Germinal Center-derived B-cell Lymphomas Research Articles

Development of Syngeneic Murine Cell Lines of Germinal Center-Derived B-Cell Lymphomas

Background. Germinal center (GC)-derived B-cell lymphomas are clinically and biologically heterogeneous. Next generation sequencing has enabled identification of genetic aberrations involved in lymphoma pathogenesis, which may provide prognostic predictions and potential therapeutic targets. However, the mechanisms driving pathogenesis are still not fully understood, especially in the context of an intact tumor microenvironment, in part because there are no available syngeneic models of lymphoma faithfully representing the pathological features of human disease. Therefore, there is a critical need for lymphoma models which can be studied in immunocompetent mice. Methods. We established eight murine cell lines derived from spontaneous B-cell lymphomas with GC origin arising in genetically engineered mouse models (GEMM). These cell lines were developed by passing an original tumor from a GEMM through several passages of Rag1-/- and wild type mice, which enabled selection of more fit/aggressive clones that grow in vitro. We also tested multiple culture conditions and characterized the lines via flow cytometry, VDJ and targeted sequencing, karyotyping, and evaluation of tumor development and its microenvironment in vivo. Results. This resource describes eight cell lines driven by recurrent genetic mutations found in diffuse large B-cell lymphoma and follicular lymphoma patients. The first cell line is defined by (1) BCL2 overexpression (hereafter B2). Four cell lines couple BCL2 overexpression with epigenetic dysregulation, defined by (2) BCL2 and gain-of-function (GOF) mutation of EZH2 Y641F (hereafter EZB), (3) EZB and MYC overexpression (hereafter EBM), (4) BCL2 with knockout of KMT2D (hereafter KB), and (5) BCL2 with knockout of SETD2 (hereafter S2B). Finally, three cell lines are defined by mutations to the Toll-like and B-cell receptors pathways, defined by (6) BCL2 and GOF mutations in Myd88 L252P and CD79b Y195H (hereafter MBC79), (7) BCL2, Myd88 L252P and GOF mutation in Card11 L251P (hereafter MBC11), and (8) BCL2 and Card11 L251P (hereafter BC11). Doubling time of these lines ranges from 30-72 hours, with EZB and EBM on the fast end and S2B on the slow end. By flow cytometry, we show that B2, MBC79, and MBC11 lose B-cell surface markers and are very plasmacytic (>95% B220- CD19- CD138+). In contrast, the EZB, EBM, KB, BC11, and S2B are mostly characterized by GC B-cell markers (B220+ CD19+ CD138- FAS+ CD38- GL7+). By VDJ sequencing, we found that EZB, BC11, and S2B are very monoclonal, with one rearrangement arising from the original tumor and taking over to represent >95% of clones. In contrast, the MCB11 and MBC79 lines are very polyclonal, with the top 10 rearrangements being equally represented, and tending to shift between passages. Thus far, S2B and EZB have been shown to grow in immunocompetent mice, representing novel syngeneic lymphoma models. While mice injected with S2B can survive for up to 30 days, mice injected with EZB die significantly earlier. We established two EZB lines from early (P1) and late (P6) passages of a tumor arising from the same original mouse. Notably, we show that mice injected with P1 survive 15-20 days, whereas mice injected with P6 survive 10-15 days. Although both P1 and P6 grow at similar rates in vitro, P1 develops high grade follicular lymphomas in C57BL6 recipients, whereas P6 develops DLBCL, suggesting additional passaging drives selection for more aggressive disease. Imaging of the tumors reveals a lack of follicular pattern. Notably, P6 tumors display less infiltration of CD4 and CD8 T-cells and low abundance or absence of FDC meshwork, as compared to P1. Imaging of mice engrafted with luciferase transduced P1 and P6 shows dissemination into spleen, lymph nodes, liver, and kidney, with more advanced disease in P6 injected mice. Conclusion. While conventional preclinical tumor mouse models are often used for drug sensitivity and treatment prediction studies, they are time-consuming and expensive. These cell lines serve as a preclinical tool to test novel therapeutic strategies and develop a deeper understanding of the molecular mechanisms driving lymphomagenesis, therapeutic response, and resistance in genetically defined subtypes of GC-derived B-cell lymphomas. Most importantly, this resource meets a critical need in the field for syngeneic models of lymphoma to study disease progression and precision therapy in immunocompetent mice.

