David W. Salzman
Yale School of Medicine
Department of Therapeutic Radiology
Fallopian Mucosa NSCLC Papillary Serous Ovari...
Need for new companion diagnostic markers in cancer therapyNeed for new companion diagnostic markers in cancer therapy
• T...
microRNA-Target Sites:
uncharted territory to identify disease biomarkers
microRNA-Target Sites:
uncharted territory to id...
miRNA-mRNA complementarity is required for target selectionmiRNA-mRNA complementarity is required for target selection
AGO...
miRNA-mRNA complementarity is required for target selectionmiRNA-mRNA complementarity is required for target selection
AGO...
miRNA-mRNA complementarity is required for target selectionmiRNA-mRNA complementarity is required for target selection
AGO...
miRNA-mRNA complementarity is required for target selectionmiRNA-mRNA complementarity is required for target selection
5'
...
Hypothesis: variants in microRNA-target sites can
deregulate gene expression and result in cancer
Hypothesis: variants in ...
Identification of a germline let-7 target site variant (rs61764370)Identification of a germline let-7 target site variant ...
rs61764370 associates with cancer riskrs61764370 associates with cancer risk
Cancer Subtype (subgroup) Genotype
Fold-incre...
rs61764370 associates with poor OS in HNSSCrs61764370 associates with poor OS in HNSSC
Christensen et al, Carcinogenesis (...
rs61764370 associates with poor OS in ovarian cancerrs61764370 associates with poor OS in ovarian cancer
Ratner et al, Onc...
rs61764370 confers platinum resistance in ovarian cancerrs61764370 confers platinum resistance in ovarian cancer
Adapted f...
rs61764370 is sufficient to up-regulate KRAS gene expressionrs61764370 is sufficient to up-regulate KRAS gene expression
p...
rs61764370 positive tumors display unique transcription patternsrs61764370 positive tumors display unique transcription pa...
Targeted therapeutics in the EGFR signaling pathwayTargeted therapeutics in the EGFR signaling pathway
EGFR
Proliferation
...
rs61764370 associates with poor OS in mCRC patients
undergoing cetuximab-irinotecan salvage therapy
rs61764370 associates ...
The germline rs61764370 3’UTR variant phenocopies
a tumor acquired KRAS ORF mutation
The germline rs61764370 3’UTR variant...
Future directions
Clinicopathology
Hypotheses
Genotype
Summary and Current Work FlowSummary and Current Work Flow
rs617643...
Generation of Isogenic KRAST/T
and KRAST/G
Cell Lines
(workflow)
Generation of Isogenic KRAST/T
and KRAST/G
Cell Lines
(wo...
Generation of Isogenic KRAST/T
and KRAST/G
Cell Lines
(workflow)
Generation of Isogenic KRAST/T
and KRAST/G
Cell Lines
(wo...
Generation of Isogenic KRAST/T
and KRAST/G
Cell Lines
(workflow)
Generation of Isogenic KRAST/T
and KRAST/G
Cell Lines
(wo...
Sequence Verification of MCF10a Isogenic Cell LinesSequence Verification of MCF10a Isogenic Cell Lines
Reverse
Sequence
Re...
MCF10a rs61764370 positive cells senesce in culture
and display a mesenchymal-like morphology
MCF10a rs61764370 positive c...
Knock-in of rs61764370 into MCF10a cells caused
an epithelial-to-mesenchymal transition (EMT)
Knock-in of rs61764370 into ...
Knock-in of rs61764370 into MCF10a caused a mild growth defectKnock-in of rs61764370 into MCF10a caused a mild growth defe...
Sequence verification of HCC1937 isogenic cell linesSequence verification of HCC1937 isogenic cell lines
‘T-allele’
Allele...
HCC1937 rs61764370 positive cells display altered platting
efficiency and cell growth
HCC1937 rs61764370 positive cells di...
