Targeted Cancer Therapy
Xiaole Shirley Liu
STAT115, STAT215, BIO298, BIST520
Hallmarks of Cancer
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Mutually Exclusivity and Co-occurrence
• Most cancers have >=2 sequential mutations developed over many years.
• Mutations in different pathways can co-occur in the same cancer, whereas those in the same pathway are rarely mutated in the same sample.
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Why Tumor Sequencing
• http://www.foundationone.com video• Chemotherapy vs targeted therapy– Chemotherapy: non-specific cytotoxic drugs, mostly
affecting dividing cells, mostly intravenous
– Targeted: inhibit a specific target, less toxic to normal cells, mostly oral
• Many major cancer hospitals in US started patient tumor sequencing
• The hope to identify the correct targeted therapy
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Cancer Profiles vs Treatment
• “The Difficulty is going to be figuring out how to use the information to help people rather than to just catalogue lots and lots of mutations.” – Bert Voglestein, John Hopkins University
• Chemotherapy vs targeted therapy– Chemotherapy: non-specific cytotoxic drugs, mostly
affecting dividing cells, mostly intravenous
– Targeted: inhibit a specific target, less toxic to normal cells, mostly oral
• http://www.foundationone.com video
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~479 genes
Limited Number of Cancer Driver GenesHalf Druggable
ALK Inhibitors
• ALK normally functions in the brain
• First rearrangement in lung cancer discovered 2007 in Japan
• Upstream of multiple cancer pathways
• 2010 starting clinical trials on ALK inhibitor
• 2011 FDA approved crizotinib
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Testing on Patients Takes Lots of Time and Money
Can we do this faster?
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Cell Line Drug Screens
• CGP: 138 drugs on 727 cell lines
• CCLE: 24 drugs on 1,036 cell lines
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Targeting a Cancer Pathway
• Why bother screening if we know the target of a drug? E.g. doesn’t ALK inhibitor inhibit ALK?
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Cell Line Drug Screens
• Cell lines: – Expression– Mutations– Drug sensitivity
measure: IC50, half maximal inhibitory concentration (IC50)
• How to find expression or mutation biomarkers for drug response?
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Drug Response BioMarkers
• Mutations
• Expression
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AHR expression high or low on MEK inhibitor (PD-0325901)
Instead of Drug-Focused, Can we Test Tumor-Specific
Therapies?
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Genome-wide Loss of Function Screens
• Get rid of a gene (DNA or RNA) in a cell
• See how it influences one specific cancer cell as compared to other cells (specificity)
• Can we do this in high throughput?
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Profile Cancer Cell Vulnerability
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CRISPR-Cas:Bacterial Adaptive Immune System• Clustered regularly interspaced short palindromic
repeats
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CRISPR-Cas9 Knockout
• Guide RNA allows Cas9 to make ds breaks at specific genomic locations in the genome
• Repairs on exonic breaks create loss-of function gene knockouts
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Genome-Wide CRISPR-Cas9 Knockout Screen
• Positive selection:
– Guide abundance up
– Knockout genes make cells grow faster
• Negative selection:
– Guide abundance down
– Knockout genes make cells grow slower
• Identify cell-specific and condition-specific essential genes and biomarkers of drug response & resistance
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Shalem et al, Science, 2014
Genome-Wide CRISPR/Cas9 Knockout Screens
• Each vector contains a guide sequence (sgRNA) knock out a gene (influence DNA) instead of knock down expression (influence RNA)
• Detection through sequencing instead of bar-coded arrays
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Shalem et al, Science 2014; Wang et al, Science 2014
Analyzing Ge-LoF Screen Data
• How to normalize raw data?
• What if one shRNA / sgRNA doesn’t work
• How to identify key genes if we have multiple shRNAs / sgRNA per gene?
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Targeted Therapy
• ENO1 and ENO2 parallel pathway
• Glioblastoma tumors with ENO1 deletion (5%) is sensitive to ENO2 inhibition
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Drug Resistance
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Targeted Therapy I
• AR and prostate cancer
• Antiandrogen resistance
• Mutation on AR LBD
• ERG expands AR cistrome
• GR over expression
• EMT
• Luminal to basal
Targeted Therapy II
• EGFR and non-small cell lung cancer• EGFR inhibitor resistance• T790M• MET amplification• HGF production• PI3K mutation• SCLC
Similarities
• Anti AR resistance– Mutation on AR LBD
– ERG expands AR cistrome
– GR over expression
– EMT
– Luminal to basal
• Anti EGFR resistance– T790M
– HGF production
– MET amplification
– PI3K mutation
– SCLC
Resistance Mechanism
• Is resistance developed before or after drug treatment?
• Point mutation rate: 10^-9
• Minority of resistance clones get selected for clonal expansion
• Possible ways to evade drug?
Tumor Heterogeneity
Mathematical Models of Resistance
• Use cell data to estimate the parameters• Test agreement between simulation and
observation• Suggest full dose neo-adjuvant chemotherapy
before surgery
Haeno et al, Cell 2012
How to Find Resistance Mechanisms
• Cell lines?• CCLE and CGP drug screens– Drug A works for cancers with Gene A mutation
– Identify informative Gene B whose expression or mutation influence cancer response to drug A
– If Gene B is also drugable, then can find combination A & B for tumor subsets
How to Find Resistance Mechanisms
• Cell lines?• CCLE and CGP drug screens• shRNA or sgRNA screens– Treat resistant cell with drug or no drug
– Does ko/kd of a gene make the cell more / less sensitive?
Effective Drug Treatment
• A perfect recipe for certain treatment failure: sequential therapy.
• Successful combination therapy must force the cancer to make at least two mutations steps.
• Is current FDA clinical trials unethical?
Summary
• Use expression and mutations as biomarkers to predict drug response
• Use high throughput screening in cell lines to identify specific targets essential for cancer cells
• Resistance to targeted therapy is extremely prevalent, and many present in initial tumor
• Tumor heterogeneity and cancer evolution• Effective treatment: neo-adjuvant and
combination therapy
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Acknolwedgement
• John Pack• James Lechner• Alex Chenchik• Natalia Kamarova• Haiyun Wang
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