SHLD1 is dispensable for 53BP1-dependent V(D)J recombination but critical for productive class switch recombination.

SHLD1 is part of the Shieldin (SHLD) complex, which acts downstream of 53BP1 to counteract DNA double-strand break (DSB) end resection and promote DNA repair via non-homologous end-joining (NHEJ). While 53BP1 is essential for immunoglobulin heavy chain class switch recombination (CSR), long-range V(D)J recombination and repair of RAG-induced DSBs in XLF-deficient cells, the function of SHLD during these processes remains elusive. Here we report that SHLD1 is dispensable for lymphocyte development and RAG-mediated V(D)J recombination, even in the absence of XLF. By contrast, SHLD1 is essential for restricting resection at AID-induced DSB ends in both NHEJ-proficient and NHEJ-deficient B cells, providing an end-protection mechanism that permits productive CSR by NHEJ and alternative end-joining. Finally, we show that this SHLD1 function is required for orientation-specific joining of AID-initiated DSBs. Our data thus suggest that 53BP1 promotes V(D)J recombination and CSR through two distinct mechanisms: SHLD-independent synapsis of V(D)J segments and switch regions within chromatin, and SHLD-dependent protection of AID-DSB ends against resection.

The Sec61 translocon is a therapeutic vulnerability in multiple myeloma.

Multiple myeloma (MM) is an incurable malignancy characterized by the uncontrolled expansion of plasma cells in the bone marrow. While proteasome inhibitors like bortezomib efficiently halt MM progression, drug resistance inevitably develop, and novel therapeutic approaches are needed. Here, we used a recently discovered Sec61 inhibitor, mycolactone, to assess the interest of disrupting MM proteostasis via protein translocation blockade. In human MM cell lines, mycolactone caused rapid defects in secretion of immunoglobulins and expression of pro-survival interleukin (IL)-6 receptor and CD40, whose activation stimulates IL-6 production. Mycolactone also triggered pro-apoptotic endoplasmic reticulum stress responses synergizing with bortezomib for induction of MM cell death and overriding acquired resistance to the proteasome inhibitor. Notably, the mycolactone-bortezomib combination rapidly killed patient-derived MM cells ex vivo, but not normal mononuclear cells. In immunodeficient mice engrafted with MM cells, it demonstrated superior therapeutic efficacy over single drug treatments, without inducing toxic side effects. Collectively, these findings establish Sec61 blockers as novel anti-MM agents and reveal the interest of targeting both the translocon and the proteasome in proteostasis-addicted tumors.

Fam72a enforces error-prone DNA repair during antibody diversification.

Efficient humoral responses rely on DNA damage, mutagenesis and error-prone DNA repair. Diversification of B cell receptors through somatic hypermutation and class-switch recombination are initiated by cytidine deamination in DNA mediated by activation-induced cytidine deaminase (AID)1 and by the subsequent excision of the resulting uracils by uracil DNA glycosylase (UNG) and by mismatch repair proteins1-3. Although uracils arising in DNA are accurately repaired1-4, how these pathways are co-opted to generate mutations and double-strand DNA breaks in the context of somatic hypermutation and class-switch recombination is unknown1-3. Here we performed a genome-wide CRISPR-Cas9 knockout screen for genes involved in class-switch recombination and identified FAM72A, a protein that interacts with the nuclear isoform of UNG (UNG2)5 and is overexpressed in several cancers5. We show that the FAM72A-UNG2 interaction controls the levels of UNG2 and that class-switch recombination is defective in Fam72a-/- B cells due to the upregulation of UNG2. Moreover, we show that somatic hypermutation is reduced in Fam72a-/- B cells and that its pattern is skewed upon upregulation of UNG2. Our results are consistent with a model in which FAM72A interacts with UNG2 to control its physiological level by triggering its degradation, regulating the level of uracil excision and thus the balance between error-prone and error-free DNA repair. Our findings have potential implications for tumorigenesis, as reduced levels of UNG2 mediated by overexpression of Fam72a would shift the balance towards mutagenic DNA repair, rendering cells more prone to acquire mutations.

MAD2L2 dimerization and TRIP13 control shieldin activity in DNA repair.

