosis Induction in OncoPanel Cell Lines Treated OncoPanel In Vitro Selectivity for EGFR and Bcr-Abl Inhibitors OncoPanel Anticancer Profiling Assay Continuous Culture Cells vs Cryo-preserved Cells with Staurosporine or Taxol AG1478, EGFR inhibitor Bcr-Abl inhibitor Cell line, Continuous Culture HT29 K562 MCF-7 NCI H1299 NCI H23 NCI H82 RPMI 8226 SKMEL28 SW48 T47D Growth rate vs EC50 The US National Cancer Institute (NCI) panel of 60 human tumor cell lines has Data Analysis For HCS 12 bit tiff images were acquired using the InCell Analyzer Cell Line # Doublings in 72 hrs Log (EC50) microM 4 5 a) sensitive resistant b) sensitive resistant become a widely used resource for in vitro anticancer drug discovery. The 1000 3.2 and analyzed with Developer Toolbox 1.6 software. EC50 and IC50 values THP1 1.55 0.1 1.3 3 4 RPMI 8226 SW48 Etoposide, EC50 microM 1.72 0.12 0.38 1.17 0.21 0.27 0.02 2.01 0.05 0.06 average ratio NCIH82 A431 average ratio T47D 1.74 1.0 extensive profiling of these cell lines now includes information on cell line specific were calculated using nonlinear regression to fit data to a sigmoidal 4 point, 4 MCF7 1.93 0.8 1.1 2 3 MCF7 K562 NCIH23 A375 THP1 Doubling time, hrs 19 20 30 19 39 24 35 41 31 46 HCT116 cancer gene mutations that are known to render cells carrying them more sensitive parameter One-Site dose response model, where: y (fit) = A + [(B – A)/(1 + ((C/x) MDA MB 468 1.96 1.0 2 NCIH1299 NCIH1299 Log (EC50) microM 1 HT29 NCIH82 ^ D))]. Curve-fitting and EC50 / IC50 calculations were performed using XLFit™ 0.9 A375 MDA MB 468 to chemotherapeutic agents. A powerful attribute of cell-based screening NCIH23 SKMEL28 2.16 2.19 0.4 1.0 0 1 THP1 SKMEL28 SKMEL28 T47D Cell line, Cryo -preserved HT29 K562 MCF7 NCI H1299 NCI H23 NCI H82 RPMI 8226 SKMEL28 SW48 T47D 0 with multiple tumor cell lines is these cells demonstrate diverse sensitivities to software (IDBS) or MathIQ based software. RPMI 8226 2.31 0.5 0.7 24 hrs 72 hrs 24 hrs 72 hrs NCIH23 T47D MDA MB 468 MCF7 HT29 HCT116 Etoposide, EC50 microM 1.55 0.28 0.43 0.72 0.27 0.46 0.06 2.45 0.05 0.16 anticancer agents. The patterns of relative drug sensitivity and resistance found NCIH82 SW48 2.74 3.15 0.6 0.9 0.5 [mitosis] / [cell count EC50] [apoptosis] / [cell count EC50] [mitosis] / [cell count EC50] [apoptosis] / [cell count EC50] SW48 A431 RPMI 8226 K562 with standard anticancer drugs across different tumor cell lines with defined Measured Parameters Cell proliferation was measured by the signal intensity Doubling time, hrs 18 21 37 19 33 23 31 33 26 41 -1.00 -0.75 -0.50 -0.25 0.00 0.50 1.00 -0.50 0.25 0.75 -1.00 -0.75 -0.25 0.00 0.50 1.00 0.25 0.75 NCIH1299 3.27 0.9 onocogenic mutations have been shown to reflect mechanisms of drug action. A431 3.36 1.1 0.3 Staurosporine Taxol of the incorporated nuclear dye. The cell proliferation output was referred to K562 3.39 1.3 ∆ log (average EC50 ) µM ∆ log (average EC50 ) µM The relative cell count EC50 (half maximal effective concentration) measures cell proliferation. Growth rate is We have assembled a 15 human tumor cell line panel to examine mechanisms as the relative cell count. To determine the cell proliferation end point, the cell A375 3.45 0.9 0.1 Average concentration ratio for apoptosis induction to cell proliferation EC 50 in treated cells at 24 and 72 hr time points across all 15 reflected as the time required for the cell population to double once. of cytotoxicity. The cell line panel was assembled with 5 common human tumor proliferation