MYC to BCL6 State-Transitions Determine Cell Size and Metabolic Fluctuations and Define a Novel Biorhythm in B-Cell Malignancies

Background: Cell size fluctuations are well-documented during critical transitions during normal B-cell development. For instance, during early stages of development, “large cycling pre-B cells” (also classified as Fraction C') eventually exit cell cycle and shrink to become “small resting pre-B cells” (Fraction D) to enable immunoglobulin light chain gene recombination. We and others had shown that this transition is marked by loss of Myc expression and gain of Bcl6. Later in development, germinal center B-cells cycle between large MYC+ “centroblast” and smaller BCL6+ “centrocyte” states between dark and light zones, respectively. MYC is involved in biomass accumulation and provides fuel for cell division, while BCL6 confers a quiescent phenotype to cells and protects B cells from DNA damage-induced apoptosis. BCL6 can suppress MYC transcription, indicating MYC and BCL6 is mutually exclusive, and their expression dictates distinct cellular states. Significance: In B-cell malignancies, transitions between mutually exclusive MYC+ and BCL6+ states seem to be important as well: B-ALL cells that are driven by oncogenic tyrosine kinases (e.g. BCR-ABL1) express predominantly MYC but can be forced into a BCL6+ state upon tyrosine kinase inhibition (e.g. imatinib). In germinal center-derived B-cell lymphoma, both MYC and BCL6 are frequently targeted by chromosomal translocation, suggesting that disruption of physiological state transitions may be part of the malignant transformation program. Results: To elucidate regulation of MYC-BCL6 state transitions and their importance in progression and development of drug resistance in B-ALL and germinal center-derived B-cell lymphoma, we developed a MYC-eGFP and BCL6-mCherry dual-reporter mouse model. In addition, we engineered human B-ALL PDX cells with MYC-mNeonGreen and BCL6mScarlet knockin fusion genes by CRISPR/Cas9-mediated HDRT. As expected, MYC+ and BCL6+ states were largely mutually exclusive during early stages of B-cell development, as well as in BCR-ABL1- and NRAS G12D-driven B-ALL. Interestingly, a large fraction of B-ALL cells expressed neither MYC nor BCL6, as validated by cell sorting and Western blot. Based on single-cell sorting and subsequent time-lapse monitoring over 12 hours, we found MYC+ cells transitioning through a double-negative state to become BCL6+ and then revert again to a MYC+ state ( Figure A), suggesting dynamic transitions between MYC and BCL6 cycles. We characterized these populations by analyzing cell size, cell proliferation, cell cycle, clone formation, and gene expression profiles. The results indicated that MYC+ cells exhibited larger cell size, active proliferation, and increased glycolytic activity. Conversely, BCL6+ cells displayed smaller cell size, activation of autophagy with suppression of glycolysis, and cell cycle arrest in the G0 phase of the cell cycle. Conclusions and future directions: Given the dynamic inter-transition between MYC and BCL6 states, and the significant roles of both MYC and BCL6 in B-ALL and germinal center-derived B-cell lymphomas, we propose MYC- and BCL6-dependent fluctuations of cellular activity (wake) and quiescence (sleep; Figure B). Thereby, MYC and BCL6 are expressed in an oscillatory manner. During the MYC state, cells exhibit high glycolysis activity, actively accumulate biomass, and undergo proliferation, representing the wake phase. In contrast, during the BCL6 state, cells suppress glycolysis metabolism, pause the cell cycle, and display a quiescent phenotype, representing the sleep phase. Based on this discovery, we will examine the ‘B-cell exhaustion’ paradigm that Bcl6 and Myc mark iterative cycles of quiescence and activation. In analogy to sleep-wake phases, we hypothesize that the Bcl6-autophagy phase is essential for recovery and regeneration. We will investigate how B-cells transition from one phase to the next and consequences of elongation of one phase at the expense of the other. Malignant B-cells may be particularly sensitive to shortening of quiescence periods (“sleep deprivation”). These and other observations will lead to new conceptual frameworks for the understanding of how MYC-BCL6 state transitions, cell-size fluctuations and the length of recovery-periods regulate energy-supply and survival of B-cell malignancies and create opportunities for therapeutic intervention.

The RNA helicase DDX3 and its role in c-MYC driven germinal center-derived B-cell lymphoma.

DDX3X is an RNA helicase with many functions in RNA metabolism such as mRNA translation, alternative pre-mRNA splicing and mRNA stability, but also plays a role as a regulator of transcription as well as in the Wnt/beta-catenin- and Nf-κB signaling pathways. The gene encoding DDX3X is located on the X-chromosome, but escapes X-inactivation. Hence females have two active copies and males only one. However, the Y chromosome contains the gene for the male DDX3 homologue, called DDX3Y, which has a very high sequence similarity and functional redundancy with DDX3X, but shows a more restricted protein expression pattern than DDX3X. High throughput sequencing of germinal center (GC)-derived B-cell malignancies such as Burkitt Lymphoma (BL) and Diffuse large B-cell lymphoma (DLBCL) samples showed a high frequency of loss-of-function (LOF) mutations in the DDX3X gene revealing several features that distinguish this gene from others. First, DDX3X mutations occur with high frequency particularly in those GC-derived B-cell lymphomas that also show translocations of the c-MYC proto-oncogene, which occurs in almost all BL and a subset of DLBCL. Second, DDX3X LOF mutations occur almost exclusively in males and is very rarely found in females. Third, mutations in the male homologue DDX3Y have never been found in any type of malignancy. Studies with human primary GC B cells from male donors showed that a loss of DDX3X function helps the initial process of B-cell lymphomagenesis by buffering the proteotoxic stress induced by c-MYC activation. However, full lymphomagenesis requires DDX3 activity since an upregulation of DDX3Y expression is invariably found in GC derived B-cell lymphoma with DDX3X LOF mutation. Other studies with male transgenic mice that lack Ddx3x, but constitutively express activated c-Myc transgenes in B cells and are therefore prone to develop B-cell malignancies, also showed upregulation of the DDX3Y protein expression during the process of lymphomagenesis. Since DDX3Y is not expressed in normal human cells, these data suggest that DDX3Y may represent a new cancer cell specific target to develop adjuvant therapies for male patients with BL and DLBCL and LOF mutations in the DDX3X gene.

