Search

T cell–independent (TI) B cell responses to nonprotein Ags involve multiple cues from the innate immune system. Neutrophils express complement receptors and activated neutrophils can release BAFF, but mechanisms effectively linking neutrophil activation to TI B cell responses are incompletely understood. Using germline and conditional knockout mice, we found that TI humoral responses involve alternative pathway complement activation and neutrophil-expressed C3a and C5a receptors (C3aR1/C5aR1) that promote BAFF-dependent B1 cell expansion and TI Ab production. Conditional absence of C3aR1/C5aR1 on neutrophils lowered serum BAFF levels, led to fewer Peyer’s patch germinal center B cells, reduced germinal center B cells IgA class-switching, and lowered fecal IgA levels. Together, the results indicate that sequential activation of complement on neutrophils crucially supports humoral TI and mucosal IgA responses through upregulating neutrophil production of BAFF.

PDF file 233K, SupplementaryTable S1. Detailed clinical and pathological features of all the cases included in the study; S2. Detailed flow cytometric, cytogenetic and molecular findings of all the cases included in the study; S3. Sequence analysis of the MTC PCR products encompassing the IGH/CCND1 junction; S4. List of differentially expressed genes between MCL and MALD1 with order of appearance in Figure 4C; S5. Summary of the main pathways differentially enriched in MALD1 and MCL lymphocytes; S6. Table of all samplesused to generatethe CART. Classification column presents the assigned group after applying the CART

PDF file 132K, Validation of gene expression analysis results by qRT-PCR

Purpose: According to current diagnostic criteria, mantle cell lymphoma (MCL) encompasses the usual, aggressive variants and rare, nonnodal cases with monoclonal asymptomatic lymphocytosis, cyclin D1–positive (MALD1). We aimed to understand the biology behind this clinical heterogeneity and to identify markers for adequate identification of MALD1 cases.Experimental Design: We compared 17 typical MCL cases with a homogeneous group of 13 untreated MALD1 cases (median follow-up, 71 months). We conducted gene expression profiling with functional analysis in five MCL and five MALD1. Results were validated in 12 MCL and 8 MALD1 additional cases by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and in 24 MCL and 13 MALD1 cases by flow cytometry. Classification and regression trees strategy was used to generate an algorithm based on CD38 and CD200 expression by flow cytometry.Results: We found 171 differentially expressed genes with enrichment of neoplastic behavior and cell proliferation signatures in MCL. Conversely, MALD1 was enriched in gene sets related to immune activation and inflammatory responses. CD38 and CD200 were differentially expressed between MCL and MALD1 and confirmed by flow cytometry (median CD38, 89% vs. 14%; median CD200, 0% vs. 24%, respectively). Assessment of both proteins allowed classifying 85% (11 of 13) of MALD1 cases whereas 15% remained unclassified. SOX11 expression by qRT-PCR was significantly different between MCL and MALD1 groups but did not improve the classification.Conclusion: We show for the first time that MALD1, in contrast to MCL, is characterized by immune activation and driven by inflammatory cues. Assessment of CD38/CD200 by flow cytometry is useful to distinguish most cases of MALD1 from MCL in the clinical setting. MALD1 should be identified and segregated from the current MCL category to avoid overdiagnosis and unnecessary treatment. Clin Cancer Res; 20(4); 1007–19. ©2013 AACR.

Maturation of B cells within germinal centers (GCs) generates diversified B cell pools and high-affinity B cell antigen receptors (BCRs) for pathogen clearance. Increased receptor affinity is achieved by iterative cycles of T cell–dependent, affinity-based B cell positive selection and clonal expansion by mechanisms hitherto incompletely understood. Here we found that, as part of a physiologic program, GC B cells repressed expression of decay-accelerating factor (DAF/CD55) and other complement C3 convertase regulators via BCL6, but increased the expression of C5b-9 inhibitor CD59. These changes permitted C3 cleavage on GC B cell surfaces without the formation of membrane attack complex and activated C3a- and C5a-receptor signals required for positive selection. Genetic disruption of this pathway in antigen-activated B cells by conditional transgenic DAF overexpression or deletion of C3a and C5a receptors limited the activation of mechanistic target of rapamycin (mTOR) in response to BCR–CD40 signaling, causing premature GC collapse and impaired affinity maturation. These results reveal that coordinated shifts in complement regulation within the GC provide crucial signals underlying GC B cell positive selection. Heeger and colleagues report that activated B cells dynamically regulate the expression of complement regulatory proteins via the transcription factor BCL6. C3 convertase activity and C3aR1–C5aR1 signaling were both necessary for optimal B cell activation and germinal center formation.

PRDM (PRDI-BF1 and RIZ homology domain containing) family members are sequence-specific transcriptional regulators involved in cell identity and fate determination, often dysregulated in cancer. The PRDM15 gene is of particular interest, given its low expression in adult tissues and its overexpression in B-cell lymphomas. Despite its well characterized role in stem cell biology and during early development, the role of PRDM15 in cancer remains obscure. Herein, we demonstrate that while PRDM15 is largely dispensable for mouse adult somatic cell homeostasis in vivo, it plays a critical role in B-cell lymphomagenesis. Mechanistically, PRDM15 regulates a transcriptional program that sustains the activity of the PI3K/AKT/mTOR pathway and glycolysis in B-cell lymphomas. Abrogation of PRDM15 induces a metabolic crisis and selective death of lymphoma cells. Collectively, our data demonstrate that PRDM15 fuels the metabolic requirement of B-cell lymphomas and validate it as an attractive and previously unrecognized target in oncology., The transcriptional regulator PRDM15 is expressed at low levels in normal tissues but overexpressed in B-cell lymphomas. Here, the authors show that PRDM15 depletion does not affect adult somatic cell homeostasis but leads to a metabolic crisis which impairs B-cell lymphomagenesis.

Cell cycle progression, or its arrest upon checkpoint activation, is directed by a complex array of cellular processes dependent on the diffusion of chemical signals. These signals regulate the onset of each cell cycle phase and prevent undesired phase transitions. Functional complementation is a robust strategy to identify such signals, by which mutant phenotypes are rescued through complementation with candidate factors. Here we describe a method that reclaims a five-decade old mammalian cell-cell fusion strategy of functional complementation to study the molecular control of cell cycle progression. The generation of cell-cell fusions (heterokaryons) allows for the analysis, via immunofluorescence, of cell cycle regulator dynamics and evaluating the effective rescue of cell cycle progression in specific genetic settings.

Cell cycle progression, or its arrest upon checkpoint activation, is directed by a complex array of cellular processes dependent on the diffusion of chemical signals. These signals regulate the onset of each cell cycle phase and prevent undesired phase transitions. Functional complementation is a robust strategy to identify such signals, by which mutant phenotypes are rescued through complementation with candidate factors. Here we describe a method that reclaims a five-decade old mammalian cell-cell fusion strategy of functional complementation to study the molecular control of cell cycle progression. The generation of cell-cell fusions (heterokaryons) allows for the analysis, via immunofluorescence, of cell cycle regulator dynamics and evaluating the effective rescue of cell cycle progression in specific genetic settings.

