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Germinal centers (GCs) are specialized compartments within the secondary lymphoid organs where B cells proliferate, differentiate, and mutate their antibody genes in response to the presence of foreign antigens. They play a central role in generating an effective immune response against infectious pathogens, and failures in their regulating mechanisms can lead to the development of autoimmune diseases and cancer. While previous works study experimental systems of the immune response with mouse models that are immunized with specific antigens, our study focuses on a real life situation, with an ongoing GC response in a human lymph node (LN) involving multiple asynchronized GCs reacting simultaneously to unknown antigens. We combined laser capture microdissection (LCM) of individual GCs from human LN with next-generation repertoire sequencing (Rep-seq) to characterize individual GCs as distinct evolutionary spaces. In line with well-characterized GC responses in mice, elicited by immunization with model antigens such as NP-CGG, we observe a relatively low sequence similarity, as well as heterogeneous clonal diversity across individual GCs from the same human LN. Still, we identify shared clones in several individual GCs, and phylogenetic tree analysis combined with paratope modeling suggest the re-engagement and rediversification of B-cell clones across GCs as well as expanded clones exhibiting shared antigen responses across distinct GCs, indicating convergent evolution of the GCs. Finally, our study allows for the characterization of non-functional clones, where frequencies of V(D)J or SHM induced stop codons are quantified.

Metabolic alterations are important cancer-associated features that allow cancer cell transformation and survival under stress conditions. Multiple myeloma (MM) plasma cells show increased glycolysis and oxidative phosphorylation (OXPHOS), which are characteristics associated with recurrent genetic aberrations that drive the proliferation and survival of MM cells. The protein kinase B/AKT acts as a central node in cellular metabolism and is constitutively active in MM cells. Despite the known role of AKT in modulating cellular metabolism, little is known about the downstream factors of AKT that control the metabolic adaptability of MM cells. Here, we demonstrate that negative regulation of the forkhead box O (FOXO) transcription factors (TFs) by AKT is crucial to prevent the metabolic shutdown in MM cells, thus contributing to their metabolic adaptability. Our results demonstrate that the expression of several key metabolic genes involved in glycolysis, the tricarboxylic acid (TCA) cycle, and OXPHOS are repressed by FOXO TFs. Moreover, the FOXO-dependent repression of glycolysis- and TCA-associated genes correlates with a favorable prognosis in a large cohort of patients with MM. Our data suggest that repression of FOXO by AKT is essential to sustain glycolysis and the TCA cycle activity in MM cells and, as such, predicts patient survival.

We recently demonstrated that normal memory B lymphocytes carry a substantial number of de novo mutations in the genome. Here, we performed exome-wide somatic mutation analyses of bona fide autoreactive rheumatoid factor (RF)-expressing memory B cells retrieved from patients with Sj?gren's syndrome (SS). The amount and repertoire of the de novo exome mutations of RF B cells were found to be essentially different from those detected in healthy donor memory B cells. In contrast to the mutation spectra of normal B cells, which appeared random and non-selected, the mutations of the RF B cells were greater in number and enriched for mutations in genes also found mutated in B-cell non-Hodgkin lymphomas. During the study, one of the SS patients developed a diffuse large B-cell lymphoma (DLBCL) out of an RF clone that was identified 2 years earlier in an inflamed salivary gland biopsy. The successive oncogenic events in the RF precursor clone and the DLBCL were assessed. In conclusion, our findings of enhanced and selected genomic damage in growth-regulating genes in RF memory B cells of SS patients together with the documented transformation of an RF-precursor clone into DLBCL provide unique novel insight into the earliest stages of B-cell derailment and lymphomagenesis. ? 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The phosphatidylinositide-3 kinases and the downstream mediator AKT drive survival and proliferation of multiple myeloma (MM) cells. AKT signaling is active in MM and has pleiotropic effects; however, the key molecular aspects of AKT dependency in MM are not fully clear. Among the various downstream AKT targets are the Forkhead box O (FOXO) transcription factors (TFs) and glycogen synthase kinase 3 (GSK3), which are negatively regulated by AKT signaling. Here we show that abrogation of AKT signaling in MM cells provokes cell death and cell cycle arrest, which crucially depends on both FOXO TFs and GSK3. Based on gene expression profiling, we defined a FOXO-repressed gene set that has prognostic significance in a large cohort of patients with MM, indicating that AKT-mediated gene activation is associated with inferior overall survival. We further show that AKT signaling stabilizes the antiapoptotic myeloid cell leukemia 1 (MCL1) protein by inhibiting FOXO- and GSK3-mediated MCL1 turnover. In concordance, abrogation of AKT signaling greatly sensitized MM cells for an MCL1-targeting BH3-mimetic, which is currently in clinical development. Taken together, our results indicate that AKT activity is required to restrain the tumor-suppressive functions of FOXO and GSK3, thereby stabilizing the antiapoptotic protein MCL1 in MM. These novel insights into the role of AKT in MM pathogenesis and MCL1 regulation provide opportunities to improve targeted therapy for patients with MM.

