Proteogenomic analysis of alternative splicing: the search for novel biomarkers for colorectal cancer Gosia Komor

Transcription

1 Proteogenomic analysis of alternative splicing: the search for novel biomarkers for colorectal cancer Gosia Komor

2 Translational Gastrointestinal Oncology Collect clinical samples (Tumor) tissue Blood Stool Perform molecular profiling DNA RNA Protein Collect clinical information Study tumor biology Preclinical models

3 Translational Gastrointestinal Oncology Collect clinical samples (Tumor) tissue Blood Stool Collect clinical information Perform molecular profiling DNA RNA Protein Molecular profiling of colon tumors Study tumor biology Preclinical models Translation of molecular knowledge into clinical tests

4 Colorectal cancer (CRC) Colorectal cancer is the 2 nd most common cancer type in the Netherlands - Incidence rate of over patients per year - Most patients between years old Colorectal tumor progression years Figure adapted from Nature Reviews Cancer 9, (2009)

5 Colorectal cancer has a high cure rate when diagnosed early 100% 80% 60% 40% 5-year survival rate by stage 94% 82% 67% 20% 0% 11% I II III IV

6 Population wide screening for colorectal cancer implemented in the Netherlands Fecal immunochemical test (FIT) Colonoscopy FIT+ FIT performance * Specificity: ~95% Sensitivity CRC : ~79% Sensitivity precursor lesions (advanced adenomas): ~27% * Lee et al, Accuracy of Fecal Immunochemical Tests for Colorectal Cancer, Annals of Internal Medicine, 2014

7 Population wide screening for colorectal cancer implemented in the Netherlands Fecal immunochemical test (FIT) Colonoscopy FIT+ FIT performance * Specificity: ~95% Sensitivity CRC : ~79% Sensitivity precursor lesions (advanced adenomas): ~27% Clinical need for novel biomarkers * Lee et al, Accuracy of Fecal Immunochemical Tests for Colorectal Cancer, Annals of Internal Medicine, 2014

8 AIM: Identify novel biomarkers for CRC screening Tumor-specific molecular changes accompany tumor progression DNA alterations, e.g.: Mutations (SNVs) Copy number aberrations Methylation RNA alterations, e.g.: RNA Splicing

9 AIM: Identify novel biomarkers for CRC screening Tumor-specific molecular changes accompany tumor progression DNA alterations, e.g.: Mutations (SNVs) Copy number aberrations Methylation RNA alterations, e.g.: RNA Splicing Figure adapted from Sveen et al. Oncogene 2016;35(19):

10 AIM: Identify novel biomarkers for CRC screening Tumor-specific molecular changes accompany tumor progression DNA alterations, e.g.: Mutations (SNVs) Copy number aberrations Methylation RNA alterations, e.g.: RNA Splicing BCL2L1: - Bcl-xL anti-apoptotic - Bcl-xS pro-apoptotic VEGFA: - pro-angiogenic - anti-angiogenic Figure adapted from Sveen et al. Oncogene 2016;35(19):

11 AIM: Identify novel biomarkers for CRC screening splicing translation pre-mrna alternatively spliced mrna protein isoforms Tumor-specific protein isoforms could complement or outperform hemoglobin in CRC screening

12 AIM: Identify novel biomarkers for CRC screening splicing translation pre-mrna alternatively spliced mrna protein isoforms Tumor-specific protein isoforms could complement or outperform hemoglobin in CRC screening Design an approach to identify tumor specific protein variants

13 In silico Experimental Figure adapted from Duncan et al. Nat Biotechnol. 2010;28: With the use of available protein sequence databases ~50% of mass spectra are still not identified

14 In silico Experimental RNA-seq Figure adapted from Duncan et al. Nat Biotechnol. 2010;28: With the use of available protein sequence databases ~50% of mass spectra are still not identified

15 Experimental design Down-modulation of splicing machinery to investigate differential splicing in a controlled setting CRC cell lines SW480 RNA-seq Illumina HiSeq 2x125bp sisrsf1 sisf3b1 sinontargeting (sint) proteomics LC-MS/MS QExactive

16 Experimental design Down-modulation of splicing machinery to investigate differential splicing in a controlled setting CRC cell lines SW480 RNA-seq Illumina HiSeq 2x125bp sisrsf1 sisf3b1 sinontargeting (sint) proteomics LC-MS/MS QExactive PacBio Iso-Seq RSII, 4 fractions 0-50kb