The transcription factor forkhead box O1 (FOXO1), which instructs the dark zone program to direct germinal center (GC) polarity, is typically inactivated by phosphatidylinositol 3-kinase (PI3K) signals. Here, we investigated how FOXO1 mutations targeting this regulatory axis in GC-derived B cell non-Hodgkin lymphomas (B-NHLs) contribute to lymphomagenesis. Examination of primary B-NHL tissues revealed that FOXO1 mutations and PI3K pathway activity were not directly correlated. Human B cell lines bearing FOXO1 mutations exhibited hyperactivation of PI3K and Stress-activated protein kinase (SAPK)/Jun amino-terminal kinase (JNK) signaling, and increased cell survival under stress conditions as a result of alterations in FOXO1 transcriptional affinities and activation of transcriptional programs characteristic of GC-positive selection. When modeled in mice, FOXO1 mutations conferred competitive advantage to B cells in response to key T-dependent immune signals, disrupting GC homeostasis. FOXO1 mutant transcriptional signatures were prevalent in human B-NHL and predicted poor clinical outcomes. Thus, rather than enforcing FOXO1 constitutive activity, FOXO1 mutations enable co-option of GC-positive selection programs during the pathogenesis of GC-derived lymphomas.

Oncogene deregulation triggers replication stress, which can be detected in precancerous lesions and is a source of subsequent genomic instability in cancer. Studies on the mechanisms underlying DNA replication stress caused by frequently deregulated oncogenes, like MYC or RAS, can identify novel mechanisms of oncogenesis and point to actionable pathways with potential for anticancer therapy. To understand the molecular mechanisms underlying this cellular response, we used high-content microscopy to monitor the dynamic behavior of >60 GFP-tagged proteins linked to replication stress or DNA damage in primary human cells. Iterations of this screen identified a proteomic signature specific to MYC- and RAS-driven replication stress that was significantly different from responses activated by other sources of replication stress (i.e. DNA damaging drugs). These findings indicated that the replication stress response to oncogenes is somewhat unique. MYC-dependent replication stress responses typically activated the INO80 chromatin-remodeling complex — a highly conserved complex necessary for maintenance of genome stability in yeast. Core components of the INO80 complex assembled onto chromatin at MYC-bound sites and were quantitatively enriched at active replication forks. These molecular events were independent of transcription, and their disruption by pharmacological or genetic means specifically altered replication dynamics and cellular growth in cells overexpressing MYC. Notably, we identified recurrent somatic mutations in several genes encoding for INO80 complex components across multiple cancer types, with an abundance of nonsense and splice-site mutations consistent with loss of function. Most of these mutant variants behaved as hypomorphs when tested in cell-based assays: they were insensitive to MYC deregulation and failed to rescue cell proliferation in knock-out cells with MYC overexpression. Computational predictions also suggested that many of these mutations had been selected as potential cancer drivers. Consistent with this notion, we found that in vivo, INO80 complex mutations shortened tumor latency in a mouse model of MYC-dependent lymphomagenesis. Collectively, these findings indicate that the replication stress response to oncogene deregulation is unique in its molecular architecture, and modification of this response by partial loss-of-function mutations in cancer facilitates tumor progression. Citation Format: Rosa Vinas-Castells, Alja Kozulic-Pirher, Tomás Aparicio, Sandra Casas Recasens, Mark P. Roberto, Rachel Sue, Francisco Martinez, Nuria López-Bigas, Jean Gautier, David Dominguez-Sola. Molecular architecture of replication stress response to oncogene deregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1517.

The transcription factor FOXO1 directs germinal center (GC) polarity and supports affinity maturation by modulating immune activation programs in GC B cells. Recurrent somatic mutations targeting FOXO1 in GC-derived B-cell non-Hodgkin lymphomas (B-NHL) are thought to disrupt its negative regulation by PI3K and function as gain-of-function, constitutively active alleles. Herein, we demonstrate that B-NHL FOXO1 mutants are instead hypomorphic alleles encoding proteins with altered transcriptional activities. Analysis of Foxo1 mutant mouse models, engineered cell lines, and primary samples shows that B-NHL FOXO1 mutations induce simultaneous hyperactivation of Stress Activated Protein Kinase -SAPK/JNK and Phosphoinositide 3-kinase -PI3K/AKT signaling pathways and gene expression programs characteristic of GC B cells undergoing positive selection. These alterations confer mutant B cells with a competitive advantage in response to key immune signals, leading to abnormal amplification of GC responses. Moreover, we find that FOXO1 mutant-driven transcriptional programs are prevalent in human B-NHL and predict poor clinical outcomes. These results imply the frequent co-option of GC positive selection programs in the pathogenesis of GC-derived lymphomas. Citation Format: Mark P. Roberto, Gabriele Varano, Rosa Viñas-Castells, Antony B. Holmes, Rahul Kumar, Pedro Farinha, David W. Scott, David Dominguez-Sola. FOXO1 mutations mimic positive selection signals to promote germinal center B-cell expansion and lymphomagenesis [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-40.

Somatic missense mutations targeting MYC's coding sequence are found in >50% of Burkitt lymphomas and in a small fraction of diffuse large B-cell lymphomas. These mutations typically arise in translocated MYC alleles and cluster at specific protein residues, particularly Threonine 58 (T58). Previous studies in non-B cell models suggested that MYC mutations enhance oncogenic properties by escaping MYC-induced apoptosis. However, the functional impact of these mutations on the biology of germinal center (GC) B cells, where these mutations arise, has never been investigated. In GC B cells, MYC expression is induced by positive selection signals during cognate interactions with T follicular helper cells, concurrent with activation of PI3K and mTOR signaling. PI3K and mTOR are activated upon CD40 and B-cell receptor co-engagement and are critically required for positive selection and clonal expansion of GC B cells. Using newly generated MYC T58A knock-in mouse models, we found that MYC T58A mutation allows B cells to bypass a cell cycle entry checkpoint dependent on PI3K and mTOR activities. Exposure of wild-type (WT) B cells to rapamycin or, less markedly, to PI3K inhibitors, prevented the proliferation upon CD40 and IL4 stimulation ex vivo (a cytokine combination mimicking T-cell help) whereas MYC T58A mutant B cells divided multiple times and maintained adequate rates of active biosynthesis. Similarly, administration of rapamycin to immunized WT mice collapsed formed GCs while MYC T58A GCs maintained normal size and proliferation rates in vivo. These phenotypes were in part explained by the effects of PI3K/mTOR signaling on GSK3-beta, a kinase that normally phosphorylates MYC T58 to control protein turnover. Differences in metabolic and gene expression profiles between MYC WT and T58A cells offered additional insights into the mechanisms by which this mutation bypasses PI3K/mTOR checkpoint. Finally, we found that GC B cell-specific overexpression of MYC T58A, resulted in abnormally enlarged GCs upon immunization, a phenotype common to lymphoma models. Altogether, these results indicate that MYC somatic mutations confer competitive advantages to B cells by bypassing a strict requirement for upstream signals during cell cycle entry, which may support the clonal expansion of MYC mutant B cells during GC responses. These results have important translational implications, particularly given the increasing interest in the potential use of PI3K and/or mTOR inhibitors in the treatment of lymphomas. Citation Format: Jongkuen Lee, David Dominguez-Sola. Functional bypass of cell cycle entry checkpoints by MYC T58A mutation in germinal center-derived lymphomas [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-36.