The BCR-ABL1 fusion gene is the driver oncogene in chronic myeloid leukemia (CML) and Philadelphia-chromosome positive (Ph+) acute lymphoblastic leukemia (ALL). The introduction of tyrosine kinase inhibitors (TKIs) targeting the ABL kinase (such as imatinib) has dramatically improved survival of CML and Ph+ ALL patients. However, primary and acquired resistance to TKIs remains a clinical challenge. Ph+ leukemia patients who achieve a complete cytogenetic (CCR) or deep molecular response (MR) (≥4.5log reduction in BCR-ABL1 transcripts) represent long-term survivors. Thus, the fast and early eradication of leukemic cells predicts MR and is the prime clinical goal for these patients. We show here that the first-in-class inhibitor of the Nedd8-activating enzyme (NAE1) MLN4924 effectively induced caspase-dependent apoptosis in Ph+ leukemia cells, and sensitized leukemic cells for ABL tyrosine kinase inhibitors (TKI) and hydroxyurea (HU). We demonstrate that MLN4924 induced DNA damage in Ph+ leukemia cells by provoking the activation of an intra S-phase checkpoint, which was enhanced by imatinib co-treatment. The combination of MLN4924 and imatinib furthermore triggered a dramatic shift in the expression of MCL1 and NOXA. Our data offers a clear rationale to explore the clinical activity of MLN4924 (alone and in combination with ABL TKI) in Ph+ leukemia patients

A major complication of primary Sjögren's syndrome (pSS) is development of mucosa associated lymphoid tissue (MALT) B-cell lymphoma, particularly in salivary glands. These lymphomas express FcRL4 and are characteristically associated with lymphoepithelial lesions. Neoplastic B-cells may be derived from non-neoplastic glandular intraductal B-cells, also virtually all expressing FcRL4. A characteristic feature of MALT lymphomas is the production of rheumatoid factors (RFs), which are largely encoded by stereotypic immunoglobulin variable heavy chain (IGHV) sequences. The aim of this study was to examine whether there is a relationship between the intraductal and periductal B-cells and whether the intraductal B-cells are selected for RF. RNA was extracted from laser-microdissected infiltrated ductal areas and periductal infiltrates from frozen parotid gland tissue sections of 5 pSS patients. PCR amplified IGHV transcripts were cloned into pCR™4-TOPO vector and subsequently sequenced. Microdissected ducts yielded 96 unique IGHV sequences derived from intraductal B-cells, while 119 unique IGHV sequences were obtained from periductal infiltrates. No major difference in VH-gene usage was observed between intraductal and periductal B-cells. Nearly all (>90%) IGHV sequences derived from both intraductal and periductal B-cells were mutated. Clonal expansions as defined by shared VDJ rearrangements were also present among both intraductal and periductal B-cells: in total 32 clones were found, from which 12 were located within ducts, 15 in periductal areas, and five clones shared members in both areas. We observed 12 IGHV rearrangements encoding for RF sequences from which two were derived from intraductal B-cells and 10 from periductal B-cells. Nine RF sequences were part of a clone. Together these findings indicate that intraductal and periductal B-cells are closely related to each other. Intraductal B-cells are most likely derived from periductal B-cells. We did not obtain evidence that RF-specific B-cells are enriched within the striated ducts. We speculate that in principle any activated B-cell can enter the striated ducts from the periductal infiltrate, irrespective of its antigenic specificity. Within the ducts, these B-cells may receive additional activation and proliferation signals, to further expand at these sites and by acquisition of driver-mutations develop toward lymphoma.

In the past years, the genomes of thousands of tumors have been elucidated. To date however, our knowledge on somatic gene alterations in normal cells is very limited. In this study, we demonstrate that tetanus-specific human memory B lymphocytes carry a substantial number of somatic mutations in the coding regions of the genome. Interestingly, we observed a statistically significant correlation between the number of exome mutations and those present in the immunoglobulin heavy variable regions. Our findings indicate that the majority of these genomic mutations arise in an antigen-dependent fashion, most likely during clonal expansion in germinal centers. The knowledge that normal B cells accumulate genomic alterations outside the immunoglobulin loci during development is relevant for our understanding of the process of lymphomagenesis.

