Role Of Somatic Hypermutation Research Articles

Negative regulation of activation-induced cytidine deaminase gene transcription in developing B cells by a PU.1-interacting intronic region

Activation-induced cytidine deaminase (AID, encoded by Aicda) plays a key role in somatic hypermutation and class switch recombination in germinal center B cells. However, off-target effects of AID are implicated in human leukemia and lymphoma. A mouse model of precursor B cell acute lymphoblastic leukemia driven by deletion of the related transcription factors PU.1 and Spi-B revealed C->T transition mutations compatible with being induced by AID. Therefore, we hypothesized that PU.1 negatively regulates Aicda during B cell development. Aicda mRNA transcript levels were increased in leukemia cells and bone marrow pre-B cells lacking PU.1 and/or Spi-B, relative to wild type cells. Using chromatin immunoprecipitation, PU.1 was found to interact with a negative regulatory region (R2–1) within the first intron of Aicda. CRISPR-Cas9-induced mutagenesis of R2–1 in cultured pre-B cells resulted in upregulation of Aicda in response to lipopolysaccharide stimulation. Mutation of the PU.1 interaction site and neighboring sequences resulted in reduced repressive ability of R2–1 in transient transfection analysis followed by luciferase assays. These results show that a PU.1-interacting intronic region negatively regulates Aicda transcription in developing B cells.

Abstract C14: A novel RAD51 inhibitor, CYT-0851, shows anticancer activity in preclinical models of pancreatic cancer

Abstract Genomic instability is recognized as a driver of tumorigenesis and cancer progression. Loss of tumor suppressors or activation of oncogenes can induce DNA damage stress, promoting genomic instability and creating dependencies upon key DNA repair pathways. These dependencies can be targeted therapeutically to induce synthetic lethality. We leverage a “gain-of-function” synthetic lethality approach to selectively target cancers that ectopically express Activation Induced Cytidine Deaminase (AID). AID is a DNA-directed cytidine deaminase normally transiently expressed in maturing B-lymphocytes where it plays a critical role in somatic hypermutation and immunoglobulin class switching. However, AID is dysregulated and inordinately expressed in many cancers, where it acts as a DNA-damaging enzyme mutating widespread locations throughout the genome. This activity leads to the accumulation of point mutations and DNA breaks and promotes DNA replication stress. In the latter context, AID-expressing cells become critically dependent on the homologous recombination factor RAD51 to survive DNA damage-induced replication stress. We have developed a novel small-molecule inhibitor of RAD51, CYT-0851, that is preferentially cytotoxic to AID expressing cells. We have shown in vitro and in vivo that CYT-0851 is efficacious in AID-expressing lymphoma and leukemia mouse models. Genome profiling and gene expression studies have shown many solid tumors, including pancreatic cancer, also aberrantly express AID. To determine whether targeting RAD51 may be an effective approach to the treatment of AID-expressing pancreatic cancer, we evaluated the antitumor activity of CYT-0851 in multiple patient-derived pancreatic cancer xenografts (PDX) models with a range of AID expression levels. All models tested showed tumor growth inhibition (TGI), ranging from 63 to 104% with CYT-0851 treatment. A crossover experiment was performed with one model to determine the activity of CYT-0851 in large, established tumors. We observed tumor regression (137% TGI) in large tumors upon treatment with CYT-0851, resulting in one partial responder and one tumor-free responder. The data presented here demonstrate that CYT-0851 exerts anticancer activity in multiple preclinical models of pancreatic cancer. Considering that recent published studies have shown that these cancers can ectopically overexpress AID, we conclude that CYT-0851 may be an effective treatment for pancreatic cancer. Overall, this provides a rationale for the continued development of CYT-0851 as a novel therapeutic for pancreatic cancer patients. Citation Format: Melinda Day, Jean-Marc Lapierre, Tom O'Shea, Kevin Mills. A novel RAD51 inhibitor, CYT-0851, shows anticancer activity in preclinical models of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C14.

Chicken MBD4 Regulates Immunoglobulin Diversification by Somatic Hypermutation.