Identification of two unannotated miRNAs in classic Hodgkin lymphoma cell lines.

MicroRNAs (miRNAs) are small non coding RNAs responsible for posttranscriptional regulation of gene expression. Even though almost 2000 precursors have been described so far, additional miRNAs are still being discovered in normal as well as malignant cells. Alike protein coding genes, miRNAs may acquire oncogenic properties in consequence of altered expression or presence of gain or loss of function mutations. In this study we mined datasets from miRNA expression profiling (miRNA-seq) of 7 classic Hodgkin Lymphoma (cHL) cell lines, 10 non-Hodgkin lymphoma (NHL) cell lines and 56 samples of germinal center derived B-cell lymphomas. Our aim was to discover potential novel cHL oncomiRs not reported in miRBase (release 22.1) and expressed in cHL cell lines but no other B-cell lymphomas. We identified six such miRNA candidates in cHL cell lines and verified the expression of two of them encoded at chr2:212678788-212678849 and chr5:168090507-168090561 (GRCh38). Interestingly, we showed that one of the validated miRNAs (located in an intron of the TENM2 gene) is expressed together with its host gene. TENM2 is characterized by hypomethylation and open chromatin around its TSS in cHL cell lines in contrast to NHL cell lines and germinal centre B-cells respectively. It indicates an epigenetic mechanism responsible for aberrant expression of both, the TENM2 gene and the novel miRNA in cHL cell lines. Despite the GO analysis performed with the input of the in silico predicted novel miRNA target genes did not reveal ontologies typically associated with cHL pathogenesis, it pointed to several interesting candidates involved in i.e. lymphopoiesis. These include the lymphoma related BCL11A gene, the IKZF2 gene involved in lymphocyte development or the transcription initiator GTF2H1.

Mutational landscape of high-grade B-cell lymphoma with MYC-, BCL2 and/or BCL6 rearrangements characterized by whole-exome sequencing.

High-grade B-cell lymphoma accompanied with double/triple-hit MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) poses a cytogenetically-defined provisional entity among aggressive B-cell lymphomas that is traditionally associated with unfavorable prognosis. In order to better understand the mutational and molecular landscape of HGBL-DH/TH we here performed whole-exome sequencing and deep panel next-generation sequencing of 47 clinically annotated cases. Oncogenic drivers, mutational signatures and perturbed pathways were compared with data from follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL). We find an accumulation of oncogenic mutations in NOTCH, IL6/JAK/STAT and NFκB signaling pathways and delineate the mutational relationship within the continuum between FL/DLBCL, HGBL-DH/TH and BL. Further, we provide evidence of a molecular divergence between BCL2 and BCL6 rearranged HGBL-DH. Beyond a significant congruency with the C3/EZB DLBCL cluster in BCL2 rearranged cases on an exome-wide level, we observe an enrichment of the SBS6 mutation signature in BCL6 rearranged cases. Differential gene set enrichment and subsequent network propagation analysis according to cytogenetically defined subgroups revealed an impairment of TP53 and MYC pathway signaling in BCL2 rearranged cases, whereas BCL6 rearranged cases lacked this enrichment, but instead showed impairment of E2F targets. Intriguingly, HGBL-TH displayed intermediate mutational features considering all three aspects. This study elucidates a recurrent pattern of mutational events driving FL into MYC-driven BCL2-rearranged HGBL, unveiling the mutational pathogenesis of this provisional entity. Through this refinement of the molecular taxonomy for aggressive, germinal center-derived B-cell lymphomas, this calls into question the current World Health Organization classification system, especially regarding the status of MYC/BCL6-rearranged HGBL.

Mouse Models of Germinal Center Derived B-Cell Lymphomas.

Over the last decades, the revolution in DNA sequencing has changed the way we understand the genetics and biology of B-cell lymphomas by uncovering a large number of recurrently mutated genes, whose aberrant function is likely to play an important role in the initiation and/or maintenance of these cancers. Dissecting how the involved genes contribute to the physiology and pathology of germinal center (GC) B cells –the origin of most B-cell lymphomas– will be key to advance our ability to diagnose and treat these patients. Genetically engineered mouse models (GEMM) that faithfully recapitulate lymphoma-associated genetic alterations offer a valuable platform to investigate the pathogenic roles of candidate oncogenes and tumor suppressors in vivo, and to pre-clinically develop new therapeutic principles in the context of an intact tumor immune microenvironment. In this review, we provide a summary of state-of-the art GEMMs obtained by accurately modelling the most common genetic alterations found in human GC B cell malignancies, with a focus on Burkitt lymphoma, follicular lymphoma, and diffuse large B-cell lymphoma, and we discuss how lessons learned from these models can help guide the design of novel therapeutic approaches for this disease.