data output was transformed to percent of control (POC) using the HT29 4.07 0.3 1.6 3 4.4 OncoPanel cell lines is shown in red: [Apoptosis] / [Cell proliferation EC50]. Average concentration ratio for cell cycle change to cell a) A431 cell line, known to over express the EGF receptor, showed the greatest sensitivity to AG 1478. b) K562 cell line, known to have proliferation EC50 in treated cells at 24 and 72 hr time points across all 15 OncoPanel cell lines is shown in blue: [Mitosis] / [Cell prolif- types including breast, lung, colon, skin and leukemia. Cell lines were chosen based following formula: HCT116 4.46 0.4 # Doublings in 72 hrs CONCLUSIONS eration EC50]. activated BCR-Abl mutation, demonstrated a ten-fold lower EC50 for cell proliferation inhibition and apoptosis induction response. on available published mutation profiling data (Sanger Institute) and represent the major mutations occurring in cancer genes. Standard cancer therapeutic The relative cell count EC50 (cell proliferation parameter) value was measured in 5-FU treated OncoPanel Percent of Control = relative cell count (compound wells) x 100 cell lines. EC50 values did not correlate with cell growth rate (# Doublings/72hrs), correlation coefficient = agents were tested for proliferative, apoptotic and cell cycle arrest responses More Sensitive Apoptosis Detection at the 72 Hour Time Point Methotrexate Treatment OncoPanel Breast Cancer Cell lines: T47D and MDA MB relative cell count (vehicle wells) 0.003. The integration of mutation profiling data (Sanger Institute) with the cellular using multiplexed high content screening (HCS) with automated fluorescence 468 Resistant and MCF-7 Sensitive response phenotypes generated with this multiparametric OncoPanel profiling microscopy and image analysis based technology (GE Healthcare INCell Analyzer assay allowed investigation of mechanisms of enhanced susceptibility to anti- Relative cell count IC50 is the test compound concentration that produces 50% of the [Apoptosis 24hrs] - [Apoptosis 72hrs], 1000). We generated cell line profiles to reveal drug sensitivity and resistance 0.50 Cell Cycle Changes Detected at 24 hrs in the Absence of Growth Inhibition Are cancer agents. Biomarkers for cell proliferation and cell death coupled with patterns using multiplexed cellular response phenotypes. The combination of cell proliferation inhibitory response or 50% cytotoxicity level. A relative cell count sensitive resistant sensitive resistant Predictive of 72 Hour Toxicity. 0.25 genomic information linking the sensitivity/resistance of cell lines to activating or mutation profiling data with cellular response phenotypes allowed investigation EC50 is the test compound concentration that produces 50% of the maximum effective SKMEL28 T47D T47D MDA MB 468 RPMI 8226 SKMEL28 inhibiting mutations in oncogenes permit screening for sensitivity and selectivity of mechanisms of enhanced susceptibility to anti-cancer agents. Time course response that occurs at the curve inflection point. The output of each biomarker is A431 microM RPMI 8226 0.00 NCIH23 A431 A375 NCIH23 of a compound towards different mutations within similar cancers. Methotrexate determinations for growth inhibition, apoptosis and cell cycle arrest detection were fold increase over vehicle background normalized to the relative cell count in each Cell Proliferation Apoptosis Cell Cycle MDA MB 468 NCIH1299 A375 NCIH1299 SK HC NC RP M A4 NC HT A3 NC M K5 T4 TH SW treated breast cancer cell lines T47D