Therapy-induced APOBEC3A drives evolution of persistent cancer cells.

Acquired drug resistance to anticancer targeted therapies remains an unsolved clinical problem. Although many drivers of acquired drug resistance have been identified1-4, the underlying molecular mechanisms shaping tumour evolution during treatment are incompletely understood. Genomic profiling of patient tumours has implicated apolipoprotein B messenger RNA editing catalytic polypeptide-like (APOBEC) cytidine deaminases in tumour evolution; however, their role during therapy and the development of acquired drug resistance is undefined. Here we report that lung cancer targeted therapies commonly used in the clinic can induce cytidine deaminase APOBEC3A (A3A), leading to sustained mutagenesis in drug-tolerant cancer cells persisting during therapy. Therapy-induced A3A promotes the formation of double-strand DNA breaks, increasing genomic instability in drug-tolerant persisters. Deletion of A3A reduces APOBEC mutations and structural variations in persister cells and delays the development of drug resistance. APOBEC mutational signatures are enriched in tumours from patients with lung cancer who progressed after extended responses to targeted therapies. This study shows that induction of A3A in response to targeted therapies drives evolution of drug-tolerant persister cells, suggesting that suppression of A3A expression or activity may represent a potential therapeutic strategy in the prevention or delay of acquired resistance to lung cancer targeted therapy.

UnAIDed Class Switching in Activated B-Cells Reveals Intrinsic Features of a Self-Cleaving IgH Locus.

Activation-induced deaminase (AID) is the major actor of immunoglobulin (Ig) gene diversification in germinal center B-cells. From its first description, it was considered as mandatory for class switch recombination (CSR), and this discovery initiated a long quest for all of the AID-interacting factors controlling its activity. The mechanisms focusing AID-mediated DNA lesions to given target sequences remain incompletely understood with regards the detailed characterization of optimal substrates in which cytidine deamination will lead to double strand breaks (DSBs) and chromosomal cleavage. In an effort to reconsider whether such CSR breaks absolutely require AID, we herein provide evidence, based on deep-sequencing approaches, showing that this dogma is not absolute in both human and mouse B lymphocytes. In activated B-cells from either AID-deficient mice or human AID-deficient patients, we report an intrinsic ability of the IgH locus to undergo "on-target" cleavage and subsequent synapsis of broken regions in conditions able to yield low-level CSR. DNA breaks occur in such conditions within the same repetitive S regions usually targeted by AID, but their repair follows a specific pathway with increased usage of microhomology-mediated repair. These data further demonstrate the role of AID machinery as not initiating de novo chromosomal cleavage but rather catalyzing a process which spontaneously initiates at low levels in an appropriately conformed IgH locus.

Activation-induced deaminase is critical for the establishment of DNA methylation patterns prior to the germinal center reaction.

Activation-induced deaminase (AID) initiates antibody diversification in germinal center B cells by deaminating cytosines, leading to somatic hypermutation and class-switch recombination. Loss-of-function mutations in AID lead to hyper-IgM syndrome type 2 (HIGM2), a rare human primary antibody deficiency. AID-mediated deamination has been proposed as leading to active demethylation of 5-methycytosines in the DNA, although evidence both supports and casts doubt on such a role. In this study, using whole-genome bisulfite sequencing of HIGM2 B cells, we investigated direct AID involvement in active DNA demethylation. HIGM2 naïve and memory B cells both display widespread DNA methylation alterations, of which ∼25% are attributable to active DNA demethylation. For genes that undergo active demethylation that is impaired in HIGM2 individuals, our analysis indicates that AID is not directly involved. We demonstrate that the widespread alterations in the DNA methylation and expression profiles of HIGM2 naïve B cells result from premature overstimulation of the B-cell receptor prior to the germinal center reaction. Our data support a role for AID in B cell central tolerance in preventing the expansion of autoreactive cell clones, affecting the correct establishment of DNA methylation patterns.

The Role of IgM Antibodies in T Cell Lymphoma Protection in a Novel Model Resembling Anaplastic Large Cell Lymphoma.