Mutational mechanisms shaping the coding and noncoding genome of germinal center derived B-cell lymphomas

B cells have the unique property to somatically alter their immunoglobulin (IG) genes by V(D)J recombination, somatic hypermutation (SHM) and class-switch recombination (CSR). Aberrant targeting of these mechanisms is implicated in lymphomagenesis, but the mutational processes are poorly understood. By performing whole genome and transcriptome sequencing of 181 germinal center derived B-cell lymphomas (gcBCL) we identified distinct mutational signatures linked to SHM and CSR. We show that not only SHM, but presumably also CSR causes off-target mutations in non-IG genes. Kataegis clusters with high mutational density mainly affected early replicating regions and were enriched for SHM- and CSR-mediated off-target mutations. Moreover, they often co-occurred in loci physically interacting in the nucleus, suggesting that mutation hotspots promote increased mutation targeting of spatially co-localized loci (termed hypermutation by proxy). Only around 1% of somatic small variants were in protein coding sequences, but in about half of the driver genes, a contribution of B-cell specific mutational processes to their mutations was found. The B-cell-specific mutational processes contribute to both lymphoma initiation and intratumoral heterogeneity. Overall, we demonstrate that mutational processes involved in the development of gcBCL are more complex than previously appreciated, and that B cell-specific mutational processes contribute via diverse mechanisms to lymphomagenesis.

Cytokine-related genes play critical roles in extrafollicular growth of follicular lymphoma cells.

Follicular lymphoma (FL) is a germinal center-derived B-cell lymphoma that is known to proliferate in the intrafollicular region. However, lymphoma cells can be identified in the extrafollicular regions, which are related to disease dissemination. We purified the intrafollicular and extrafollicular regions of FL cells by laser microdissection and conducted microarray analysis in order to characterize the gene expression profiles of FL cells from both regions. BCL2 and genes of germinal B-cell markers clearly separated intrafollicular and extrafollicular regions of reactive follicular hyperplasia, suggesting the adequacy of the current analysis. In FL cases, cytokine-related genes were significantly enriched in extrafollicular regions compared with those in the intrafollicular regions. In intrafollicular regions of FL, cell-cycle-related genes were enriched. We found that the FL cells in the extrafollicular region more strongly expressed IL3RA and CXCL12 than those of intrafollicular regions. The cytokines might be also derived from stroma cells in the extrafollicular regions, which may initiate activation and migration of the tumor cells to this region. Our results suggest that FL cell interaction with surrounding stroma cells plays an important role in the pathophysiology of FL and that such interactions should be a good target for therapy.

Abemaciclib, a CDK4/6 inhibitor, exerts preclinical activity against aggressive germinal center-derived B-cell lymphomas.

The revised WHO classification newly defined the entities “High‐grade B‐cell lymphoma with MYC and BCL2, and/or BCL6 rearrangements (HGBL‐DH/TH)” and “HGBL, NOS.” Standard immunochemotherapy for diffuse large B‐cell lymphoma (DLBCL), R‐CHOP, is insufficient for HGBL patients, and there are currently no optimized therapeutic regimens for HGBL. We previously reported that CCND3, which encodes cyclin D3, harbored high mutation rates in Burkitt lymphoma (BL), HGBL and a subset of DLBCL. Furthermore, the knockdown of cyclin D3 expression was toxic to germinal center (GC)‐derived B‐cell lymphomas. Thus, the fundamental function of cyclin D3 is important for the pathogenesis of GC‐derived B‐cell lymphoma. We herein used two structurally different CDK4/6 inhibitors, palbociclib and abemaciclib, and examined their suppressive effects on cell proliferation and their ability to induce apoptosis in various aggressive B‐cell lymphoma cell lines. The results obtained demonstrated that abemaciclib more strongly suppressed cell proliferation and induced apoptosis in GC‐derived B‐cell lymphoma cell lines than the control, but only slightly inhibited those features in activated B‐cell (ABC)‐like DLBCL cell lines. Palbociclib exerted partial or incomplete effects compared with the control and the effect was intermediate between abemaciclib and the control. Moreover, the effects of abemaciclib appeared to depend on cyclin D3 expression levels based on the results of the expression analysis of primary aggressive B‐cell lymphoma samples. Therefore, abemaciclib has potential as a therapeutic agent for aggressive GC‐derived B‐cell lymphomas.