and MDA MB 468 were resistant to treatment DA CF HT29 HT29 7D 31 75 62 29 P1 M IH M T1 IH IH 48 evaluated for all fifteen tumor cell lines. To determine the feasibility of using cryo- well. Concentrations of test compound that cause a 5 or more fold induction in the MCF7 7 I8 EL -0.25 MCF7 12 23 82 16 M 22 NCIH82 B 99 NCIH82 28 while MCF-7 demonstrated significant sensitivity. Highly selective inhibitors for 46 6 THP1 THP1 preserved cells as ready-to-use reagents for routine screening, cellular responses caspase-3 signal were determined to induce significant apoptotic induction. When 8 SW48 K562 140 K562 SW48 specific oncogene mutations demonstrated greater cell proliferation inhibition and Percent of Control 40 8 -0.50 HCT116 HCT116 24 hrs 120 from continuous culture and cryo-preserved cells in regards to growth inhibition, the fold induction of the phospho-histone-3 signal over background is ~1, there 7 over Background over Background 100 Fold Induction 0.75 0.50 0.00 1.00 0.25 -0.25 -0.50 -0.75 -1.00 -0.25 0.75 0.25 -0.50 -0.75 -1.00 Fold Induction 1.00 0.50 0.00 apoptosis induction in the tumor cell lines in the panel possessing the targeted 30 6 80 number of doublings, apoptosis and cell cycle arrest were compared for the 15 was “no effect” on the cell cycle. Two or more fold increase in phospho-histone- 5 60 20 4 Apoptosis inducing concentration at 24 hrs subtracted from apoptosis inducing concentration at 72 hrs for staurosporine treated 3 Δ log (average EC50 ) µM Δ log (average IC50 ) µM oncogene. Development of melanoma treatments have been based on the known tumor cell lines included in this panel. This automated multiplexed cell image based 3 signal over vehicle background indicated significant test compound induction 40 10 2 OncoPanelcell lines are shown. Positive values indicate apoptosis induction is more sensitive at the 72 hour time point for all cell selective sensitivity to MEK inhibition in BRAF mutant cells. Melanoma cell lines 20 1 lines tested. technology coupled with genetic analysis offers a robust and sensitive approach of mitotic block. Two or more fold decrease in the phospho-histone-3 signal may 0 0 0 MCF-7 -12 -11 -10 -9 -8 -7 -6 -5 -4 -12 -11 -10 -9 -8 -7 -6 -5 -4 -12 -11 -10 -9 -8 -7 -6 -5 -4 140 T47D 140 MDA MB 468 140 SK-MEL-28 and A375 demonstrated an enhanced sensitivity to U0126, a selective Percent of Control Percent of Control Percent of Control [Staurosporine], M [Staurosporine], M 120 [Staurosporine], M 120 120 EC50= 16.4 nM to assessing mechanism of action and sensitivity to chemotherapeutics agents. indicate G1/S block only when cytotoxicity levels are below the measured relative 100 EC50= 8.49 nM 100 EC50= 26.3 nM 100 IC50= 19.8 nM 80 IC50> 30 µM IC50> 30µM inhibitor of MEK in BRAF mutant cells. The cell line A431, known to over express Further the use of cryo-preserved cells enables this cost-effective multiplexed cell count IC80. When 2 or more fold decrease in the phospho-histone-3 signal are 80 80 EGF receptors, demonstrated the greatest sensitivity to EGFR inhibitor treatment. 140 60 60 60 Percent of Control 8 observed at concentrations higher than the relative cell count IC80, the decrease in 40 OncoPanel screening platform to be conducted with enhanced efficiency. 