Molecular Biology of CML Overview: Research into chronic myeloid leukemia (CML) over the last 40 years has lent insight into the role of altered cellular biochemistry (deregulated regulatory kinases), chromosome biology (genomic instability) and the emerging influence of neoplastic stem cells on leukemogenesis and, possible, other cancers. From this knowledge significant improvement in CML patient outcomes has been achieved and has ushered in the era of molecularly targeted therapeutics. For this section of the CML module, we will discuss the following learning objectives: Appreciate the concept of “chromosome territory” and how it influences common chromosomal recombination events observed in leukemia Understand the impact of altered genetic recombination leading to altered master regulatory kinase activity Know the impact of a deregulated kinase activity resulting in survival of the leukemic cell Understand the stem cell basis of CML and how this will influence treatment. Hemat CML Module: Perkins, May 20, 2008

Chronic Myeloid Leukemia Why do good chromosomes go bad? Why do good kinases go bad? Why do good stem cells go bad? www.pathology.unibe.ch/.../ zyt_gen/zg_cml.htm

Why do good chromosomes go bad? Cytogenetic alterations observed in chronic myeloproliferative disorders Some chromosome rearrangements are observed in other disorders Commonality of chromosome rearrangement events Recipricol translocations observed in other leukemias Cytology: Detection by FISH

Why do good chromosomes go bad? Arrangement of chromosomes within a nucleus. During interphase, the two arms of a chromosome are confined to a discrete region called a domain (speckled blue area). Some of the DNA in this chromosome is extruded as loops that extend either within or beyond the domain. These loops can extend into channels between different domains (pale blue), sometimes reaching as far as the pores of the nuclear membrane. Extruded loops can form specific associations either with other loops from the same domain (yellow) or with loops from different chromosomes in different domains (red). Such associations could provide the proximity necessary for the intrachange (exchange within the same chromosome) or interchange (exchange between different chromosomes) induced when a cell is exposed to ionizing radiation. Why do good chromosomes go bad? Concept of chromosome domains or territories Savage (2000) Science 290: 62-63

Why do good chromosomes go bad? Tethering of chromosome territories to nuclear structures restricts their movement and contributes to maintenance of chromosome positioning. Chromosome territories might be positioned within the nucleus in order to place specific genes in particular nuclear neighborhoods that favor the expression or silencing of genes. Chromosome territories may also be cell type specific The major component of the lamina, the intermediate-filament-like lamins, can bind directly to Dna and also to core histones, suggesting that chromatin can be tethered to the lamina. Further constraint mediated by subnuclear compartments. Parada and Mistel (2002) Trends in Cell Biology 12: 425-432

Why do good chromosomes go bad? Potential gene positioning–function relationships. Gene positioning might contribute to gene activity. Active genes have been proposed to be preferentially localized: towards the nuclear interior (a) on loops extending away from the chromosome territory (CT) (b) distant from centromeres (cen) (c) Relative positioning of chromosomes and genes contribute to translocation frequencies (d) Proximally positioned translocation partners are more likely to undergo illegitimate rejoining upon chromosome breaks than distantly located partners. A-C) Gene positioning might contribue to gene activity. Parada et al., (2004) Experimental Cell Research 296: 64-70

Why do good chromosomes go bad? Chromosome territories that are close together in the interphase nucleus have a higher chance of undergoing reciprocal translocations than chromosomes that are far apart. Chromosome translocations in therapy-related leukemias (result of alkylating agent/topoisomerase II mediated therapies) support the idea of proximity influencing translocation frequency Parada and Mistel (2002) Trends in Cell Biology 12: 425-432

Cytological Techniques to Study Chromosome Structure Cells swelled in hypotonic solution and dropped onto slide Prepare labeled probe from plasmid/PCR product Denature Combine denature probe with denatured slide and hybridize Cells with metaphase chromosomes in nucleus Denature Wash Counter stain chromosomes Develop signal (amplification with coupled Abs and fluorochromes) Microscopy (fluorescent) Metaphase Spread

76 Kb duplicon (2 copy DNA repeat) common to BCR and ABL. BCR = breakpoint cluster region ABL= Abelson murine leukemia viral homolog 1

Why do good kinases go bad? BCR (Chr 22) 23 exons; 130 kb; 5' centromere - 3' telomere TX into various mRNA, of which are 4.5 kb and 7 kb 130 KDa, 190 KDa; mainly 160 KDa (1271 amino acids) Oligomerization domaim responsible for homotetramerization of BCR-ABL necessary for its transforming potential; protein, Involved in DNA repair Activation of Rac (AKT) Why do good kinases go bad? ABL protooncogene encodes a cytoplasmic and nuclear protein tyrosine kinase involved in cell differentiation, cell division, cell adhesion and stress response. ABL (Chr 9) 12 exons; 230 kb; TX alternate splicing: 1a and 1b are 5' alternative exons; mRNA of 6 and 7 kb (with 1a and 1b respectively), giving rise to 2 protein of 145 kDa 1130-1143 amino acids; 4 domains: of which are SH (SRC homology) domains; ABL exhibit a permanent nuclear and cytoplasmic shuttling activity, Nuclear ABL plays a major role in the regulation of cell death after DNA damage. Cytoplasmic ABL : possible function in adhesion signaling

PCR-based Assay for BCR-ABL Fusion Gene Isolate cells from blood sample Lyse cells and purify total RNA Reverse transcribe RNA into cDNA PCR amplification to detect fusion gene product www.medscape.com

Why do good kinases go bad? p190 BCR breakpoint at intron 1 BCR exon 1 fused to ABL exons 2 -11 p210 BCR breakpoints at introns 13 and 14 BCR exons 1 - 13 or 1 - 14 fused to ABL exons 2 -11 Variations to this theme Introduction into mice leads to oncogenesis Invariably found in chronic myeloid leukemia Also seen in normal cells as well!! Detection via RT-PCR Expressed in primitive hematopoietic cells BCR-ABL gene product necessary and prime cause of CML additional genetic changes

Why do good kinases go bad?

Why do good kinases go bad? BCR-ABL: Constitutive kinase activation!

Why do good kinases go bad? BCR-ABL: Altered signal transduction pathways

Why do good stem cells go bad? CML is a disease of altered stem cells Recapitulate CML phenotype in neoplastic stem cell transplant Reya et al., Nature 414: 105 - 111 (2001)

Why do good stem cells go bad? Cancer cells can take advantage of pre-existing stem cell properties ability to self-renewal high proliferation potential form a myriad of cell types Clarke, MF (2004) NEJM 351: 634-636

Model of the Role of Activated b-Catenin in the Progression of CML Compared to healthy subjects, CML patients in chronic phases have elevated proliferative capacity due to increased expression of BCR-ABL (myeloproliferatie syndrome) Progression to blast crisis results from additional events activation of b-catenin in the granlocyte-macrophage progenitor population leads to increased self-renewal capacity generation of leukemic stem cell avoidance of cell death, evasion of innate immune response, block in differentiation must also occur for CML to progress failure of DNA repair mechanisms Jamieson et al., (2004) NEJM 351: 657-667

Why do good stem cells go bad?

Why do good stem cells go bad? However, some primitive HSCs exhibit high drug efflux! Reya et al., Nature 414: 105 - 111 (2001)