The mutational landscape of Burkitt-like lymphoma with 11q aberration is distinct from that of Burkitt lymphoma

AbstractThe new recently described provisional lymphoma category Burkitt-like lymphoma with 11q aberration comprises cases similar to Burkitt lymphoma (BL) on morphological, immunophenotypic and gene-expression levels but lacking the IG-MYC translocation. They are characterized by a peculiar imbalance pattern on chromosome 11, but the landscape of mutations is not yet described. Thus, we investigated 15 MYC-negative Burkitt-like lymphoma with 11q aberration (mnBLL,11q,) cases by copy-number analysis and whole-exome sequencing. We refined the regions of 11q imbalance and identified the INO80 complex-associated gene NFRKB as a positional candidate in 11q24.3. Next to recurrent gains in 12q13.11-q24.32 and 7q34-qter as well as losses in 13q32.3-q34, we identified 47 genes recurrently affected by protein-changing mutations (each ≥3 of 15 cases). Strikingly, we did not detect recurrent mutations in genes of the ID3-TCF3 axis or the SWI/SNF complex that are frequently altered in BL, or in genes frequently mutated in germinal center–derived B-cell lymphomas like KMT2D or CREBBP. An exception is GNA13, which was mutated in 7 of 15 cases. We conclude that the genomic landscape of mnBLL,11q, differs from that of BL both at the chromosomal and mutational levels. Our findings implicate that mnBLL,11q, is a lymphoma category distinct from BL at the molecular level.

Development of First-in-Class Histone Acetyltransferase (HAT) Activators for Precision Targeting of Epigenetic Derangements in Lymphoma

Monoalleleic inactivating mutations in histone acetyltransferase (HAT) enzymes promote lymphomagenesis in germinal center derived B-cell lymphomas, follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL), occurring in about 40% of patients. The intact wild-type allele offers an opportunity to leverage the normal enzyme to overcome the pathogenic impact of the mutated allele. We hypothesize that if inactivating mutations in HATs are critical to FL and DLBCL lymphomagenesis, then drugs capable of inducing enhanced function of the wild-type HAT allele product should be cytotoxic in cells harboring HAT mutations.We designed and synthesized a library of new chemical entities with HAT activating properties (N=70). The cytotoxic effects of the compounds (N=29) were evaluated via medium-throughput screening in 4 DLBCL cell lines. IC50 ranged from 3.6 to 43.2 µM. Focusing on 6 analogue compounds, which share the same Nphenylbenzamide scaffold, we evaluated cytotoxicity across an expanded panel of 11 DLBCL cell lines. The median IC50 of 6 analogues tested was lower in the EP300 mutated cell lines (median 9.6 µM, range 5 - 11 µM) compared to the wildtype lines (median, 17 µM, range 15 - 24 µM). YF2 was chosen as the lead compound because it was the most selective of the analogues in inducing cytotoxicity in cell lines harboring EP300 mutations compared to wildtype (IC50 5 µM and 19 µM respectively, p<0.0005).To determine YF2's functional effect on activating p300 in a cell free assay, p300-mediated histone and p53 acetylation was measured by combining recombinant p300, substrate and acetyl-CoA. YF2 increased p300-mediated histone H3 lysine27 acetylation (EC50=38.64 nM) and H3 lysine18 acetylation (EC50=1.656 nM). YF2 also induced acetylation of p53 by 5-fold in a dose dependent manner. In cellular assays, YF2 induces acetylation of histone (H3K27 2-fold and H3K14 1.6-fold) after exposure in the SUDHL-6 cell line (EP300 mutated) as measured by mass spectrometry and confirmed by immunoblot of histone extracts.To assess the pharmacokinetics (PK) and preliminary in vivo efficacy of YF2, SUDHL-6 (EP300-mutated) xenograft bearing mice were treated once daily i.p. for 6 days with YF2 doses of 40mg/kg or 60mg/kg. Serum and tumor samples were collected at sequential time points. The Cmax of YF2 60mg/kg was 2424 ng/mL (5.1 µM) whereas Cmax of 40mg/kg was 2091.96 ng/mL (4.5 µM) which is consistent with the cellular IC50. Both concentrations of YF2, 40mg/kg and 60mg/kg, accumulated in tumor with Cmax 9536.71 and 9858.15 ng/g, respectively. YF2 is rapidly absorbed in the serum (Tmax 0.25 h) and sustained in the tumor (Tmax 4h). Significant effects on tumor size were observed in 13 of 19 mice demonstrating decreased tumor volume following only 6 days of YF2 treatment. Mice treated with YF2 40mg/kg induced H3K27 acetylation in tumor specimens as determined by mass spectrometry.YF2 40mg/kg was well tolerated in SCID/beige mice for 30 days without significant weight loss, while 60mg/kg YF2 led to 20% weight loss during 6-days of treatment. Additionally, YF2 demonstrated cytotoxic effects in 2 primary patient lymphoma samples but were non-cytotoxic to peripheral blood mononuclear cells from healthy donors.Furthermore, we hypothesized that if DLBCL is sensitive to an enhanced acetylation state, then combined targeting of epigenetic machinery with HAT activators and HDAC inhibitors may induce profound epigenetic modification leading to synergistic induction of programmed cell death. The concentration : effect relationship of YF2 and the pan-HDAC inhibitor, romidepsin, was evaluated over time across a panel of lymphoma cell lines (N=7). Synergy was calculated by Excess over Bliss (EOB>10 connotes synergy). Combination of YF2 and romidepsin demonstrated strong synergism in DLBCL lines (EOB = 48). The combination led to enhanced histone acetylation compared to either single agents. The combination is safe in mice and murine xenograft studies of the combination are underway.In summary, YF2 induces HAT-mediated acetylation of histone and p53. It demonstrates selective cytotoxic effects in EP300-mutated DLBCL cell lines, and is both well tolerated and effective in xenograft mouse models of lymphoma suggesting potential clinical application and precision medicine opportunities for patients harboring this mutation. [Display omitted] DisclosuresO'Connor:Seattle Genetics: Research Funding; ADC Therapeutics: Research Funding; Celgene: Research Funding.