Immunoglobulin (Ig) diversification occurs via somatic hypermutation (SHM) and class switch recombination (CSR), and is initiated by activation-induced deaminase (AID), which converts cytosine to uracil. Variable (V) region genes undergo SHM to create amino acid substitutions that produce antibodies with higher affinity for antigen. The conversion of cytosine to uracil in DNA promotes mutagenesis. Two distinct DNA repair mechanisms regulate uracil processing in Ig genes. The first involves base removal by the uracil DNA glycosylase (UNG), and the second detects uracil via the mismatch repair (MMR) complex. Methyl binding domain protein 4 (MBD4) is a uracil glycosylase and an intriguing candidate for involvement in somatic hypermutation because of its interaction with the MMR MutL homolog 1 (MLH1). We found that the DNA uracil glycosylase domain of MBD4 is highly conserved among mammals, birds, shark, and insects. Conservation of the human and chicken MBD4 uracil glycosylase domain structure is striking. Here we examined the function of MBD4 in chicken DT40 B cells which undergo constitutive SHM. We constructed structural variants of MBD4 DT40 cells using CRISPR/Cas9 genome editing. Disruption of the MBD4 uracil glycosylase catalytic region increased SHM frequency in IgM loss assays. We propose that MBD4 plays a role in SHM.

Abstract 2566: CYT01B suppresses RAD51 focus formation and induces replication catastrophe following AID induced DNA damage

Abstract Activation Induced Cytidine Deaminase (AID) is a DNA-directed cytidine deaminase that plays a critical role in somatic hypermutation and immunoglobulin class switching in activated B- lymphocytes. AID is often ectopically expressed and promotes genomic hypermutation in a range of lymphoid malignancies. Deamination of genomic cytidines results in point mutations, single strand DNA breaks (SSB), and double strand DNA breaks (DSB). The resulting DNA damage stress leads to an obligate dependency on homologous recombination for cellular survival. RAD51 is a critical component of the homologous recombination pathway, forming nucleoprotein filaments at sites of DNA damage or replication fork stalls. RAD51 mediates homologous DNA strand exchange to promote recombinational repair of breaks and damaged replication forks. We have previously shown that RAD51 depletion leads to accumulation of DNA breaks, and ultimately cell death, in AID expressing cells. We now describe the mechanism of action for a novel small molecule, CYT01B, that targets RAD51 mediated recombination. Treatment of AID-expressing cells with CYT01B, attenuates RAD51 focus formation, reduces the nuclear concentration of RAD51, and promotes RAD51 protein degradation. CYT01B reduces homologous recombination activity in cells, culminating in cytologically visible chromosome fragmentation following DNA damage. Finally, CYT01B treated cells undergo replication catastrophe associated with caspase 3/7 activation and persistent PCNA nuclear signal. We conclude that CYT01B is a novel homologous recombination inhibitor, and propose a new synthetic lethality mechanism, induced by CYT01B, mediated by DNA replication stress and replication catastrophe. Citation Format: Melinda Day, Amber Cyr, Tyler Maclay, Kevin Mills. CYT01B suppresses RAD51 focus formation and induces replication catastrophe following AID induced DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2566.

Abstract 4730: A novel RAD51 inhibitor, CYT01B, shows anti-cancer activity in preclinical models of AID expressing solid tumors

Abstract AID, a DNA-directed cytidine deaminase, plays a critical role in somatic hypermutation and immunoglobulin class switching in maturing B-lymphocytes. Unlike site specific recombinases such as RAG1/2, AID is a promiscuous DNA damaging enzyme that deaminates cytidines at sites throughout the genome. AID expressing cells become critically dependent on the homologous recombination factor RAD51 to repair DNA double strand breaks that result from these off target deamination events. We have developed a novel small molecule, CYT01B, which inhibits RAD51 response to DSBs and is potent to a much greater extent in AID expressing cells. Previously, we have shown this molecule to have activity in mouse lymphoma xenograft models that constitutively express AID. Here we present new preclinical characterization data of CYT01B in solid cancer models. We first analyzed the frequency and levels of AID overexpression in solid tumor data in The Cancer Genome Atlas. Multiple solid tumor types displayed ectopic AID expression, including breast cancer, sarcoma, melanoma, pancreatic cancer, lung cancer, and head and neck cancers with about 30% of the patient samples 4-fold above the baseline expression level. We then tested CYT01B in several solid tumor derived human cell lines. We observed a correlation between AID expression and activity. Cell lines with low ectopic expression of AID had EC50 values in the low micromolar range (~2µM), while those without AID expression gave EC50 values of about 5µM and higher. Next, we examined the activity of our small molecule in 14 different PDX models with various levels of AID expression. These models included renal, head and neck, lung, pancreatic, ovarian, colorectal, and breast cancer samples. We observed a wide range of tumor growth inhibition across the different models (0% to 60%+), which tended to correspond with AID expression; the higher the AID expression the greater the observed tumor growth inhibition. Taken together, these data demonstrate that CYT01B shows anti-cancer activity in a range of solid tumor models. Overall, this provides the basis for continued development of CYT01B as an AID/RAD51 synthetic lethal therapeutic for solid cancers. Citation Format: Melinda Day, Tyler Maclay, Kevin Mills. A novel RAD51 inhibitor, CYT01B, shows anti-cancer activity in preclinical models of AID expressing solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4730.

