Mammalian cells possess sophisticated and intricate mechanisms to detect and repair DNA double-strand breaks, serious genetic lesions that can cause a cell to die or become cancerous. Our laboratory is exploring the mechanisms that maintain genome stability and how they relate to human pathology. We are investigating the two predominant double-strand break repair pathways: nonhomologous end joining, which repairs two broken ends without respect to sequence integrity, and homologous recombination, which uses a template to catalyze high fidelity repair. Another goal is to clarify the mechanisms of oncogenic chromosomal translocations, where the exchange of genetic material between chromosomes leads to tumorigenesis. We are examining how nuclear architecture, genome structure and DNA repair mechanisms all influence this process as well as working with a mouse model of human leukemia/lymphoma to better understand the molecular pathobiology of the human disease. We are also investigating the connection between DNA damage and natural variations in aging, using the mouse as a model system to explore how genome stability control changes with age.
Replication, Recombination, and Repair: The 3 R's of Tumor Suppression
Nonhomologous End Joining: Multifaceted Roles in Cancer
Nonhomologous end joining (NHEJ) is a critical DNA repair pathway, with proposed tumor suppression functions in many tissues. Mutations in the NHEJ factor ART/DCLRE1C cause radiation sensitive severe combined immunodeficiency (RS-SCID) in humans and may increase susceptibility to lymphoma in some settings. We previously found that lack of Art/Dclre1c provokes loss of heterozygosity (LOH) for Trp53, which encodes the critical tumor suppressor p53, leading to the formation of lymphomas. This led us to propose a new model for NHEJ-mediated tumor suppression, in which the interplay between DNA repair and Trp53 LOH influences the sequence and timing of subsequent events in tumor development/progression.
It has also been known for some time that NHEJ Trp53 double null mice rapidly develop aggressive progenitor B-cell lymphomas. These tumors arise with defining cytogenetic features, including a type of complex oncogenic lesion that we term complicons, which harbor amplification of Myc family oncogenes. We have found that DNA double-strand breaks (DSB) occurring within a tiny portion of the immunoglobulin heavy chain (Igh) gene are necessary to initiate complicon formation, but not other types of oncogenic chromosome rearrangements. Surprisingly, we also found that the powerful Em transcriptional enhance, previously implicated in hyperactivation of Myc (after translocation with Igh), is actually not required for Myc overexpression in B-cell tumors. This study is important, because it is the first to delineate molecular mechanisms of complex versus simple instability and the first to identify specific chromosomal elements required for complex chromosomal aberrations.
Homologous Recombination: New Roles in Lymphocyte Development
It has long been recognized that genomic instability is a hallmark of many tumor types, including lymphoid tumors. However, the cellular functions that normally prevent such instability, and the precise role of instability in driving tumor formation, remain poorly understood. Several lines of evidence have implicated NHEJ as a key suppressor of oncogenic genome instability, but the contribution of homologous recombination (HR) has been less well characterized. In this context, recurrent translocations or deletions affecting Chromosome 7q32-36 are often noted in myeloid and lymphoblastic leukemias, especially those with complex karyotypes. The X-ray cross-complementing 2 (Xrcc2) gene is an intriguing candidate at 7q36, as a member of the Rad51-family of HR factors required for chromosomal stability, ionizing radiation resistance and embryonic viability in mice. We recently showed that Xrcc2 is essential for normal B-cell development. B-cell differentiation/maturation is accompanied by several rounds of rapid proliferative expansion. We found that Xrcc2 is critical for developing B-cells to successfully transit the S-phase of the cell cycle. In the absence of Xrcc2, developing B-cells undergo a dramatic, p53-dependent S-phase arrest. If this S-phase arrest is alleviated, B-cell development proceeds, but at the cost of high genomic instability. From these and other findings we propose that XRCC2 is important for recombination mediated restart of stalled or collapsed replication forks. We are currently taking both genetic and biochemical approaches to test this model. We are also investigating whether XRCC2, or homologous recombination in general, has roles in other stages of B or T cell development.
