Liposarcoma Genomic Alterations Define New Targets for Therapy

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1 Liposarcoma Genomic Alterations Define New Targets for Therapy Samuel Singer, MD Memorial Sloan-Kettering Cancer Center Sarcoma Disease Management Program Chief Gastric and Mixed Tumor Service

2 Liposarcoma ~20% of 12,000 new STS cases in US each year, the majority are sporadic Distinct cytogenetic subgroups comprising 5 subtypes Simple (translocation-associated) Myxoid and round cell liposarcoma: >90% t(12;16) Complex rearrangements (alterations in cell cycle genes and checkpoint defects) Well-differentiated and Dedifferentiated liposarcoma: ~90% 12q amplification Pleomorphic liposarcoma Genetic events associated with liposarcomagenesis remain undiscovered

3 Therapeutic Challenges More than 60% of patients with newly diagnosed liposarcoma eventually die of disease Diverse histopathology and biological behavior WDLS and DDLS are not very responsive to chemotherapy Pressing need to develop subtype specific molecularly targeted therapeutics for patients with advanced/recurrent disease

4 Histologic distribution of Liposarcoma subtype MSKCC Clinical Sarcoma Database 1772 liposarcoma patients over 29 years ( ) Site specific histologic subtype distribution WD

5 Sarcoma Genome Project (Phase I) Genome-wide molecular genetic analysis of 7 sarcoma types (Phase I) DNA copy number + LOH Sequencing 225 genes and 451 micrornas Transcriptional changes (protein-coding genes) Clinical annotation database (MSKCC) 207 sarcomas and matched normals Functional genetic screen of amplified genes in DDLPS

6 Sarcoma Genome Project (Phase I) Analysis of DNA sequence in 6 sarcoma types Kit in GIST was the most frequently mutated gene Next most frequently mutated genes in more than 5% of samples within a subtype were: genes mutated PIK3CA TP53 sarcoma type myxoid/round cell liposarcoma pleomorphic liposarcoma % of samples mutated drug able target 18 % PI3K inhibitors 17 % Small molecules that reactivate mutant p53 (PRIMA-1) NF1 myxofibrosarcoma 10.5 % MEK or mtor inhibitors pleomorphic liposarcoma 8 %

7 PIK3CA mutations in myxoid/round cell liposarcoma are associated with shorter diseasespecific survival compared with wildtype (p-value = 0.036)

8 E545K helical-domain mutations were associated with increased Akt phosphorylation relative to wildtype in myxoid/round cell liposarcoma TORC2 phosphorylation site PDK1 phosphorylation site

9 Nucleotide and Copy-number alterations in Dedifferentiated Liposarcoma (n=50) and myxoid/round cell liposarcoma (n=21) Consensus plot of statistically significant genome-wide copy-number alterations assessed by RAE

10 Knockdown of the Dedifferentiated Liposarcoma Amplicome as a Functional Screen for Potential Driver Genes Which amplified genes in DDLS are necessary for cancer cell proliferation and survival? Genomics-driven RNAi screen in 3 genotype-matched DDLS cell lines Systematic knockdown with shrnas on 385 significantly amplified genes 99 genes whose knockdown decreased cell growth, 27 of 99 were amplified in at least one cell line

11 Functional Validation of CDK4 as a Therapeutic Target in DDLS Effect of three validated shrnas targeting CDK4 on the proliferation of two DDLS cell lines 1 m selective CDK4/CDK6 inhibitor PD induces a G1 growth arrest and senescence

12 Therapeutics for DDLS Potential targets: MDM2 MDM2 antagonists Nutlin-3 Roche (R7112) prevents MDM2-p53 interaction % Apoptosis / Annexin Apoptosis Following 48hr Nutlin Treatment NADIP DDLS LS141 No Drug 5uM Nutlin CDK4 CDK4/CDK6 inhibitor PD G1 arrest and senescence senescence associated heterochromatic foci AURKA AURKA inhibitor MLN8237 oral, ATPcompetitive, disrupts assembly of mitotic spindle mitotic delay other kinases (IRAK3, PCTK2)? DDLS ND DDLS AK (5 µm)