MRL/lpr mice typically succumb to immune complex-mediated nephritis within the first year of life. However, MRL/lpr mice that only secrete IgM Abs because of activation-induced deaminase deficiency (AID-/-MRL/lpr mice) experienced a dramatic increase in survival. Further crossing of these mice to those incapable of making secretory IgM (µS mice) generated mice lacking any secreted Abs but with normal B cell receptors. Both strains revealed no kidney pathology, yet Ab-deficient mice still experienced high mortality. In this article, we report Ab-deficient MRL/lpr mice progressed to high-grade T cell lymphoma that can be reversed with injection of autoreactive IgM Abs or following adoptive transfer of IgM-secreting MRL/lpr B cells. Anti-nuclear Abs, particularly anti-dsDNA IgM Abs, exhibited tumor-killing activities against a murine T cell lymphoma cell line. Passive transfers of autoreactive IgM Abs into p53-deficient mice increased survival by delaying onset of T cell lymphoma. The lymphoma originated from a double-negative aberrant T cell population seen in MRL/lpr mice and most closely resembled human anaplastic large cell lymphoma. Combined, these results strongly implicate autoreactive IgM Abs in protection against T cell lymphoma.

Deficiency of activation-induced cytidine deaminase in a murine model of ulcerative colitis.

Patients with inflammatory bowel disease (IBD) have an increased risk of colorectal cancer, particularly in ulcerative colitis (UC) when the majority of colon epithelial cells may be exposed to inflammation-associated mutagenesis. In addition to mutagenesis generated by oxidative stress, inflammation can induce activation-induced cytidine deaminase (Aicda), a mutator enzyme in the APOBEC family, within colon epithelial cells. This study tested the hypothesis that deletion of the Aicda gene could protect against the development of inflammation-associated colorectal cancers, using a model of UC-like colitis in "T/I" mice deficient in TNF and IL10. Results showed that T/I mice that were additionally Aicda-deficient ("TIA" mice) spontaneously developed moderate to severe UC-like colitis soon after weaning, with histologic features and colon inflammation severity scores similar those in T/I mice. Although the mean survival of TIA mice was decreased compared to T/I mice, multivariable analysis that adjusted for age when neoplasia was ascertained showed a decreased numbers of neoplastic colorectal lesions in TIA mice, with a trend toward decreased incidence of neoplasia. Aicda deficiency increased serum IL1α and slightly decreased IL12p40 and M-CSF, as compared with T/I mice, and led to undetectable levels of IgA, IgG1, IgG2a, IgG2b, and IgG3. Taken together, these studies show that Aicda deficiency can decrease the number of neoplastic lesions but is not sufficient to prevent the risk of inflammation-associated colorectal neoplasia in the setting of severe UC-like inflammation. The TIA model may also be useful for assessing the roles of antibody class-switch recombination deficiency and somatic hypermutation on regulation of microbiota and inflammation in the small intestine and colon, as well as the pathogenesis of colitis associated with hyper-IgM syndrome in humans. Further studies will be required to determine the mechanisms that drive early mortality in TIA mice.

A decrease in NAMPT activity impairs basal PARP-1 activity in cytidine deaminase deficient-cells, independently of NAD.

Cytidine deaminase (CDA) deficiency causes pyrimidine pool disequilibrium. We previously reported that the excess cellular dC and dCTP resulting from CDA deficiency jeopardizes genome stability, decreasing basal poly(ADP-ribose) polymerase 1 (PARP-1) activity and increasing ultrafine anaphase bridge (UFB) formation. Here, we investigated the mechanism underlying the decrease in PARP-1 activity in CDA-deficient cells. PARP-1 activity is dependent on intracellular NAD+ concentration. We therefore hypothesized that defects of the NAD+ salvage pathway might result in decreases in PARP-1 activity. We found that the inhibition or depletion of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD+ salvage biosynthesis pathway, mimicked CDA deficiency, resulting in a decrease in basal PARP-1 activity, regardless of NAD+ levels. Furthermore, the expression of exogenous wild-type NAMPT fully restored basal PARP-1 activity and prevented the increase in UFB frequency in CDA-deficient cells. No such effect was observed with the catalytic mutant. Our findings demonstrate that (1) the inhibition of NAMPT activity in CDA-proficient cells lowers basal PARP-1 activity, and (2) the expression of exogenous wild-type NAMPT, but not of the catalytic mutant, fully restores basal PARP-1 activity in CDA-deficient cells; these results strongly suggest that basal PARP-1 activity in CDA-deficient cells decreases due to a reduction of NAMPT activity.