Germinal centers are short-lived microanatomical compartments with essential roles in adaptive immunity. These lymphoid structures can be identified in secondary lymphoid organs using both flow cytometry and immunohistological analyses, but only the latter provides useful architectural and spatial information. Here we describe how to use immunofluorescence and immunohistochemistry with specific antibodies to precisely highlight the cellular and architectural features of germinal centers, both in human and mouse secondary lymphoid organs, and to study their normal development and disturbance in disease.

Germinal centers are short-lived microanatomical compartments with essential roles in adaptive immunity. These lymphoid structures can be identified in secondary lymphoid organs using both flow cytometry and immunohistological analyses, but only the latter provides useful architectural and spatial information. Here we describe how to use immunofluorescence and immunohistochemistry with specific antibodies to precisely highlight the cellular and architectural features of germinal centers, both in human and mouse secondary lymphoid organs, and to study their normal development and disturbance in disease.

SUMMARY c-Myc oncogenic activity is thought to be mediated in part by its ability to generate DNA replication stress and subsequent genomic instability when deregulated. Previous studies have demonstrated a nontranscriptional role for c-Myc in regulating DNA replication. Here, we analyze the mechanisms by which c-Myc deregulation generates DNA replication stress. We find that overexpression of c-Myc alters the spatiotemporal program of replication initiation by increasing the density of early-replicating origins. We further show that c-Myc deregulation results in elevated replication-fork stalling or collapse and subsequent DNA damage. Notably, these phenotypes are independent of RNA transcription. Finally, we demonstrate that overexpression of Cdc45 recapitulates all c-Myc-induced replication and damage phenotypes and that Cdc45 and GINS function downstream of Myc.

During antibody affinity maturation, germinal center (GC) B cells cycle between affinity-driven selection in the light zone (LZ) and proliferation and somatic hypermutation in the dark zone (DZ). Although selection of GC B cells is triggered by antigen-dependent signals delivered in the LZ, DZ proliferation occurs in the absence of such signals. We show that positive selection triggered by T cell help activates the mechanistic target of rapamycin complex 1 (mTORC1), which promotes the anabolic program that supports DZ proliferation. Blocking mTORC1 prior to growth prevented clonal expansion, whereas blockade after cells reached peak size had little to no effect. Conversely, constitutively active mTORC1 led to DZ enrichment but loss of competitiveness and impaired affinity maturation. Thus, mTORC1 activation is required for fueling B cells prior to DZ proliferation rather than for allowing cell-cycle progression itself and must be regulated dynamically during cyclic re-entry to ensure efficient affinity-based selection. During germinal center selection, signals from Tfh cells in the light zone dictate the extent of B cell proliferation in the dark zone. Ersching et al. (2017) show that Tfh help induces mTORC1 activation in light zone B cells, leading to cell growth that sustains the subsequent dark zone proliferative burst.

After antigenic challenge, B cells enter the dark zone (DZ) of germinal centers (GCs) to proliferate and hypermutate their immunoglobulin genes. Mutants with greater affinity for the antigen are positively selected in the light zone (LZ) to either differentiate into plasma and memory cells or reenter the DZ. The molecular circuits that govern positive selection in the GC are not known. We show here that the GC reaction required biphasic regulation of expression of the cell-cycle regulator c-Myc that involved its transient induction during early GC commitment, its repression by Bcl-6 in DZ B cells and its reinduction in B cells selected for reentry into the DZ. Inhibition of c-Myc in vivo led to GC collapse, which indicated an essential role for c-Myc in GCs. Our results have implications for the mechanism of GC selection and the role of c-Myc in lymphomagenesis.

Alterations of the c-MYC (MYC) locus, mainly in the form of translocations or amplifications, are common in hematologic malignancies and result in deregulated MYC expression, disrupting cellular proliferation and differentiation and promoting leukemogenesis. While amplified MYC loci typically maintain all native cis-regulatory elements, translocated loci are instead driven by heterologous regulatory regions and are therefore expected to be unresponsive to external signals. However, no studies have directly compared the effects of different modes of MYC deregulation in cellular phenotypes and functions. Consistent with the idea that different modes of MYC deregulation may respond differentially to external mitogenic signals, we found that treatment with DMSO resulted in rapid downregulation of MYC protein expression in hematopoietic cell lines with amplified MYC loci (HL-60, BJAB), but failed to do so in cell lines harboring a t(8;14) MYC translocation (Ramos, LY8) despite the induction of cell cycle arrest in all lines. To precisely model these two genetic mechanisms of MYC deregulation during hematopoiesis and study their effects in phenotypes relevant to oncogenesis (differentiation and proliferation), we used genetic engineering of human embryonic stem cells (hESCs). First, MYC deregulation upon translocation was modeled using a doxycycline (DOX)-inducible lentiviral construct to ectopically express MYC with GFP (MYC-DoxOE). Transduced single cell clones of the H1 hESC line were selected to ensure uniform MYC expression. Second, we created a conditional MYC gene amplification model in H1 hESCs by using CRISPR/Cas9 to insert a floxed cassette consisting of three tandem MYC cDNA copies and GFP in the MYC locus (MYC-Amp). Correctly edited clones were identified via PCR and Southern Blot, and one clone with bi-allelic targeting (yielding 8 inducible MYC copies) was selected. All clones were confirmed to be karyotypically normal. Upon in vitro hematopoietic differentiation of unmodified H1 hESCs, we identified a peak of MYC expression (day 12) coinciding with the emergence of CD34+/CD45+ hematopoietic progenitor cells (HPCs). MYC expression subsequently declined as cells matured further down the myeloid lineage and lost CD34 expression (days 14-20). To test the effects of MYC deregulation in the two models we induced MYC expression by doxycycline (MYC-DoxOE) or cell-permeant TAT-Cre recombinase (MYC-Amp) on day 10 of hematopoietic differentiation. Induction of MYC expression above normal levels was found to be comparable between the two models during peak MYC expression (day 12). However, while ectopically expressed MYC in the MYC-DoxOE model maintained high levels throughout the culture period, amplified MYC in the MYC-Amp model - which is subject to regulation by the native MYC locus cis regulatory elements - was downregulated after day 12, in a pattern similar to that observed in uninduced H1 cells. MYC induction in the MYC-DoxOE model caused a block in differentiation with accumulation of CD34+ HPCs and reduction of more mature CD34-/CD45+ hematopoietic cells. Wright-Giemsa staining corroborated these results showing a predominance of morphologically immature myeloid cells. Strikingly, MYC-DoxOE HPCs could be maintained in culture for over 90 days, while H1-derived HPCs without doxycycline treatment completely arrested their proliferation after approximately 28 days of culture. This phenotype is dramatically distinct from the finite growth potential of HPCs derived from normal human pluripotent stem cells, documented in multiple hESC and induced pluripotent stem cell (iPSC) lines, and reminiscent of the extended proliferation we previously reported in HPCs derived from iPSCs generated from patients with acute myeloid leukemia (AML) (Kotini et al. Cell Stem Cell 2017). Induction of MYC in the MYC-Amp model had similar effects in differentiation and proliferation, albeit much less pronounced, extending the growth of HPCs to approximately 50 days. This milder phenotypic perturbation is likely due to the downregulation of amplified MYC, as opposed to the continuous high expression of the ectopically expressed MYC in the MYC-DoxOE model. In summary, we have generated a novel model that offers for the first time the opportunity to study the effects of MYC deregulation by different genetic mechanisms in human hematopoiesis in a dynamic fashion. Disclosures No relevant conflicts of interest to declare.