MEK inhibitors target HCC1937 rs61764370 positive cellsMEK inhibitors target HCC1937 rs61764370 positive cells
anti-MEK
Se...
BATTLE1: Biomarker-integrated Approaches of Targeted Therapy
of Lung Cancer Elimination
BATTLE1: Biomarker-integrated Appr...
AcknowledgementsAcknowledgements
The Weidhaas Lab
Collaborators
Frank Slack, Roy Herbst – Yale University
Nicola Miller, M...
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miRNA-Target Site SNPs as Predictors for Cancer Risk and Treatment Response

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  • Joanne conflict of interest
  • Patients w/ G-allele are otherwise KRAS ORF WT - ORF mutations in G-allele carries is very rare EGFR mutations in G-allele carriers is also very rare Looked in virtually every cancer type - and found KRAS variant - especially in non-informatiave
  • Over 6,750 KRAS WT lung cancer patients were tested for the KRAS- variant in this initial report
  • Brock Christensen - in Karl Kelsey’s lab at Brown
  • Everyone gets the same treatment - platinum
  • Gene expression profiling from 74 TNBC samples Stratified by transcriptional units Over expression of angiogenesis pathways
  • Sorafenib = RAF inhibitor - FDA approved for liver and kidney cancer Vemurafenib = RAF V600E specific inhibitor - FDA approved for late stage melanoma MEK inhibitors = none in phase III yet...but moving in BRAF V600E mutant melanoma
  • Metastatic Colorectal Cancer Patients KRAS (ORF) WT BRAF (ORF) WT TREATED with Cetuximab
  • Metastatic Colorectal Cancer Patients KRAS (ORF) WT BRAF (ORF) WT TREATED with Cetuximab
  • Normal media for MCF10a cells is: BPE, EGF, Insulin, Hydrocortisone, cholera toxin MCF10a cells senesce in culture They require serum – when we gave them serum – they grew like crazy and had altered morphology
  • miRNA-Target Site SNPs as Predictors for Cancer Risk and Treatment Response

    1. 1. David W. Salzman Yale School of Medicine Department of Therapeutic Radiology Fallopian Mucosa NSCLC Papillary Serous Ovarian Tumor DicermAb(13D6) miRNA-Target Site SNPs as Predictors of Cancer Risk and Treatment Response
    2. 2. Need for new companion diagnostic markers in cancer therapyNeed for new companion diagnostic markers in cancer therapy • Tumors contain a heterogeneous array of inherited and acquired mutations • Best cure rates are achieved when specific drugs are used to target tumors with particular mutations (targeted therapeutics) • Current strategies to identify targeted therapeutics rely heavily on the identification of tumor acquired mutations • May only be represented in a small population of cells – elude identification • Short-term response is good, long-term response is poor due to drug resistance Therefore: we need a new paradigm to identify companion diagnostics that do NOT rely on identifying tumor acquired mutations but where do you find such mutations in the genome?