KLHL6 Is Preferentially Expressed in Germinal Center-Derived B-Cell Lymphomas.

KLHL6 is a recently described BTB-Kelch protein with selective expression in lymphoid tissues and is most strongly expressed in germinal center B cells. Using gene expression profiling as well as immunohistochemistry with an anti-KLHL6 monoclonal antibody, we have characterized the expression of this molecule in normal and neoplastic tissues. Protein expression was evaluated in 1,058 hematopoietic neoplasms. Consistent with its discovery as a germinal center marker, KLHL6 was positive mainly in B-cell neoplasms of germinal center derivation, including 95% of follicular lymphomas (106/112). B-cell lymphomas of non-germinal center derivation were generally negative (0/33 chronic lymphocytic leukemias/small lymphocytic lymphomas, 3/49 marginal zone lymphomas, and 2/66 mantle cell lymphomas). In addition to other germinal center markers, including BCL6, CD10, HGAL, and LMO2, KLHL6 immunohistochemistry may prove a useful adjunct in the diagnosis and future classification of B-cell lymphomas.

T(14;18)(q32;q21) in chronic lymphocytic leukemia patients: Report of two cases and a literature review

The chromosomal abnormality t(14;18)(q32;q21) is most commonly associated with germinal center-derived B-cell lymphomas, particularly follicular lymphoma (FL). Generally, it is considered a hallmark of FL. The t(14;18)(q32;q21) translocation is rare in chronic lymphocytic leukemia (CLL) and its prognostic significance remains unclear. In the present study, two cases of CLL with t(14;18)(q32;q21) were diagnosed using conventional cytogenetic analysis and fluorescence in situ hybridization. Both patients presented with leukemia and the morphological features and immunophenotypes were typical of CLL. Case 2 underwent a further lymph node biopsy, which established a diagnosis of CD5– CLL/small lymphocyte lymphoma. In addition to t(14;18)(q32;q21), trisomy 12 was identified in the same clone in Case 2. Both cases exhibited immunoglobulin heavy chain variable mutations, and heavy-chain variable region gene (VH) 4–39 and VH3-62 were used in Case 1 and Case 2, respectively. In addition, direct Sanger sequencing of exons 4–9 revealed that Case 2 harbored the tumor protein p53 mutation, c.829T>G. Both cases had indications for therapy. Case 1 responded well to chlorambucil treatment, and was still alive at the last follow-up. Conversely, Case 2 exhibited aggressive disease that appeared refractory to treatment, and eventually succumbed to the disease.

Identification of Blut, a Novel Long-Noncoding RNA Differentially Expressed in Burkitt Lymphoma

The class of the so-called long non-coding RNAs (lncRNAs) is defined by their lacking coding potential and their length of more than 200 nucleotides. LncRNAs are readily detected in transcriptome analysis but have so far not been characterized or catalogued exhaustively. Although lncRNAs make up a major portion of the mammalian non-coding transcriptome, it is challenging to predict their functions due to their diversity in terms of size, sequence and biogenesis. However, several lncRNAs have already been shown to be involved in crucial cellular processes such as imprinting, regulation of cell proliferation and cellular differentiation. Their mode of action generally involves epigenetic modifications as well as transcriptional and post-transcriptional regulation.Within the ICGC (International Cancer Genome Consortium)-MMML-Seq (Molecular Mechanisms in Malignant Lymphoma by Sequencing) Consortium, which aims at fully characterizing a total of 250 germinal center derived B-cell lymphomas, including Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), we conducted a search for yet unidentified genes.We here report the identification of a novel transcript, which we have named BLUT (Burkitt Lymphoma Unknown Transcript). BLUT is highly expressed in BL, with only very low expression levels in FL and no detectable expression in DLBCL. It is around 1050 nucleotides long with splice evidence for three exons, whereby exons 1 and 3 are shared between all isoforms.BLUT represents a putative lncRNA since it lacks conserved protein domains and has a splice site conservation pattern typical of non-coding transcripts. Furthermore, it is predicted as non-coding by the software Coding Potential Calculator and there was not a single peptide match between a published peptide database (Human Proteome Map) and any potential BLUT peptide (all isoforms). Moreover, BLUT shares a high sequence identity with two (so far) uncharacterized non-coding RNAs (Pan Troglodyte, Chlorocebus sabeus).We tested a cell line panel (consisting of 16 different cell lines including non-lymphoma cell lines) for BLUT expression but could only detect it in the two BL cell lines Namalwa and Raji.Cellular fractionation experiments showed that BLUT is predominantly localized in the nucleus in both Namalwa and Raji.Overexpression of BLUT in SU-DHL-4 resulted in the identification of 222 differentially regulated genes (FDR < 0.05) with a logFC of ± 0.25, among which 49 genes had a logFC of ± 0.50. Several lymphoma relevant genes were part of this list including BIN1, CCL22, CCR7, EBI3, ID2, JAG1, NQO1, and RGS1.Given its nuclear localization, we currently perform chromatin isolation by RNA purification experiments to identify the genomic regions which BLUT binds to. In combination with our overexpression data this will allow further insights into the functions of BLUT.In summary, we have identified a novel long-noncoding RNA termed BLUT, which is differentially expressed between lymphoma subtypes with a high expression in Burkitt lymphoma. Overexpression of BLUT resulted in the deregulation of several genes with known roles in lymphomagenesis. Given its predominantly nuclear localization, we are currently investigating its functions on chromatin regulation.(Supported by BMBF through 01KU1002A-J and by the Duesseldorf School of Oncology (funded by the Comprehensive Cancer Center Duesseldorf/Deutsche Krebshilfe and the Medical Faculty HHU Düsseldorf)) DisclosuresTruemper:Amgen, roche, Mundipharma: Research Funding; Sandoz, Celgene, AMGEN, Nordic Nanovector: Other: Advisory board.