The BCL6 proto-oncogene encodes a transcriptional repressor that is required for the germinal center (GC) reaction and is implicated in lymphomagenesis. BCL6 protein stability is regulated by F-box protein 11 (FBXO11)–mediated ubiquitination and degradation, which is impaired in ∼6% of diffuse large B-cell lymphomas that carry inactivating genetic alterations targeting the FBXO11 gene. In order to investigate the role of FBXO11 in vivo, we analyzed GC-specific FBXO11 knockout mice. FBXO11 reduction or loss led to an increased number of GC B cells, to an altered ratio of GC dark zone to light zone cells, and to higher levels of BCL6 protein in GC B cells. B-cell receptor–mediated degradation of BCL6 was reduced in the absence of FBXO11, suggesting that FBXO11 contributes to the physiologic downregulation of BCL6 at the end of the GC reaction. Finally, FBXO11 inactivation was associated with the development of lymphoproliferative disorders in mice.

SummaryThe pathways regulating formation of the germinal center (GC) dark zone (DZ) and light zone (LZ) are unknown. In this study we show that FOXO1 transcription factor expression was restricted to the GC DZ and was required for DZ formation, since its absence in mice led to the loss of DZ gene programs and the formation of LZ-only GCs. FOXO1-negative GC B cells displayed normal somatic hypermutation but defective affinity maturation and class switch recombination. The function of FOXO1 in sustaining the DZ program involved the trans-activation of the chemokine receptor CXCR4, and cooperation with the BCL6 transcription factor in the trans-repression of genes involved in immune activation, DNA repair, and plasma cell differentiation. These results also have implications for the role of FOXO1 in lymphomagenesis because they suggest that constitutive FOXO1 activity might be required for the oncogenic activity of deregulated BCL6 expression.

Mutations in the gene encoding the KMT2D (also called MLL2) methyltransferase are highly recurrent and occur early during tumorigenesis in diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL). However, the functional consequences of KMT2D mutations and their role in lymphomagenesis are unknown. Here we show that FL/DLBCL-associated KMT2D mutations impair KMT2D enzymatic activity, leading to diminished global H3K4 methylation in germinal-center (GC) B-cells and DLBCL cells. Conditional deletion of Kmt2d early during B cell development, but not after initiation of the GC reaction, results in an increase in GC B-cells and enhances B cell proliferation in mice. In mice overexpressing BCL2, which develop GC-derived lymphomas resembling human tumors, genetic ablation of Kmt2d leads to a further increase in tumor incidence. These findings suggest that KMT2D acts as a tumor suppressor gene whose early loss facilitates lymphomagenesis by remodeling the epigenetic landscape of the cancer precursor cells. Eradication of KMT2D-deficient cells may represent a rational therapeutic approach for targeting early tumorigenic events.

Myc is a well known proto-oncogene encoding for a transcription factor whose activity is tightly regulated in the cellular context. Myc was the first oncogene recognized to activate the ARF tumor suppressor gene which suppresses cell proliferation partly through stabilization of the p53 tumor suppressor protein but which also has p53-independent growth-suppressive functions. Recent studies have indicated that mouse p19ARF negatively regulates Myc's transcriptional activity. We here show that the human p14ARF directly associates with Myc and relocates Myc from the nucleoplasm to the nucleolus. We found that p14ARF interacts with the Myc-Max complex and the binding of p14ARF does not interfere with Myc-Max interaction in vitro. Protein interaction assays define the Myc BoxII as a critical domain required for interaction with p14ARF. Moreover, we identify 30 amino acids encompassing Myc BoxII domain required for p14ARF interaction and colocalization in vivo. Finally, we show that p14ARF down regulates Myc activated transcription and that this activity cannot be addressed to an intrinsic p14ARF repressor domain.

PMCID: PMC4488901.-- et al., [Purpose]: According to current diagnostic criteria, mantle cell lymphoma (MCL) encompasses the usual, aggressive variants and rare, nonnodal cases with monoclonal asymptomatic lymphocytosis, cyclin D1- positive (MALD1). We aimed to understand the biology behind this clinical heterogeneity and to identify markers for adequate identification of MALD1 cases. [Experimental Design]: We compared 17 typical MCL cases with a homogeneous group of 13 untreated MALD1 cases (median follow-up, 71 months). We conducted gene expression profiling with functional analysis in five MCL and five MALD1. Results were validated in 12 MCL and 8 MALD1 additional cases by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and in 24MCLand 13MALD1 cases by flow cytometry. Classification and regression trees strategy was used to generate an algorithm based on CD38 and CD200 expression by flow cytometry. [Results]: We found 171 differentially expressed genes with enrichment of neoplastic behavior and cell proliferation signatures in MCL. Conversely, MALD1 was enriched in gene sets related to immune activation and inflammatory responses. CD38 and CD200 were differentially expressed betweenMCLandMALD1and confirmed by flow cytometry (median CD38, 89% vs. 14%; median CD200, 0% vs. 24%, respectively). Assessment of both proteins allowed classifying 85% (11 of 13) of MALD1 cases whereas 15% remained unclassified. SOX11 expression by qRT-PCR was significantly different between MCL and MALD1 groups but did not improve the classification. [Conclusion]: We show for the first time that MALD1, in contrast to MCL, is characterized by immune activation and driven by inflammatory cues. Assessment of CD38/CD200 by flow cytometry is useful to distinguish most cases of MALD1 from MCL in the clinical setting. MALD1 should be identified and segregated from the current MCL category to avoid overdiagnosis and unnecessary treatment., This work has been supported, in part, by grants from Instituto de Salud Carlos III RD07/0020/2004, RD09/0076/00036, RD12/0036/0044, (RTICC, FEDER), Generalitat de Catalunya 2009/SGR541, and the “Xarxa de Bancs de Tumors” sponsored by Pla Director d'Oncologia de Catalunya (XBTC).

MEF2B encodes a transcriptional activator and is mutated in ∼11% of diffuse large B cell lymphomas (DLBCLs) and ∼12% of follicular lymphomas (FLs). Here we found that MEF2B directly activated the transcription of the proto-oncogene BCL6 in normal germinal-center (GC) B cells and was required for DLBCL proliferation. Mutation of MEF2B resulted in enhanced transcriptional activity of MEF2B either through disruption of its interaction with the corepressor CABIN1 or by rendering it insensitive to inhibitory signaling events mediated by phosphorylation and sumoylation. Consequently, the transcriptional activity of Bcl-6 was deregulated in DLBCLs with MEF2B mutations. Thus, somatic mutations of MEF2B may contribute to lymphomagenesis by deregulating BCL6 expression, and MEF2B may represent an alternative target for blocking Bcl-6 activity in DLBCLs.

