Architecture and evolution of neochromosomes
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We isolated and analyzed, at single-nucleotide resolution, cancer-associated neochromosomes from well- and/or dedifferentiated liposarcomas. Neochromosomes, which can exceed 600 Mb in size, initially arise as circular structures following chromothripsis involving chromosome 12. The core of the neochromosome is amplified, rearranged, and corroded through hundreds of breakage-fusion-bridge cycles. Under selective pressure, amplified oncogenes are overexpressed, while coamplified passenger genes may be silenced epigenetically. New material may be captured during punctuated chromothriptic events. Centromeric corro- sion leads to crisis, which is resolved through neocentromere formation or native centromere capture. Finally, amplification terminates, and the neochromosome core is stabilized in linear form by telomere capture. This study investigates the dynamic mutational processes underlying the life history of a special form of cancer mutation.
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Architecture and evolution of neochromosomes
- 1. Architecture and evolution of cancer neochromosomes Tony Papenfuss Bioinformatics Division The Walter and Eliza Hall Institute of Medical Research Bioinformatics and Cancer Genomics Lab Peter MacCallum Cancer Centre
- 2. What are chromosome-scale mutations? • Large-scale changes to chromosomes found in cancers (& congenitally) • Includes structures and processes • Often complex; require ways of thinking and analysing data to make sense of them
- 3. Kataegis Single nucleotide variant index Genomic distance • Greek for thunderstorm • Localized hypermutation Alexandrov et al, Nature 2013
- 4. Chromothripsis: chromosomal shattering Stephens et al. Cell 2011
- 5. “Criteria” for chromothripsis Korbel & Campbell, Cell 2013
- 6. Linear Breakage-Fusion-Bridge cycle Gisselsson et al, Hum Genet 1999 McClintock, Genetics 1941 Telomere loss Replication Di-centric Di-centric Torn apart at cell division Daughter cells inherit different chromosomes lacking telomeres BFB generates inverted duplications
- 8. Giant cancer-associated neochromosomes • Giant super-numerary chromosomes found in some cancers • Little studied • Linear or circular (rings) • Gigantic • NCs have centromeres & telomeres Sandberg, Cancer Genet Cytogenet (2004) • Harbour known oncogenes
- 9. Prevalence of neochromosomes in cancers Class Tumour type NC prevalence Mesenchymal Parosteal osteosarcoma 90% Well-differentiated liposarcoma 85% De-differentiated liposarcoma 82% Dermatofibrosarcoma 67% protuberans Overall 14% Haematological Dendritic cell neoplasm 24% Large B cell lymphoma 18% Overall 3% Garsed, Holloway & Thomas, Bioessays (2009)
- 10. Hereditas, Volume 42, Issue 3-4, 1956 The remarkable case of four interlocked rings forming a chain (Fig.2v) was found in a cell with quadruple chromosome number, the rest of the chromosomes being arranged in quartets. This structure is unparalleled in chromosome experience, as far as we know…
- 11. Low resolution studies of neochromosomes in well-differentiated liposarcoma (WD-LPS) • Neochromosomes are composed of material from multiple chromosomes • Chr12 always present • High level of amplification of known oncogenes: MDM2, CDK4, HMGA2 mFISH 778 WD-LPS cell line Pedeutour et al. Genes Chromosomes Cancer, 1999
- 12. Amplification of neochromosomal material may involve circular breakage-fusion-bridge cycles Gisselsson et al, Hum Genet 1999
- 13. Sequencing of WD-LPS neochromosomes • Flow enriched neochromosome isoforms from 5 cell lines: 449 (primary), 778 (recurrence), GOT3, T1000, LPS141 • Sequenced enriched neochromosomes to 5X-30X coverage per cell line • Performed RNA-seq in all cell lines • Sequenced 2 patient primary WD-LPS tumours to ~30X coverage • FISH and ChIP-seq (CENP-A) studies of centromeres in cell lines 778 chrA 778 chrB Chromosome size GC content 778 cell line Normal chromosomes
- 14. Initial analysis • Align the reads back to the human reference genome (hg19) • No recurrent single nucleotide variants or indels on the neochromsome • Several fusion genes containing novel exons • No recurrent fusions genes
- 15. Estimation of copy number GC bias GC (%) Number of reads 778 Chr12 - Read depth Position (Mb) Number of reads (5kb windows)
- 16. Estimation of copy number: Background correction and calibration Copy number 778 Chr12 - Copy number Position (Mb) 778 Chr22 - Copy number Position (Mb)
- 17. Neochromosomes are composed of 100s of highly amplified genomic intervals derived focally from nearly every chromosome 778 Chr12 - Copy number ST059 Chr12 - Copy number Position (Mb) Position (Mb) Copy number
- 18. Amplified genomic intervals show high level of internal rearrangement 778 Chr12 How are these connected?
