Genomic Instability And Chromosome Architecture Kevin Mills, Ph.D. Associate Professor, The Jackson Laboratory
Genomic Instability is a Hallmark of Cancer Angiogenesis Invasion and Metastasis Metabolism Genomic Instability Perpetual Replication " Inflammation Resisting Apoptosis Evading Growth Inhibition Growth Signaling
Cancer Genomes Can Be Highly Aberrant and Variable Normal Genome Cancer Genome
Chromosome Lesions in Cancer acentric ring chromosome isochromosome pericentric inversion paracentric inversions reciprocal translocations Robertsonian translocations dicentric insertion deletion duplication double minute homogeneously staining region
Chromosome Lesions in Cancer acentric ring chromosome isochromosome pericentric inversion Abnormalities affecting a single chromosome paracentric inversions reciprocal translocations Robertsonian translocations dicentric Abnormalities affecting two or more chromosomes insertion deletion duplication double minute homogeneously staining region Copy number abnormalities
Genomic Instability The Good, The Bad, and The Ugly Tumor Initiation
Genomic Instability The Good, The Bad, and The Ugly Tumor Initiation Resistance and Relapse
Genomic Instability The Good, The Bad, and The Ugly Tumor Initiation Neoantigens Synthetic Lethality Resistance and Relapse
B-Cell Development and Transformation Germinal Center Immature B-cells AID AID Mature B-cells (memory, plasma) Somatic hypermutation Ig class switching
B-Cell Development and Transformation Germinal Center Immature B-cells AID AID Mature B-cells (memory, plasma) Somatic hypermutation Ig class switching GCB lymphoma ABC lymphoma myeloma
T(12;15) translocations involving IgH and c-myc in pro-b cell lymphomas from XRCC4 -/- p53 -/- mice! SKY! FISH for c-myc and IgH! c-myc! IgH! c-myc! IgH!
Complex Translocations and Gene Amplification! in NHEJ/p53 deficient pro-b cell Lymphomas! SKY! FISH! chromosome 12! chromosome 15! T162! IgH! c-myc! C(12;15)! C(15;12;6)! Chr. 12! T(12;15)! T(15;12;6)! Chr. 15! T(12;15)! T(15;12;6)! T554! -12! IgH! c-myc! C(12;15)! C(15;12;3)! Chr. 12! T(12;15)! T(15;12;3)! Chr. 15! T(12;15)! T(15;12;3)! Complicon! Complicon! Complicon! Zhu, et al. Cell 2002.
Chr. 12 IgH Chr. 15 c-myc c-myc c-myc c-myc c-myc IgH IgH IgH IgH 1. Why two translocated products from a single copy of Chr. 12? 2. Why not both copies of 12 and 15? 3. Why not other der(12) or der(15) translocations?
Chr. 12 IgH Chr. 15 c-myc c-myc c-myc c-myc c-myc IgH IgH IgH IgH Does proximity in the nucleus restrict possible translocation partners or function in target site selection? Does chromosome morphology influence tranlsocation susceptibility or function?
Chr. 12 IgH Chr. 15 c-myc c-myc c-myc c-myc c-myc IgH IgH IgH IgH Does proximity in the nucleus restrict possible translocation partners or function in target site selection? Does chromosome morphology influence tranlsocation susceptibility or function?
Nuclear Distribution of Chromosomes 12 and 15! 0 15 30 45 60 75 90 0 15 30 45 60 75 90 Bone Marrow C57BL/6 Chr. 12 Chr. 15
Nuclear Distribution of Chromosomes 12 and 15! 2-D maximum intensity projection Manual Object Identification Bone Marrow Chr. 12 C57BL/6 Chr. 15
Nuclear Distribution of Chromosomes 12 and 15! 2-D maximum intensity projection Manual Object Identification Bone Marrow C57BL/6 Chr. 12 Chr. 15
Nuclear Distribution of Chromosomes 12 and 15! 2-D maximum intensity projection Manual Object Identification Bone Marrow C57BL/6 Chr. 12 Chr. 15
Nuclear Distribution of Chromosomes 12 and 15! 2-D maximum intensity projection: category frequencies 0.7 0.6 0.5 0.4 0.3 Cat 1 Cat 2 Cat 3 Cat 4 0.2 0.1 0 1
Chromosome territory proximity relationships 12 + 15 N=194 12 + 1 N=200 12 + 19 N=234 R G G R 5.2% (10) 7.0% (14) 7.7% (18) R R G G 12.4% (24) 18.0% (36) 15.8% (37) R G 79.9% (155) 69.5% (139) 69.7% (163) R G G R 2.6% (5) 5.5% (11) 6.8% (16)
Subcategories of heterologous proximal pairs R G G R R G R G G R R G R R G R G G
Subcategories of heterologous proximal pairs 12 + 15 12 + 1 12 + 19 G R G R 44.5% 48.9% 39.9% R G 23.2% 11.5% 19.7% G R R G R G R G 32.3% 39.6% 40.4%
Nuclear Distribution of Chromosomes 12 and 15! Confocal Z-stack: 200nm step size -4.8-4.6-4.4-4.2-4.0-3.8-3.6-3.4-3.2-3.0-2.8-2.6-2.4-2.2-2.0-1.8-1.6-1.4-1.2 --1.0-0.8-0.6-0.4-0.2 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 Bone Marrow C57BL/6 Chr. 12 Chr. 15
Nuclear Distribution of Chromosomes 12 and 15! Confocal Z-stack: 3-D chromosome positioning 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 Formaldehyde fixation Bone Marrow C57BL/6 Chr. 12 Chr. 15
We Needed a Better Approach to Image Analysis Objective Quantitative Sensitive Accurate High Throughput
Automated image segmentation and object identification! Wavelet-based multifractal analysis algorithm
Automated image segmentation and object identification! Objective analysis of image features A B A B D(A,B) = 0.0 D(A,A) = 15.0 D(A,B) = 29.4 D(B,B) = 39.8 D(A,B) = 65.8 D(A,B) = 87.2 Object Perimeter Diameter Area Filament Index Center of Mass A 152 73 1683 1.65 (89,87) B 105 44 945 1.23 (157,99) A 87 37 690 1.16 (152,71) B 94 34 757 1.07 (186,161)
Automated image segmentation and object identification! Objective analysis of image features A B A B D(A,B) = 0.0 D(A,A) = 15.0 D(A,B) = 29.4 D(B,B) = 39.8 D(A,B) = 65.8 D(A,B) = 87.2 Object Perimeter Diameter Area Filament Index Center of Mass A 152 73 1683 1.65 (89,87) B 105 44 945 1.23 (157,99) A 87 37 690 1.16 (152,71) B 94 34 757 1.07 (186,161)
Ultra high resolution 4Pi microscopy! Superior resolution in the Z axis Widefield Confocal 4Pi Y X X Z