Abstract IA47: Epigenetic programming and therapy in germinal center derived B-cell lymphomas.

Abstract Diffuse Large B-cell Lymphomas (DLBCLs) are a heterogeneous group of diseases initiating from germinal center (GC) B cells. GC B cells are uniquely specialized to tolerate rapid proliferation, and physiological genomic instability in order to generate a diverse set of clones of cells encoding high affinity antibodies. This accelerated micro-evolutionary process requires the concerted action of transcription and epigenetic factors that collectively reprogram B cells to overcome many of their normal checkpoint restraints and tolerate the various stresses associated with affinity maturation. The GC phenotype poses a significant risk of malignant transformation to B cells, which must accordingly balance suppression of checkpoints with suppression of pro-oncogenic genes. Epigenetic regulatory complexes play a critical role in lymphomagenesis. EZH2, which epigenetically silences genes through histone 3 lysine 27 methylation is upregulated in normal and malignant GC B cells. EZH2 is often affected by gain of function mutations in lymphomas that alter its enzymatic specificity. EZH2 mediates GC formation by transiently suppressing checkpoint genes and terminal differentiation genes through formation of bivalent chromatin domains. EZH2 somatic mutations induce germinal center hyperplasia and malignant transformation, and cooperate with other oncogenes such as BCL2. EZH2 specific inhibitors can suppress the growth of GC derived lymphoma cells in vitro and in vivo, and are currently being evaluated in early phase clinical trials. DNA methyltransferase 1 (DNMT1) is required for B cells to form GCs, and GC B cells display cytosine methylation redistribution as compared to resting or naïve B cells. DLBCLs in turn exhibit prominent and heterogeneous disruption of cytosine methylation distribution, with specific and distinct DNA methylation profiles occurring in different lymphoma subtypes. Epigenetic heterogeneity is associated with unfavorable outcome in B-cell lymphomas, suggesting that epigenetic diversity may provide a survival advantage to lymphoma cell populations. DNA methyltransferase inhibitors can reprogram lymphoma cells to develop a form of incomplete senescence that sensitizes lymphoma cells to more readily respond to chemotherapy. DNA methyltransferase inhibitors can be safely combined with standard lymphoma therapies for the up-front treatment of patients with high-risk lymphomas. Citation Format: Ari M. Melnick. Epigenetic programming and therapy in germinal center derived B-cell lymphomas. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr IA47.

LMP1 mediates multinuclearity through downregulation of shelterin proteins and formation of telomeric aggregates

AbstractHodgkin lymphoma (HL) and Burkitt lymphoma are both germinal center–derived B-cell lymphomas. To assess the consequences of permanent latent membrane protein 1 (LMP1) expression as observed in tumor cells of Epstein-Barr virus (EBV) –associated HL, we analyzed 3-dimensional (3D) telomere dynamics and measured the expression of shelterin proteins at the transcriptional and translational level and their topographic distribution in the EBV-negative Burkitt cell line BJAB stably transfected with an inducible LMP1 system. Stable LMP1 expression led to a highly significant increase of multinucleated cells, nuclear volume, and 3D telomeric aggregates when compared with the LMP1-suppressed BJAB controls. Most importantly, LMP1 induced a significant downregulation of the shelterin components TRF1, TRF2, and POT1 at the transcriptional and translational level, and this downregulation was reversed after resuppression of LMP1. In addition, as revealed by spectral karyotyping, LMP1 induced “outré” giant cells and hypoploid “ghost” cells. This LMP1-induced multinucleation was blocked upon LMP1-independent TRF2 expression. These results show that LMP1-dependent deregulation of telomere stability and nuclear organization via shelterin downregulation, in particular TRF2, favors chromosomal rearrangements. We speculate that telomeric aggregates and ongoing breakage-bridge-fusion cycles lead to disturbed cytokinesis and finally to multinuclearity, as observed in EBV-associated HL.

