Targeted Cancer Therapy Xiaole Shirley Liu STAT115, STAT215, BIO298, BIST520

Hallmarks of Cancer 2

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. 3

Why Tumor Sequencing videohttp:// 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 4

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 videohttp:// 5

6 ~479 genes Limited Number of Cancer Driver Genes Half 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 7

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Testing on Patients Takes Lots of Time and Money Can we do this faster? 9

Cell Line Drug Screens CGP: 138 drugs on 727 cell lines CCLE: 24 drugs on 1,036 cell lines 10

Targeting a Cancer Pathway Why bother screening if we know the target of a drug? E.g. doesn’t ALK inhibitor inhibit ALK? 11

Cell Line Drug Screens Cell lines: –Expression –Mutations –Drug sensitivity measure: IC50, half maximal inhibitory concentration (IC 50 ) How to find expression or mutation biomarkers for drug response? 12

Drug Response BioMarkers Mutations Expression 13 AHR expression high or low on MEK inhibitor (PD )

Instead of Drug-Focused, Can we Test Tumor-Specific Therapies? 14

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? 15

Profile Cancer Cell Vulnerability 16

CRISPR-Cas: Bacterial Adaptive Immune System Clustered regularly interspaced short palindromic repeats 17

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 18

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 19 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 20 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? 21

Targeted Therapy ENO1 and ENO2 parallel pathway Glioblastoma tumors with ENO1 deletion (5%) is sensitive to ENO2 inhibition 22

Drug Resistance 23

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 33

Acknolwedgement John Pack James Lechner Alex Chenchik Natalia Kamarova Haiyun Wang 34