Discovery of Drug Sensitizing Genotypes in Discovery of Drug Sensitizing Genotypes in Cancer Cells Mathew Garnett NCT Conference Heidelberg, Sept 2013
Precision Cancer Medicine Precision Cancer Medicine • Using targeted drugs to exploit specific vulnerabilities and dependencies within cancer cells. • Genomic alterations can be used as biomarkers to Genomic alterations can be used as biomarkers to identify patients most likely to benefit from treatment treatment.
Combined BRAF and MEK inhibitors for the treatment of BRAF mutant melanoma l Flaherty et al, NEJM 2012
Targeted Molecular Therapies Targeted Molecular Therapies Mutated cancer genes as biomarkers of drug response: Mutated cancer genes as biomarkers of drug response: FDA ‐ approved targeted therapies Molecular biomarker FDA-approved drug M l l bi k FDA d d Cli i Clinical indication(s) l i di ti ( ) Th Therapeutic target ti t t BCR-ABL Imatinib, Dasatinib, Nilotinib CML, AML ABL1 KIT, PDGFR Imatinib Gastrointestinal stromal tumour KIT, PDGFRA EGFR Gefitinib, Erlotinib Non-small cell lung cancer, pancreatic EGFR ERBB2/HER2 ERBB2/HER2 T Trastuzumab, Lapatinib t b L ti ib HER+ breast cancer HER b t HER2 HER2 BRAF Vemurafinib melanoma BRAF EML4-ALK Crizotinib Non-small cell lung cancer ALK ER+ Tamoxifen ER+ breast cancer ER
Preclinical Biomarker Discovery Preclinical Biomarker Discovery • To systematically explore pre ‐ clinically the diversity To systematically explore pre clinically the diversity of cancer for biomarkers that predict drug sensitivity sensitivity. • To understand the landscape of drug response in relation to cancer genes. g • To identify effective combinatorial therapies to T id tif ff ti bi t i l th i t circumvent resistance.
High ‐ throughput Drug Screening in Cancer Cells High throughput Drug Screening in Cancer Cells a b c [drug] [drug] IC50 concentration (uM) 72 hour drug treatment Fluorescence based viability assay Fluorescence based viability assay
High throughput Drug Screening in Cancer Cells High ‐ throughput Drug Screening in Cancer Cells Single drug screens Single drug screens Cancer cell lines Cancer cell lines Cancer cell lines Cancer cell lines Combinatorial screens Patient ‐ derived cultures Patient ‐ derived cultures siRNA +/ ‐ drug Cancer organoids Cancer organoids Drug resistant clones Drug resistant clones Drug resistant clones Drug resistant clones a b c [drug] [drug] IC50 concentration (uM) 72 hour drug treatment Fluorescence based viability assay Fluorescence based viability assay Link drug response with genomic features
Screening compounds are selected to t target cancer pathways t th Energy/ATP Synthesis Amino Acid Synthesis Ser/Thr Kinase (mTOR) Translation Nucleotide Synthesis Gene Expression Gene Expression Chromatin (EZH2) Differentiation (Wnt) Tyrosine Kinase (EGFR) Tyrosine Kinase (EGFR) Ser/Thr Kinase (BRAF) Cell Cycle (Aurora) DNA Damage ER Stress Senescence Senescence Autophagy Apoptosis (Bcl2, IAP) Other Cell Death
The GDSC1000 Cell Line Collection Soft tissue Testis Bone Hodgkin lymphoma Burkitt lymphoma Other Other Cell lines are grouped according to the TCGA classification system
Pharmacogenomic Characterisation of the GDSC1000 h GDSC1000
Cancer Cell Lines as an Experimental Model Biological relevance Experimental tractability
Systematic Analysis of Drug Sensitivity Systematic Analysis of Drug Sensitivity IC50 value heatmap • 551 anti ‐ cancer drugs • Mean of 432 cell lines screened per drug (range 7 – 672) • 238,000 drug ‐ cell line combinations g • Correlated drug response with coding mutation, amplification and deletion in 71 frequently mutated cancer genes.