In B cells, the error-prone repair of activation-induced cytidine deaminase (AID)-induced lesions in immunoglobulin variable genes cause somatic hypermutation (SHM) of antibody genes. Due to clonal selection in the germinal centers (GC) this active mutation process provides the molecular basis for antibody affinity maturation. AID deaminates cytosine (C) to create uracil (U) in DNA. Typically, the short patch base excision repair (spBER) effectively restores genomic U lesions. We here demonstrate that GC B cells actively degrade DNA polymerase β (Polβ), resulting in the inactivation of the gap-filling step of spBER. Consequently, lesions instigated by AID, and likely other base damages, are channeled towards mutagenic non-canonical mismatch repair (mncMMR), responsible for the vast majority of mutations at adenine and thymine (A:T) bases. Apparently, GC B cells prohibit faithful spBER, thereby favoring A:T mutagenesis during SHM. Lastly, our data suggest that the loss of Polβ relates to hypoxia that characterizes the GC microenvironment.

The phosphatidylinositide-3 kinases (PI3K) and the downstream mediator AKT drive survival and proliferation of multiple myeloma (MM) cells and several AKT inhibitors are currently being tested in clinical trials for MM patients. AKT inhibition has pleiotropic effects, and the key aspects that determine therapeutic efficacy are not fully clear. Therefore, we investigated the antimyeloma mechanism(s) of AKT inhibition. Among the various downstream AKT targets are Forkhead box O (FOXO) transcription factors, and we demonstrate that they are crucial for changes in gene expression upon AKT inhibition. Based on gene expression profiling we defined an AKT-induced FOXO-dependent gene set that has prognostic significance in a large cohort of MM patients, where low FOXO activity correlates with inferior survival. We show that cell cycle exit and cell death of MM cells after AKT inhibition required FOXO. In addition, glycogen synthase kinase 3 (GSK3), a negatively regulated AKT substrate, proved to be pivotal to induce cell death and to inhibit cell cycle progression after AKT inhibition. Finally, we demonstrate that FOXO and GSK3 induced cell death by increasing the turnover of the myeloid cell leukemia 1 (MCL1) protein. In concordance, the AKT inhibitor MK2206 greatly sensitized MM cells for the MCL1 inhibitor S63845. Thus, our results indicate that FOXO and GSK3 are crucial mediators of the antimyeloma effects of AKT inhibition, and suggest combination therapies that may have therapeutic potential in MM.KEYPOINTSFOXO transcription factors and the GSK3 kinase are pivotal tumor suppressors downstream of AKT inhibition in MM cells.FOXO and GSK3 activation after AKT inhibition leads to a decrease in MCL1 levels in MM cells resulting in cell death.

OBJECTIVE: Patients with Sjӧgren's syndrome (SS) have an increased risk of developing malignant B cell lymphomas, particularly mucosa-associated lymphoid tissue (MALT)-type lymphomas. We have previously shown that a predominant proportion of patients with SS-associated salivary gland MALT lymphoma express somatically hypermutated IgM with strong amino acid sequence homology with stereotypic rheumatoid factors (RFs). The present study was undertaken in a larger cohort of patients with SS-associated MALT lymphoma to more firmly assess the frequency of RF reactivity and the significance of somatic IGV-region mutations for RF reactivity.METHODS: B cell antigen receptors (BCRs) of 16 patients with SS-associated salivary gland MALT lymphoma were analyzed. Soluble recombinant IgM was produced of 12 MALT lymphoma samples, including 1 MALT lymphoma sample that expressed an IgM antibody fitting in a novel IGHV3-30-encoded stereotypic IGHV subset. For 4 of the 12 IgM antibodies from MALT lymphoma samples, the somatically mutated IGHV and IGKV gene sequences were reverted to germline configurations. Their RF activity and binding affinity were determined by enzyme-linked immunosorbent assay and surface plasmon resonance, respectively.RESULTS: Nine (75%) of the 12 IgM antibodies identified in patients with SS-associated salivary gland MALT lymphoma displayed strong monoreactive RF activity. Reversion of the IGHV and IGKV mutations to germline configuration resulted in RF affinities for IgG that were significantly lower for 3 of the 4 somatically mutated IgM antibodies. In stereotypic IGHV3-7/IGKV3-15-encoded RFs, a recurrent replacement mutation in the IGKV3-15-third complementarity-determining region was found to play a pivotal role in the affinity for IgG-Fc.CONCLUSION: A majority of patients with SS-associated salivary gland MALT lymphoma express somatically mutated BCRs that are selected for monoreactive, high-affinity binding of IgG-Fc. These data underscore the notion that soluble IgG, most likely in immune complexes in inflamed tissues, is the principal autoantigen in the pathogenesis of a variety of B cell lymphomas, particularly SS-associated MALT lymphomas.