MAD2L2 (REV7) plays an important role in DNA double-strand break repair. As a member of the shieldin complex, consisting of MAD2L2, SHLD1, SHLD2 and SHLD3, it controls DNA repair pathway choice by counteracting DNA end-resection. Here we investigated the requirements for shieldin complex assembly and activity. Besides a dimerization-surface, HORMA-domain protein MAD2L2 has the extraordinary ability to wrap its C-terminus around SHLD3, likely creating a very stable complex. We show that appropriate function of MAD2L2 within shieldin requires its dimerization, mediated by SHLD2 and accelerating MAD2L2-SHLD3 interaction. Dimerization-defective MAD2L2 impairs shieldin assembly and fails to promote NHEJ. Moreover, MAD2L2 dimerization, along with the presence of SHLD3, allows shieldin to interact with the TRIP13 ATPase, known to drive topological switches in HORMA-domain proteins. We find that appropriate levels of TRIP13 are important for proper shieldin (dis)assembly and activity in DNA repair. Together our data provide important insights in the dependencies for shieldin activity.

The (Lack of) DNA Double-Strand Break Repair Pathway Choice During V(D)J Recombination.

DNA double-strand breaks (DSBs) are highly toxic lesions that can be mended via several DNA repair pathways. Multiple factors can influence the choice and the restrictiveness of repair towards a given pathway in order to warrant the maintenance of genome integrity. During V(D)J recombination, RAG-induced DSBs are (almost) exclusively repaired by the non-homologous end-joining (NHEJ) pathway for the benefit of antigen receptor gene diversity. Here, we review the various parameters that constrain repair of RAG-generated DSBs to NHEJ, including the peculiarity of DNA DSB ends generated by the RAG nuclease, the establishment and maintenance of a post-cleavage synaptic complex, and the protection of DNA ends against resection and (micro)homology-directed repair. In this physiological context, we highlight that certain DSBs have limited DNA repair pathway choice options.

Repair of G1 induced DNA double-strand breaks in S-G2/M by alternative NHEJ.

The alternative non-homologous end-joining (NHEJ) pathway promotes DNA double-strand break (DSB) repair in cells deficient for NHEJ or homologous recombination, suggesting that it operates at all stages of the cell cycle. Here, we use an approach in which DNA breaks can be induced in G1 cells and their repair tracked, enabling us to show that joining of DSBs is not functional in G1-arrested XRCC4-deficient cells. Cell cycle entry into S-G2/M restores DSB repair by Pol θ-dependent and PARP1-independent alternative NHEJ with repair products bearing kilo-base long DNA end resection, micro-homologies and chromosome translocations. We identify a synthetic lethal interaction between XRCC4 and Pol θ under conditions of G1 DSBs, associated with accumulation of unresolved DNA ends in S-G2/M. Collectively, our results support the conclusion that the repair of G1 DSBs progressing to S-G2/M by alternative NHEJ drives genomic instability and represent an attractive target for future DNA repair-based cancer therapies.

Breakage-Fusion-Bridge Events Trigger Complex Genome Rearrangements and Amplifications in Developmentally Arrested T Cell Lymphomas.

To reveal the relative contribution of the recombination activating gene (RAG)1/2 nuclease to lymphomagenesis, we conducted a genome-wide analysis of T cell lymphomas from p53-deficient mice expressing or lacking RAG2. We found that while p53-/- lymphoblastic T cells harbor primarily ectopic DNA deletions, Rag2-/-p53-/- T cell lymphomas display complex genomic rearrangements associated with amplification of the chromosomal location 9qA4-5.3. We show that this amplicon is generated by breakage-fusion-bridge during mitosis and arises distinctly in T cell lymphomas originating from an early progenitor stage. Notably, we report amplification of the corresponding syntenic region (11q23) in a subset of human leukemia leading to the overexpression of several cancer genes, including MLL/KMT2A. Our findings provide direct evidence that lymphocytes undergo malignant transformation through distinct genome architectural routes that are determined by both RAG-dependent and RAG-independent DNA damage and a block in cell development.

Coordinated signals from the DNA repair enzymes PARP-1 and PARP-2 promotes B-cell development and function.

