1. Integrative and Functional Characterization of the Soft Tissue Sarcoma Genome The Sarcoma Genome Project Jordi Barretina / Barry S. Taylor CTOS 15 th Annual Meeting 6 th November 2009

2. Derived from connective tissues (mesenchymal tumors). Rare (<1% of all cancers) and heterogeneous. STS have not yet been a focus of large-scale genomic efforts (low TCGA, ICGC priority). Most of them caused by somatic mutations. Gleevec as a paradigm of targeted cancer therapies. Soft Tissue Sarcomas

3. The need: 50% of patients with newly diagnosed sarcoma eventually die of metastatic disease. The majority of sarcoma subtypes are not very responsive to chemotherapy. The solution: Conduct a detailed genomic characterization of soft tissue sarcoma tumor samples. Selective therapies against genomically altered targets can be highly effective and can exert fewer side effects. From Cancer Genomics to Targeted Therapies

4. Computational analysis Data integration Targeted functional validation Gene selection The Sarcoma Genome Project Sample collection RNA and DNA extraction U133A Affy Expression arrays (n=141) Sample collection RNA and DNA extraction U133A Affy Expression arrays (n=141) 250K Affy (StyI) SNP arrays (n= 207) Sequencing (n=48) Candidate mutation validation (n=48 + 159*) 250K Affy (StyI) SNP arrays (n= 207) Sequencing (n=48) Candidate mutation validation (n=48 + 159*)

5. Elucidate the genetic alterations and signaling pathways associated with specific sarcoma subtypes. Improve sarcoma classification. Identify new therapeutic targets in sarcoma through integrative analysis. TSGP Goals

6. … + matched normals (205) Paired expression data for 141 of them. Samples SubtypeKaryotypeTotal Dedifferentiated liposarcoma complex50 Myxoid/round cell liposarcoma simple, t(12;16), t(12;22)21 Pleomorphic liposarcoma complex24 Myxofibrosarcomacomplex38 GISTsimple22 Synovial sarcomasimple, t(X;18)25 Leiomyosarcomacomplex27 TOTAL207

7. 48 Soft Tissue Sarcomas: –21 liposarcomas –10 myxofibrosarcomas – 6 GISTs –11 synovial sarcomas Genes chosen for exon resequencing: –Group 1: All Tyrosine Kinases –Group 2: Selected Cancer and Sarcoma genes –Group 3: All microRNAs ( 224 genes + 496 microRNAs, 3587 exons, 3830 amplicons) Sequencing Summary

8. Title line 1 Title line 2 GeneNo. of mut. a Frequency b SubtypemRNAProtein CDH122DDLPS712A>AGN238D 4.5GIST1849G>AGA617T e CTNNB122DDLPS122C>CTT41I d 4Synovial95A>ATD32V d EPHA112DDLPS634G>GAA212T EPHA514.2Pleomorphic2386A>AGY796H EPHA712.6MYXF1649C>CTS550N ERBB422.6MYXF3437A>ATD1146V 4.2Pleomorphic1558A>ATC520S FBXW722DDLPS338_342delTCATC>TCE113fs 4.5GIST563G>GTC188F IRS114.5GIST3406C>CTE1136K KIT623GIST1727T>CTL576P d GIST1961T>CTV654A d GIST1667_1674delAGTGGAAG>AGQ556fs GIST1667_1687del c Q556_I563>Q GIST1670_1675delGGAAGGW557_V559>F e 4.8MRC2334G>CGK778N LTK14Synovial2243_2244delTT>TC748fs MOS14.5GIST898A>AGS300P MST1R14.5GIST1229G>AGP410L NF1710.5MYXF7972C>CTH2658Y MYXF7790C>CTS2597L MYXF910C>TR304* d MYXF910C>TR304* d MYXF7010T>TGL2337R 8.3Pleomorphic1105C>CTQ369* d Pleomorphic4006C>CTQ1336* NTRK112.6MYXF2338C>CTR780W PI4KA22.6MYXF4081_4088delTCTTATCT>TCT1361fs 4Synovial4081_4088delTCTTATCT>TCT1361fs PIK3CA619MRC1633G>AGE545K e MRC1633G>AGE545K e MRC3140A>AGH1047R e MRC3140A>AGH1047R e 4.2Pleomorphic1660delCH554fs 4Synovial1659delTS553fs PTEN22.6MYXFG>CGSplice site 4Synovial106G>AAG36R e PTK2B14.2PleomorphicG>AGSplice site RB114.2Pleomorphic1818T>TAY606* e SYK14.2Pleomorphic52G>AAG18S TP53416.7Pleomorphic404C>AAC135F e Pleomorphic464G>AAT155I PleomorphicC>CTSplice site PleomorphicC>TTSplice site