Diffuse large B cell lymphoma (DLBCL) comprises two distinct molecular subtypes: germinal center B cell (GCB) subtype and activated B cell (ABC) subtype. The pathogenesis of ABC-DLBCL is characterized by two processes - the activation of NF-KB and a block in terminal B-cell differentiation. However, in GCB-DLBCL only a few biologically relevant pathways have been identified, which has hampered the development of targeted therapies with specific efficacy in this subtype. Recurrent genetic alterations involved in PI3K-AKT signaling pathway have been identified in the patients with solid cancers, and dramatic responses have been observed to PI3K inhibitors in clinical trials. The prevalence and clinical significances of genetic alterations in PI3K-AKT pathway have not been well studied in DLBCL. Previous studies have reported that loss of PTEN expression was observed in almost half of GCB-DLBCL cases but were largely absent in the ABC-DLBCL. In addition, functional studies have recently shown that the inactivation of Gα13 signaling pathway genes may also activate AKT in germinal center driven lymphoma, raising the possibility that additional genes within these pathways are affected in GCB-DLBCL. Herein, we identified genetic alterations involved in the PI3K-AKT pathway and evaluated their clinical impact. We analyzed biopsies from 347 patients newly diagnosed with de novo DLBCL uniformly treated with R-CHOP at the BC Cancer Agency. High-resolution copy number analyses were performed using Affymetrix SNP 6.0 arrays. Mutation status was determined using deep targeted re-sequencing of the coding exons of 61 genes with a Truseq Custom Amplicon assay (Illumina) and/or Fluidigm Access Array chips, and RNAseq. Immunohistochemical staining of phospho-AKT (pAKT) was performed on tissue microarrays (n=332). Cell-of-origin (COO) was assigned by gene expression (Lymph2Cx assay) in 323 cases - 183 GCB, 104 ABC and 36 unclassifiable. GISTIC analysis revealed several COO-specific peaks with copy number changes. Among them, focal 10q23.3 deletion including PTEN was detected in GCB-DLBCL (q-value=1.7e-7), but not in ABC-DLBCL. We also identified two focal amplification peaks in GCB-DLBCL containing microRNAs MIR17HG (13q31.1;q=1.01e-24) and MIR21 (17q23.3; q=1.45e-6), which are known to down-regulate PTEN and result in activation of PI3K-AKT signaling. Furthermore, we detected recurrent INPP4B deletion (4q21.23) in GCB-DLBCL (q= 0.004) only. Of note, the lipid phosphatase INPP4B has been shown to play a role as a tumor suppressor that controls the levels of PI3K lipid products leading to AKT activation and metastasis of some solid cancers. This is consistent with the observation that PTEN and INPP4B deletions were individually associated with increased pAKT protein expression in our cohort (p=0.015 and p With respect to mutations, GNA13, P2RY8 and ARHGEF1 were more frequently mutated in the GCB than ABC subtype (26% vs 0.6%; p Based on these genetic data, we found that the PI3K-AKT signaling pathway was more commonly altered in GCB-DLBCL (64% (114/177)) than in ABC-DLBCL (23% (23/98) p In conclusion, genetic alterations in the PI3K-AKT pathway were frequently observed in GCB-DLBCL and were associated with activation of the pathway and inferior outcomes. These results suggested that AKT and mTOR inhibitors might be beneficial in a greater proportion of GCB-DLBCL than expected. Fig 1. The distribution of genetic alterations of PI3K-AKT pathway and pAKT protein expression. Fig 1. The distribution of genetic alterations of PI3K-AKT pathway and pAKT protein expression. KM curves according to pAKT protein expression (Fig 2) and PTEN and INPP4B deletions (Fig 3). KM curves according to pAKT protein expression (Fig 2) and PTEN and INPP4B deletions (Fig 3). Disclosures Sehn: roche/genentech: Consultancy, Honoraria; amgen: Consultancy, Honoraria; seattle genetics: Consultancy, Honoraria; abbvie: Consultancy, Honoraria; TG therapeutics: Consultancy, Honoraria; celgene: Consultancy, Honoraria; lundbeck: Consultancy, Honoraria; janssen: Consultancy, Honoraria. Connors:F Hoffmann-La Roche: Research Funding; Millennium Takeda: Research Funding; Seattle Genetics: Research Funding; Bristol Myers Squib: Research Funding; NanoString Technologies: Research Funding. Scott:NanoString Technologies: Patents & Royalties: named inventor on a patent for molecular subtyping of DLBCL that has been licensed to NanoString Technologies.

Modulation of chromatin accessibility through histone modification is a key step in the regulation of gene transcription and its disruption by genetic lesions has been implicated in malignant transformation. Indeed, a consistent theme in recent cancer genome studies has been the discovery of recurrent mutations in multiple histone/chromatin modifier genes, including methyltransferases, acetyltransferases and histone themselves. Among these, KMT2D (MLL2 or MLL4), encoding for a histone H3K4 methyltransferase, emerged as one of the most common targets of genetic lesion in B cell non-Hodgkin lymphoma, being found in ~30% of diffuse large B cell lymphoma (DLBCL) and ~90% of follicular lymphoma (FL), which together account for over 70% of all lymphoma diagnoses (Pasqualucci et al, Nat Genetics 2011; Morin et al, Nature 2011). KMT2D mutations are mostly represented by truncating events that are predicted to remove the protein C-terminal enzymatic domains, thus inactivating its function; however, missense mutations were also found in a subset of cases, suggesting selection for a functional role. These events are biallelically distributed in one third of mutated cases, while the remaining >60% harbor monoallelic mutations, consistent with a role as a tumor suppressor. Interestingly, analysis of the history of clonal evolution during FL transformation to DLBCL suggests that KMT2D mutations may be already present in a common precursor clone before divergent evolution to FL or DLBCL, suggesting an early role during B cell clonal expansion (Pasqualucci et al, Cell Rep, 2014; Green et al, Blood, 2013). To elucidate the functional consequences of KMT2D mutations, we first examined the effects of 16 DLBCL/FL-derived KMT2D missense mutant alleles on its enzymatic activity in vitro. The results showed that all 8 mutants located in the C-terminal portion of the protein were associated with significantly diminished H3K4 mono-, di- and tri-methylation activity. Consistently, a significant reduction in global methylation was observed in Kmt2d deficient murine B-cells, as well as in 4 biallelically truncated cell lines, indicating that this methyltransferase can influence all three H3K4 modifications. To gain further insights into the program regulated by KMT2D in germinal center (GC) B cells (i.e. the normal counterpart of FL/DLBCL) and the mechanism by which its loss contributes to lymphomagenesis, we crossed mice carrying a conditional Kmt2d knockout allele (Lee J et al, Elife, 2013) with either CD19Cre or Cγ1Cre deletor mice, leading to gene inactivation early during B-cell development (Kmt2dCD19KO), thus mimicking the postulated “common precursor model,” or specifically in mature, GC B-cells (Kmt2dCγ1KO). Notably, deletion of Kmt2d before, but not after initiation of the GC reaction led to a significant increase in GC B-cells and enhanced B cell proliferation. These changes were accompanied by the acquisition of distinct transcriptional signatures enriched in cell cycle regulation and apoptosis genes. A cross-species strategy combining gene expression profile analysis of murine GC B-cells and Kmt2d chromatin immunoprecipitation and sequencing of purified human GC B cells identified a core of KMT2D direct targets genes involved in biological programs with critical functions in B cells physiology, including B cell receptor signaling, lymphocyte migration, and chemokine signaling components. Finally, while loss of Kmt2d alone in Kmt2dCγ1KO was not sufficient to induce tumor development, its combination with VavP-BCL2 transgenic mice increased the incidence of GC-derived lymphomas resembling the features of the human tumors. These data support a role for KMT2D as a tumor suppressor gene whose early loss during B cell development facilitates lymphomagenesis by remodeling the epigenetic landscape of the cancer precursor cell. Citation Format: Jiyuan Zhang, David Dominguez-Sola, Shafinaz Hussein, Ji-Eun Lee, Antony B. Holmes, Mukesh Bansal, Sofija Vlasevska, Tongwei Mo, Hongyan Tang, Katia Basso, Kai Ge, Riccardo Dalla-Favera, Laura Pasqualucci. Disruption of KMT2D-dependent histone methylation perturbs GC B cell development and cooperates with BCL2 deregulation in lymphomagenesis. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B25.

