miRNA-Target Site SNPs as Predictors for Cancer Risk and Treatment Response
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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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
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. 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
Hinweis der Redaktion
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