Evolutionary effects of the AID/APOBEC family of mutagenic enzymes on human gamma-herpesviruses.

The human gamma-herpesviruses, Epstein–Barr virus and Kaposi's sarcoma-associated herpesvirus, establish lifelong latency in B cells and are associated with multiple malignancies. Virus-host coevolution often drive changes in both host immunity and in the viral genome. We consider one host immune mechanism, the activation-induced deaminase (AID)/APOBEC family of cytidine deaminases, that induces mutations in viral DNA. AID, the ancestral gene in the family has a conserved role in somatic hypermutation, a key step in antibody affinity maturation. The APOBEC3 subfamily, of which there are seven genes in human, have evolved antiviral functions and have diversified in terms of their expression pattern, subcellular localization, and DNA mutation motifs (hotspots). In this study, we investigated how the human gamma-herpesviruses have evolved to avoid the action of the AID/APOBEC enzymes and determine if these enzymes are contributing to the ongoing evolution of the viruses. We used computational methods to evaluate observed versus expected frequency of AID/APOBEC hotspots in viral genomes and found that the viruses have evolved to limit the representation of AID and certain APOBEC3 motifs. At the same time, the remaining hotspots were highly likely to cause amino acid changes, suggesting prolonged evolutionary pressure of the enzymes on the viruses. To study current hypermutation, as opposed to historical mutation processes, we also analyzed putative mutations derived from alignments of published viral genomes and found again that AID and APOBEC3 appear to target the genome most frequently. New protein variants resulting from AID/APOBEC activity may have important consequences in health, including vaccine development (epitope evolution) and host immune evasion.

Critical Role for Apobec and Its Interacting Partners in Mediating Mutations and Cell Growth in Multiple Myeloma (MM)

The APOBEC family of cytidine deaminases include AID (activity induced deaminase) and 10 related APOBEC enzymes (A1,A2,A3A,A3B,A3C,A3D,A3F,A3G,A3H and A4). AID is well studied for its role in somatic hyper mutation and class switch recombination of immunoglobulin genes. APOBECs (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) have been shown to have roles in mRNA editing and in antiviral immunity. Recently, a causal role for the AID/APOBECs in inducing somatic mutations in myeloma has been proposed and we have previously published that APOBEC signature mutations as a frequent event in Myeloma. We have also observed that APOBEC-mediated mutations may account for mutations associated with progression of smoldering myeloma to MM.We further investigated the role of APOBEC in genomic changes in MM and observed that APOBEC expression and activity is elevated in myeloma cell lines as well as patient samples. Using knockdown and over expression approaches, we showed that depletion of APOBECs in myeloma cell lines reduces genomic instability. Following APOBEC3G knock down we observed decreased DNA damage (by g-H2AX), decrease in acquisition of new copy number events over time, and reduced mutational load, strongly suggesting that inhibiting APOBECs could be a potential approach to reduce genome evolution in MM.We next investigated the effect of APOBEC inhibition on myeloma cell proliferation. We observed that Sh-RNA-based APOBEC knock down in MM1S and H929 MM cell lines, led to significant inhibition of MM cell proliferation, and induction of apoptotic cell death. Associated with APOBEC knockdown, we also observed increased levels of Cyclin-dependent kinase inhibitor 1B (p27Kip1) at both RNA and protein level. By immunoprecipitation we found that APOBEC3G interacts and inhibits the RNA binding protein DEAD-END 1 (DND1), thereby preventing it from inhibiting miR-221-mediated targeting of p27 transcripts. Knockdown of DND1, or over-expression of miR-221 in APOBEC-depleted cells rescued the cell proliferation defects with concomitant decrease in p27 levels. These results show that APOBCs bind to and sequester DND1, leading to miR-221-mediated depletion of p27. In the absence of APOBEC, DND1 prevents the degradation of p27 mRNA, leading to elevated p27 levels and inhibition of cell cycle, suggesting a role for APOBECs in regulating MM cell proliferation that might be independent of its RNA/DNA mutator function. Taken together, these results indicate a significant functional role for APOBECs both in genome evolution as well as cell growth in myeloma and may constitute an important therapeutic target. DisclosuresMunshi:OncoPep: Other: Board of director.