Murine Leukemia Virus P50 Protein Counteracts APOBEC3 by Blocking Its Packaging.

Apolipoprotein B editing enzyme, catalytic polypeptide 3 (APOBEC3) family members are cytidine deaminases that play important roles in intrinsic responses to retrovirus infection. Complex retroviruses like human immunodeficiency virus type 1 (HIV-1) encode the viral infectivity factor (Vif) protein to counteract APOBEC3 proteins. Vif induces degradation of APOBEC3G and other APOBEC3 proteins and thereby prevents their packaging into virions. It is not known if murine leukemia virus (MLV) encodes a Vif-like protein. Here, we show that the MLV P50 protein, produced from an alternatively spliced gag RNA, interacts with the C terminus of mouse APOBEC3 and prevents its packaging without causing its degradation. By infecting APOBEC3 knockout (KO) and wild-type (WT) mice with Friend or Moloney MLV P50-deficient viruses, we found that APOBEC3 restricts the mutant viruses more than WT viruses in vivo Replication of P50-mutant viruses in an APOBEC3-expressing stable cell line was also much slower than that of WT viruses, and overexpressing P50 in this cell line enhanced mutant virus replication. Thus, MLV encodes a protein, P50, that overcomes APOBEC3 restriction by preventing its packaging into virions.IMPORTANCE MLV has existed in mice for at least a million years, in spite of the existence of host restriction factors that block infection. Although MLV is considered a simple retrovirus compared to lentiviruses, it does encode proteins generated from alternatively spliced RNAs. Here, we show that P50, generated from an alternatively spliced RNA encoded in gag, counteracts APOBEC3 by blocking its packaging. MLV also encodes a protein, glycoGag, that increases capsid stability and limits APOBEC3 access to the reverse transcription complex (RTC). Thus, MLV has evolved multiple means of preventing APOBEC3 from blocking infection, explaining its survival as an infectious pathogen in mice.

Estrogen Reverses HDAC Inhibitor-Mediated Repression of Aicda and Class-Switching in Antibody and Autoantibody Responses by Downregulation of miR-26a.

Estrogen contributes to females' strong antibody response to microbial vaccines and proneness to autoimmunity, particularly antibody-mediated systemic autoimmunity, in females. We have hypothesized that this is due to estrogen-mediated potentiation of class switch DNA recombination (CSR) and somatic hypermutation (SHM). As we have shown, estrogen boosts AID expression, which is critical for both CSR and SHM, through upregulation of HoxC4, which together with NF-κB critically mediates Aicda (AID gene) promoter activation. We contend here that additional regulation of Aicda expression by estrogen occurs through epigenetic mechanisms. As we have shown, histone deacetylase inhibitors (HDIs) short-chain fatty acid (SCFA) butyrate and propionate as well as the pharmacologic HDI valproic acid upregulate miRNAs that silence AID expression, thereby modulating specific antibody responses in C57BL/6 mice and autoantibody responses in lupus-prone MRL/Faslpr/lpr mice. Here, using constitutive knockout Esr1-/- mice and B cells as well as conditional knockout Aicdacre/creEsr1flox/flox mice and B cells, we showed that the HDI-mediated downregulation of Aicda expression as well as the maturation of antibody and autoantibody responses is reversed by estrogen and enhanced by deletion of ERα or E2 inhibition. Estrogen's reversion of HDI-mediated inhibition of Aicda and CSR in antibody and autoantibody responses occurred through downregulation of B cell miR-26a, which, as we showed, targets Aicda mRNA 3'UTR. miR-26a was significantly upregulated by HDIs. Accordingly, enforced expression of miR-26a reduced Aicda expression and CSR, while miR-26a-sponges (competitive inhibitors of miR-26a) increased Aicda expression and CSR. Thus, our findings show that estrogen reverses the HDI-mediated downregulation of AID expression and CSR through selective modulation of miR-26a. They also provide mechanistic insights into the immunomodulatory activity of this hormone and a proof-of-principle for using combined ER inhibitor-HDI as a potential therapeutic approach.