BRAF Mutations Confer Sensitivity to BRAF and MEK inhibitor combo C ll li Cell line IC50s to combination of BRAF and MEK inhibitor IC50 t bi ti f BRAF d MEK i hibit
Drug response associated with BRAF mutational status Dabrafenib + Trametinib ‐ vlaue) Multiple MEK and BRAF inhibitors Multiple MEK and BRAF inhibitors ficance (p Signif sensitivity resistance
Landscape of drug response in relation to cancer genes Target(s) drug g 1e − 55 SB590885 BRAF 9e − 50 Nilotinib ABL 1e − 50 (BRAF) (BCR ‐ ABL) 1e − 45 gene g PLX4720 BRAF PLX4720 BRAF 9e − 40 1e − 40 (BRAF) GDSC SC vlaue) 1e − 35 30 cance (p ‐ v 1e 30 1e − 30 9e − 1e − 25 G 9e − 20 1e − 20 Signifi 1e − 15 9e − 10 1e − 10 1e − 05 20% fdr = 1 18e − 02 20% fdr = 1.18e 02 p = 0.05 0 05 1e+00 1e+00 1e − 07 1e − 06 1e − 05 1e − 04 1e − 03 1e − 02 1e − 01 1e+00 1e+01 1e+02 1e+03 sensitivity resistance 1924 significant gene drug interactions (p<0.05, 20% FDR)
Cell line models capture clinical markers of drug sensitivity k f d i i i FDA ‐ approved targeted therapies pp g p Molecular biomarker FDA-approved drug Clinical indication(s) Therapeutic target ✔ BCR-ABL Imatinib, Dasatinib, Nilotinib CML, AML ABL1 KIT, PDGFR Imatinib Gastrointestinal stromal tumour KIT, PDGFRA ND ✔ ✔ EGFR EGFR G fiti ib E l ti ib Gefitinib, Erlotinib N Non-small cell lung cancer, pancreatic EGFR ll ll l ti EGFR ✔ ERBB2/HER2 Trastuzumab, Lapatinib HER+ breast cancer HER2 ✔ BRAF Vemurafinib melanoma BRAF ND EML4-ALK Crizotinib Non-small cell lung cancer ALK ✔ ✔ ER+ ER T Tamoxifen if ER ER+ breast cancer b t ER ER Targeted therapies in clinical development Molecular biomarker Drugs in clinical development Clinical indication(s) Molecular biomarker Drugs in clinical development Clinical indication(s) Therapeutic target Therapeutic target ✔ BRAF e.g. PD0325907 melanoma, NSCLC MEK ✔ KRAS e.g. PD0325908 NSCLC MEK ✔ NRAS e.g. PD0325909 melanoma MEK ✔ ✔ FGFR2 e.g. PD173074 g FGFR ✔ PIK3CA e.g. AZD6482 PI3K ✔ PIK3CA e.g. AKT inhibitor VIII AKT ✔ FLT3 e.g. sunitinib FLT3 ✔ BRCA1/2 e.g. Olaparib g p Breast, ovarian , PARP Many novel association identified some of which may represent new therapeutic avenues
The Majority of Cancer Genes are Correlated With Drug Response 200 CDKN2A orrelations 150 significant co APC 100 sensitivity iti it Number of s resistance 50 N median 0 0 1 11 21 31 41 51 61 71 Cancer Genes
The Activity of Most Drugs is Correlated with Cancer Genes 25 tions 20 cant correla 15 er of signific Sensitivity 10 Resistance Numbe 5 median 0 1 1 51 51 101 101 151 151 201 201 251 251 301 301 351 351 401 401 451 451 501 501 Drugs
EWS-FLI1 Mutated Cells are Sensitive to PARP Inhibitors Olaparib AG ‐ 014699 (PARP1/2) (PARP1/2) C 50 (uM) 50 (uM) IC IC n = 14 n = 544 n = 13 n = 467 Mutations of BRCA1 or BRCA2 are not present in these EWS ‐ FLI1 mutated cell lines
EWS FLI1 EWS ‐ FLI1 • Characteristic of Ewing’s sarcoma, a malignant bone tumour that affects g , g children. • A chromosomal translocation (11:22)(q24;q12) fusing the EWSR1 gene to the A chromosomal translocation (11:22)(q24;q12) fusing the EWSR1 gene to the FLI1 gene. • Fusion proteins act as aberrant transcription factors that bind DNA through • Fusion proteins act as aberrant transcription factors that bind DNA through their ETS DNA binding domain. • Current treatment is aggressive chemotherapy, surgery and radiotherapy. C t t t t i i h th d di th • Poor prognosis in the 15 ‐ 25% of patients with metastatic or recurrent disease.
Olaparib Selectively Induces Apoptosis p y p p in Ewing’s Cells B$ FN " L 6 3 7 4L / 9 &' 9 2 3 4546 7 8 M M #9 #9 &' 9 &' 9 2 3 2 3 4546 4546 7 7 8 8 7 7 / # L 4K ,J9 @ H 54 / $ " 4H I 9 @ @ # H 7 G43 EF6 E $ D / C + , - + + + + 2 4 ) ) 3 , @ B ) , ? , ? ? A A ? ? ' Ewing’s
PARP Inhibitors Induce DNA Damage in Ewing’ Cells H2AX Nuclei 8h 5uM AZD2281 5uM AZD2281 15 Ctrl sponders ES8 ncrease in res 10 5 Fold in Olaparib 0 - 2 4 8 4 2 Time (hours) Time (hours) H2AX – Marker of DSBs H2AX – Marker of DSBs
Is PARP inhibitor sensitivity is dependent on the y p EWS-FLI1 translocation? mouse mesenchymal cells e 1.2 1.0 ability 0.8 elative via 0.6 0.4 0 4 R EWS-FLI1 FUS-CHOP 0.2 SKNMC 0.0 0 0 9 8 6 3 5 5 0 3 7 5 1 2 . . . . . . . 2 0 0 0 1 3 6 1 [Olaparib] (uM) [Olaparib] (uM)
PARP Inhibitors Trials in Ewing’s Patients N No single agent activity? Why not? i l t ti it ? Wh t?
Modeling Drug Resistance Modeling Drug Resistance 1000 cell line drug sensitivity screen 1000 cell line drug sensitivity screen Sensitive Intrinsic resistance Acquired resistance • drug combinations • Prolonged drug exp. • RNAi +/ ‐ drug • Insertional Mutagenesis • Clinically observed resistance
Combinatorial Strategies to Overcome Drug Resistance BRAF inhibitor PLX4720 Transform of BRAF_PLX4720 10000 1000 IC50 uM M 100 10 1 0 1 0.1 WT V600 V600 melanoma All tissues colorectal
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