Abstract 347: Targeting the AICDA/RAD51 axis: A novel gain-of-function synthetic lethal therapy for the treatment of AICDA-expressing cancers

Abstract A key characteristic of many cancers is genomic instability, and is often associated with poor prognosis. While genomic instability can promote tumorigenesis, it also provides a therapeutic opportunity for synthetic lethality. The induction of DNA damage creates stress that necessitates highly active DNA repair as a critical survival system for many transformed cells. Recently, therapeutics that target PARP1 have been approved by the FDA as synthetic lethal therapeutics for cancers with deficiencies in BRCA1/2 and associated pathways. Here we present the early development of a new synthetic lethal therapy that leverages gain-of-function abnormalities to selectively target cancer cells. Activation Induced Cytidine Deaminase (AICDA or AID) is a DNA-directed cytidine deaminase that is normally expressed exclusively and transiently in activated B-lymphocytes, where it plays critical roles in somatic hypermutation and immunoglobulin class switching. AICDA is a DNA damaging enzyme, producing DNA base pair mismatches which can subsequently be converted into mutations, DNA single strand breaks (SSB), or DNA double strand breaks (DSB). Numerous cancers show constitutive overexpression of AICDA, leading to hypermutation, genomic instability, and tumor evolution. We have previously demonstrated that cells expressing AICDA are critically dependent upon the DNA repair factor RAD51. Here we present data illustrating the effectiveness of a novel small molecule, CYT02A, in targeting the AICDA/RAD51 axis. Cell culture assays showed CYT02A to be a potent DNA repair inhibitor that targets RAD51 subcellular localization and filament formation. Although CYT02A was stable when incubated with liver microsomes. Although CYT-02A showed limited oral bioavailability, it was stable in liver microsome assyas assays and the pharmacokinetic profile following intravenous injection revealed dose proportionality and acceptable in vivo exposures. CYT02A was efficacious in a human-to-mouse xenograft model of chronic lymphocytic leukemia (CLL). CYT02A was administered intravenously at a concentration of 50 mg/kg to AID+ CLL xenograft mice daily for up to 9 days. Treated mice showed a significant reduction in CLL burden in the bone marrow compared to vehicle control animals. CYT02A was well tolerated by the animals, with no observable change in behavior or complete blood counts (CBC). Taken together, these data validate a novel gain of function synthetic lethal approach targeting the RAD51/AICDA axis. Preclinical efficacy data support this strategy for the treatment of AICDA-expressing cancers. We continue to build upon this foundation to produce a new therapeutic paradigm that may be effective in a wide range of both hematologic malignancies and solid tumors. Citation Format: Muneer Hasham, Kin-hoe Chow, Tyler Maclay, Amber Cyr, Darryl Patrick, Melinda Day, Kevin D. Mills. Targeting the AICDA/RAD51 axis: A novel gain-of-function synthetic lethal therapy for the treatment of AICDA-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 347.

Regulation of the DNA Repair Complex during Somatic Hypermutation and Class-Switch Recombination.

B lymphocytes optimize Ab responses by somatic hypermutation (SH), which introduces point mutations in the variable regions of the Ab genes and by class-switch recombination (CSR), which changes the expressed C region exon of the IgH. These Ab diversification processes are initiated by the deaminating enzyme activation-induced cytidine deaminase followed by many DNA repair enzymes, ultimately leading to deletions and a high mutation rate in the Ab genes, whereas DNA lesions made by activation-induced cytidine deaminase are repaired with low error rate on most other genes. This indicates an advanced regulation of DNA repair. In this study, we show that initiation of Ab diversification in B lymphocytes of mouse spleen leads to formation of a complex between many proteins in DNA repair. We show also that BCR activation, which signals the end of successful SH, reduces interactions between some proteins in the complex and increases other interactions in the complex with varying kinetics. Furthermore, we show increased localization of SH- and CSR-coupled proteins on switch regions of the Igh locus upon initiation of SH/CSR and differential changes in the localization upon BCR signaling, which terminates SH. These findings provide early evidence for a DNA repair complex or complexes that may be of functional significance for carrying out essential roles in SH and/or CSR in B cells.