Fundamental roles of chromatin loop extrusion in antibody class switching.

Antibody class switch recombination (CSR) in B lymphocytes replaces immunoglobulin heavy chain locus (Igh) Cµ constant region exons (CHs) with one of six CHs lying 100-200 kb downstream1. Each CH is flanked upstream by an I promoter and long repetitive switch (S) region1. Cytokines and activators induce activation-induced cytidine deaminase (AID)2 and I-promoter transcription, with 3' IgH regulatory region (3' IgHRR) enhancers controlling the latter via I-promoter competition for long-range 3' IgHRR interactions3-8. Transcription through donor Sµ and an activated downstream acceptor S-region targets AID-generated deamination lesions at, potentially, any of hundreds of individual S-region deamination motifs9-11. General DNA repair pathways convert these lesions to double-stranded breaks (DSBs) and join an Sµ-upstream DSB-end to an acceptor S-region-downstream DSB-end for deletional CSR12. AID-initiated DSBs at targets spread across activated S regions routinely participate in such deletional CSR joining11. Here we report that chromatin loop extrusion underlies the mechanism11 by which IgH organization in cis promotes deletional CSR. In naive B cells, loop extrusion dynamically juxtaposes 3' IgHRR enhancers with the 200-kb upstream Sµ to generate a CSR centre (CSRC). In CSR-activated primary B cells, I-promoter transcription activates cohesin loading, leading to generation of dynamic subdomains that directionally align a downstream S region with Sµ for deletional CSR. During constitutive Sα CSR in CH12F3 B lymphoma cells, inversional CSR can be activated by insertion of a CTCF-binding element (CBE)-based impediment in the extrusion path. CBE insertion also inactivates upstream S-region CSR and converts adjacent downstream sequences into an ectopic S region by inhibiting and promoting their dynamic alignment with Sµ in the CSRC, respectively. Our findings suggest that, in a CSRC, dynamically impeded cohesin-mediated loop extrusion juxtaposes proper ends of AID-initiated donor and acceptor S-region DSBs for deletional CSR. Such a mechanism might also contribute to pathogenic DSB joining genome-wide.

A lentivirus-based system for Cas9/gRNA expression and subsequent removal by Cre-mediated recombination.

A major concern of CRISPR and related genome engineering technologies is off-target mutagenesis from prolonged exposure to Cas9 and related editing enzymes. To help mitigate this concern we added a loxP site to the 3'-LTR of an HIV-based lentiviral vector capable of expressing Cas9/gRNA complexes in a wide variety of mammalian cell types. Transduction of susceptible target cells yields an integrated provirus that expresses the desired Cas9/gRNA complex. The reverse transcription process also results in duplication of the 3'-LTR such that the integrated provirus becomes flanked by loxP sites (floxed). Subsequent expression of Cre recombinase results in loxP-to-loxP site-specific recombination that deletes the Cas9/gRNA payload and effectively prevents additional Cas9-mediated mutations. This construct also expresses a gRNA with a single transcription termination sequence, which results in higher expression levels and more efficient genome engineering as evidenced by disruption of the SAMHD1 gene. This hit-and-run CRISPR approach was validated by recreating a natural APOBEC3B deletion and by disrupting the mismatch repair gene MSH2. This hit-and-run strategy may have broad utility in many areas and especially those where cell types are difficult to engineer by transient delivery of ribonucleoprotein complexes.

The circulating immunoglobulins negatively impact on the parasite clearance in the liver of Leishmania donovani-infected mice via dampening ROS activity.

