Exome sequencing analysis of the mutational spectrum in carcinogen and genetic models of Kras-driven lung cancer Peter Westcott, Kyle Halliwill, Minh To, David Quigley, Reyno Delrosario, Erik Fredlund, David Adams 1, and Allan Balmain UCSF Helen Diller Family Comprehensive Cancer Center, rd Street, San Francisco. 1 Wellcome Trust Sanger Centre, Cambridge, England.

Why sequence tumors from mice?  Timing of initiation  collection  Initiating gene(s), carcinogen(s)  Can distinguish mutations involved in initiation from progression Control!

Specific goals of this study Part of the MMHCC TCGA Pilot Project  What is the effect of the causative carcinogen on mutation spectrum? Characterize the utility of sequencing mouse tumors:  Clean genetic induction (GEM) vs. carcinogen induction?  What mutations arise after Kras initiation?

Exome sequencing Urethane MNU Kras LA2 (GEM) 44 lung tumors from 17 mice 26 lung tumors from 7 mice 13 lung tumors from 4 mice Kras +/- (FVB/Ola) Kras +/- Kras +/+ Kras LA2 (FVB/Ola) Control tail DNA: 2 Kras +/+ tails Spontaneous lung tumors

Exome sequencing  Have a confident list of somatic variants  Have aligned reads to mouse genome, called against multiple controls and performed extensive QC (Kyle Hallilwill)  Illumina paired-end sequencing (Wellcome Trust Sanger Centre)

Exome sequencing

Carcinogen models of Kras-driven lung cancer  ~90% of lung tumors harbor Kras mutations. Urethane (ethyl carbamate)  Adenosine and cytidine DNA adducts lead to mispairing:  Kras Q61L (CAA  CTA), Q61R (CAA  CGA). A T Replication Mispairing

Carcinogen models of Kras-driven lung cancer MNU (methyl-nitroso urea)  ~90% of lung tumors harbor Kras mutations  Guanosine DNA adducts lead to G  A transitions  Kras G12D (GGT  GAT) Genome-wide spectrum of these carcinogen mutations not known G GG A Replication Mispairing

Mutation spectrum Urethane MNU LA2 Light shade = Kras +/-

Mutation spectrum Slight bias for mutations at G/C nucleotide Strong bias for mutations at G nucleotide with flanking G or A Strong bias for mutations at A/T nucleotide

Mutation spectrum Average counts per tumor  Purine bias at 5’ flanking base 5’ A 5’ G

Mutation spectrum  Are non-carcinogen mutations separable? Average counts per tumor For the most part NCG->T Other G->A A->T A->G A->C G->C G->T Urethane MNU LA2

ARE CARCINOGEN MUTATIONS RELEVANT?

Other driver mutations?  Analysis complicated: High mutation rates: MNU – 21.2/Mb Urethane – 6.4/Mb LA2 – 1.9/Mb Correlation between gene length and mutations  Start with variants within Vogelstein’s 2013 list of drivers: Selected only consequential mutations at highly conserved sites in expressed genes

Other driver mutations? GENEEXON_LENGTHNONSYN_MUT Mll Sf3b Crebbp75074 Asxl Pdgfra65533 Met66523 Cic60993 Atm Arid1b Alk59183 Gnas37172 Notch Arid1a81752 Fgfr Hnf1a31862 Flt Brca Akt Rb None of these mutations occur in LA2 tumors Slight enrichment for longer genes Modest increase in NS mutation ratio One S367 to F – required for autophosph. and activity Subclonal Myc T58P?

Conclusions  Clear recapitulation of expected carcinogen mutations Mutation Spectrum  GEM shows few mutations  Mutations highly specific and distinguishable Driver Mutations  Kras  Interesting candidates in carcinogen-induced tumors

Future work  InDel analysis.  Optimize list of potential driver mutations (relevant sites?).  Validate top 1000 interesting variants by Sequenom (Wellcome Trust Sanger Centre).  Array CGH (copy number analysis). Inverse correlation of point mutational burden and copy number changes?

Acknowledgments $: NSF Kyle Halliwill Minh To David Quigley Reyno Del Rosario Erik Fredlund ALLAN BALMAIN DAVID ADAMS (WELLCOME TRUST SANGER CENTRE) $: NIH Training Grant T32 GM $: MMHCC

Supplemental (Kyle’s Pipeline) Capture using Agilent mouse whole exome kit Sequenced on illumina HiSeq – Paired end, 75 bp each, average read span of 180 bp Converted back to FASTQ, then followed QC pipeline (next slide)

Supplemental (Kyle’s Pipeline) Align to Mm10 with BWA Mark duplicates and fix mate information with picard Base recalibration and realignment with GATK Alignment and coverage information with picard Variant calling with MuTect Filter for depth and previously observed variants with vcftools QC and Variant Calling Strategy

Supplemental (Kyle’s Pipeline) Sample.bam Sample.bam Control 1.bam Control 2.bam Intersect Variant List1.vcf Variant List2.vcf Variant Calling via MuTect Candidate Variant List.vcf Candidate Variant List.vcf Candidate Variants Filter, Annotate Variant Calling Details