Poly (ADP-ribose) polymerase (PARP)-1 and PARP-2 regulate the function of various DNA-interacting proteins by transferring ADP-ribose emerging from catalytic cleavage of cellular ß-NAD+. Hence, mice lacking PARP-1 or PARP-2 show DNA perturbations ranging from altered DNA integrity to impaired DNA repair. These effects stem from the central role that PARP-1 and PARP-2 have on the cellular response to DNA damage. Failure to mount a proper response culminates in cell death. Accordingly, PARP inhibitors are emerging as promising drugs in cancer therapy. However, the full impact of these inhibitors on immunity, including B-cell antibody production, remains elusive. Given that mice carrying dual PARP-1 and PARP-2 deficiency develop early embryonic lethality, we crossed PARP-1-deficient mice with mice carrying a B-cell-conditional PARP-2 gene deletion. We found that the resulting dually PARP-1 and PARP-2-deficient mice had perturbed bone-marrow B-cell development as well as profound peripheral depletion of transitional and follicular but not marginal zone B-cells. Of note, bone-marrow B-cell progenitors and peripheral mature B-cells were conserved in mice carrying either PARP-1 or PARP-2 deficiency. In dually PARP-1 and PARP-2-deficient mice, B-cell lymphopenia was associated with increased DNA damage and accentuated death in actively proliferating B-cells. Moreover, dual PARP-1 and PARP-2 deficiency impaired antibody responses to T-independent carbohydrate but not to T-dependent protein antigens. Notwithstanding the pivotal role of PARP-1 and PARP-2 in DNA repair, combined PARP-1 and PARP-2 deficiency did not perturb the DNA-editing processes required for the generation of a protective antibody repertoire, including Ig V(D)J gene recombination and IgM-to-IgG class switching. These findings provide key information as to the potential impact of PARP inhibitors on humoral immunity, which will facilitate the development of safer PARP-targeting regimens against cancer.

The immune system profoundly restricts intratumor genetic heterogeneity.

Tumors develop under the selective pressure of the immune system. However, it remains critical to establish how the immune system affects the clonal heterogeneity of tumors that often display cell-to-cell variation in genetic alterations and antigenic expression. To address these questions, we introduced a multicolor barcoding strategy to study the growth of a MYC-driven B cell lymphoma harboring a large degree of intratumor genetic diversity. Using intravital imaging, we visualized that lymphoma subclones grow as patches of sessile cells in the bone marrow, creating a spatially compartmentalized architecture for tumor diversity. Using multicolor barcoding and whole-exome sequencing, we demonstrated that immune responses strongly restrict intratumor genomic diversity and favor clonal dominance, a process mediated by the selective elimination of more immunogenic cells and amplified by epitope spreading. Anti-PD-1 treatment also narrowed intratumor diversity. Our results provide direct evidence that immune pressure shapes the level of intratumor genetic heterogeneity and have important implications for the design of therapeutic strategies.

Chromosomal Translocation Formation Is Sufficient to Produce Fusion Circular RNAs Specific to Patient Tumor Cells.

Circular RNAs constitute a unique class of RNAs whose precise functions remain to be elucidated. In particular, cancer-associated chromosomal translocations can give rise to fusion circular RNAs that play a role in leukemia progression. However, how and when fusion circular RNAs are formed and whether they are being selected in cancer cells remains unknown. Here, we used CRISPR/Cas9 to generate physiological translocation models of NPM1-ALK fusion gene. We showed that, in addition to generating fusion proteins and activating specific oncogenic pathways, chromosomal translocation induced by CRISPR/Cas9 led to the formation of de novo fusion circular RNAs. Specifically, we could recover different classes of circular RNAs composed of different circularization junctions, mainly back-spliced species. In addition, we identified fusion circular RNAs identical to those found in related patient tumor cells providing evidence that fusion circular RNAs arise early after chromosomal formation and are not just a consequence of the oncogenesis process.

Shieldin complex promotes DNA end-joining and counters homologous recombination in BRCA1-null cells.

BRCA1 deficiencies cause breast, ovarian, prostate and other cancers, and render tumours hypersensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. To understand the resistance mechanisms, we conducted whole-genome CRISPR-Cas9 synthetic-viability/resistance screens in BRCA1-deficient breast cancer cells treated with PARP inhibitors. We identified two previously uncharacterized proteins, C20orf196 and FAM35A, whose inactivation confers strong PARP-inhibitor resistance. Mechanistically, we show that C20orf196 and FAM35A form a complex, 'Shieldin' (SHLD1/2), with FAM35A interacting with single-stranded DNA through its C-terminal oligonucleotide/oligosaccharide-binding fold region. We establish that Shieldin acts as the downstream effector of 53BP1/RIF1/MAD2L2 to promote DNA double-strand break (DSB) end-joining by restricting DSB resection and to counteract homologous recombination by antagonizing BRCA2/RAD51 loading in BRCA1-deficient cells. Notably, Shieldin inactivation further sensitizes BRCA1-deficient cells to cisplatin, suggesting how defining the SHLD1/2 status of BRCA1-deficient tumours might aid patient stratification and yield new treatment opportunities. Highlighting this potential, we document reduced SHLD1/2 expression in human breast cancers displaying intrinsic or acquired PARP-inhibitor resistance.