Visceral leishmaniasis, the most severe form of leishmaniasis, is caused by Leishmania donovani and L. infantum. Immunity to Leishmania infection has been shown to depend on the development of Th1 cells; however, the roles of B cells and antibodies during infection remain unclear. In the present study, we showed that AID and µs double-deficient mice (DKO), which have B cells but not circulating immunoglobulins (cIgs), became resistant to L. donovani infection, whereas µs or AID single-deficient mice did not. This resistance in DKO mice occurred in the liver from an early stage of the infection. The depletion of IFN-γ did not affect the rapid reduction of parasite burden, whereas NADPH oxidases was up-regulated in the livers of infected DKO mice. The inhibition of the reactive oxygen species pathway in vivo by apocynin, a NADPH oxidase inhibitor, resulted in a significant increase in the parasite burden in DKO mice. These results indicate that a circulating Ig deficiency induces a protective response against L. donovani infection by elevating IFN-γ-independent NADPH oxidase activity, and also that cIgs play a regulatory role in controlling L. donovani infection in mice.

Circulating Follicular Helper and Follicular Regulatory T Cells Are Severely Compromised in Human CD40 Deficiency: A Case Report.

Mutations in genes that control class switch recombination and somatic hypermutation during the germinal center (GC) response can cause diverse immune dysfunctions. In particular, mutations in CD40LG, CD40, AICDA, or UNG cause hyper-IgM (HIGM) syndrome, a heterogeneous group of primary immunodeficiencies. Follicular helper (Tfh) and follicular regulatory (Tfr) T cells play a key role in the formation and regulation of GCs, but their role in HIGM pathogenesis is still limited. Here, we found that compared to CD40 ligand (CD40L)- and activation-induced cytidine deaminase (AICDA)-deficient patients, circulating Tfh and Tfr cells were severely compromised in terms of frequency and activation phenotype in a child with CD40 deficiency. These findings offer useful insight for human Tfh biology, with potential implications for understanding the molecular basis of HIGM syndrome caused by mutations in CD40.

Human-APOBEC3G-dependent restriction of porcine endogenous retrovirus replication is mediated by cytidine deamination and inhibition of DNA strand transfer during reverse transcription.

Although human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G, hA3G)-mediated deamination is the major mechanism used to restrict the infectivity of a broad range of retroviruses, it is unclear whether porcine endogenous retrovirus (PERV) is affected by hA3G or porcine A3F (poA3F). To determine whether DNA deamination is required for hA3G- and poA3F-dependent inhibition of PERV transmission, we developed VSV-pseudotype PERV-B expressing hA3G, mutant hA3G-E67Q (encapsidation and RNA binding activity-deficient), mutant hA3G-E259Q (deaminase-deficient), or poA3F. hA3G-E67Q decreased virus infectivity by ~ 93% compared to the ~ 99% decrease of viral infectivity by wild-type hA3G, while hA3G-E259Q decreased the infectivity of PERV-B by ~ 35%. These data suggest that cytidine deamination activity is crucial for efficient restriction of PERV by hA3G, but cytidine deamination cannot fully explain the inactivation of PERV by hA3G. Furthermore, differential DNA denaturation PCR (3D-PCR) products from 293T cells infected with PERV-B expressing hA3G mutants were sequenced. G-to-A hypermutation was detected at a frequency of 4.1% for hA3G, 3.4% for hA3G-E67Q, and 4.7% for poA3F. These results also suggest that hA3G and poA3F inhibit PERV by a deamination-dependent mechanism. To examine the effect of hA3G on the production of PERV DNA, genomic DNA was extracted from 293T cells 12 h after infection with PERV expressing hA3G, and this DNA was used as template for real-time PCR. A 50% decrease in minus strand strong stop (-sss) DNA synthesis/transfer was observed in the presence of hA3G. Based on these results, we conclude that hA3G may restrict PERV by both deamination-dependent mechanisms and inhibition of DNA strand transfer during PERV reverse transcription.

Germ Line IgM Is Sufficient, but Not Required, for Antibody-Mediated Alphavirus Clearance from the Central Nervous System.