120 7 40 40 40 over Background K562 cells contain a Philadelphia chromosome which forms due to translocation 72 hrs over Background Fold Induction 100 Fold Induction 30 6 20 20 20 mitotic cell counts are most likely due to a more general cytotoxicity effect rather 80 5 OncoPanel Cell Line Gene Mutation Chart* 0 0 0 -10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 between chromosomes 22 and 9. This translocation leads to fusion of the Bcr 4 60 20 -10 -9 -8 -7 -6 -5 -4 -3 than a true G1/S phase block. Wells with concentrations higher than the relative [Methotrexate], M 3 40 [Methotrexate], M [Methotrexate], M 2 and Abl genes to generate a chimeric oncogene, bcr-abl, which causes chronic 10 Cell Lines Organ BRC-ABL CDKN2A PDGFRA TP53 EGFR KRAS NRAS ER+ PIK3CA RB1 MADH4 PTEN STK II bRAF BRCA2 CTNNB1 APC METHODS 20 1 cell count nuclear IC80 are eliminated from the phospho-histone-3 analysis. 0 0 0 myelogenous leukemia. Bcr-Abl inhibitor treatment led to a ten fold higher cell MDA MB 468 Breast X X X X The relative cell count IC50 (half maximal inhibitory concentration) and EC50 (half maximal effective concentration) values measure cell -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 [Staurosporine], M [Staurosporine], M [Staurosporine], M MCF-7 Breast X X X proliferation. Methotrexate, a folic acid analog and a potent inhibitor of the enzyme DHFR, is commonly used for treatment of breast proliferation inhibition and apoptosis induction in the K562 cell line than the ONCOPANEL ANTICANCER PROFILING T47D Breast X X X cancer. Several mechanisms associated with increased resistance to methotrexate have been identified, such as, diminished drug Cell culture Breast cancer cell lines MCF7, MDA MB 468, T47D, colon cancer cell 140 HT-29 Colon X X X X X uptake, expression of an altered DHFR enzyme with a reduced affinity for methotrexate, and increased levels of the target enzyme, DHFR, and conversion of methotrexate to polyglutamates. other oncopanel cell lines. The high selectivity of the Bcr-Abl inhibitor for the ASSAY K562 cancer cell line growth inhibition could be directly attributed to inhibition 40 8 lines HT-29, HCT-116, SW48, lymphoid cancer cell lines K562, RPMI 8226, THP1, skin 120 SW48 Colon X X X over Background Percent of Control 7 24 hrs over Background Fold Induction Fold Induction 100 30 6 cancer cell lines SK-MEL-28, A375, A431 and lung cancer cell lines NCI-H23, NCI- 80 60 20 5 4 HCT-116 Colon X X X X X of the activated oncogene product found in this particular cell line. Time course H1299, and NCI-H82 were grown in RPMI1640, 10% FBS, 1% L-Alanyl-L-Glutamine 40 20 10 3 2 RPMI 8226 Lymphoid X X X determinations for the multiparametric OncoPanel profiling assay showed that K562 Lymphoid X X X X cell cycle detection with phospho-histone-3 was an early indicator of cell arrest 1 Assay Procedure Seed Cells in 384-well plate and 1% sodium pyruvate in tissue culture flasks in a humidified atmosphere of 0 -13 -12 -11 -10 -9 -8 -7 -6 -5 0 -13 -12 -11 -10 -9 -8 -7 -6 -5 0 -13 -12 -11 -10 -9 -8 -7 -6 -5 THP1 Lymphoid X X X Rapamycin Treatment OncoPanel Cell Lines: Breast Cancer (MCF-7) and Myeloma 5% CO2 at 37oC. Working stocks of log-phase cells were cryo-preserved using [Taxol], M [Taxol], M [Taxol], M with similar 24 and 72 hour endpoints. Where cell proliferation was not inhibited 24 hrs NCI-H1299 Lung X Cancer (RPMI 8226) Most Sensitive a standard slow cooling in 10% DMSO protocol. For plating continuous culture NCI-H23 Lung X X during the 24 hours of compound exposure, detection of cell cycle block and/or Tipless Compound Addition (ECHO-550) apoptosis with activated caspase-3 were indicative of potential cell proliferation 140 40 cells the cells were thawed from working stocks and passed