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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