Sindbis virus (SINV) infection of neurons in the brain and spinal cord in mice provides a model system for investigating recovery from encephalomyelitis and antibody-mediated clearance of virus from the central nervous system (CNS). To determine the roles of IgM and IgG in recovery, we compared the responses of immunoglobulin-deficient activation-induced adenosine deaminase-deficient (AID-/-), secretory IgM-deficient (sIgM-/-), and AID-/- sIgM-/- double-knockout (DKO) mice with those of wild-type (WT) C57BL/6 mice for disease, clearance of infectious virus and viral RNA from brain and spinal cord, antibody responses, and B cell infiltration into the CNS. Because AID is essential for immunoglobulin class switch recombination and somatic hypermutation, AID-/- mice produce only germ line IgM, while sIgM-/- mice secrete IgG but no IgM and DKO mice produce no secreted immunoglobulin. After intracerebral infection with the TE strain of SINV, most mice recovered. Development of neurologic disease occurred slightly later in sIgM-/- mice, but disease severity, weight loss, and survival were similar between the groups. AID-/- mice produced high levels of SINV-specific IgM, while sIgM-/- mice produced no IgM and high levels of IgG2a compared to WT mice. All mice cleared infectious virus from the spinal cord, but DKO mice failed to clear infectious virus from brain and had higher levels of viral RNA in the CNS late after infection. The numbers of infected cells and the amount of cell death in brain were comparable. We conclude that antibody is required and that either germ line IgM or IgG is sufficient for clearance of virus from the CNS.IMPORTANCE Mosquito-borne alphaviruses that infect neurons can cause fatal encephalomyelitis. Recovery requires a mechanism for the immune system to clear virus from infected neurons without harming the infected cells. Antiviral antibody has previously been shown to be a noncytolytic means for alphavirus clearance. Antibody-secreting cells enter the nervous system after infection and produce antiviral IgM before IgG. Clinical studies of human viral encephalomyelitis suggest that prompt production of IgM is associated with recovery, but it was not known whether IgM is effective for clearance. Our studies used mice deficient in production of IgM, IgG, or both to characterize the antibody necessary for alphavirus clearance. All mice developed similar signs of neurologic disease and recovered from infection. Antibody was necessary for virus clearance from the brain, and either early germ line IgM or IgG was sufficient. These studies support the clinical observation that prompt production of antiviral antibody is a determinant of outcome.

A role for Tau protein in maintaining ribosomal DNA stability and cytidine deaminase-deficient cell survival.

Cells from Bloom's syndrome patients display genome instability due to a defective BLM and the downregulation of cytidine deaminase. Here, we use a genome-wide RNAi-synthetic lethal screen and transcriptomic profiling to identify genes enabling BLM-deficient and/or cytidine deaminase-deficient cells to tolerate constitutive DNA damage and replication stress. We found a synthetic lethal interaction between cytidine deaminase and microtubule-associated protein Tau deficiencies. Tau is overexpressed in cytidine deaminase-deficient cells, and its depletion worsens genome instability, compromising cell survival. Tau is recruited, along with upstream-binding factor, to ribosomal DNA loci. Tau downregulation decreases upstream binding factor recruitment, ribosomal RNA synthesis, ribonucleotide levels, and affects ribosomal DNA stability, leading to the formation of a new subclass of human ribosomal ultrafine anaphase bridges. We describe here Tau functions in maintaining survival of cytidine deaminase-deficient cells, and ribosomal DNA transcription and stability. Moreover, our findings for cancer tissues presenting concomitant cytidine deaminase underexpression and Tau upregulation open up new possibilities for anti-cancer treatment.Cytidine deaminase (CDA) deficiency leads to genome instability. Here the authors find a synthetic lethal interaction between CDA and the microtubule-associated protein Tau deficiencies, and report that Tau depletion affects rRNA synthesis, ribonucleotide pool balance, and rDNA stability.

Cytidine deaminase deficiency impairs sister chromatid disjunction by decreasing PARP-1 activity.