Generation and CRISPR/Cas9 editing of transformed progenitor B cells as a pseudo-physiological system to study DNA repair gene function in V(D)J recombination.

Antigen receptor gene assembly is accomplished in developing lymphocytes by the V(D)J recombination reaction, which can be separated into two steps: DNA cleavage by the recombination-activating gene (RAG) nuclease and joining of DNA double strand breaks (DSBs) by components of the nonhomologous end joining (NHEJ) pathway. Deficiencies for NHEJ factors can result in immunodeficiency and a propensity to accumulate genomic instability, thus highlighting the importance of identifying all players in this process and deciphering their functions. Bcl2 transgenic v-Abl kinase-transformed pro-B cells provide a pseudo-physiological cellular system to study V(D)J recombination. Treatment of v-Abl/Bcl2 pro-B cells with the Abl kinase inhibitor Imatinib leads to G1 cell cycle arrest, the rapid induction of Rag1/2 gene expression and V(D)J recombination. In this system, the Bcl2 transgene alleviates Imatinib-induced apoptosis enabling the analysis of induced V(D)J recombination. Although powerful, the use of mouse models carrying the Bcl2 transgene for the generation of v-Abl pro-B cell lines is time and money consuming. Here, we describe a method for generating v-Abl/Bcl2 pro-B cell lines from wild type mice and for performing gene knock-out using episomal CRISPR/Cas9 targeting vectors. Using this approach, we generated distinct NHEJ-deficient pro-B cell lines and quantified V(D)J recombination levels in these cells. Furthermore, this methodology can be adapted to generate pro-B cell lines deficient for any gene suspected to play a role in V(D)J recombination, and more generally DSB repair.

The RAG recombinase: Beyond breaking.

DNA double-strand breaks (DSBs) are commonly seen as lesions that threaten genome integrity and contribute to cancer and aging processes. However, in the context of antigen receptor gene assembly, known as V(D)J recombination, DSBs are obligatory intermediates that allow the establishment of genetic diversity and adaptive immunity. V(D)J recombination is initiated when the lymphoid-restricted recombination-activating genes RAG1 and RAG2 are expressed and form a site-specific endonuclease (the RAG nuclease or RAG recombinase). Here, we discuss the ability of the RAG nuclease to minimize the risks of genome disruption by coupling the breakage and repair steps of the V(D)J reaction. This implies that the RAG genes, derived from an ancient transposon, have undergone strong selective pressure to prohibit transposition in favor of promoting controlled DNA end joining in cis by the ubiquitous DNA damage response and DNA repair machineries. We also discuss the idea that, in addition to being essential for the rearrangement of antigen receptor genes, RAG-mediated DSBs could impact cellular processes and outcomes by affecting genetic and epigenetic programs.

Synthetic lethality between PAXX and XLF in mammalian development.

PAXX was identified recently as a novel nonhomologous end-joining DNA repair factor in human cells. To characterize its physiological roles, we generated Paxx-deficient mice. Like Xlf-/- mice, Paxx-/- mice are viable, grow normally, and are fertile but show mild radiosensitivity. Strikingly, while Paxx loss is epistatic with Ku80, Lig4, and Atm deficiency, Paxx/Xlf double-knockout mice display embryonic lethality associated with genomic instability, cell death in the central nervous system, and an almost complete block in lymphogenesis, phenotypes that closely resemble those of Xrcc4-/- and Lig4-/- mice. Thus, combined loss of Paxx and Xlf is synthetic-lethal in mammals.

Specific Roles of XRCC4 Paralogs PAXX and XLF during V(D)J Recombination.

Paralog of XRCC4 and XLF (PAXX) is a member of the XRCC4 superfamily and plays a role in nonhomologous end-joining (NHEJ), a DNA repair pathway critical for lymphocyte antigen receptor gene assembly. Here, we find that the functions of PAXX and XLF in V(D)J recombination are masked by redundant joining activities. Thus, combined PAXX and XLF deficiency leads to an inability to join RAG-cleaved DNA ends. Additionally, we demonstrate that PAXX function in V(D)J recombination depends on its interaction with Ku. Importantly, we show that, unlike XLF, the role of PAXX during the repair of DNA breaks does not overlap with ATM and the RAG complex. Our findings illuminate the role of PAXX in V(D)J recombination and support a model in which PAXX and XLF function during NHEJ repair of DNA breaks, whereas XLF, the RAG complex, and the ATM-dependent DNA damage response promote end joining by stabilizing DNA ends.