The Damage Accumulation Model of Aging
One theory of aging posits that accumulation of DNA damage or chromosomal abnormalities with age leads to decline or derangement of normal cellular processes. In support of this hypothesis, mice deficient for DNA damage response or repair often show phenotypes resembling accelerated aging, especially tumor development. However, natural aging and age-specific pathologies, such as cancer, are complex and variable phenomena that may be considered quantitative traits.
We have taken advantage of a comprehensive collection of aging inbred mouse strains that were generated at The Jackson Laboratory—with support from The Ellison Foundation and the NIH Nathan Shock Center of Excellence in the Basic Biology of Aging—to probe the connection between DNA damage and natural aging. We have found that endogenous genotoxic stress, and the magnitude of the resulting apoptotic response, varies dramatically among different inbred strains of mice, at least in the tissues we tested. This suggests that either the underlying susceptibility to DNA damage or the nature of the response is under genetic control. Intriguingly, we have found that apoptosis and spontaneous chromosomal instability appear to be inversely correlated, suggesting that some strains more effectively avoid genomic instability by mounting a stronger apoptotic reaction to damage. Moreover, we have found that this apoptosis/instability relationship also correlates with the average lifespan characteristics of individual strains, suggesting that DNA damage, chromosomal instability and cellular genotoxicity may relate to some aspects of overall organismal aging. We are now taking a genetics approach to begin dissecting the pathways governing these phenotypes.
Associate Professor: Kevin Mills, Ph.D.
Associate Research Scientist: Muneer Hasham, Ph.D.
Postdoctoral Fellows: Kristin Lamont, Ph.D.
Research Assistant II: Nina Donghia, B.A.
Biomedical Technologist I: Jane Maynard
Research Administrative Assistant: Patricia Cherry
Lamont KR, Hasham MG, Donghia NM, Branca J, Chavaree M, Chase B, Breggia A, Hedlund J, Emery I, Cavallo F, Jasin M, Rüter J, Mills KD. 2013. Attenuating homologous recombination stimulates an AID-induced antileukemic effect. J Exp Med 210(5):1021-1033. PMCID: PMC3646491
Mills KD. 2013. Tumor suppression: Puting p53 in context. Cell Cycle 12(22):3461-3462. (Review)
Vuong BQ, Herrick-Reynolds K, Vaidyanathan B, Pucella JN, Ucher AJ, Donghia NM, Gu X, Nicolas L, Nowak U, Rahman N, Strout MP, Mills KD, Stavnezer J, Chaudhuri J. 2013. A DNA break- and phosphorylation-dependent positive feedback loop promotes immunoglobulin class-switch recombination. Nat Immunol.14(11):1183-1189.
Hasham MG, Snow KJ, Donghia NM, Branca JA, Lessard MD, Stavnezer J, Shopland LS, Mills KD. 2012. AID-initiated off-target DNA breaks are detected and resolved during S-phase. J Immunol 189(5):2374-2382. PMCID: PMC3424338
Snow KJ, Wright SM, Woo Y, Titus LC, Mills KD, Shopland LS. 2011. Nuclear positioning, higher-order folding, and gene expression of Mmu15 sequences are refractory to chromosomal translocation. Chromosoma 120:61-71. PMCID: PMC3057431
Sher RB, Cox GA, Mills KD, Sundberg JP. Rhabdomyosarcomas in aging a/j mice. PLoS One 6(8):e23498. PMCID: PMC3154500
Hasham MG, Donghia NM, Coffey E, Maynard J, Snow KJ, Ames J, Wilpan RY, He Y, King BL, Mills KD. 2010. Widespread genomic breaks generated by activation induced cytidine deaminase are prevented by homologous recombination. Nat Immunol 11(9):820-826. PMCID: PMC2930818
