Reconstructing tumour evolution: reconstructing the evolution of a drug-resistant liposarcoma -...

21
Reconstructing the evolution of a drug-resistant liposarcoma David Goode Sarcoma Genetics & Genomics Peter MacCallum Cancer Centre March 28, 2014

description

Tumours are a collection of genetically distinct cellular lineages related that must compete against each other and the external environment. Many cell lineages die out, while those with phenotypes that are advantageous expand. A major clinical consequence of this evolutionary process is the emergence of drug resistant tumour cells. We have established an in vitro model system in which we can induce the human well-differentiated liposarcoma (WDLPS) cell line 778 to acquire resistance to the MDM2 inhibitor Nutlin-3a. I will detail we have applied bioinformatics and evolutionary principles to reconstruct major evolutionary events that occurred as this line acquired drug resistance. Integration of SNP array and exome sequencing data from different time points during the evolution of Nutlin resistance allow us to infer the relative order of genetic changes and how the rate of evolution fluctuated during the course of the experiment.

Transcript of Reconstructing tumour evolution: reconstructing the evolution of a drug-resistant liposarcoma -...

Page 1: Reconstructing tumour evolution: reconstructing the evolution of a drug-resistant liposarcoma - David Goode

Reconstructing the evolution of a

drug-resistant liposarcoma

David Goode

Sarcoma Genetics & Genomics

Peter MacCallum Cancer Centre

March 28, 2014

Page 2: Reconstructing tumour evolution: reconstructing the evolution of a drug-resistant liposarcoma - David Goode

Evolution of Drug Resistance in Tumours

“Clonal cell lineage (clone)”

Page 3: Reconstructing tumour evolution: reconstructing the evolution of a drug-resistant liposarcoma - David Goode

+2.5 μM Nutlin

95 days

in Nutlin

252 days

in Nutlin

778

(liposarcoma)

Experimental Outline

R95

R252

(‘resistant’)

Copy-number (CN)

profiling using SNP

genotyping arrays

778

(‘parental’)

Page 4: Reconstructing tumour evolution: reconstructing the evolution of a drug-resistant liposarcoma - David Goode

SNP genotyping arrays

• B Allele Frequency (BAF)

• Relative frequency of the B allele [0 – 1]

• Log R Ratio (LRR)

• Copy-number relative to normal diploid genomes

A/B

B A

Page 5: Reconstructing tumour evolution: reconstructing the evolution of a drug-resistant liposarcoma - David Goode

Localised but frequent changes in

CN observed in Nutlin-resistant lines

R252 - 778

R95 - 778

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y

Page 6: Reconstructing tumour evolution: reconstructing the evolution of a drug-resistant liposarcoma - David Goode

Change in allelic ratios on chr15 in R252

(red) from 778 (black)

Page 7: Reconstructing tumour evolution: reconstructing the evolution of a drug-resistant liposarcoma - David Goode

Change in allelic ratios on chr15 occurred between R95 & R252

Page 8: Reconstructing tumour evolution: reconstructing the evolution of a drug-resistant liposarcoma - David Goode

+2.5 μM Nutlin

Loss of one

copy of chr15

778

(‘parental’)

Ordering chromosomal CN changes

R95

R252

(‘resistant’)

Page 9: Reconstructing tumour evolution: reconstructing the evolution of a drug-resistant liposarcoma - David Goode

Gain of section of chr11q in resistant lines

Page 10: Reconstructing tumour evolution: reconstructing the evolution of a drug-resistant liposarcoma - David Goode

+2.5 μM Nutlin

Gain of part

of 11q

Gain of one

copy of chr15

778

(‘parental’)

Ordering chromosomal CN changes

R95

R252

(‘resistant’)

Page 11: Reconstructing tumour evolution: reconstructing the evolution of a drug-resistant liposarcoma - David Goode

Exome sequencing

• 12 clones each from R252 (resistant) and 778 (parental)

