B lymphocytes are the immune system cells that recognize and dispose pathogens such as viruses and bacteria though special receptors on their cell surface known as antibodies. How the immune system recognizes and eliminates pathogens via antibody molecules depends to a great extent on two genetic processes targeting B cell antibody genes: somatic hypermutation and class switch recombination. The first mechanism introduces random point mutations at the N terminal portion of the antibody gene. Mutations coupled to cell selection during the immune response increase the binding affinity of the antibody for the pathogen. The second mechanism changes (via gene recombination) the C terminal portion of the antibody gene, which in turns dictates the strategy used by the immune system to eliminate the pathogen in question. Both somatic hypermutation and switch recombination are carried out by a B cell specific enzyme: Activation-Induced cytidine Deaminase or AID. The AID enzyme modifies the chemical nature of DNA, converting cytidines into another base called uracil, a process known as cytidine deamination. Because uracils are mutagenic, AID activity attracts a plethora of repair enzymes to the immunoglobulin loci, which attempt to revert uracils into cytidines. One critical factor involved in this repair process is the uracyl glycosilase I (UNG). However, while spontaneous cytidine deamination is faithfully repaired, in a manner not fully understood AID-mediated deamination leads to the formation mutations and DNA breaks, which are at the basis of somatic hypermutation and switch recombination respectively. The importance of AID in the immune response is highlighted in AID deficient humans and animals, which are highly susceptible to infection and exhibit gut flora-dependent hyperplasia of intestinal villi. Conversely, complex diseases such as autoimmunity have long been associated with AID-dependent hypermutation. Moreover, AID is not only recruited to antibody genes, but increasing evidence indicate that many other genes, including oncogenes (tumor-inducing genes) can be physiological targets of AID. Because AID activity leads to DNA mutations, oncogenes can become deregulated by AID-targeting, resulting in malignant transformation of AID expressing cells in susceptible individuals. In addition, AID-mediated DNA breaks can also recombine or bring oncogenes into close proximity of the immunoglobulin loci, a chromosomal irregularity known as a translocation. Chromosomal translocations are responsible for the formation of B cell lymphomas in humans. Burkits and multiple myeloma are prime examples of AID-induced translocations leading to tumor development. These arguments underscore the important of unraveling how AID activity is regulated under normal conditions and deregulated during tumorigenesis. This fiscal year we have furthered our understanding of AID physiological and pathological activity in two separate studies: i) As stated above, a significant fraction of AID-mediated deamination events is faithfully reverted to cytidines through a process initiated by UNG. This pathway is dubbed error-free repair, which occurs through single stranded DNA breaks. However, contiguous uracyls in opposite DNA strands of Ig genes can also be recognized as staggered DNA double-stranded breaks by the ATM repair pathway. Ig gene recombination and chromosomal translocations are believed to result from such staggered DNA break intermediates. This step is referred to as error-prone repair. We have investigated the role of UNG in the formation of translocations and B cell tumor development in BalbC mice carrying the anti-apoptotic transgene BclXL. In a manuscript under preparation we now describe that similar to AID-/- mice, UNG-/- are refractory to the formation of Ig-gene specific DNA breaks, canonical IgH-cMyc translocations, and B cell tumor development. This data supports the idea that at the Ig loci, UNG functions upstream of the DNA lesions that are substrates for both Ig gene switch recombination and IgH-cMyc chromosomal translocations. Surprisingly however, UNG heterozygous mice show little or no error-prone repair happloinsufficiency, whereas error-free repair is severely compromised in a manner directly proportional to UNG expression. This is in stark contrast to AID+/- B cells, which we have shown display happloinsufficiency both at the level of error-free and error-prone repair. Although a mechanistic explanation for this last observation is lacking at this point, the data for the first time differentiates UNG activity in the two types of repair steps, which are believed to target Ig genes simultaneously. In analogy to AID-/- mice however, we have found that the few tumors developing in the absence of UNG do so via IgH-nMyc inversions (in chromosome 12) and non-canonical IgH-cMyc translocations, which do not directly involve Ig genes. Finally, within the peritoneal granuloma, we have pinpointed the step at which plasma cell tumor development is arrested in UNG knock-out mice. In summary, our new study sheds light on the steps involved in the formation of chromosomal translocations, which are etiologic to B cell tumor development both in human and mice. ii) For decades the pharmaceutical industry has been screening thousands of chemicals to battle cancer, in the hope of finding miracle drugs that specifically target cancerous cells but leaves healthy ones untouched. In may 2001, the FDA approved the first example of such a drug, a small chemical marketed by Novartis as Gleevec, which can successfully arrest the development of chronic myeloid leukemia (CML), gastrointestinal stromal tumors (GISTs) and other cancers. Following a series of clinical trials that showed the full recovery of terminally ill CML patients, TIME magazine broadcasted it in its cover as the magic bullet to cure cancer. While for the most part Gleevec has lived up to its fame, a considerable number of patients, particularly those with advanced CML, rapidly develop resistance to Gleevec and die within a few months of treatment initiation. How these tumors bypass the inhibitory effects of Gleevec has been a mystery. In collaboration with Markus Muschens lab from the University of Southern California at Los Angeles, have now discovered that resistance to Gleevec treatment is provided by the AID mutator. It turns out that CML tumor cells develop from hematopoietic stem cells, a cell type that has the capacity to differentiate into a variety of blood cells, including those of the immune system. In some cases, the CML tumor will partially develop into an antibody forming cell, which will in turn begin expressing AID. Whereas under normal circumstances the activity of AID is tightly regulated, in CML cells AID runs awry and rapidly mutates the cancer DNA leading indirectly to Gleevec resistance and the demise of the patient. As our findings provide a rationale on how CML tumors develop resistance to current chemotherapy treatments, we entertain the possibility that new drugs could be develop to counteract these mechanisms and thus extent life of terminally ill patients.

Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2009
Total Cost
$1,051,854
Indirect Cost
Name
National Institute of Arthritis and Musculoskeletal and Skin Diseases
Department
Type
DUNS #
City
State
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Zip Code
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