RAG2 and XLF/Cernunnos interplay reveals a novel role for the RAG complex in DNA repair.

XRCC4-like factor (XLF) functions in classical non-homologous end-joining (cNHEJ) but is dispensable for the repair of DNA double-strand breaks (DSBs) generated during V(D)J recombination. A long-standing hypothesis proposes that, in addition to its canonical nuclease activity, the RAG1/2 proteins participate in the DNA repair phase of V(D)J recombination. Here we show that in the context of RAG2 lacking the C-terminus domain (Rag2(c/c) mice), XLF deficiency leads to a profound lymphopenia associated with a severe defect in V(D)J recombination and, in the absence of p53, increased genomic instability at V(D)J sites. In addition, Rag2(c/c) XLF(-/-) p53(-/-) mice develop aggressive pro-B cell lymphomas bearing complex chromosomal translocations and gene amplifications involving Igh and c-myc/pvt1 loci. Our results reveal an unanticipated functional interplay between the RAG complex and XLF in repairing RAG-induced DSBs and maintaining genome integrity during antigen receptor gene assembly.

Listeria monocytogenes dampens the DNA damage response.

The DNA damage response (DDR) is an essential signaling pathway that detects DNA lesions, which constantly occur upon either endogenous or exogenous assaults, and maintains genetic integrity. An infection by an invading pathogen is one such assault, but how bacteria impact the cellular DDR is poorly documented. Here, we report that infection with Listeria monocytogenes induces host DNA breaks. Strikingly, the signature response to these breaks is only moderately activated. We uncover the role of the listerial toxin listeriolysin O (LLO) in blocking the signaling response to DNA breaks through degradation of the sensor Mre11. Knocking out or inactivating proteins involved in the DDR promotes bacterial replication showing the importance of this mechanism for the control of infection. Together, our data highlight that bacterial dampening of the DDR is critical for a successful listerial infection.

RAG2 mutants alter DSB repair pathway choice in vivo and illuminate the nature of 'alternative NHEJ'.

DNA double-stranded breaks (DSBs) can be repaired by several mechanisms, including classical NHEJ (c-NHEJ) and a poorly defined, error-prone process termed alternative NHEJ (a-NHEJ). How cells choose between these alternatives to join physiologic DSBs remains unknown. Here, we show that deletion of RAG2's C-terminus allows a-NHEJ to repair RAG-mediated DSBs in developing lymphocytes from both c-NHEJ-proficient and c-NHEJ-deficient mice, demonstrating that the V(D)J recombinase influences repair pathway choice in vivo. Analysis of V(D)J junctions revealed that, contrary to expectation, junctional characteristics alone do not reliably distinguish between a-NHEJ and c-NHEJ. These data suggest that a-NHEJ is not necessarily mutagenic, and may be more prevalent than previously appreciated. Whole genome sequencing of a lymphoma arising in a p53(-/-) mouse bearing a C-terminal RAG2 truncation reveals evidence of a-NHEJ and also of aberrant recognition of DNA sequences resembling RAG recognition sites.

Modernizing the nonhomologous end-joining repertoire: alternative and classical NHEJ share the stage.

DNA double-strand breaks (DSBs) are common lesions that continually threaten genomic integrity. Failure to repair a DSB has deleterious consequences, including cell death. Misrepair is also fraught with danger, especially inappropriate end-joining events, which commonly underlie oncogenic transformation and can scramble the genome. Canonically, cells employ two basic mechanisms to repair DSBs: homologous recombination (HR) and the classical nonhomologous end-joining pathway (cNHEJ). More recent experiments identified a highly error-prone NHEJ pathway, termed alternative NHEJ (aNHEJ), which operates in both cNHEJ-proficient and cNHEJ-deficient cells. aNHEJ is now recognized to catalyze many genome rearrangements, some leading to oncogenic transformation. Here, we review the mechanisms of cNHEJ and aNHEJ, their interconnections with the DNA damage response (DDR), and the mechanisms used to determine which of the three DSB repair pathways is used to heal a particular DSB. We briefly review recent clinical applications involving NHEJ and NHEJ inhibitors.