17 Experimental design Down-modulation of splicing machinery to investigate differential splicing in a controlled setting CRC cell lines SW480 RNA-seq Illumina HiSeq 2x125bp sisrsf1 sisf3b1 sinontargeting (sint) proteomics LC-MS/MS QExactive PacBio Iso-Seq RSII, 4 fractions 0-50kb Proteogenomic pipeline

18 SPLICIFY proteogenomic pipeline for differential splice variant identification Short RNA-seq reads Mass spectra

19 SPLICIFY proteogenomic pipeline for differential splice variant identification Short RNA-seq reads Quality and adapter trimming (Trimmomatic) Reads mapping (STAR) Differential splicing analysis (rmats) Reference annotation Differential splice variants on RNA level Mass spectra

20 SPLICIFY proteogenomic pipeline for differential splice variant identification Short RNA-seq reads Quality and adapter trimming (Trimmomatic) Reads mapping (STAR) Differential splicing analysis (rmats) Reference annotation Enriched protein database Human protein database Potential protein variants 3-frame translation Differential splice variants on RNA level Mass spectra

21 SPLICIFY proteogenomic pipeline for differential splice variant identification Short RNA-seq reads Quality and adapter trimming (Trimmomatic) Reads mapping (STAR) Differential splicing analysis (rmats) Reference annotation Enriched protein database Human protein database Potential protein variants 3-frame translation Differential splice variants on RNA level Mass spectra Identify MS/MS Extract variant peptides Differential protein isoforms Differential peptide expression (limma)

22 SPLICIFY proteogenomic pipeline for differential splice variant identification Short RNA-seq reads Quality and adapter trimming (Trimmomatic) Reads mapping (STAR) Differential splicing analysis (rmats) Reference PacBio annotation full-length transcripts Enriched protein database Human protein database Potential protein variants 3-frame translation Differential splice variants on RNA level Mass spectra Identify MS/MS Extract variant peptides Differential protein isoforms Differential peptide expression (limma)

23 Differential splice variants identified on RNA level Exon skipping: inclusion exclusion - exclusion spanning reads - inclusion spanning reads

24 Differential splice variants identified on RNA level Exon skipping: inclusion exclusion - exclusion spanning reads - inclusion spanning reads Alternatively splicing events Skipped exon (SE) Mutually exclusive exons (MXE) Alternatively 5 splice site (A5SS) Alternatively 3 splice site (A3SS) Retained intron (RI)

25 RT-qPCR validation of SPLICIFY results on RNA level RNA-seq

26 RT-qPCR validation of SPLICIFY results on RNA level Relative expression RNA-seq RT-qPCR OSBPL3 inclusion OSBPL3 exclusion sint sisf3b1

27 RNA to protein translation : isoform-specific peptides Exon skipping inclusion exclusion exon

28 RNA to protein translation : isoform-specific peptides Exon skipping XXXXX ZZZZZ inclusion exclusion XXXXXZZZZZ exon XXXXXZZZZZ exclusion specific split peptide

29 RNA to protein translation : isoform-specific peptides Exon skipping XXXXX ZZZZZ XXXXX------YYYY inclusion XXXXXYYYY exclusion XXXXXZZZZZ exon XXXXXZZZZZ XXXXXYYYY exclusion specific split peptide inclusion specific split peptide

30 RNA to protein translation : isoform-specific peptides Exon skipping XXXXX ZZZZZ XXXXX------YYYY YYVV inclusion XXXXXYYYY YYVV exclusion XXXXXZZZZZ exon XXXXXZZZZZ XXXXXYYYY YYVV exclusion specific split peptide inclusion specific split peptide inclusion specific peptide on target

31 RNA to protein translation : isoform-specific peptides Exon skipping Retained intron XXXXX ZZZZZ XXXXX ZZZZZ XXXXX------YYYY YYVV XXXXXAAAA AABB inclusion exclusion inclusion exclusion XXXXXYYYY YYVV XXXXXZZZZZ XXXXXAAAA AABB XXXXXZZZZZ exon intron XXXXXZZZZZ exclusion specific split peptide XXXXXZZZZZ exclusion specific split peptide XXXXXYYYY inclusion specific split peptide XXXXXAAAA inclusion specific spanning peptide YYVV inclusion specific peptide on target AABB inclusion specific peptide on target

32 Differential isoform identified on protein level Experiment On target Spanning peptide Split peptide sisf3b1 vs sint sisrsf1 vs sint