once prior to seeding 8 NCI-H82 Lung X 120 over Background 7 over Background Percent of Control Fold Induction 72 hrs Fold Induction inhibition at the 72 hour assay endpoint. The doubling time varied among the 30 100 6 SK-MEL-28 Skin X X X into plates. For cryo-preserved studies the cells were thawed, washed in media 24 – 72 hrs 80 20 5 sensitive resistant sensitive resistant oncopanel cell lines, however, no correlation was found between inherent growth 4 60 A375 Skin X X THP1 SW48 without DMSO, and then seeded into 384-well plates. 3 T47D Cell Fixation 40 10 2 A431 Skin X THP1 SKMEL28 SW48 20 0 0 -13 -12 -11 -10 -9 -8 -7 -6 -5 1 0 NCIH82 A375 A431 SKMEL28 NCIH82 NCIH23 rate and the cell proliferation measurement. For all 15 human tumor cell lines *Sanger Institute, http://www.sanger.ac.uk/genetics/CGP/CellLines/ -13 -12 -11 -10 -9 -8 -7 -6 -5 included in the OncoPanel profiling assay the EC50 values for cell proliferation -13 -12 -11 -10 -9 -8 -7 -6 -5 [Taxol], M [Taxol], M [Taxol], M HCT116 NCIH1299 Immuno-label and Stain Cells HT29 MDA MB 468 OncoPanel Anticancer Profiling Assay Caspase 3 activation (a marker of apoptosis), (60 plates – 4 hrs) NCIH1299 K562 K562 HT29 and growth rate in cryo-preserved cells highly correlated with values generated in Data Acquisition / NCIH23 HCT116 phospho-histone-3 (a marker of mitosis), and relative cell count (an index of cell T47D MCF7 A431 A375 RPMI 8226 continuous culture cells. The use of cryo-preserved cells enhances the efficiency RPMI 8226 proliferation) were measured. Each cell line was seeded at an experimentally OncoPanel Anticancer Profiling Assay, representative curves at 24 and 72 hrs for cell proliferation, apoptosis Analysis MDA MB 468 MCF7 induction, and cell cycle block induced with staurosporine in the T47D cell line and Taxol in the NCI H82 cell of this cost-effective automated multiplexed OncoPanel screening platform in determined optimal density with complete growth media into 384-well plates and 1.5 0.5 1.0 0.0 -1.0 1.0 0.0 -0.5 -3.0 1.5 0.5 -0.5 -1.5 -3.0 -2.5 -1.5 -2.5 -2.0 -1.0 -2.0 Read plates on the InCell 1000 Analyzer InCell Developer AIM curve-fitting Automated Report line are shown. U0126, MEK Inhibitor, Selectively Inhibits the Growth of BRAF Mutant Cell Lines assessing mechanism of action and sensitivity to chemotherapeutic agents. Drug incubated in a humidified atmosphere of 5%CO2 at 37oC. Test compounds were (30 min / plate, Software Analysis analysis (MathIQ Generation ∆log (average EC 50 ) µ M ∆log (average IC 50 ) µ M combination analysis using this OncoPanel profiling assay to determine synergistic (60 plates – 10 hrs) based software) serially diluted 3-fold over 10 concentrations and added 24 hours post cell seeding 60 plates – 20 hrs) UO126, MEK inhibitor Inhibitors of the PI3K/Akt/mammalian target of the rapamycin (mTOR) pathway are promising therapeutics for breast and myeloma cancer. activity of compound cocktails could provide pharmacologically relevant data for with a final assay concentration of 0.002% DMSO. Following an additional 24 or 72 sensitive resistant improving cancer treatment efficacy. hour incubation in the humidified atmosphere of 5%CO2 at 37oC, cells were fixed A summary of OncoPanel Anticancer Profiling parameters at 24 and 72 hrs in RPMI 8226 RAS* GTP GDP and immunolabeled with anti-active caspase-3 for detection of apoptosis, anti- T47D and NCI H82 cell lines NCIH82 T47D RAF* 5-FU Treatment OncoPanel Cell Lines: Non-Melanoma Skin Cancer (A431) Most