9. We found 37 somatic point mutations and 9 indels (involving 21 genes across 6 sarcoma subtypes). Several previously described in sarcoma and other cancers (COSMIC). 30 not previously reported. 24 affected kinases. 18 predicted to have a functional effect by in silico analysis Sequencing Summary EPHA5NTRK1

10. PIK3CA mutations in ~18% of myxoid/round cell liposarcomas

11. Diverse NF1 alterations in soft tissue sarcoma

12. Nucleotide and copy number alterations in soft tissue sarcoma

13. Question: Which amplified genes in DDLPS are necessary for cancer cell proliferation/survival? Answer: Genomics-driven RNAi screen in DDLPS Systematic knockdown of ~400 significantly amplified genes with shRNAs in 3 genotype-matched cell lines Functional annotation of the Cancer Genome

14. Functional annotation of the Dedifferentiated Liposarcoma “Amplicome” Genes essential for DDLPS cell proliferation.

15. PD-0332991 CDK4/CDK6 inhibitor CDK4 as a target in Dedifferentiated Liposarcoma

16. Functional annotation of the Dedifferentiated Liposarcoma “Amplicome” Genes essential for DDLPS cell proliferation.

17. YEATS4 and MDM2 co-amplification and potential cooperation in p53 pathway regulation

18. Our study yields the most detailed map of molecular alterations across diverse sarcoma subtypes to date. Subtype-specific genomic alterations define new targets for soft tissue sarcoma therapy. - PI3K pathway in PIK3CA-mutant myxoid/round cell liposarcomas - mTOR pathway in NF1-deficient soft tissue sarcomas Summary

19. Next-Gen sequencing is replacing capillary and array-based technologies DNA Point mutations (substitutions/indels) Chromosomal aberrations Copy gains and losses Loss of heterozygosity (LOH) Rearrangements & fusion genes Epigenetic modifications RNA Protein Transcript expression level changes Differential alternative splicing Allele-specific expression changes Protein expression level changes Protein modification changes Protein degradation/stability changes standard technologyMolecular Alterations in Cancer CANNOT BE DETECTED NO GENOME-WIDE HIGH THROUGHPUT TECHNOLOGY new technology NO GENOME-WIDE HIGH THROUGHPUT TECHNOLOGY

20. Significantly Amplified Genes Significantly Amplified Genes Genes Essential for Proliferation Recurrent Mutations and Fusion Genes 50 Paired Patient Samples Affymetrix 250K SNP Array 50 Paired Patient Samples Affymetrix 250K SNP Array 5 Cell Lines 54,020 lentiviral shRNA pool screen (~11,000 genes) 5 Cell Lines 54,020 lentiviral shRNA pool screen (~11,000 genes) 12 Paired Patient Samples Whole Exome Sequencing ~186,000 baits (~16,000 target genes) & RNA-Seq 12 Paired Patient Samples Whole Exome Sequencing ~186,000 baits (~16,000 target genes) & RNA-Seq Shantanu Banerji Next steps in DDLPS…

21. MSKCC Pennelope DeCarolis Mariana Lagos-Quintana Alan Ho Tsuyoshi Saito Neerav Shukla Christopher Lau Comp Biology Center Barry Taylor John Major Boris Reva Nick Socci Alex Lash Genomics Core Lab Agnes Viale Biological Samples Platform Scott Mahan Jennifer Franklin Jennifer Chen Alex Thomson Kristin Ardlie Genetic Analysis Platform Brendan Blumensteil Kristian Cibulskis Liuda Ziaugra Carrie Sougnez Stacey Gabriel Sequencing Platform Robb Onofrio Jen Baldwin RNAi Platform Hanh Le Pat Lizotte Brian Wong Alan Derr Jen Grenier Serena Silver David Root Acknowledgements Cancer Genome Analysis Shantanu Banerji Alex Ramos Rameen Beroukhim Gaddy Getz Craig Mermel Derek Chiang Barbara Weir Kinjal Shah Lauren Ambrogio Tzu-Hsiu Chen Megan Hanna Laura MacConaill Project Management Wendy Winckler Comp Biology & Bioinf Jim Robinson David Twomey Ted Liefeld Michael Reich Pablo Tamayo Heidi Greulich Todd Golub Bill Hahn Levi Garraway Bill Sellers Eric Lander Matthew Meyerson Robert Maki Gary Schwartz Cristina Antonescu Chris Sander Marc Ladanyi Harold Varmus Sam Singer