- 19. Detecting genomic fusions (ii) Discordant reads (iii) Split reads
- 20. 778 Chr12 Copy number profile Discordant read counts Discordantly aligned read pairs Breakdancer Segmentation DRCluster
- 21. 778 neochromosome • 260 genomic intervals • >500 genomic fusions • Nearly every chromosome involved
- 22. 778 GOT3 LPS141 ST059 ST079 T1000
- 23. Identifying material on the neochromosome Initial segmentation by thresholding
- 24. Scaffolding amplified genomic intervals
- 25. Large-scale structure of neochromosomes • 778 NC contains a 221Mb core region • Derived from 31Mb of the genome & includes 289 genes • Low copy regions derived from Chr7 & 22 provide telomeric caps • Similar structures in the other lines • Only 1.4Mb of sequence, containing 24 genes, is recurrently amplified
- 26. How did the neochromosomes form?
- 27. Circular versus linear breakage fusion bridge • Linear breakage fusion bridge: • Expected to generate an excess of inversions—not observed • Generates inverted duplications—not observed • Additionally, in 778/449 ring chromosomes were observed in the patient primary
- 28. Fusions at the edges of amplified genomic regions on Chr12 frequently connect to other other edges on Chr12 Edge to edge fusions A B C B A C Assembly
- 29. We can construct a walks across most of Chr12 via intra-chromosomal edge to edge fusions Edge to edge fusions Amplified genomic intervals 778 Chr12 ST059
- 30. Edge to edge walks involving Chr12 suggest chromothripsis prior to amplification and initially affecting only Chr12
- 31. T1000: initiated from a fusion chromosome?
- 32. Inferring chromothripsis A. Non-uniform clustering of breakpoints (✔︎; qq-plot) B. Regularity of oscillating copy number (½; not Chr12) C. Interspersed LOH (½; mono-allelic amplification) D. Rearrangements affect 1 haplotype (✗) E. Random ordering of fragments and fusion types (✔︎) F. Walks (✔︎; edge to edge walk & formal statistical tests)
- 33. Captured telomeres of the 778 NC bear the classic signatures of chromothripsis 778 Chr7 778 Chr22 Position (Mb) Position (Mb)
- 34. Other chromothripsis-like signatures on unenriched chromosomes T1000 Chr4 ST059 Chr3 GC corrected read depth Copy number Position (Mb) Position (Mb)
- 35. Can we test whether chromothripsis occurred prior to breakage fusion bridge? Can the patterns be explained without resorting to chromothripsis?
