Abnormal developmental control of replication-timing domains in pediatric acute lymphoblastic leukemia

DNA replication in human cells proceeds according to a defined temporal order (Hiratani et al. 2009). Several studies have identified abnormal temporal control of replication in many cancers (Amiel et al. 2001, 2002; Smith et al. 2001; Sun et al. 2001; Korenstein-Ilan et al. 2002). For example, specific chromosome translocations result in a chromosome-wide delay in replication timing (Breger et al. 2005; Chang et al. 2007) that is found frequently in cancer cells (Smith et al. 2001). Some cancer-specific replication-timing changes appear to be epigenetic in that, similar to developmental changes, they are mitotically stable but do not involve detectable genetic lesions (Eul et al. 1988; Adolph et al. 1992). A far-reaching aspect of epigenetic abnormalities is that they are potentially reversible. In fact, in a mouse lymphoma model showing aberrant replication timing, fusion of affected cells with normal mouse fibroblasts restored the normal pattern of replication timing and reversed the malignant phenotype (Eul et al. 1988; Adolph et al. 1992). Despite these observations, there has not been a comprehensive study to evaluate the extent of replication-timing abnormalities in cancer. We recently generated genome-wide replication-timing profiles for a wide collection of human and mouse cell lines and embryonic stem cell (ESC) differentiation intermediates, revealing developmentally regulated changes in replication timing that encompass at least half of the genome (ReplicationDomain.org). Developmentally regulated changes take place in units of 400–800 kb and are associated with changes in subnuclear 3D organization of the affected domains (Hiratani et al. 2008, 2010). This replication-timing program is a highly stable epigenetic characteristic of a given cell type that is indistinguishable between the same cell types from different individuals (Pope et al. 2011). This stability has allowed for the development of tools to unambiguously determine cellular identity using their specific “replication fingerprints” (Ryba et al. 2011b). Intriguingly, replication-timing profiles correlate more strongly with genome-wide maps of the sites and frequencies of chromatin interactions (Hi-C) (Lieberman-Aiden et al. 2009) than with any other chromosomal property identified to date (Ryba et al. 2010), indicating that replication domains reflect the structural architecture of chromosomes and support the model of replication-timing domains as structural and functional large-scale units (the replication domain model). In summary, replication-timing profiles are unique to specific cell types and define an unexplored level of chromosome domain organization with intriguing potential for epigenetic fingerprinting. We reasoned that just as specific cell types display unique replication-timing fingerprints, specific cancers may also be definable by their replication-timing fingerprints. Acute lymphoblastic leukemia (ALL) is an excellent model cancer to investigate this hypothesis due to the availability of relatively homogeneous cancer tissue from affected patients and several well-characterized genetic subtypes linked to prognosis. Current clinical risk stratification for pediatric ALL includes factors such as age, leukocyte count at time of diagnosis, and recurrent chromosomal abnormalities detected in malignant lymphoblasts (Yeoh et al. 2002; Jeha and Pui 2009; Luo et al. 2009). Chromosomal abnormalities with prognostic significance include aneuploidies, such as hypodiploidy (<44 chromosomes) and hyperdiploidy (with trisomies 4, 10, and 17), translocations, and deletions (Pui et al. 2011). However, only a minority of these abnormalities such as t(9;22) show a direct activation of an oncogene, and the underlying mechanisms of tumorigenesis for the majority of ALL subtypes remain elusive. Furthermore, ∼20% of ALL and 50% of AML cases present with a normal karyotype (Bienz et al. 2005; Kearney and Horsley 2005; Usvasalo et al. 2009; Collins-Underwood and Mullighan 2010; Pui et al. 2011) but have widely varying clinical outcomes, underscoring the need for additional epigenetic markers. Here we query the replication program genome-wide in 17 primary childhood leukemias and three ALL cell lines and report widespread instability, with some changes in common to all leukemias and others unique to specific patients. The differences that distinguish different cancers also align with the boundaries of normal developmentally regulated replication domains, supporting the replication domain model. In addition, the timing changes that distinguish cancers from normal cells do not resemble any particular tissue, extending a model derived from DNA methylation studies that cancers are characterized by widespread epigenetic instability (Hansen et al. 2011; Pujadas and Feinberg 2012).