    3. 3. microRNA-Target Sites: uncharted territory to identify disease biomarkers microRNA-Target Sites: uncharted territory to identify disease biomarkers • The 3’UTR of mRNAs contain cis-regulatory elements that regulate the nature and timing of gene expression in conjunction with a requisite trans-acting factor • MicroRNAs are a class of non-coding, trans-acting RNAs that negatively regulate gene expression by binding to complementary elements in the 3’UTR of a target mRNA 5' 3'AAAAAA ORF5’UTR 3’UTR Protein
    4. 4. miRNA-mRNA complementarity is required for target selectionmiRNA-mRNA complementarity is required for target selection AGO 3' 5' • Seed pairing = complementarity between nucleotides 2-7 • ± complementarity of nucleotide 8 • ± Adenosine residue opposite nucleotide 1 • ± 3’ end complementarity 5' 3'AAAAAA ORF5’UTR 3’UTR Protein 3’ NNNNNNNNNNNNNNNNNNNNNN 5’ |||||||||| ||||||| 5’...NNNNNNNNNNNNNNNNNNNNNNNNNNNN...3’ 3’ NNNNNNNNNNNNNNNNNNNNNN 5’ |||||||||| ||||||| 5’...NNNNNNNNNNNNNNNNNNNNNNNNNNNN...3’
    5. 5. miRNA-mRNA complementarity is required for target selectionmiRNA-mRNA complementarity is required for target selection AGO 3' 5' 5' 3'AAAAAA ORF5’UTR 3’UTR Protein Translation Inhibition X • Seed pairing = complementarity between nucleotides 2-7 • ± complementarity of nucleotide 8 • ± Adenosine residue opposite nucleotide 1 • ± 3’ end complementarity
    6. 6. miRNA-mRNA complementarity is required for target selectionmiRNA-mRNA complementarity is required for target selection AGO 3' 5' • Centered pairing = complementarity between nucleotides (approx.) 6-16 • Must include Watson-Crick base-pairing between nucleotides 9-12 5' 3'AAAAAA ORF5’UTR 3’UTR Protein 3’ NNNNNNNNNNNNNNNNNNNNNN 5’ |||||||||||||| 5’...NNNNNNNNNNNNNNNNNNNNNNNNNNNN...3’ 3’ NNNNNNNNNNNNNNNNNNNNNN 5’ |||||||||||||| 5’...NNNNNNNNNNNNNNNNNNNNNNNNNNNN...3’
    7. 7. miRNA-mRNA complementarity is required for target selectionmiRNA-mRNA complementarity is required for target selection 5' 3' AAAAAA ORF 5’UTR 3’UTR Protein X mRNA cleavage • Centered pairing = complementarity between nucleotides (approx.) 6-16 • Must include Watson-Crick base-pairing between nucleotides 9-12
    8. 8. Hypothesis: variants in microRNA-target sites can deregulate gene expression and result in cancer Hypothesis: variants in microRNA-target sites can deregulate gene expression and result in cancer X SNP inhibit oncogene targeting enhance (or lead to aberrant) targeting of a tumor suppressor X SNP over expression of oncogene under expression of tumor suppressor
    9. 9. Identification of a germline let-7 target site variant (rs61764370)Identification of a germline let-7 target site variant (rs61764370) 5' 3'AAAAAA KRAS5’UTR 3’UTR 1 9 2 3 4 5 610 78 LCS6 Genotype Tumor and NAT Tumor TT 35 24 TG/GG 8 7 n=74 (NCSLC patients) G-allele present in (approx.) 20% of lung cancer patients (otherwise KRAS ORF WT) Chin et al, Cancer Research (2008) • let-7 targets the KRAS 3’UTR • 10 predicted let-7 complementary sites
