DNA Repair, Genome stability, and Cancer

Research interests

DNA Repair, Genome Stability, and Cancer

Research Interests

DNA double strand breaks (DSB) are serious chromosomal lesions that can lead to cell death or neoplastic transformation. Mammalian cells possess sophisticated and intricate mechanisms to sense and repair DNA breaks, thus maintaining genomic integrity and ensuring faithful and efficient replication of genetic material. The research interest of our laboratory is to understand the mechanisms that maintain genome stability and how they relate to human pathology, broadly focusing on three main topics:

1. Basic Mechanisms of Genome Stability

Mammalian cells employ two predominant DSB repair pathways: nonhomologous end joining (NHEJ), which repairs DNA breaks by the ligation of two broken ends without respect to sequence homology; and homologous recombination (HR), which uses homology on an undamaged template to catalyze high fidelity repair.

Nonhomologous end joining and chromosomal fragile site stability

One type of chromosomal damage thought to be involved in tumorigenesis is fragile site breakage. Recent evidence has indicated that fragile site breakage may be related to DNA replication stress, but almost nothing is known about the DNA repair pathways that respond after breakage has occurred. We are currently examining the role of NHEJ in repair of chromosomal fragile sites, using classical cytogenetic methods, fluorescent in situ hybridization (FISH), and spectral karyotyping (SKY) to monitor fragile site stability, and the consequences of fragile site breakage, in cell lines and primary cells derived from NHEJ-deficient mice.

Homologous recombination in lymphocyte development and DSBR

Because NHEJ is important for general genome stability, it is not surprising the lymphomas that develop in an NHEJ-deficient setting exhibit numerous chromosomal abnormalities. Unexpectedly, however, pre-malignant, NHEJ-deficient pro-B cells show very little genomic instability. This is an interesting paradox, which may be explained by the reliance of pro-B cells on another DSB repair pathway such as HR. We are testing this hypothesis, in culture and in vivo, by taking advantage of cells deficient for the XRCC2 homologous recombination factor.

2. Genome Instability in Tumorigenesis

The role of nuclear architecture in oncogenic genome instability

An emerging picture of oncogenic genome instability indicates that nuclear architecture influences oncogenic instability by favoring certain chromosomal translocations while disfavoring others. This model posits that non-random distribution of chromosomes within the nuclear volume results in proximity of certain chromosome pairs, and thus predisposes these pairs to involvement in translocations. We are examining how nuclear architecture influences the acquisition of characteristic chromosome abnormalities in NHEJ Trp53-deficient lymphomas. A major goal is to elucidate the mechanisms for oncogenic chromosomal translocations and to understand the molecular etiology of complex cytogenetic features commonly seen in cancer.

New mouse tumor models

With the exception of medulloblastomas, NHEJ Trp53 doubly deficient animals do not develop other malignancies. This narrow tumor spectrum is surprising because NHEJ is required for V(D)J recombination in T, as well as B cells, and functions in general genome stability in many, if not all, somatic cells. It was reasoned that lack of the J_H region should prevent or delay pro-B cell tumorigenesis, and possibly allow the development of tumors in other cell types. Such non-B lineage tumors may represent important new mouse cancer models, and will be a valuable resource in evaluating the role of DSB repair in tumor suppression in other tissues.

3. Mouse Model of Human Leukemia/Lymphoma

It is estimated that more than 87,000 new cases of leukemia and non-Hodgkin’s lymphoma were diagnosed last year in the United States (Leukemia and Lymphoma Society). The pro-B cell malignancy that develops in Lig4 Trp53 double null mice recapitulates many features of human B-lineage lymphomas or leukemias, including chromosomal translocations involving immunoglobulin (Ig) loci and gross overall karyotypic complexity. Therefore, another important focus of the lab is the exploitation of this B-ALL mouse model to understand the molecular pathobiology of the human disease. Several different approaches are being employed.

Array comparative genomic hrbridization

It is well known that, like chromosomal translocations, gene deletion and gene amplification are common chromosomal abnormalities affecting tumor cells. Comparative genomic hybridization (CGH) on an array of genomic clones (array CGH) is a powerful tool for identification of such copy number abnormalities. In addition to the well-characterized oncogenic translocation/amplification seen in Lig4-/- Trp53-/- lymphomas, numerous other chromosomal abnormalities typically occur, including recurrent deletions and gene amplification. In collaboration with Dr. Gary Churchill’s laboratory, we are developing a high resolution array CGH platform to enable the precise identification of affected regions and their boundaries. Preliminary data suggest that we will be able to interrogate tumors with high sensitivity and resolution by this approach. Using this approach, we hope to (1) define chromosomal deletion and amplification regions and identify the genes that reside in the affected intervals; and (2) accurately map gene deletion and amplification boundaries, and elucidate mechanistic aspects of copy number aberrations in these mice.

Gene expression profiling

While it is typical in B-ALL to find widespread dissemination in secondary lymphoid organs, the mechanism for this dissemination remains obscure. Lig4 Trp53 double null tumors are similar in this regard, typically locating to bone marrow, spleen, lymph nodes, and thymus. This is a highly aberrant situation, as progenitor B cells normally reside in the bone marrow, with very few entering circulation. To dissect the constellation of adaptive gene expression changes that allow transformed progenitor B cells in this model to home to ectopic locations, we are employing gene expression profile analysis, in collaboration with Dr. Churchill’s group. As another approach, we are also implementing a gene expression study in collaboration with Dr. Derry Roopenian’s group, using the group’s 384-well format CancerQuantArray platform to analyze defined sets of genes of interest.

Publications

• Mills KD, Ferguson DO, Essers J, Eckersdorff M, Kanaar R, Alt FW. 2004. Rad54 and DNA Ligase IV cooperate to maintain mammalian chromatid stability. Genes Dev 18:1283-1292.
• Couedel C, Mills KD, Marco B, Shen L, Olshen A, Johnson RD, Nussenzweig A, Essers J, Kanaar R, Li GC, Alt FW, Jasin M. 2004. Collaboration of homologous recombination and nonhomologous end-joining factors for the survival and integrity of mice and cells. Genes Dev 18:1293-1304.
• Mostoslavsky R, Chua KF, Lombard DB, Pang WW, Fischer MR, Gellon L, Liu P, Mostoslavsky G, Franco S, Murphy MM, Mills KD, Patel P, Hsu JT, Hong AL, Ford E, Cheng HL, Kennedy C, Nunez N, Bronson R, Frendewey D, Auerbach W, Valenzuela D, Karow M, Hottiger MO, Hursting S, Barrett JC, Guarente L, Mulligan R, Demple B, Yancopoulos GD, Alt FW. 2006. Genomic instability and aging-like phenotype in the absence os mammalian SIRT6. Cell 124:315-329.

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