POLD3 Plays an Essential Role in Somatic Hypermutation

Abstract Somatic hypermutation (SHM) increases the affinity of antibodies to antigen. While the initial steps of SHM, including development of the lesion, its excision and formation of an abasic site, are well established, the repair and mutation events are still under investigation. Multiple DNA repair pathways have been suggested for the SHM. POLD3, as a part of a scaffold complex, connects error free or error prone DNA polymerases with the processivity factor PCNA and this interaction plays an essential role in mutagenesis and recombination. In this study we tested the role of POLD3 in SHM. We demonstrated elevated expression of POLD3 in activated mice B cells and accumulation of this protein at DNA damaged sites. We created a Burkett’s lymphoma Raji cell clone (Raji 2C9 clone) expressing endogenous mutant POLD3 lacking C-terminus PCNA interaction site, using the CRISPR system. Functional studies shown that the Raji cells expressing mutant variant demonstrated abnormal cell cycle distribution, hyper-sensitivity to genotoxic agents and compromised SHM, compare to the control cells. The frequency of mutations in locus encoding immunoglobulin heavy chain variable region was are five times lower in cells expressing mutant POLD3 compare to control cells. In control cells, mutations accumulated at the RGYW/WRCY “hot spots”, and majority of mutations were C to T or G to A transitions. In cells expressing mutant POLD3 mutations accumulated outside “hot spots” and are represented by transversions. The overall results indicate that POLD3 plays an important role in SHM.

Investigation of AID, Dicer, and Drosha Expressions in Patients with Chronic Lymphocytic Leukemia

ABSTRACTChronic lymphocytic leukemia (CLL) is the most common leukemia in Western countries. Cytogenetic lesions such as del13q14, del11q22.3, and del17p13 are identified in 50–60% of patients. Activation-induced cytidine deaminase (AID) plays a central role in somatic hyper mutation (SHM) and class switch recombination (CSR) and functions on Ig genes, but also target non-Ig genes, and over-expression of AID can lead to point mutations or translocations in non-Ig genes such as IgH/Myc translocation. Dicer and Drosha, which have a role in activation process of miRNA, also act in a double-strand DNA break (DSB) repair mechanism. In this study, whether the changes of AID, Dicer and Drosha expressions may be associated with both deletions and clinical outcomes in patients with CLL were investigated. AID expressions were increased in patients with CLL. However, cell lysate AID protein levels were only increased in patients with del17p or del11q who have poor prognosis. Decreased Dicer expressions were found in patients with deletion, whereas increased Drosha expressions were found in patients without deletion and with del13q. According to Rai and Binet staging systems, advanced-stage patients showed increased AID protein levels, decreased Dicer and Drosha expressions. Our findings may suggest that high AID expression and lower Dicer expression were observed in patients with CLL especially del17p and del11q and might associated with deletions such as del17p and del11q. AID, Dicer, and Drosha expressions might be used as an indicator of prognosis for CLL.

Somatic hypermutation of immunoglobulin genes in Rad18 knockout mice

Somatic hypermutation (SHM) of immunoglobulin (Ig) genes is triggered by the activity of activation-induced cytidine deaminase (AID). AID induces DNA lesions in variable regions of Ig genes, and error-prone DNA repair mechanisms initiated in response to these lesions introduce the mutations that characterize SHM. Error-prone DNA repair in SHM is proposed to be mediated by low-fidelity DNA polymerases such as those that mediate trans-lesion synthesis (TLS); however, the mechanism by which these enzymes are recruited to AID-induced lesions remains unclear. Proliferating cell nuclear antigen (PCNA), the sliding clamp for multiple DNA polymerases, undergoes Rad6/Rad18-dependent ubiquitination in response to DNA damage. Ubiquitinated PCNA promotes the replacement of the replicative DNA polymerase stalled at the site of a DNA lesion with a TLS polymerase. To examine the potential role of Rad18-dependent PCNA ubiquitination in SHM, we analyzed Ig gene mutations in Rad18 knockout (KO) mice immunized with T cell-dependent antigens. We found that SHM in Rad18 KO mice was similar to wild-type mice, suggesting that Rad18 is dispensable for SHM. However, residual levels of ubiquitinated PCNA were observed in Rad18 KO cells, indicating that Rad18-independent PCNA ubiquitination might play a role in SHM.