    10. 10. rs61764370 associates with cancer riskrs61764370 associates with cancer risk Cancer Subtype (subgroup) Genotype Fold-increased risk (OR, 95% CI; p-value) Reference Lung Non-small cell lung cancer (NSCLC) TG/GG 2.3 (1.1-4.6; p=0.02) Chin et al, 2008 Ovarian Hereditary breast ovarian (HBOC) TG/GG 2.46 (1.14-5.29; p=0.02) Ratner et al, 2010 Breast Triple negative (ER- /PR- /Her2- ) TG/GG 2.307 (1.261-4.219; p=0.0067) Paranjape et al, 2011 multivariate analysis – adjusted for age and race • Over 40,000+ individuals studies worldwide • Represented in (approx.) 6% of world populations • More frequently associated with cancer in women • Associated with later onset for most patients
    11. 11. rs61764370 associates with poor OS in HNSSCrs61764370 associates with poor OS in HNSSC Christensen et al, Carcinogenesis (2009) TT (wild type) TG/GG (variant) n=344 All sites (oral, pharyngeal, laryngeal) HR (CI 95%): 1.6 (1.0-2.5, p=0.20) TT (wild type) TG/GG (variant) n=190 Oral cancer HR (CI 95%): 2.7 (1.4-5.3, p=0.06) • rs61764370 is present in (approx.) 17.5% of this HNSSC cohort • Treatment not detailed
    12. 12. rs61764370 associates with poor OS in ovarian cancerrs61764370 associates with poor OS in ovarian cancer Ratner et al, Oncogene (2011) Variable HR 95% CI p-value KRAS mutation 1.671 1.087 - 2.568 0.0192 Age 1.025 1.002-1.049 0.0307 Stage 1.380 1.185-1.607 <0.0001 Grade 1.341 0.912-1.972 0.1360 Histology 0.970 0.900-1.045 0.4168 Center (Yale vs non-Yale) 1.868 1.438-2.427 <0.0001 TT (wild type) TG/GG (variant) n=279 Ovarian cancer HR (CI 95%): 1.671 (1.087-2.568, p=0.0192)
    13. 13. rs61764370 confers platinum resistance in ovarian cancerrs61764370 confers platinum resistance in ovarian cancer Adapted from Ratner et al, Oncogene (2011) Genotype Univariate Multivariate OR 95% CI p-value OR 95% CI p-value Wild type (TT) (n=225) 1.00 1.00 Variant (TG/GG) (n=66) 2.45 1.08-5.53 0.0313 3.18 1.31-7.72 0.0106 Multivariate analysis: adjusted for age, stage, grade, histology, residual disease after cytoreductive surgery and treatment center Cell Line Gemcitabin e Doxorubicin Topotecan BG1 (TG variant) 30.4uM 307.5nM 161.8nM CAOV3 (TT wild type) 2.2nM 75.9nM 30.8nM p=<0.0001 p=<0.04 (TG variant) (TT wild type) (TG variant/ BRCA1 MT) 1st line therapy 2nd line therapy
    14. 14. rs61764370 is sufficient to up-regulate KRAS gene expressionrs61764370 is sufficient to up-regulate KRAS gene expression p=0.007 p=0.036 TT TG KRAS Actin TT TG Lung Normal Lung 1o Tumor
    15. 15. rs61764370 positive tumors display unique transcription patternsrs61764370 positive tumors display unique transcription patterns Expression Signature TT vs TG/GG TNBC Tumor pK-S Test NRAS up 0.02 BRCA mutant-like up 0.04 Luminal Progenitor up 0.04 MAPK Creighton up 0.06 PCA Estrogen down 0.04 Adapted from Paranjape et al, Lancet Oncology (2010) The let-7 family of microRNAs is also consistently and significantly down-regulated in rs61764370 positive (NSCLC, TNBC, ovarian and HNSSC) tumors Adapted from Ratner et al, Oncogene (2011) TTTG/GG n=10, p=0.095 TTTG/GG n=10, p=0.095 TTTG/GG n=10, p=0.05
    16. 16. Targeted therapeutics in the EGFR signaling pathwayTargeted therapeutics in the EGFR signaling pathway EGFR Proliferation RASRAS RAFRAF MEKMEK MAPKMAPK anti-EGFR Gefitinib Erlotinib Cetuximab T790M L858R G12D Q61L V600E anti-BRAF Sorafenib anti-MEK Selumetinib Trametinib AZD6244