Germinal centers (GCs) are sites of B-cell clonal expansion, hypermutation, and selection. GCs are polarized into dark (DZ) and light zones (LZ), a distinction that is of key importance to GC selection. However, the difference between the B cells in each of these zones in humans remains unclear. We show that, as in mice, CXCR4 and CD83 can be used to distinguish human LZ and DZ cells. Using these markers, we show that LZ and DZ cells in mice and humans differ only in the expression of characteristic “activation” and “proliferation” programs, suggesting that these populations represent alternating states of a single-cell type rather than distinct differentiation stages. In addition, LZ/DZ transcriptional profiling shows that, with the exception of “molecular” Burkitt lymphomas, nearly all human B-cell malignancies closely resemble LZ cells, which has important implications for our understanding of the molecular programs of lymphomagenesis.

B-cell non-Hodgkin’s lymphoma comprises biologically and clinically distinct diseases the pathogenesis of which is associated with genetic lesions affecting oncogenes and tumour-suppressor genes. We report here that the two most common types—follicular lymphoma and diffuse large B-cell lymphoma—harbour frequent structural alterations inactivating CREBBP and, more rarely, EP300, two highly related histone and non-histone acetyltransferases (HATs) that act as transcriptional co-activators in multiple signalling pathways. Overall, about 39% of diffuse large B-cell lymphoma and 41% of follicular lymphoma cases display genomic deletions and/or somatic mutations that remove or inactivate the HAT coding domain of these two genes. These lesions usually affect one allele, suggesting that reduction in HAT dosage is important for lymphomagenesis. We demonstrate specific defects in acetylation-mediated inactivation of the BCL6 oncoprotein and activation of the p53 tumour suppressor. These results identify CREBBP/EP300 mutations as a major pathogenetic mechanism shared by common forms of B-cell non-Hodgkin’s lymphoma, with direct implications for the use of drugs targeting acetylation/deacetylation mechanisms. In three different subtypes of B-cell lymphomas, two papers report frequent somatic mutations in the genes CREBBP and EP300, which are present in primary tumours or acquired at relapse. These genes encode related acetyltransferases that mainly function to regulate gene expression by acetylating histones and other transcriptional regulators. The mutations disrupt these activities and thus alter chromatin regulation of gene expression, as well as proliferation and potentially the response to anticancer drugs. These studies may provide a rationale for the use of histone deacetylase inhibitors in certain B-cell lymphomas. In three different subtypes of B-cell lymphomas, two papers now report frequent somatic mutations in CREBBP and EP300, present in primary tumours or acquired at relapse. These genes encode related acetyltransferases that mainly function to regulate gene expression by acetylating histones and other transcriptional regulators. The mutations found inactivate these activities and thus alter chromatin regulation of gene expression, as well as proliferation and potentially the response to therapeutic drugs.

Abstract 264 Splenic marginal zone lymphoma (SMZL) is one of few B-cell lymphoma types that remain orphan of molecular lesions in cancer related genes. Detection of active NF-κB signaling by immunohistochemical assays for nuclear NFKB1 (p50) and NFKB2 (p52) in 14/24 (58%) SMZL prompted the investigation of NF-κB molecular alterations in SMZL (Fig.1A). To this aim, NF-κB pathway genes (n=20) were screened in 101 SMZL for mutations by Sanger sequencing and for copy number abnormalities (CNAs) by FISH and SNP array (GeneChip Human Mapping 250K NspI, Affymetrix). Mutations targeting multiple NF-κB genes were detected in 20/101 (20%) SMZL. Mutations affected key regulators of both canonical (IKBKB, TNFAIP3) and non-canonical (BIRC3, TRAF3, MAP3K14) NF-κB pathways and were mutually exclusive, pointing to multiple molecular mechanisms targeting NF-κB in SMZL (Fig.1B). By combining mutations and CNAs, NF-κB was affected in 36/101 (36%) SMZL. The TRAF3/MAP3K14-TRAF2/BIRC3 regulatory complex of the non-canonical NF-κB pathway was targeted in 25/101 (25%) SMZL. BIRC3 was affected in 11/101 (11%) SMZL by inactivating mutations (3 frameshift and 2 non-sense), missense mutations (n=1), and gene deletions (n=5). All inactivating mutations were somatically acquired, were predicted to generate aberrant transcripts carrying premature stop codons, and caused elimination or truncation of the C-terminal RING domain, whose E3 ubiquitin ligase activity is essential for MAP3K14 proteosomal degradation by BIRC3 (Fig. 1C). TRAF3 was affected in 10/101 (10%) SMZL by inactivating mutations (2 frameshift and 1 non-sense), missense mutations (n=1) and deletions (n=7). Inactivating mutations were predicted to generate aberrant transcripts carrying premature stop codons and causing elimination or truncation of the C-terminal MATH domain that provides the docking site for MAP3K14, and is required for MAP3K14 recruitment to BIRC3 degradation. MAP3K14 was affected by gain (n=7) or missense mutations (n=1) in 8/101 (8%) SMZL. The missense mutation was somatically acquired and mapped near the kinase domain, suggesting a potential role in MAP3K14 downstream signaling activation. SMZL primary cells harboring BIRC3, TRAF3 or MAP3K14 mutations displayed constitutive NF-κB activation, including MAP3K14 accumulation and active NFKB2 processing by Western blot, and/or nuclear NF-κB localization by immunohistochemistry (Fig. 1D). In addition to non-canonical signaling, also the canonical NF-κB pathway was targeted in SMZL (15/101, 15%) by genetic lesions of TNFAIP3 and IKBKB, which encode for negative and positive regulators of NF-κB signaling, respectively. TNFAIP3 was affected in 13/101 (13%) SMZL by inactivating mutations (6 frameshift) and deletions (n=9), including a focal loss pointing to TNFAIP3 as the specific target of deletions. IKBKB was mutated in 3/101 (3%) SMZL carrying a recurrent, somatically acquired missense mutation (K171E) targeting the IKBKB kinase domain near the activation loop. This mutation leads to an amino acid substitution within a site that is conserved both intra- and inter-species, and is predicted as possibly damaging according to PolyPhen-2. The occurrence of NF-κB molecular lesions in SMZL was observed independent of HCV infection (p=.402), IGHV gene mutation status (p=.065), IGHV1-2 gene usage (p=.761), stereotyped VH CDR3 (p=.969), 7q deletion (p=.621), or TP53 disruption (p=.638), suggesting that genetic deregulation of NF-κB is not restricted to specific SMZL clinico-molecular subgroups. The identification of NF-κB mutations in SMZL elucidates the molecular basis of a fraction of these lymphomas, and expands the genetic mechanisms activating NF-κB in B-cell neoplasia. Beside its pathogenetic relevance, the identification of NF-κB as a pathway recurrently involved in SMZL provides the rationale for targeted anti-NF-κB therapeutic approaches in this lymphoma. Disclosures: No relevant conflicts of interest to declare.