Dysregulated Aid/Apobec Family Proteins Promote Genomic Instability in Multiple Myeloma

The AID/APOBEC family of cytidine deaminase proteins includes AID (activity induced deaminase), and 10 related APOBEC enzymes (A1, A2, A3A, A3B, A3C, A3D, A3F, A3G, A3H and A4). AID has been well-studied for its role in somatic hyper mutation and class switch recombination of immunoglobulin genes whereas APOBECs (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) have been shown to have roles in mRNA editing and in antiviral immunity. Dysregulated activity of APOBECs causes C >T transitions or C>G, C>A transversions in DNA. We have recently shown APOBEC signature mutation pattern in multiple myeloma (MM) genomes (Bolli et al Nat. Comm. 2014), and interestingly, the APOBEC mutation signature correlates with sub clonal diversity in myeloma. A role for the AID/APOBECs in generation of somatic mutations has also been proposed in a variety of other cancers based on identification of APOBEC signature mutationsIn order to understand which APOBECs are dysregulated in myeloma, we performed RNA sequencing analysis of primary myeloma cells from 409 newly-diagnosed MM patients and myeloma cell lines. Our analysis showed elevated expression of several APOBEC family members; mainly A3A, A3B, A3C, and A3G. We then optimized a plasmid-based functional assay and found high cytidine deaminase activity in extracts from a number of myeloma cell lines and patient derived CD138+ cells compared to CD138+ cells from healthy donors, suggesting that APOBECs are dysregulated in myeloma. We then investigated the impact of elevated APOBEC expression/function on overall genome maintenance and acquisition of genomic changes (such as amplifications, deletions) overtime. We used shRNA-mediated knockdown of specific APOBEC proteins in myeloma cell lines and investigated the acquisition of genomic changes in control and knockdown cells during their growth in culture, using SNP (Single Nucleotide Polymorphism) arrays and WGS (whole genome sequencing) platforms. Our results with both approaches showed significant reduction in the accumulation of copy number changes (both amplifications and deletions) and overall mutation load after APOBEC knockdown. Evaluation with both the SNP and WGS showed that when control and APOBEC knockdown cells were cultured for three weeks, the acquisition of new copy number and mutational changes throughout genome were reduced by ~50%.We next investigated the relationship between APOBEC expression/activity in MM and other DNA repair pathways. Using an in vitro HR activity assay, we measured HR activity in extracts from control and APOBEC knockdown cells. Depletion of APOBEC proteins resulted in 50-80% reduction in in vitro HR activity of the extracts. We also evaluated correlation between HR activity and gene expression using RNA-seq data from myeloma cells derived from 100 patients at diagnosis and identified the genes whose expression correlated with HR activity. Elevated expression of APOBECs 3D, 3G and 3F significantly correlated with high HR activity (R=0.3; P≤0.02), suggesting their relevance to HR. Analyzing genomic copy number information for each patient we have also observed significant correlation between higher expression of A3G and increased genomic instability in this dataset (P=0.0045).In summary, our study shows that dysregulated APOBECs induce mutations and genomic instability, and inhibiting APOBEC activity could reduce the rate of accumulation of ongoing genomic changes. This data sheds light on biology of the disease as well as clonal evolution. DisclosuresMunshi:Amgen: Consultancy; Oncopep: Patents & Royalties; Celgene: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Merck: Consultancy; Pfizer: Consultancy.

Aberrant activation-induced cytidine deaminase expression in Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia

Activation-induced cytidine deaminase (AID) is expressed in germinal center B cells and plays a critical role in somatic hypermutation and class-switch recombination of immunoglobulin genes. Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) carries a poor prognosis and is specifically treated with tyrosine kinase inhibitors. Interestingly, AID has been shown to be aberrantly expressed and functional in Ph+ ALL and is thought to contribute to genetic instability. We hypothesized that AID might be detectable in routinely processed bone marrow biopsies by immunohistochemistry (IHC) and assist in identifying Ph+ ALL. We found that AID was expressed in 26 (70%) of 37 cases of Ph+ ALL but only 1 (2.9%) of 38 cases of Ph- ALL cases. There was a significant difference in AID expression between these 2 ALL groups (P < .001, Fisher exact test). The expression of AID was confirmed by RT-PCR (reverse-transcriptase polymerase chain reaction) and correlated with IHC scoring. AID protein is expressed in a large proportion of Ph+ ALL cases at levels detectable by IHC in clinical samples and might be useful to rapidly identify cases likely to have a BCR/ABL1 fusion.