    17. 17. rs61764370 associates with poor OS in mCRC patients undergoing cetuximab-irinotecan salvage therapy rs61764370 associates with poor OS in mCRC patients undergoing cetuximab-irinotecan salvage therapy anti-EGFR Cetuximab TT (wild type) n=100 TG/GG (variant) n=34 p=0.001 Graziano et al, Pharmacogenomics J. (2010) *Patient cohort was otherwise KRAS ORF WT and BRAF ORF WT EGFR Proliferation RASRAS RAFRAF MEKMEK MAPKMAPK rs61764370
    18. 18. The germline rs61764370 3’UTR variant phenocopies a tumor acquired KRAS ORF mutation The germline rs61764370 3’UTR variant phenocopies a tumor acquired KRAS ORF mutation KRASKRAS5' 3' Tumor Acquired KRAS ORF Mutation X X Cancer anti-EGFR Rx resistance KRASKRAS5' 3' Germ-line KRAS rs61764370 T>G X X Cancer anti-EGFR Rx resistance KRASKRAS5' 3' WT KRAS Normal anti-EGFR Rx sensitive let-7 RISC let-7 RISClet-7 RISC
    19. 19. Future directions Clinicopathology Hypotheses Genotype Summary and Current Work FlowSummary and Current Work Flow rs61764370rs61764370 Increased cancer riskIncreased cancer risk Altered Response to Therapy Altered Response to Therapy rs61764370 positive cells will display enhanced cancer- associated phenotypes rs61764370 positive cells will display enhanced cancer- associated phenotypes We can selectively target rs61764370 positive cells We can selectively target rs61764370 positive cells rs61764370 is a predictive biomarker to direct cancer therapy rs61764370 is a predictive biomarker to direct cancer therapy Clinical trialsClinical trials Isogenic cell lines High throughput screening of FDA- approved compounds High throughput screening of FDA- approved compounds Cell biology: Transformation, growth, Mobility, Invation, EMT Cell biology: Transformation, growth, Mobility, Invation, EMT
    20. 20. Generation of Isogenic KRAST/T and KRAST/G Cell Lines (workflow) Generation of Isogenic KRAST/T and KRAST/G Cell Lines (workflow) Obtain cell line from NCI (KRAST/T ) Transfect with •zinc-finger plasmids (x2) •donor plasmid A B C 3 2 1 { (3) zinc-fingers/nuclease (highly specific DNA binding) bidentate nucleases (dsDNA cleavage) KRAS 3’UTR dsDNA cleavage DNA repair Homologous recombination Donor (mutant KRAS 3’UTR) A B C 3 2 1 DNA binding
    21. 21. Generation of Isogenic KRAST/T and KRAST/G Cell Lines (workflow) Generation of Isogenic KRAST/T and KRAST/G Cell Lines (workflow) Obtain cell line from NCI (KRAST/T ) Transfect with •zinc-finger plasmids (x2) •donor plasmid Single cell clone Expand 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H X X X X X Exclude wells w/ >1 cell Expand Extract gDNA
    22. 22. Generation of Isogenic KRAST/T and KRAST/G Cell Lines (workflow) Generation of Isogenic KRAST/T and KRAST/G Cell Lines (workflow) Obtain cell line from NCI (KRAST/T ) Transfect with •zinc-finger plasmids (x2) •donor plasmid Single cell clone Expand Screen for rs61764370 insertion into KRAS 3’UTR ID KRAST/G •sequence verify •expand •store TaqMan genotype for rs61764370 (ID positive clones) PCR amplify KRAS 3’UTR (1kb +/- donor sequence) Topo clone PCR amplicon TaqMan genotype bacterial colonies for rs61764370 Cell Line Cell type rs61764370 (T:G) Cal27 (+ control) Lung 12:16 MCF10a-luc Parental Normal breast epithelial 12:0 MCF10a-luc Isogenic Normal breast epithelial 15:15 HCC1937 Parental TNBC (BRCA1-/- ) 12:0 HCC1937 Isogenic TNBC (BRCA1-/- ) 22:19 H1299 Parental Lung (P53-/- ) 10:0 H1299 Isogenic Lung (P53-/- ) 15:16 If KRAST/G then allele frequency = 50:50 (T:G) 1 4 cell lines failed to make isogenic pairs 2 isogenic cell line generation is ongoing process analysisof positionalinsertion