The c-Myc proto-oncogene encodes a transcription factor that is essential for cell growth and proliferation and is broadly implicated in tumorigenesis. However, the biological functions required by c-Myc to induce oncogenesis remain elusive. Here we show that c-Myc has a direct role in the control of DNA replication. c-Myc interacts with the pre-replicative complex and localizes to early sites of DNA synthesis. Depletion of c-Myc from mammalian (human and mouse) cells as well as from Xenopus cell-free extracts, which are devoid of RNA transcription, demonstrates a non-transcriptional role for c-Myc in the initiation of DNA replication. Overexpression of c-Myc causes increased replication origin activity with subsequent DNA damage and checkpoint activation. These findings identify a critical function of c-Myc in DNA replication and suggest a novel mechanism for its normal and oncogenic functions.

Chromosomal translocations causing deregulated c-MYC expression are detectable in most Burkitt lymphoma cases. However, little is known about the additional lesions necessary for lymphomagenesis. Now, two independent studies, one of which was performed by Sander et al. in this issue of Cancer Cell, identify constitutive PI3K signaling and CyclinD3 mutations as cooperating lesions in both mice and humans. The results have directly actionable therapeutic implications.

A significant fraction of B cell non-Hodgkin lymphomas (B-NHL) of germinal center origin carry heterozygous missense mutations in FOXO1, a member of the FOXO family of transcription factors. FOXO1 is a central component of the PI3K signaling cascade engaged by the B cell receptor and is essential for B cell homeostasis and survival (Dengler et al, Nat Immunol 2008; Srinivasan et al, Cell 2009; Lin et al, Nat Immunol 2010). In response to PI3K activation, AKT phosphorylates FOXO1 leading to its nuclear-cytoplasmic translocation and inactivation. Missense mutations of the FOXO1 gene are detectable in germinal center (GC)-derived B-NHL, including ~12% of Burkitt Lymphoma (BL) and ~9% of Diffuse Large B Cell Lymphoma (DLBCL) cases (Schmitz et al, Nature 2012; Trinh et al, Blood 2013; Pasqualucci et al, Cell Rep 2014). The role of FOXO1 in normal GC development as well as the contribution of its mutations to lymphomagenesis is unclear. We show that FOXO1 expression is restricted to the dark zone of GCs, where its nuclear localization is detectable in most B cells. Mice carrying the conditional inactivation of FOXO1 in GC B cells display normal GC in number and size. However, these GCs lack phenotypically defined (CXCR4hi/CD86lo) dark zones and are entirely composed by light zone B cells (CXCR4lo/CD86hi). FOXO1-/- GC B cells express AICDA and carry a normal number of mutations in their immunonoglobulin genes, but do not undergo affinity maturation, resulting in severely impaired antigen responses. In order to identify the biological program controlled by FOXO1 in GC B cells, we identified candidate transcriptional target genes by integrating ChIP-seq and gene expression data. These analyses showed that that the establishment of the dark zone fate relies on a FOXO1-dependent transcriptional network that is enriched for genes involved in immune signaling cascades triggered by the B cell receptor and by a variety of cytokines controlling GC polarity. Notably, a majority of these target genes are co-bound and co-regulated, in a FOXO1-dependent manner, by BCL6, a well characterized GC master regulator. To assess the role of BL- and DLBCL-associated mutations, we first investigated the subcellular localization of FOXO1 mutant proteins by transfecting wild type and mutant GFP-tagged FOXO1 alleles into HeLa cells. As previously shown (Trinh et al, Blood 2013), this analysis showed that mutant FOXO1 proteins, but not the wild-type one, readily localize in the nucleus. Analogously, immunofluorescence analysis of BL and DLBCL samples showed the presence of nuclear FOXO1 in all tumors carrying mutations in the FOXO1 gene. However, nuclear localization was also detectable in virtually all cases carrying normal FOXO1 genes. Accordingly, in vitro experiments testing the ability of normal and mutated FOXO1 proteins to respond to various signals activating the PI3K pathway in multiple BL and DLBCL cell lines, failed to display a correlation between the presence of mutations and responsiveness to these signals. Taken together, these results suggest that other mechanisms in addition to direct gene mutation are responsible for the constitutive nuclear localization of FOXO1 in tumors. We are now examining the consequences of FOXO1 missense mutations in vivo, by reconstituting FOXO1-/- GC B cells with FOXO1 mutants using bone marrow chimeras. Disclosures No relevant conflicts of interest to declare.

Diffuse large B cell lymphoma (DLBCL) is the most common form of B cell non-Hodgkin lymphoma (B-NHL), accounting for ~25-40% of all lymphoid tumors. DLBCL comprises genetically, phenotypically and clinically distinct subtypes, including the prognostically favorable germinal center B cell like (GCB)-DLBCL and the more aggressive activated B cell like (ABC)-DLBCL. We have shown that >60% of DLBCL, independent of molecular subtype, lack cell surface expression of HLA-class I (HLA-I), suggesting that these tumors may escape immune recognition by cytotoxic T cells (CTL) (Challa-Malladi, Lieu et al., Cancer Cell, 2011). HLA-I loss also represents a common lesion acquired at transformation of follicular lymphoma (FL) to DLBCL (Pasqualucci et al., Cell Reports 2014). We have investigated the expression of HLA-I across the clinico-pathological spectrum of mature B cell tumors, and found that HLA-I loss is significantly less common in other mature B-NHL, including Burkitt lymphoma (13/43, 30.2%; p=.002), FL (12/60, 20.0%; p One mechanism of HLA-I loss, identified by exome-sequencing and copy number analysis, is represented by genomic deletions and/or mutational inactivation of the B2M gene, which are found in ~50% of HLA-I negative cases (29% of all DLBCL). These lesions lead to the complete loss of B2-microglobulin, a required component for the assembly and cell surface expression of the HLA-I complex (Pasqualucci et al. Nat Genet, 2011; Challa-Malladi, Lieu et al. Cancer Cell, 2011). However, the remaining ~50% of patients lack surface HLA-I despite the absence of B2M genetic lesions, suggesting the existence of additional underlying mechanisms. In particular, a fraction of patients express an intact B2M protein, which is mislocalized to the cytoplasm. To investigate whether direct genetic disruption of the HLA-I genes could be responsible for the lack of surface HLA-I in these cases, we performed Sanger sequencing and SNP6.0 array analysis of the HLA-I heavy chain genes (HLA-A and HLA-B) in two DLBCL cell lines (Ly10 and RCK8) with wild-type B2M alleles, but cytoplasmic B2M protein. In both lines, we found the presence of biallelic mutations or deletions in the HLA-I loci. Accordingly, transduction with a retrovirus expressing either HLA-I gene was sufficient to restore cell surface B2M and HLA-I in both lines, documenting that DLBCL can exploit genetic disruption of HLA-I as an alternative mechanism to impair the assembly of a membrane HLA-I complex. The overall contribution of this mechanism to HLA-I loss is currently being determined by using a custom capture/next generation sequencing approach of the HLA-I loci in a large panel of paired tumor/normal biopsies with negative or mislocalized B2M/HLA-I. We also examined the role of B2M (HLA-I) loss in lymphomagenesis in vivo. Particularly, since constitutional B2m deletion is not tumorigenic per se (Koller et al., Science 1990), and B2M loss is frequently acquired during FL transformation to DLBCL, we investigated whether the absence of major histocompatibility complex on the cell surface of mature B cells accelerates tumorigenesis in the presence of other oncogenic lesions. To this end, we generated a conditional knock-out mouse model in which the B2m gene is specifically deleted in germinal center B cells upon expression of a Cγ1-Cre allele, and crossed them with IµHABCL6 knock-in mice, which develop DLBCL due to deregulated expression of the BCL6 oncogene (Cattoretti, Pasqualucci et al., Cancer Cell 2006). Disclosures No relevant conflicts of interest to declare.