The recombination activating gene (RAG) 1 and RAG2 protein complex introduces DNA breaks at Tcr and Ig gene segments that are required for V(D)J recombination in developing lymphocytes. Proper regulation of RAG1/2 expression safeguards the ordered assembly of Ag receptors and the development of lymphocytes, while minimizing the risk for collateral damage. The ataxia telangiectasia mutated (ATM) kinase is involved in the repair of RAG1/2-mediated DNA breaks and prevents their propagation. The simultaneous occurrence of RAG1/2-dependent and -independent DNA breaks in developing lymphocytes exposed to genotoxic stress increases the risk for aberrant recombinations. In this study, we assessed the effect of genotoxic stress on RAG1/2 expression in pre–B cells and show that activation of the DNA damage response resulted in the rapid ATM-dependent downregulation of RAG1/2 mRNA and protein expression. We show that DNA damage led to the loss of FOXO1 binding to the enhancer region of the RAG1/2 locus (Erag) and provoked FOXO1 cleavage. We also show that DNA damage caused by RAG1/2 activity in pre–B cells was able to downmodulate RAG1/2 expression and activity, confirming the existence of a negative feedback regulatory mechanism. Our data suggest that pre–B cells are endowed with a protective mechanism that reduces the risk for aberrant recombinations and chromosomal translocations when exposed to DNA damage, involving the ATM-dependent regulation of FOXO1 binding to the Erag enhancer region.

Background Sjogren’s syndrome (SS) is the autoimmune disease with the highest risk of lymphoma development. SS patients develop most frequently MALT lymphoma and to a lesser extent diffuse large B-cell lymphoma (DLBCL). Previously, we have shown that at least 40% of salivary gland MALT lymphomas express groups of near identical (also called stereotypic) B-cell receptors, which display in vitro mono-specific rheumatoid factor (RF) reactivity1. Recently, we have analysed the B-cell immunoglobulin heavy variable (IGHV) repertoire in 4 SS labial salivary glands. In 2 out of 4 salivary glands only one minor stereotypic RF B-cell clone was detected. Interestingly, in one salivary gland of patient SG22 a highly expanded stereotypic RF-expressing B-cell clone was present, which was also detected in peripheral blood. Twenty six months later, a clonally-related DLBCL was diagnosed2. Objectives To study the molecular alterations present in the RF-expressing B cell clones of salivary gland, peripheral blood and in the DLBCL of SS patient SG22. Methods From peripheral blood 4 RF-expressing immortalised B-cell clones with identical IGHV rearrangements were isolated. These 4 RF-clones were analysed by whole exome sequencing and the identified non-synonymous exome mutations were traced in the salivary gland and in the DLBCL, using targeted next generation sequencing. Results In total we identified 56 exome mutations in the 4 RF B-cell clones. Twelve non-synonymous mutations were shared between all 4 RF-clones, of which one was a missense mutation in CARD11, a well-known oncogenic mutation of DLBCL. In the salivary gland all shared 12 non-synonymous mutations were detected, whereas in the DLBCL only 4 of these mutations were detected. Remarkably, the CARD11 mutation was not detected in the DLBCL. The exome mutations were analysed for typical characteristics of induction by the B-cell specific mutator AID. This revealed that the large majority of mutations (80%) were (i) not located within 1,5 kB of the transcription initiation site of the genes, (ii) did not concern C/G nucleotides in AID hotspots (WRC/GYW) and (iii) were not in genes expressed by B cells. This lack of evidence for AID involvement, suggests that the exome mutations of the RF-clones were due to replication errors, obtained during their clonal expansion. Interestingly, as compared to the RF-clones of peripheral blood, the DLBCL accumulated 44 extra somatic mutations together in IGHV and IGKV. As expected, these new IGHV/IGKV mutations showed the characteristics of AID involvement, indicating that in an ancestor RF-clone, AID activity was induced. Conclusions We have documented the clonal outgrowth and diversification of a RF-specific B-cell clone in a SS patient and the evolution of this clone into a DLBCL. Our analysis are indicative for AID activity in the lymphoma cells, represented by accumulation of IGHV/IGKV mutations. However, we have no evidence that AID is instrumental in the accumulation of the non-IG mutations in the RF-clones from peripheral blood. References [1] Bende, et al. J. Exp. Med2005;201:1229–1241. [2] Bende, et al. Arthritis Rheumatol2015;67:1074–1083. Disclosure of Interest None declared