- 36. Computational model of circular BFB • Uses a uniform probability distribution to model breakpoints • Run without selection, it erodes the neochromosome away
- 37. Simple model of fitness and selection • Assume fitness is proportional to number of copies of key genes • Require at least one copy of each selected gene to be preserved • Scale the fitness so that it saturates at some CN • Choose daughter cell randomly, weighted according to fitness fitness=3 fitness=1 fitness=5
- 39. Combining multiple model runs • At each cycle, we select 1 daughter cell (or the parent) and discard others • Each simulation traces a single lineage in the tree of possible cell fates • Run this 500-1000 times and look at the distributions of summary statistics. e.g. Number of genomic intervals, interval lengths, … • We explored different models of fitness/selection & different spatial distributions for the breakpoint • Run model with and without chromothripsis • Used uniform, biased and empirical distributions of chromothripsis breakage
- 40. Breakage-fusion-bridge only model Number of genomic intervals
- 41. Breakage-fusion-bridge with chromothripsis model Number of genomic intervals
- 42. BFB-only model do not generate edge to edge walks; BFB+chromothripsis does
- 43. Model
- 44. Model of the life history of well-differentiated liposarcoma neochromosomes
- 45. Conclusions • Sequencing flow isolated neochromosomes revealed the dynamic processes involved in their formation • The approaches used likely have broader relevance • The combination of mechanisms is also likely to be used in other scenarios • For example, the emergence of resistance to targeted therapies is sometimes driven by extreme amplification of the target
- 46. Acknowledgements Papenfuss lab, WEHI Dale Garsed Vincent Corbin Arthur Hsu Leon di Stefano Jan Schroeder Peter MacCallum Cancer Centre David Thomas Owen Marshall, MCRI Andrew Holloway Zhi-Ping Feng Mark Lansdell Ben Lansdell The Lorenzo & Pamela Galli Melanoma Research Fellowship
Editor's Notes
- FISH image by Owen Marshall, MCRI
- Linear BFB is another chromosome-scale mutation. It begins with loss of 1 telomere. This is duplicated at replication and fusion of the naked DNA ends leads to the formation of a chromosome with 2 centromeres. At anaphase, the centromeres are pulled apart by the mitotic spindle, causing a DNA break between the centromeres. The daughter cells then inherit different chromosomes, both lacking centromeres and the cycle continues
- Based on low res data, it has been proposed that NCs form through the breakage fusion bridge mechanism. I described linear breakage-fusion-bridge earlier. There is a second flavour which operates on ring chromosomes. This is initiated by loss of both telomeres, leading to ring formation. At replication, an odd number of crossover events will cause the rings to become intertwined and a dicentric ring can be formed. Again these are torn apart causing 2 DNA breaks and leading to duplication and deletion of material.
- A simple observation put us onto the explanation. We found that fusions at the edges of the intervals frequently connect to other intervals. Non-edge to edge fusions dominated by inter-chromosomal fusions
- On the 778 neochromosome, we can construct a walk across most of Chr12 via intra-chromosomal edge to edge fusions Clear walks across 449, T1000 & ST059 also exist Similar patterns are evident in GOT3, LPS141 & ST079 These walks most likely assemble fragments of the original neochromosome This suggests that all neochromosome were formed by catastrophic, copy number neutral rearrangement involving only chr12 occurred prior to amplification
- To test whether chromothripsis is required to generate the patterns we observe in the data, we developed a computational model of circular breakage fusion bridge. The model uses a uniform probability distribution to select the two breaks. Run without selection, it quickly erodes away the NC.
- We adopt a simple model of fitness and selection. Assume fitness is proportional to number of copies of key genes Conserve at least one copy of each gene Scale the fitness so that it saturates at some CN Choose NC randomly, weighted according to fitness
- Every line is one run of the model. On the left is the best fitting model with BFB only. We could not identify a model and parameter set for which BFB only fitted the data.
- Every line is one run of the model. On the left is the best fitting model with BFB only. We could not identify a model and parameter set for which BFB only fitted the data. On the right is chromothripsis and BFB.
- Our analyses reveal the remarkably dynamic life history of neochromosomes in well-differentiated liposarcomas. The data supports 3 phases, an initiation phase in which Chr12 (and possibly fused chromosomes) undergo chromothripsis forming a rearranged ring chromosome. Circular BFB drives amplification and loss of material. New chromosomes are added, possibly through involving chromothripsis. Erosion of the centromere can lead to a crisis that is resolved by either capturing a non-native centromere or forming a neo-centromere. Finally the neochromosome is linearised and stabilised by telomere capture.