    23. 23. Sequence Verification of MCF10a Isogenic Cell LinesSequence Verification of MCF10a Isogenic Cell Lines Reverse Sequence Reads Allele-1 Allele-2 MCF10a WT (KRAS 3’UTRT/T ) Forward Sequence Reads ‘T-allele’ Allele-1 ‘G-allele’ Allele-2 MCF10a MT (KRAS 3’UTRT/G ) Allele-1 A T C Allele-2 T TC Allele-1 A T C Allele-2 G TC * * * * * * * * rs612587 rs61764370 rs2966 rs612587 rs61764370 rs2966
    24. 24. MCF10a rs61764370 positive cells senesce in culture and display a mesenchymal-like morphology MCF10a rs61764370 positive cells senesce in culture and display a mesenchymal-like morphology
    25. 25. Knock-in of rs61764370 into MCF10a cells caused an epithelial-to-mesenchymal transition (EMT) Knock-in of rs61764370 into MCF10a cells caused an epithelial-to-mesenchymal transition (EMT)
    26. 26. Knock-in of rs61764370 into MCF10a caused a mild growth defectKnock-in of rs61764370 into MCF10a caused a mild growth defect 0 0.2 0.4 0.6 0.8 1 1.2 0 20000 40000 60000 80000 100000 Cell number Absorbance MCF10a WT MCF10a MT
    27. 27. Sequence verification of HCC1937 isogenic cell linesSequence verification of HCC1937 isogenic cell lines ‘T-allele’ Allele-1 ‘G-allele’ Allele-2 HCC1937 MT (KRAS 3’UTRT/G ) Reverse Sequence Reads Allele-1 Allele-2 HCC1937 WT (KRAS 3’UTRT/T ) Forward Sequence Reads Allele-1 T C Allele-2 T TC T-Allele T C G-Allele G TC CC * ** * * ** * rs612587 rs61764370 rs2966 rs612587 rs61764370 rs2966
    28. 28. HCC1937 rs61764370 positive cells display altered platting efficiency and cell growth HCC1937 rs61764370 positive cells display altered platting efficiency and cell growth 2D Numberofcolonies HCC1937 WT HCC1937 MT HCC1937 WT HCC1937 MT 100 3D Numberofcolonies HCC1937 WT HCC1937 MT HCC1937 WT HCC1937 MT
    29. 29. MEK inhibitors target HCC1937 rs61764370 positive cellsMEK inhibitors target HCC1937 rs61764370 positive cells anti-MEK Selumetinib Trametinib AZD6244 HCC1937 MT (TG) HCC1937 WT (TT) EGFR Proliferation RASRAS RAFRAF MEKMEK MAPKMAPK rs61764370
    30. 30. BATTLE1: Biomarker-integrated Approaches of Targeted Therapy of Lung Cancer Elimination BATTLE1: Biomarker-integrated Approaches of Targeted Therapy of Lung Cancer Elimination 0-wt (Event/N = 32/32) 1-Variant (Event/N = 4/4) p=0.001 (Erlotinib) 0-wt (Event/N = 39/61) 1-Variant (Event/N = 3/8) p=0.056 (Sorafenib) anti-EGFR Erlotinib anti-BRAF Sorafenib EGFR Proliferation RASRAS RAFRAF MEKMEK MAPKMAPK rs61764370
    31. 31. AcknowledgementsAcknowledgements The Weidhaas Lab Collaborators Frank Slack, Roy Herbst – Yale University Nicola Miller, Michael Kerin – National University of Ireland Kim Smits, Manon van England - Maastricht University Jeffrey Weitzel – City of Hope Rob Pilarski – OHSU Ken Offit – MSKCC Christine Chung – JHMI Sabine Tejpar – Leuven Xifeng Wu, Hai Tran - MDACC Funding NIH/NCI RO1 NIH K08 Yale Cancer Center Mary K. Ashe Foundation Shanon Foundation RTOG Seed Grants CT State Funding

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