Multiplexed cell proliferation, apoptosis and cell cycle image data REFERENCES NCIH23 phospho-histone-3 for detection of cell cycle and stained with nuclei dye for cell MDA MB 468 MEK1 K562 A431 Sensitive proliferation quantification. Twenty eight standard cancer therapeutic agents were MEK2 MCF7 Relative cell count Relative cell count Apoptosis Mitosis Inhibition of mitosis NCIH1299 MEK inhibition; U0126 Time, tested. A non-contact dispensing automation system accommodated cell plating Compound hrs IC50 EC50 induction cell cycle block (G1/S cell cycle HCT116 THP1 SKMEL28 ERK1/2 sensitive resistant sensitive resistant Sanger Institute, http:/www.sanger.ac.uk/genetics/CGP/CellLines/ (microM) (microM) (microM) (microM) block) (microM) and reagent additions (Thermo Combidrops or Multidrops) and test compound HT29 C-MYC C-MYC MNK1 T47D Staurosporine 24 >3 >3 − − 0.03 A375 SW48 MSK1 RSK MNK2 ELK-1 STAT3 K562 NCIH82 MDA MB 468 Rodrigues RA, Alfonso J, O,Day C, Ovechkina Y, Ward C. 297 Distinct Cell Lines: A High-Content additions (Labcyte ECHO-550). Automated fluorescence microscopy was carried Staurosporine 72 0.05 0.04 0.31 − 0.02 SKMEL28 T47D K562 SKMEL28 Analysis Assay and a Full Automation Design Solely Using Noncontact Liquid Dispensing. -0.25 -1.00 -0.75 -0.50 0.00 0.25 0.75 0.50 1.00 MDA MB 468 NCIH82 Taxol 24 > 0.3 0.011 0.009 0.012 − out using a GE Healthcare INCell Analyzer 1000, and images were collected with Taxol 72 0.002 0.002 0.002 0.002 − Transcription/Proliferation NCIH1299 A375 MCF7 MCF7 NCIH1299 A375 Journal of the Association for Laboratory Automation. 2007;12(5):318-326. a 4X objective. ? log (average EC50 ) µM SW48 NCIH23 HCT116 SW48 Vehicle Taxol Staurosporine NCIH23 HCT116 HT29 Shoemaker RH. The NCI60 human tumour cell line anticancer drug screen. Nature Reviews. The relative cell count IC50 (half maximal inhibitory concentration) and EC50 (half maximal effective concentration) HT29 THP1 2006;6:813-823. THP1 Activated BRAF mutants increase sensitivity to U0126 induced MEK inhibition in SK-MEL-28, A375 and HT29 cell lines. RPMI 8226 RPMI 8226 values measure cell proliferation. Compound concentrations are indicative of a 5-fold apoptosis induction in BRAF mutation has been shown to predict sensitivity to MEK inhibition in melanoma cell lines (D. B. Solitet al.). Development A431 A431 activated caspase-3 signal and a 2-fold change in phospho-histone3 signal (mitosis marker) over vehicle back- Ikediobi ON, Davies H, Bignell G, et al. Mutation analysis of 24 known cancer genes in the -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 Representative images are shown for T47D cell line. Labels: Nuclei - blue; Apoptotic cells - green; Mitotic 0.0 0.5 -1.5 -1.0 -0.5 1.0 -2.0 of MEK inhibitors for treatment of melanoma has resulted from this in vitro selectivity translating to in vivo xenograph efficacy. cells - red ground. ∆log (average EC 50 ) µ M ∆log (average IC 50 ) µ M NCI-60 cell line set. Mol Cancer Ther, 2006;5(11):2606-2612. Huang R, Wallqvist A, Covell DG. Assessment of in vitro and in vivo activities in the National 5-fluorouracil (5-FU) is an effective treatment for non-melanoma skin cancer. A431, derived from non-melanoma epidermoid carcinoma, showed the greatest sensitivity to 5-FU treatment. Cancer Institute’s anticancer screen with respect to chemical structure, target specificity, and mechanism of action. J Med. Chem., 2006;49:1964-1979. Solit DB, et al. BRAF mutation predicts sensitivity to MEK inhibition. Nature, 2006;439:358-362.