Opinion: uracil DNA glycosylase (UNG) plays distinct and non-canonical roles in somatic hypermutation and class switch recombination.

Activation-induced cytidine deaminase (AID) is essential to class switch recombination (CSR) and somatic hypermutation (SHM). Uracil DNA glycosylase (UNG), a member of the base excision repair complex, is required for CSR. The role of UNG in CSR and SHM is extremely controversial. AID deficiency in mice abolishes both CSR and SHM, while UNG-deficient mice have drastically reduced CSR but augmented SHM raising a possibility of differential functions of UNG in CSR and SHM. Interestingly, UNG has been associated with a CSR-specific repair adapter protein Brd4, which interacts with acetyl histone 4, γH2AX and 53BP1 to promote non-homologous end joining during CSR. A non-canonical scaffold function of UNG, but not the catalytic activity, can be attributed to the recruitment of essential repair proteins associated with the error-free repair during SHM, and the end joining during CSR.

DNA polymerases β and λ do not directly affect Ig variable region somatic hypermutation although their absence reduces the frequency of mutations

During somatic hypermutation (SHM) of antibody variable (V) region genes, activation-induced cytidine deaminase (AID) converts dC to dU, and dUs can either be excised by uracil DNA glycosylase (UNG), by mismatch repair, or replicated over. If UNG excises the dU, the abasic site could be cleaved by AP-endonuclease (APE), introducing the single-strand DNA breaks (SSBs) required for generating mutations at A:T bp, which are known to depend upon mismatch repair and DNA Pol η. DNA Pol β or λ could instead repair the lesion correctly. To assess the involvement of Pols β and λ in SHM of antibody genes, we analyzed mutations in the VDJh4 3' flanking region in Peyer's patch germinal center (GC) B cells from polβ(-/-)polλ(-/-), polλ(-/-), and polβ(-/-) mice. We find that deficiency of either or both polymerases results in a modest but significant decrease in V region SHM, with Pol β having a greater effect, but there is no effect on mutation specificity, suggesting they have no direct role in SHM. Instead, the effect on SHM appears to be due to a role for these enzymes in GC B cell proliferation or viability. The results suggest that the BER pathway is not important during V region SHM for generating mutations at A:T bp. Furthermore, this implies that most of the SSBs required for Pol η to enter and create A:T mutations are likely generated during replication instead. These results contrast with the inhibitory effect of Pol β on mutations at the Ig Sμ locus, Sμ DSBs and class switch recombination (CSR) reported previously. We show here that B cells deficient in Pol λ or both Pol β and λ proliferate normally in culture and undergo slightly elevated CSR, as shown previously for Pol β-deficient B cells.

The translesion DNA synthesis polymerase Rev1 recruits uracil DNA glycosylase to switch regions and enhances dU glycosylation for immunoglobulin class switch DNA recombination (P1478)

Abstract Immunoglobulin (Ig) class switch DNA recombination (CSR) plays a critical role in the maturation of the immune response by diversifying the biological effector functions of antibodies. CSR is initiated by activation-induced cytidine deaminase (AID)-mediated deoxycytosine (dC) deamination, yielding deoxyuridine (dU), and dU glycosylation by uracil DNA glycosylase (Ung) in Ig switch (S) region DNA. Translesion DNA synthesis (TLS) polymerases, such as Rev1, play important roles in somatic hypermutation (SHM), but their role in CSR has not been defined. We found that Rev1 directly interacts with Ung to recruit it to S region DNA and enhance dU glycosylation for CSR. Rev1 targets S regions undergoing CSR in an AID-dependent and Ung-independent fashion. Ung recruitment to S regions, DNA-dU glycosylation and CSR are reduced in Rev1-/- B cells. Together with a mutation spectrum in S region DNA that is similar to that of Ung-/- B cells, our findings suggest that Rev1 operates in the same pathway as Ung during CSR, as emphasized by further decreased CSR in Rev1-/- Msh2-/- B cells. Rescue of CSR by a catalytically inactive Rev1 mutant in Rev1-/- B cells shows that the role of Rev1 in CSR is mediated by Rev1’s scaffolding function, not its enzymatic function. Thus, we describe a novel and important scaffolding function for Rev1 in CSR, which together with its known enzymatic function in SHM highlights a central role of this TLS polymerase in the maturation of the antibody response.