Objective Among autoimmune diseases, Sjogren's syndrome (SS) is most strongly associated with the development of malignant B cell lymphoma, in particular mucosa-associated lymphoid tissue (MALT)–type lymphoma. Previously, we have shown that in ∼40% of cases of salivary gland MALT lymphoma, high-affinity stereotypic rheumatoid factor (RF) B cell receptors, specific for IgG-Fc, are expressed. This study was undertaken to investigate whether in the inflamed salivary glands of patients with SS, a similar RF-biased Ig repertoire is present. Methods Extensive analyses of the B cell Ig VH region repertoire were performed on microdissected tissue samples from the labial salivary glands of 4 patients with SS. Results All SS labial salivary glands harbored expanded B cell clones, of which 1 or 2 were highly expanded and detected in >50% of the microdissected samples. However, among the identified 464 distinct Ig clonotypes, only 3 stereotypic RF–expressing clones were detected. In 2 patients with SS, an RF-expressing clone was detected at low frequency in 1 of the microdissected samples, whereas 1 patient with SS harbored a highly expanded RF-expressing clone that was detected in all microdissected samples and also detected in the peripheral blood. Two years after analysis of this sample, the latter patient developed a diffuse large B cell lymphoma originating from the same RF clone. Conclusion Inflamed labial salivary glands in patients with SS generally harbor 1 or 2 highly expanded B cell clones. The repertoire strongly biased toward stereotypic RFs in salivary gland MALT lymphomas is not a reflection of a similar repertoire in the inflamed salivary glands of patients with SS; rather, in the latter, the repertoire is based on a strong selection advantage of incidental stereotypic RF–expressing B cells.

Follicular lymphoma (FL) is an indolent B-cell non-Hodgkin lymphoma able to transform into germinal center-type diffuse large B-cell lymphoma. We describe four extraordinary cases of FL, which progressed to TdT+CD20- precursor B-lymphoblastic lymphoma (B-LBL). Fluorescence in situ hybridization analysis showed that all four B-LBLs had acquired a MYC translocation on transformation. Comparative genomic hybridization analysis of one case demonstrated that in addition to 26 numerical aberrations that were shared between the FL and B-LBL, deletion of CDKN2A/B and 17q11, 14q32 amplification, and copy-neutral loss of heterozygosity of 9p were gained in the B-LBL cells. Whole-exome sequencing revealed mutations in FMN2, NEB, and SYNE1 and a nonsense mutation in KMT2D, all shared by the FL and B-LBL, and TNFRSF14, SMARCA2, CCND3 mutations uniquely present in the B-LBL. Remarkably, all four FL-B-LBL pairs expressed IgG. In two B-LBLs, evidence was obtained for ongoing rearrangement of IG light chain variable genes and expression of the surrogate light chain. IGHV mutation analysis showed that all FL-B-LBL pairs harbored identical or near-identical somatic mutations. From the somatic gene alterations found in the IG and non-IG genes, we conclude that the FLs and B-LBLs did not develop in parallel from early t(14;18)-positive IG-unmutated precursors, but that the B-LBLs developed from preexistent FL subclones that accumulated additional genetic damage.

In developing lymphocytes, expression and activity of the recombination activation gene protein 1 (RAG1) and RAG2 endonuclease complex is tightly regulated to ensure ordered recombination of the immunoglobulin genes and to avoid genomic instability. Aberrant RAG activity has been implicated in the generation of secondary genetic events in human B-cell acute lymphoblastic leukemias (B-ALLs), illustrating the oncogenic potential of the RAG complex. Several layers of regulation prevent collateral genomic DNA damage by restricting RAG activity to the G1 phase of the cell cycle. In this study, we show a novel pathway that suppresses RAG expression in cycling-transformed mouse pre-B cells and human pre-B B-ALL cells that involves the negative regulation of FOXO1 by nuclear factor κB (NF-κB). Inhibition of NF-κB in cycling pre-B cells resulted in upregulation of RAG expression and recombination activity, which provoked RAG-dependent DNA damage. In agreement, we observe a negative correlation between NF-κB activity and the expression of RAG1, RAG2, and TdT in B-ALL patients. Our data suggest that targeting NF-κB in B-ALL increases the risk of RAG-dependent genomic instability.