13 Multi-center phase I trial of R7112 in patients with advanced malignancies R7112 (Roche) is a small molecule antagonist of MDM2 binds to the p53 site on the surface of MDM2 and blocks protein-protein interaction between MDM2 and p53 wt protein Oral administration MTD: 2500 mg/day, BID x 10 days with 18 days of rest associated with significant thrombocytopenia Dosing schedule revised to 2500 mg/day, QD dosing x 5 days with 23 days rest 20 patients with WDLS and DDLS treated to date on BID dosing schedule 8 with stable disease at 8 weeks Molecular and pharmacokinetic analysis p53 mutation, MDM2 levels

14 Phase II clinical trial of PD in patients with WDLS/DDLS that have CDK4 amplification by FISH and express the Rb protein by IHC Patients with locally advanced, recurrent or metastatic WDLS or DDLS and disease progression on one prior systemic therapy PD dose of 200 mg PO QD for 14 days, followed by 7 days of rest. safe and tolerable in a phase I study In phase 1 study two patients with advanced WDLS had stable disease for 3.1 and 2.3 years Primary endpoint: patients who are progression-free at 12 weeks

15 Phase II clinical trial of MLN8237 in patients with WDLS/DDLS and pleomorphic liposarcoma Test a second-generation selective AURKA inhibitor in patients with advanced / metastatic liposarcoma Primary objective: response rate (CR + PR) assessed at 12 weeks Based on a completed phase I study, patients will be treated with MLN mg PO BID for 7 days, followed by 14 days of rest. Treatment is repeated every 3 weeks (one cycle)

16 Sarcoma Genome Project (Phase II) Perform a genome-wide genetic and functional analysis of well-differentiated (WDLS) and dedifferentiated (DDLS) Liposarcoma to identify: distinct genomic subtypes molecular markers that associate with outcome and pathologic features genetic alterations associated with sarcoma progression subtype specific therapeutic targets MSKCC Samuel Singer Marc Ladanyi Chris Sander Barry Taylor Rockefeller Thomas Tuschl Markus Hafner BROAD Matthew Meyerson Jordi Barretina Alex Ramos Shantanu Banerji Sample procurement and selection / RNA and DNA extraction U133A Affy transcript arrays 244K Agilent CGH array, fusion array microrna profiling microrna cloning and sequencing microrna ISH Systematic shrna screens Solexa single molecule cdna sequencing Validation of candidate mutations Agilent microrna array microrna functional analysis Computational analysis for protein-coding genes, micrornas, amplified genes, gene rearrangements, activating mutations and activated pathways Validate genetic, genomic and functional alterations

17 Well-differentiated and Dedifferentiated Liposarcoma Disease-specific survival for primary retroperitoneal liposarcoma: WDLS vs. DDLS Data from MSKCC Sarcoma Database (n=345, 7/1/82 to 6/30/2010) Local recurrence-free survival for primary retroperitoneal liposarcoma: WDLS vs. DDLS Resistant to chemotherapy Can we exploit differentially expressed micrornas as therapeutic targets for this deadly disease?

18 Role of mirnas in liposarcomagenesis mirnas regulate cell proliferation, apoptosis, and differentiation mirnas are abnormally regulated in cancer may serve as oncogenes but generally down-regulated in tumors compared to normal tissue inhibiting mirna processing enhances tumorigenesis suggest mirnas act mainly as tumorsuppressors Profile mirnas in normal fat (NF), WDLS and DDLS tissue samples to identify mirnas that associate tissue type and tumor progression Agilent microarrays (17 NF, 32 WDLS, 30 DDLS) Deep sequencing of small RNA cdna libraries (11 NF, 22 WDLS, 22 DDLS)

19 Small RNA cdna library preparation for sequencing Developed a set of bar-coded sequencing adapters parallel sequencing of up to 20 samples in one Solexa sequencing run, from < 2 µg total RNA / sample > 200,000 sequence reads / sample at a lower cost than mirna microarrays map small RNAs to the genome; annotate by functional type convert cloning frequencies to mirna expression levels Hafner, M., Tuschl, T. Methods :3-12