Diversity Against Adversity: How Adaptive Immune System Evolves Potent Antibodies

Adaptive immunity is an amazing mechanism, whereby new protein functions—affinity of antibodies (Immunoglobulins) to new antigens—evolve through mutation and selection in a matter of a few days. Despite numerous experimental studies, the fundamental physical principles underlying immune response are still poorly understood. In considerable departure from past approaches, here, we propose a microscopic multiscale model of adaptive immune response, which consists of three essential players: The host cells, viruses, and B-cells in Germinal Centers (GC). Each moiety carries a genome, which encodes proteins whose stability and interactions are determined from their sequences using laws of Statistical Mechanics, providing an exact relationship between genomic sequences and strength of interactions between pathogens and antibodies and antibodies and host proteins (autoimmunity). We find that evolution of potent antibodies (the process known as Affinity Maturation (AM)) is a delicate balancing act, which has to reconcile the conflicting requirements of protein stability, lack of autoimmunity, and high affinity of antibodies to incoming antigens. This becomes possible only when antibody producing B cells elevate their mutation rates (process known as Somatic Hypermutation (SHM)) to fall into a certain range—not too low to find potency increasing mutations but not too high to destroy stable Immunoglobulins and/or already achieved affinity. Potent antibodies develop through clonal expansion of initial B cells expressing marginally potent antibodies followed by their subsequent affinity maturation through mutation and selection. As a result, in each GC the population of mature potent Immunoglobulins is monoclonal being ancestors of a single cell from initial (germline) pool. We developed a simple analytical theory, which provides further rationale to our findings. The model and theory reveal the molecular factors that determine the efficiency of affinity maturation, thereby providing insight into the variability of the immune response to cytopathic viruses (the direct response by germline antibodies) and poorly cytopathic viruses (a crucial role of SHM in the response).

Apex2 is required for efficient somatic hypermutation but not for class switch recombination of immunoglobulin genes

The DNA cleavage step in both the class switch recombination (CSR) and somatic hypermutation (SHM) of Ig genes is initiated by activation-induced cytidine deaminase (AID). However, the detailed mechanisms of the DNA strand cleavage in SHM and CSR are still largely unknown. Recently, the apurinic/apyrimidinic endonucleases, Apex1 and Apex2, were reported to be involved in the DNA cleavage step of CSR. Here, we examined the role of Apex2 in SHM using Apex2-deficient mice and found that the Apex2 deficiency caused a drastic reduction in the frequency of SHM and the number of mutations per mutated clone without affecting the pattern of base substitution. These results suggest that Apex2 may play a critical role in SHM through its 3'-5' exonuclease activity. Unexpectedly, the efficiency of CSR was not reduced in Apex2-deficient B cells. In addition, Apex1 knockdown in CH12F3-2 B lymphoma cells did not affect the CSR frequency, suggesting that neither Apex1 nor Apex2 plays a major role in CSR.

Interference of mismatch and base excision repair during the processing of adjacent U/G mispairs may play a key role in somatic hypermutation

In eukaryotic mismatch repair (MMR), degradation of the error-containing strand initiates at nicks or gaps that can be up to a kilobase away from the mispair. These discontinuities may be the ends of Okazaki fragments or the 3'-termini of the leading strands during replication, whereas the termini of invading strands may fulfill this role during recombination. Here we show that, in extracts of human cells, MMR can initiate also at sites of ongoing base excision repair. Although unlikely under normal circumstances, this situation may arise in vivo during somatic hypermutation (SHM) and class switch recombination of Ig genes, where activation-induced cytidine deaminase (AID) generates multiple U/G mismatches in the variable or switch regions. Uracil should normally be excised by base excision repair (BER), but we show here that MMR proteins activated by a nearby mismatch interfere with uracil processing to generate long single-stranded gaps. We postulate that, in a subset of the repair events, filling-in of the MMR-generated gaps might be catalyzed by the error-prone polymerase-eta, rather than by the high-fidelity polymerase-delta. Because polymerase-eta has a propensity to misinsertions opposite adenine residues, the above mechanism would help explain why SHM affects not only C/G, but also A/T base pairs.