Maas SA, Donghia NM, Tompkins K, Foreman O, Mills KD. 2010. ARTEMIS stabilizes the genome and restrains proliferation in multipotent mesenchymal cells. BMC Biol Oct 27 [Epub ahead of print] 8(1):132. PMCID: PMC2984387
Ng SH, Maas SA, Petkov PM, Mills KD, Paigen K. 2009. Co-localization of somatic and meiotic double strand breaks near the Myc oncogene on mouse chromosome 15. Genes Chromosome Cancer 48(10):925-930. PMCID: PMC2821716
Singh P, Alley TL, Wright SM, Kamdar S, Schott W, Wilpan RY, Mills KD, Graber JH. 2009. Global changes in processing of mRNA 3' untranslated regions characterize clinically distinct cancer subtypes. Cancer Res 69(24):9422-9430. PMCID: PMC2794997
Wright SM, Woo YH, Alley TL, Shirley BJ, Akeson EC, Snow KJ, Maas SA, Elwell RL, Foreman O, Mills KD. 2009. Complex oncogenic translocation with gene amplification are initiated by specific DNA breaks in lymphocytes. Cancer Res 69(10):4454-460. PMCID: PMC2724672
Yuan R, Tsaih SW, Petkova SB, Marin de Evsikova C, Xing S, Marion MA, Bogue MA, Mills KD, Peters LL, Bult CJ, Rosen CJ, Sundberg JP, Harrison DE, Churchill GA, Paigen B. 2009. Aging in inbred strains of mice: study design and interim report on median lifespans and circulating IGF1 levels. Aging Cell 8:277-287. PMCID: PMC2768517
Maas S, Caddle LB, Mills KD. 2008. Mechanisms of DNA double strand break repair in hematopoietic homeostasis and oncogenesis. In Mouse Model of Human Blood Cancers. Li S, Editor. Springer Press.
Oberdoerffer P, Michan S, McVay M, Mostoslavsky R, Vann J, Park SK, Hartlerode A, Stegmuller J, Hafner A, Loerch P, Wright SM, Mills KD, Bonni A, Yankner BA, Scully R, Prolla TA, Alt FW, Sinclair DA. 2008. SIRT1 redistribution on chromatin promotes genomic stability but alters gene expression during aging. Cell 135:907-918. PMCID: PMC2853975
Caddle LB, Hasham MG, Schott W, Shirley BJ, Mills KD. 2008. Homologous recombination is necessary for normal lymphocyte development. Mol Cell Biol 28(7):2295-2303. PMCID: PMC2268416
Caddle LB, Grant JL, Szatkiewicz J, van Hase J, Shirley BJ, Bewersdorf J, Cremer C, Arneodo A, Khalil A, Mills KD. 2007. Chromosome neighborhood composition determines translocation outcomes after exposure to high-dose radiation in primary cells. Chromosome Res. 15:1061-1073.
Hess ST, Gould TJ, Gudheti MV, Maas SA, Mills KD, Zimmerberg J. 2007. Dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories. Proc Natl Acad Sci USA. 104:17370-17375.
Khalil A, Grant JL, Caddle LB, Atzema E, Mills KD, Arneodo A. 2007. Chromosome territories have a highly nonspherical morphology and nonrandom positioning. Chromosome Res. 15:899-916.
Woo Y, Wright SM, Maas SS, Alley TL, Caddle LB, Kamdar S, Affourtit J, Foreman O, Akeson EC, Shaffer D, Bronson RT, Morse 3rd, Roopenian D, Mills KD. 2007. The nonhomologous end joining factor Artemis suppresses multi-tissue tumor formation and prevents loss of heterozygosity. Oncogene 26:6010-6020.
Yan CT, Kaushal D, Murphy M. Zhang Y, Datta A, Chen C, Monroe B, Mostoslavsky G, Coakley K, Gao Y, Mills KD, Fazeli AP, Tepsuporn S, Hall G, Mulligan R, Rox E, Bronson R, De Girolami U, Lee C, Alt FW. 2006. XRCC4 suppresses medulloblastomas with recurrent translocations in p53-deficient mice. Proc Natl Acad Sci USA. 103:7378-7383.
Morales JC, Franco S, Murphy MM, Bassing CH, Mills KD, Adams MM, Walsh NC, Manis JP, Rassidakis GZ, Alt FW, Carpenter PB. 2006. 53BP1 and p53 synergize to suppress genomic instability and lymphomagenesis. Proc Natl Acad Sci USA. 103:3310-3315.
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.
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.
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