20 Unsupervised clustering of mirnas in normal fat, WDLS and DDLS samples analyzed by deep sequencing DDLPS Normal fat tissue WDLPS

21 Deep sequencing compared to Agilent microarray (DDLS / NF)

22 mir-21 and and mir-26a are significantly overexpressed in DDLS compared to normal fat mir-21 mir-26a Normalized Counts PTEN expression NF WD DD NF WD DD NF WD DD mir-26a-2 encoded in the intron of CTDSP2, adjacent to CDK4 on chr12, is amplified in 88% of DDLS

23 mir-143 and mir-145 are down-regulated in WDLS and DDLS compared to NF mir most strongly expressed mirna in normal fat (8% of total) mir-143 and mir-145 located within 1.8 kb of each other on 5q mir 143 mir 145 Normalized Counts N F mir 143 WD DD N WD DD F Fold Change FDR WD/NF e 5 DD/NF e 12 mir 145 Fold Change FDR WD/NF 2.8 > 0.05 DD/NF e 7 FDR= false discovery rate

24 mir-143 and mir-145 are down-regulated in WDLS and DDLS cell lines compared to ASCs by deep sequencing and quantitative PCR In situ hybridization for mir-143 DDLS cell lines WDLS cell lines DDLS cell lines WDLS cell lines

25 A lentiviral system was used for stable re-expression of mir-143 and mir-145 in liposarcoma cell lines mirna precursor in lentiviral vector Tranform E. coli cells Culture and extract plasmid DNA Transfect HEK293T Cells Functional Studies mir 143; mir 145 reexpression Harvest viral supernatant Select with Puromycin (Day 1) Infect cells of interest (Day 0)

26 Re-expression of mir-143 inhibits proliferation in two DDLS cell lines DNA content (relative to day 2) Time (Days) Time (Days)

27 Re-expression of mir-143, but not mir-145, induces apoptosis in DDLS cells DDLS14 1 DDLS8817

28 mir-143 target gene identification in DDLS cells Identify genes with 3 UTR sequences complementary to the mir-143 but not mir-145 seed sequence Treated 2 DDLS cell lines with mir-143 v. Scr/Untx Generated triplicate mrna profiles (Illumina array) 143 upregulated genes 125 downregulated genes Automated network-based computational approach, NetBox 1 1. Cerami E et al., PLoS One, 2010.

29 Network Analysis identified a 24-gene module regulated by mir-143 re-expression Direct target Downregulated Upregulated

30 Expression of 10 of the 24 genes in the mir-143 network are associated with decreased distantrecurrence free survival (DFRS) Expression profiling of 140 primary liposarcoma patients (95 training set, 45 test set) Expression of 588 genes used to calculate a genomic risk score (GRS) for each patient In validation: 3-year DRFS of 83% for low GRS vs. 45% for high GRS patients (P=0.001) The hazard ratio for GRS was 4.4, adjusted for histologic subtype Gobble R et al., Cancer Research, In Press

31 TOP2a, a gene involved in DNA replication, is a predicted direct target of mir-143 DRFS for primary liposarcoma by TOP2A expression 43-fold in DDLS vs. NF TOP2A expression associated with metastasis in DDLS 1 Direct target Downregulated Upregulated 1. Gobble R et al., Cancer Research, In Press

32 Re-expression of mir-143 decreases TOP2a expression and regulates the 3 UTR of TOP2a DDLS14 1 DDLS8817 DDLS141 DDLS8817

33 TOP2A is up-regulated in WDLS and DDLS compared to normal Fat Liposarcoma Subtype Fold Increase compared to Normal Fat False Discover Rate Well-differentiated E-05 Dedifferentiated E-19 Increased expression of TOP2A in DDLS cell lines compared to Adipose Derived Stem Cells (ASC) TOP2A ASC LPS141 DDLS8817 α-tubulin DDLS cell lines Gobble R et al., Cancer Research, In Press

34 TOP2A knockdown inhibits proliferation and induces apoptosis in DDLS cells TOP2A DDLS8817 Scr #7 #8 α-tubulin Cell proliferation day 6 after TOP2A knockdown % apoptosis day 6 after TOP2A knockdown