• Clones derived from single cells

• Nimblegen exome capture

• Illumina Hi-Seq (100X+ coverage)

• SNVs called with GATK’s Unified Genotyper

• Use frequency of alternate (non-reference) allele to

identify CN polymorphisms

Page 12: Reconstructing tumour evolution: reconstructing the evolution of a drug-resistant liposarcoma - David Goode

BAF pattern on chr11 not present in

any clones from 778

778 Clone 1 (8/12) 778 Clone 2 (4/12) All R252 clones

Page 13: Reconstructing tumour evolution: reconstructing the evolution of a drug-resistant liposarcoma - David Goode

+2.5 μM Nutlin

Gain of part

of 11q

Gain of one

copy of chr15

778

(‘parental’)

Ordering chromosomal CN changes

R95

R252

(‘resistant’)

Page 14: Reconstructing tumour evolution: reconstructing the evolution of a drug-resistant liposarcoma - David Goode

BAF pattern on chr5 present in

minority 778 clone

778 Clone 1 (11/12) 778 Clone 2 (1/12) All R252 clones

Most likely ancestor

of resistant clones

Page 15: Reconstructing tumour evolution: reconstructing the evolution of a drug-resistant liposarcoma - David Goode

+2.5 μM Nutlin

Gain of part

of 11q

Gain of one

copy of chr15

778

(‘parental’)

Ordering chromosomal CN changes

R95

R252

(‘resistant’)

Gain one copy

of chr5

Page 16: Reconstructing tumour evolution: reconstructing the evolution of a drug-resistant liposarcoma - David Goode

778

(‘parental’)

Ordering observed copy-number (CN) changes [relative to 778 SNP arrays]

R95

R252

(‘resistant’)

8 (4, 3, 1)

3 (2, 1, 0)

6 (3, 2, 1)

# of CN changes

(Loss, Gain, Other)

+2.5 μM Nutlin

Page 17: Reconstructing tumour evolution: reconstructing the evolution of a drug-resistant liposarcoma - David Goode

Phylogeny of 778 and R252 clones

Phylip (DNApars)

Page 18: Reconstructing tumour evolution: reconstructing the evolution of a drug-resistant liposarcoma - David Goode

0.87

1.93

0.66

2.79

2.99

2.65

2.45

3.31

2.38

2.16

2.45

2.42

2.32

2.12

1.2

0.7

0.99

0.88

0.96

0.94

1.33

1.06

0.95

1.005

Clone phylogeny labeled with Nutlin IC50 values

*share a non-syn

TP53 SNV (C238F)

Arcadi Cipponi

Page 19: Reconstructing tumour evolution: reconstructing the evolution of a drug-resistant liposarcoma - David Goode

+2.5 μM Nutlin

Slowing of CN

mutation rate

778

(‘parental’)

R95

R252

(‘resistant’)

Minor clones

potentially

Nutlin resistant

Evolutionary history of R252

Clones

Acquired null

TP53 mutation Many CN changes

in quick succession

Page 20: Reconstructing tumour evolution: reconstructing the evolution of a drug-resistant liposarcoma - David Goode

Summary

• BAFs to identify major CN changes occuring during

evolution of Nutlin resistance

• Sequencing individual clones improves resolution of

evolutionary events and reveals population substructure

• Selection for CN changes early in evolution

• Point mutations in clonal subpopulations later on

• Next: WGS on experimental replicates and on other cell

line/drug combinations

Page 21: Reconstructing tumour evolution: reconstructing the evolution of a drug-resistant liposarcoma - David Goode

Acknowledgements

• David Thomas

• Arcadi Cipponi

• Tiffany Pang

• Kevin Mills

• Natnicha Inthavong

• Bioinformatics Core

• Maria Doyle

• Joshy George

• Jason Li

• Jason Ellul

• Molecular Genomics Core

• LSCC/VLSCI

• Gayle Philip

• Jessica Chung

• Andrew Lonie

• Leonardo Meza-Zapeda (Oslo) • microarrays