Homebrew Gene Expression Assays Using Luminex Technology

BADGE, BeadsArray for the Detection of Gene Expression, a high throughput diagnostic bioassay – Genome Res. 2001 A method for high-throughput gene expression signature analysis Genome Biology 2006 Signature-based small molecule screening identifies cytosine arabinoside as an EWS/FLI modulator in ewing sarcoma – PLoS Medicine 2007 Gene expression signature-based chemical genomic prediction identifies a novel class of HSP90 pathway modulators – Cancer Cell 2006

BADGE Method

BADGE, BeadsArray for the Detection of Gene Expression, a high-throughput diagnostic bioassay –

Genome Res. 2001 Li Yang, Diem K. Tran, Xun Wang Torrey Mesa Research Institute, Syngenta Research and Technology

BADGE

•Flexible •Affordable •Multiplexed •Rapid Process •High Throughput Total RNA OligodT reverse transcription, double-stranded cDNA synthesized T7 RNA polymerase and Biotin UTP B B B B B B B B Hybridize - 1 hr.

BADGE

Reproducibility: Standard Deviation 5-20% of the mean LOD: 1 fmol target gene

LMF

A method for high-throughput gene expression signature analysis -

Genome Biology 2006 David Peck, Emily D Crawford, Kenneth N Ross, Kimberly Stegmaier, Todd R Golub, Justin Lamb The Broad Institute Dana-Farber Cancer Institute Howard Hughes Medical Institute

LMF method Developed at the Broad Institute

•Flexible •High-Throughput •Cost Effective •Up to 500 transcripts T3 mRNA capture Reverse Transcription - cDNA Ligate B B B Universal primer PCR Amplicon Capture

LMF Reproducibility

Transcriptional analysis of a cell culture model of hematoppoietic differentiation. HL60 cells treated with tretinoin or vehicle (DMSO) N = 1800: (two conditions x 90 transcripts x 10 replicates) 97.9% of data points within twofold of corresponding means Single transcript caused 34% of outliers

LMF Correlation to Microarray

LMF Correlation to Microarray

•0.924 Correlation of log ratios between microarray and bead platform •13.8% coefficient of variation of mean expression level •100% Classification accuracy for 94 samples, using KNN algorithm trained on 90 gene microarray LOOCV study on positive and negative control wells Accuracy Signature # of Genes # of Wells 98.8% 99.6% 100.0% 99.5% androgen sarcoma adipocyte nueroblastoma 30 11 21 11 642 766 324 191

LMF – Cost Effectiveness

“Even the most inexpensive custom low-density microarray solutions have detection costs at least one order of magnitude greater than that of LMF and they also commonly suffer from high initial set-up charges.”

LMF Application to Ewing Sarcoma

Signature-based small molecule screening identifies cytosine arabinoside as an EWS/FLI modulator in Ewing Sarcoma –

PLoS Medicine 2007 Kimberly Stegmaier, Jenny S. Wong, Kenneth N. Ross, Kwan T. Chow, David Peck, Renee D. Wright, Stephen L. Lessnick, Andrew L. Kung, Todd R. Golub Dana Farber Cancer Institute and Children’s Hospital Boston The Broad Institute Howard Hughes Medical Institute The Center for Children, Huntsman Cancer Institute

GE-HTS uses gene expression signatures as surrogates for cellular states

Define gene expression-based signatures through genome-wide gene expression.

Develop a multiplexed high-throughput, low cost assay Screen a small-molecule library for modulators of oncogenic transcription factors Not Needed Specialized Assays A priori knowledge of a target Knowledge of mutant transcription factor’s transforming mechanism

Findings

ARA-C identified as matching EWS/FLI offstate signature All cells exposed to ARA-C do not non-specifically regulate the EWS/FLI signature.

Inhibition of cell viability alone does not induce the whole genome effects of EWS/FLI modulation.

A dose-responsive increase in EWS/FLI transcript was observed EWS/FLI protein abundance decreased.

ARA-C reduced EWS/FLI protein abundance and accordingly diminished cell viability and transformation and abrogated tumor growth in a xenograft model.

LMF Application to Prostate Cancer

Gene expression signature-based chemical genomic prediction identifies a novel class of HSP90 pathway modulators –

Cancer Cell 2006 Haley Hieronymus, Justin Lamb, Kenneth N. Ross, Xiao P. Peng, Cristina Clement, Anna Rodina Maria Nieto, Jinyan Du, Kimberly Stegmaier, Srilakshmi Raj, Katherine N. Maloney, Jon Clardy William C. Hahn, Gabriela Chiosis, Todd R. Golub The Broad Institute Dana-Farber Cancer Institute Howard Hughes Medical Institute Memorial Sloan-Kettering Cancer Center Harvard Medical School

Chemical genomic discovery and prediction in mammalian cells

Define gene expression signature of AR activation in the LNCaP prostate cancer cell line Use traditional microarray to identify genes that are activated or repressed by androgen stimulation or deprivation Use GE-HTS approach using Luminex technology to test 27 gene signature on 2500 compounds Identification of celastrol and gedunin natural products as inhibitors of androgen signaling Test new compounds for dose dependant inhibition, decreased cell growth, and compare genome-wide gene expression profiles to androgen deprived cells.

Chemical genomic discovery and prediction in mammalian cells

Use a collection of genome-wide affymetrix signatures of compounds of known mechanism to compare with new compounds – Connectivity Map Celestrol and gedunin show similarity to four HSP90 inhibitors – a client protein to AR Decreased the protein levels of a host of HSP90 clients Decreased the ATP-binding activity of HSP90 and reduced its interaction with cochaperones Shown to function via a distinct mechanism than existing HSP90 inhibitors, and showed synergistic inhibition when combined