Bloom Syndrome (BS) is a rare genetic disease characterized by high levels of chromosomal instability and an increase in cancer risk. Cytidine deaminase (CDA) expression is downregulated in BS cells, leading to an excess of cellular dC and dCTP that reduces basal PARP-1 activity, compromising optimal Chk1 activation and reducing the efficiency of downstream checkpoints. This process leads to the accumulation of unreplicated DNA during mitosis and, ultimately, ultrafine anaphase bridge (UFB) formation. BS cells also display incomplete sister chromatid disjunction when depleted of cohesin. Using a combination of fluorescence in situ hybridization and chromosome spreads, we investigated the possible role of CDA deficiency in the incomplete sister chromatid disjunction in cohesin-depleted BS cells. The decrease in basal PARP-1 activity in CDA-deficient cells compromised sister chromatid disjunction in cohesin-depleted cells, regardless of BLM expression status. The observed incomplete sister chromatid disjunction may be due to the accumulation of unreplicated DNA during mitosis in CDA-deficient cells, as reflected in the changes in centromeric DNA structure associated with the decrease in basal PARP-1 activity. Our findings reveal a new function of PARP-1 in sister chromatid disjunction during mitosis.

A double-strand break can trigger immunoglobulin gene conversion.

All three B cell-specific activities of the immunoglobulin (Ig) gene re-modeling system-gene conversion, somatic hypermutation and class switch recombination-require activation-induced deaminase (AID). AID-induced DNA lesions must be further processed and dissected into different DNA recombination pathways. In order to characterize potential intermediates for Ig gene conversion, we inserted an I-SceI recognition site into the complementarity determining region 1 (CDR1) of the Ig light chain locus of the AID knockout DT40 cell line, and conditionally expressed I-SceI endonuclease. Here, we show that a double-strand break (DSB) in CDR1 is sufficient to trigger Ig gene conversion in the absence of AID. The pattern and pseudogene usage of DSB-induced gene conversion were comparable to those of AID-induced gene conversion; surprisingly, sometimes a single DSB induced multiple gene conversion events. These constitute direct evidence that a DSB in the V region can be an intermediate for gene conversion. The fate of the DNA lesion downstream of a DSB had more flexibility than that of AID, suggesting two alternative models: (i) DSBs during the physiological gene conversion are in the minority compared to single-strand breaks (SSBs), which are frequently generated following DNA deamination, or (ii) the physiological gene conversion is mediated by a tightly regulated DSB that is locally protected from non-homologous end joining (NHEJ) or other non-homologous DNA recombination machineries.

Cytidine Deaminase Deficiency Reveals New Therapeutic Opportunities against Cancer.

Purpose: One of the main challenges in cancer therapy is the identification of molecular mechanisms mediating resistance or sensitivity to treatment. Cytidine deaminase (CDA) was reported to be downregulated in cells derived from patients with Bloom syndrome, a genetic disease associated with a strong predisposition to a wide range of cancers. The purpose of this study was to determine whether CDA deficiency could be associated with tumors from the general population and could constitute a predictive marker of susceptibility to antitumor drugs.Experimental Design: We analyzed CDA expression in silico, in large datasets for cancer cell lines and tumors and in various cancer cell lines and primary tumor tissues using IHC, PDXs, qRT-PCR, and Western blotting. We also studied the mechanism underlying CDA silencing and searched for molecules that might target specifically CDA-deficient tumor cells using in silico analysis coupled to classical cellular experimental approaches.Results: We found that CDA expression is downregulated in about 60% of cancer cells and tissues. We demonstrate that DNA methylation is a prevalent mechanism of CDA silencing in tumors. Finally, we show that CDA-deficient tumor cells can be specifically targeted with epigenetic treatments and with the anticancer drug aminoflavone.Conclusions: CDA expression status identifies new subgroups of cancers, and CDA deficiency appears to be a novel and relevant predictive marker of susceptibility to antitumor drugs, opening up new possibilities for treating cancer. Clin Cancer Res; 23(8); 2116-26. ©2016 AACR.