35 mir-143 re-expression downregulates genes involved in mitosis and cytokinesis: PRC1, PLK1, CDC25B, ECT2, and CDC2 (CDK1) In early anaphase, CDC2 (CDK1) activity falls, PRC1 can now dock to PLK1 and localize it to central spindle for cytokinesis PRC1 22-fold in DDLS v. NF Direct target Downregulated Upregulated In metaphase, CDC25B is recruited to PLK1 and helps maintain activity of CDC2 (CDK1) CDC25B 8-fold in DDLS v. NF Neef, R. et al., Nature Cell Biology, 2007

36 Re expression of mir 143 decreases PRC1 and PLK1 expression and regulates the 3 UTR of PRC1 DDLS141 DDLS8817 DDLS141 DDLS8817 DDLS8817

37 PLK1 inhibition with BI 2536 decreases proliferation in DDLS cell lines ASC DNA content (relative to Day 0) DDLS141 DDLS8817 DNA content (relative to Day 0) DNA content (relative to Day 0)

38 PLK1 inhibition induces apoptosis and a G2/M arrest in DDLS cells DDLS141 DDLS8817 ASC DDLS141 DDLS141 DDLS8817

39 Kinases that regulate spindle assembly, mitosis and cytokinesis may be attractive therapeutic targets for DDLS AURKA is amplified in 30% of DDLS and is a candidate driver gene Overexpressed 3-fold in DDLS and 10-fold in pleomorphic liposarcoma compared to normal fat AURKA is prognostic for DRFS in primary liposarcoma (HR=3.1) PLK1 docking partners PRC1, CDC25B and CDC2 all highly overexpressed in DDLS compared to normal fat PLK1, PRC1, and CDC2 expression are prognostic for DRFS in primary liposarcoma Both PLK1 and AURKA interfere with spindle assembly PLK1 inhibition induces prolonged mitotic arrest and cell death AURKA inhibition leads to a more retracted mitotic arrest with chromosome segregation errors and aneuploidy

40 Summary Discovered PIK3CA helical and kinase domain mutations in 18% of myxoid / round-cell liposarcoma Helical PIK3CA mutations are associated with Akt activation in vivo and reduced patient survival Discovered NF-1 mutations in 10% of myxofibrosarcoma and 8% of pleomorphic liposarcoma TP53 mutations in 17% of pleomorphic liposarcoma These mutations identify a subset of tumors likely to respond to specific pathway inhibitors

41 Summary mirna profiles discriminate liposarcoma from normal fat and WDLS from DDLS mir-143 is downregulated in WDLS and DDLS compared to normal fat Re-expression of mir-143 inhibits proliferation and induces apoptosis in DDLS cells mir-143 regulates components of a gene network involved in cell proliferation, apoptosis, and cytokinesis in DDLS cells Target genes include TOP2a, PRC1, and PLK1 Targeted inhibition of PLK1 induces apoptosis and cell cycle arrest in DDLS cells

42 Conclusions mir-143 downregulation may be an important early event in liposarcomagenesis mir-143 may function as a tumor suppressor in liposarcoma and mir-143 re-expression vectors show promise as a multi-targeted mirna-based therapeutics for WDLS and DDLS Selective agents directed at gene products or pathways regulated by mir-143 may have therapeutic value in liposarcoma

43 Acknowledgements Sarcoma Genome Project Singer Lab Aimee Crago Penelope DeCarolis Stacy Ugras Ann Lee Christina Angeles Elliott Brill Ryan Gobble Comp Biology Center Barry Taylor Nick Socci Anders Jacobsen Robert Sheridan Raya Khanin Genomics Core Lab Agnes Viale Cristina Antonescu Robert Maki Gary Schwartz Marc Ladanyi Chris Sander Harold Varmus MSKCC Tuschl Lab Markus Hafner Sara Hakim Thomas Tuschl Rockefeller University Meyerson Lab Alex Ramos Shantanu Banerji Rameen Beroukhim Gaddy Getz Craig Mermel Wendy Winckler RNAi Platform Serena Silver David Root Jordi Barretina Heidi Greulich Todd Golub Bill Hahn Levi Garraway Bill Sellers Eric Lander Matthew Meyerson BROAD Institute