The MYC oncogene is a multifunctional protein that is aberrantly expressed in a significant fraction of tumors from diverse tissue origins. Because of its multifunctional nature, it has been difficult to delineate the exact contributions of MYC’s diverse roles to tumorigenesis. Here, we review the normal role of MYC in regulating DNA replication as well as its ability to generate DNA replication stress when overexpressed. Finally, we discuss the possible mechanisms by which replication stress induced by aberrant MYC expression could contribute to genomic instability and cancer.

The proto-oncogenic transcription factor c-Myc is a central regulator of the cell cycle and cell growth. Amino-terminal phosphorylation of c-Myc results in its proteasomal degradation. A new study shows that its dephosphorylation is regulated by the Pin1 prolyl isomerase and PP2A phosphatase, and that stabilized c-Myc can replace SV40 small T antigen in the oncogenic transformation of human cells.

Abstract 1284 Diffuse large B-cell lymphoma (DLBCL) and Follicular Lymphoma (FL) are the most common forms of non-Hodgkin's lymphoma in the adult, accounting for approximately 75% of lymphoma diagnoses. Recent technological advances, including whole-genome DNA and RNA sequencing and gene copy-number analysis, have provided a comprehensive view of the genomic landscape of DLBCL, allowing new insights in the somatic genetic lesions that are associated with the pathogenesis of this malignancy. Among the genetic alterations that are recurrently found in DLBCL and FL, but remain of unclear functional significance, are the mutations involving the MEF2B gene. MEF2B is a member of the myocyte enhancer-binding factor 2 (MEF2) family of transcription factors whose activity is dependent on association with specific co-repressors (including CABIN1 and HDACs) and co-activators in response to multiple signaling pathways. Overall, ∼11% of DLBCL and ∼12% of FL cases reported carry mutations in MEF2B (Morin Nature 2011; Pasqualucci Nat Genet 2011; Lohr PNAS 2012). We showed that within the mature B-cell lineage, MEF2B expression is restricted to germinal center (GC) B-cells. The analysis of the B-cell interactome, a network of protein-protein and protein-DNA interactions generated by reverse-engineering a large dataset of B-cell phenotypes, showed that MEF2B was uniquely connected to BCL6, a proto-oncogene and well-characterized master regulator of the GC reaction. We demonstrated that MEF2B directly binds to the promoter of BCL6 and leads to its trans-activation in GC B-cells. Consistently, silencing of MEF2B in GC-derived lymphoma cell lines led to BCL6 down-regulation and impairment of cell cycle progression and proliferation, suggesting that normal and malignant GC cells are dependent on MEF2B expression. Approximately 80% of the DLBCL and FL mutated cases carry missense mutations clustered in the N-terminal conserved MADS-box and MEF2 functional domains, suggesting that they may have a relevant impact on MEF2B function. In a second group of cases (∼20%), mutations affect the C-terminal half of the MEF2B protein, and are mostly represented by frameshift and nonsense mutations, which truncate or modify the C-terminus of the protein. In order to functionally characterize these mutations, we first investigated whether DLBCL- and FL-associated MEF2B mutations affected the ability to regulate the transcription of BCL6. Using a reporter construct containing the native BCL6 promoter region responsive to MEF2B, we demonstrated that most of the N-terminal mutations cause increased transcriptional activity as tested on the BCL6 promoter. The analysis of the N-terminal MEF2B crystal structure, upon mapping the mutated residues, predicted that these mutations may interfere with the ability of MEF2B to heterodimerize with the CABIN1 co-repressor. Indeed, we showed that these MEF2B mutant proteins fail to bind CABIN1 and are resistant to its transrepressive activity. Conversely, C-terminal MEF2B mutations lead to truncated MEF2B proteins lacking the domains responsive to two independent post-transcriptional modifications, namely PKA-mediated phosphorylation and sumoylation. We showed that MEF2B is in fact phosphorylated by PKA and sumoylated in vivo, that both of these modifications lead to negative regulation of MEF2B transcriptional activity, and that lymphoma-associated C-terminal mutants fail to be negatively regulated by PKA-mediated phosphorylation and sumoylation. In summary, these results identify MEF2B as an upstream regulator of BCL6 and GC formation, which is required for lymphoma proliferation. Lymphoma-associated MEF2B mutations may contribute to lymphomagenesis, at least in part, by deregulating the expression of the BCL6 oncogene. Thus, targeting MEF2B may represent an alternative therapeutic approach to block BCL6 and cell proliferation in DLBCL and FL. Disclosures: No relevant conflicts of interest to declare.

Abstract 474 B cell non-Hodgkin lymphoma (B-NHL) comprises a variety of biologically and clinically distinct diseases whose pathogenesis is associated with largely unique genetic lesions affecting oncogenes and tumor suppressor genes. The identification of the complete set of genes and cellular pathways that are altered in the lymphoma cell is critical for a full understanding of the genesis and maintenance of these malignancies. Using genome-wide copy number analysis and high throughput whole exome sequencing, we have recently found that the two most common types of B-NHL, follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL), are associated with frequent structural alterations inactivating the genes that encode CREBBP and, in a smaller fraction of cases, EP300, two highly related histone and non-histone acetyltranferases (HAT) that act as transcriptional coactivators in multiple signaling pathways. Overall, ~29% (n=39/134) of DLBCL and 32% (n=15/47) of FL samples display genomic deletions and/or somatic point mutations that remove or inactivate the HAT domain of CREBBP. In 19 additional cases (15 DLBCL and 4 FL), mutations and deletions were found in the EP300 gene, also predicting its functional loss. With few exceptions, structural alterations of CREBBP and EP300 were mutually exclusive, suggesting that inactivation of these loci is at least in part functionally equivalent. Thus, in total ~36% of all DLBCL (n=49/134) and at least 40% of FL cases (n=19/47) display genetic aberrations targeting acetyltransferase genes. Interestingly, while these lesions were observed in both DLBCL phenotypic subtypes, their frequency appears to be significantly higher in germinal center B cell type DLBCL, where they account for ~46% of the cases (n=30/65) as compared to 27% in activated B cell type DLBCL (n=11/69; p Disclosures: No relevant conflicts of interest to declare.