33 Differential isoform identified on protein level Experiment On target Spanning peptide Split peptide sisf3b1 vs sint sisrsf1 vs sint RNA translation Protein

34 Quantitative differences on RNA and protein level RefSeq Genes RNA isoforms

35 Quantitative differences on RNA and protein level RefSeq Genes RNA isoforms RNA-seq

36 Quantitative differences on RNA and protein level RefSeq Genes RNA isoforms Isoform-specific peptides RNA-seq

37 Quantitative differences on RNA and protein level RefSeq Genes RNA isoforms 1 2 Isoform-specific peptides 3 RNA-seq translation LC-MS/MS inclusion exclusion 1 2 3

38 PacBio full length transcripts used as annotation to identify novel events sisf3b1 vs sint SPLICIFY with reference annotation SPLICIFY with PacBio full-length transcripts Full length transcript used as annotation to quantify Illumina reads Comparison to the standard SPLICIFY with reference annotation Both approaches include Illumina reads for the differential analysis

39 PacBio Iso-Seq provides a number of novel alternatively spliced events differential splicing analysis of sisf3b1 vs sint1 Alternatively splicing events Skipped exon (SE) Mutually exclusive exons (MXE) Alternatively 5 splice site (A5SS) Alternatively 3 splice site (A3SS) Retained intron (RI)

40 PacBio Iso-Seq provides a number of novel alternatively spliced events differential splicing analysis of sisf3b1 vs sint1 Alternatively splicing events Skipped exon (SE) Mutually exclusive exons (MXE) Alternatively 5 splice site (A5SS) Alternatively 3 splice site (A3SS) Retained intron (RI)

41 PacBio Iso-Seq provides a number of novel alternatively spliced events differential splicing analysis of sisf3b1 vs sint1 Alternatively splicing events Skipped exon (SE) Mutually exclusive exons (MXE) Alternatively 5 splice site (A5SS) Alternatively 3 splice site (A3SS) Retained intron (RI)

42 PacBio Iso-Seq provides a number of novel alternatively spliced events differential splicing analysis of sisf3b1 vs sint1 Alternatively splicing events Skipped exon (SE) Mutually exclusive exons (MXE) Alternatively 5 splice site (A5SS) Alternatively 3 splice site (A3SS) Retained intron (RI)

43 Novel isoforms identified with PacBio Iso-seq are expressed on protein level Isoform-specific peptides On target Spanning peptides Split peptides PacBio transcripts Reference Annotation

44 Novel isoforms identified with PacBio Iso-seq are expressed on protein level Retained intron Read coverage in sisf3b1 Read coverage in sint RefSeq Genes PacBio transcripts RNA-seq isoforms Isoform-specific peptides

45 Novel isoforms identified with PacBio Iso-seq are expressed on protein level Alternative 3 splice site Read coverage in sisf3b1 Read coverage in sint RefSeq Genes PacBio transcripts RNA-seq isoforms Isoform-specific peptides

46 Conclusions Established SPLICIFY Proteogenomic pipeline combining RNA-seq and LC-MS/MS data for differential splice variant identification Confirmation of the splice variants on RNA level RT-qPCR by PacBio full-length transcripts will be available soon Novel splicing events identified with PacBio Iso-seq confirmation on the protein level

47 Future plans Organoids Human tissues Healthy colon tissue adenomas CRCs mrna isolation Illumina RNA-seq protein isolation LC-MS/MS SPLICIFY proteogenomic pipeline

48 Future plans Organoids Human tissues Healthy colon tissue adenomas CRCs mrna isolation Illumina RNA-seq protein isolation LC-MS/MS Stool samples FIT samples SPLICIFY proteogenomic pipeline Antibody-based assay for the best candidates

49 Acknowledgements Department of Pathology Translational Gastrointestinal Oncology Annemieke Hiemstra Anne Bolijn Marianne Tijssen Pien Delis-van Diemen Meike de Wit Beatriz Carvalho Remond JA Fijneman Gerrit A Meijer Pacific Biosciences Bo Han Elisabeth Tseng Sarah Kingan Meredith Ashby Icahn School of Medicine at Mount Sinai Robert P Sebra Medical Oncology Oncoproteomics Laboratory Tim Schelfhorst Sander Piersma Thang Pham Connie R Jimenez This research was financially supported by a grant from the Dutch Cancer Society Grant number: NKI