In-Depth miRNA Profiling Of Germinal Center Derived B-Cell Lymphomas By Next Generation Sequencing: A Report From The German Icgc-Mmml-Seq Project

The discovery of microRNAs (miRNAs), a group of small non-coding regulatory RNAs, revolutionized the field of posttranscriptional gene regulation. Mature miRNAs are single-stranded RNA molecules of 20- to 23-nucleotide length that control gene expression. They typically reduce the translation and stability of mRNAs important in tumorigenesis mediating processes such as inflammation, cell cycle regulation, stress response, differentiation, apoptosis, and invasion.Germinal center (GC) derived B-cell lymphomas, including Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), are the most frequent malignant lymphomas. Although clear distinctions on histologic and genetic grounds exist, there is also a large number of cases with intermediate features, not unequivocally attributable to one of these entities.Recently, a signature of 38 miRNAs containing MYC regulated and nuclear factor-kB pathway-associated miRNAs was published, which differentiated BL from DLBCL. However, available data is preliminary as published profiles are not derived from large sample collections and also originate mostly from PCR-based approaches and microarrays. Yet, only sequencing-based approaches allow for an unbiased analysis and the discovery of novel miRNAs and small RNA classes.The ICGC (International Cancer Genome Consortium)-MMML-Seq (Molecular Mechanisms in Malignant Lymphoma by Sequencing) Consortium aims at fully characterizing a total of 250 GC derived B-cell lymphomas over a period of five years. We here report the miRNA analysis of the first 49 patient samples including BL, DLBCL and FL using Illumina technology. We are recording the classical miRNA fraction (18-35 nucleotides) as well as a larger size fraction (35-90 nucleotides).Initial differential expression analyses comparing BL against non-BL showed eight miRNAs to be differentially expressed. This comprises hsa-miR-150, which has been ascribed a role in the adaptive immune response and which was shown to be upregulated in cutaneous marginal zone B-cell lymphomas. Further differentially expressed miRNAs include e.g. hsa-miR-211, hsa-miR-548ac and hsa-miR-1244-1/3, which represent novel targets in lymphomagenesis.Among the many bioinformatically predicted novel miRNAs, we have been able to validate four candidates by Northern Blot experiments and are currently performing functional studies.Mutational analysis is ongoing, but so far has yielded two miRNAs with mutations in the mature sequences (hsa-miR-4537, hsa-miR-758).In conclusion, our initial series of 49 cases from the ICGC-MMML-Seq cohort has already given exciting insights into the role of miRNAs in GC derived B-cell lymphomas. (Supported by BMBF through 01KU1002A-J) Disclosures:No relevant conflicts of interest to declare.

Role of activation‐induced cytidine deaminase in the progression of follicular lymphoma

Activation-induced cytidine deaminase (AID/AICDA) is required for somatic hypermutation and class-switch recombination of the immunoglobulin gene, and for c-myc translocation of germinal center-derived B-cell lymphoma. In the present study, we attempted to clarify the significance of AID associated with c-myc in the progression of follicular lymphoma (FL) using RT-PCR and quantitative real-time PCR. Tissues from the patients with grade 3 FL expressed relatively higher levels of c-myc and AID. The samples taken from a patient with FL who died within 2 years after the start of treatment showed either no or low expression of AID, despite expressing high levels of c-myc. In order to examine the role of AID expression in rapidly progressive FL, the full-length AID transcript was transfected into AID-negative cell lines established from different patients with rapidly progressive FL. This led to the establishment of AID-expressing transfectants with a low proliferation rate and a significantly increased incidence of G(0)/G(1) arrest compared with controls. Our results indicate that AID may act as a negative regulator of cell survival in FL when sufficient c-myc is expressed. Switch-off or low expression of AID after c-myc amplification may correlate with the clinical outcomes of FL.

LITAF, a BCL6 Target Gene, Regulates Autophagia in B Cells and Is Essential for T-Cell Dependent Humoral Responses

Abstract 1391LITAF was discovered as a p53-induced transcript that promoted TNFa secretion in monocytes in response to LPS. We previously reported that LITAF is inactivated by deletion or promoter hypermethylation in germinal center-derived B-cell lymphomas. However, the function of LITAF in B lymphocytes is unknown. Using gene expression analysis of isolated B-cell subpopulation and immunohistochemical studies of tonsil lymphoid follicles we found that LITAF is expressed in naïve B lymphocytes and is repressed within the germinal centers (GCs). Thus, LITAF showed an opposite expression to BCL6, an essential regulator of GC development and function. Likewise, expression of LITAF and BCL6 were inversely correlated in cell lines and biopsies from patients with B-cell lymphoma, further suggesting a link between LITAF and BCL6. ChIP-on-chip and ChIP-sequencing analyses of B cells coupled with luciferase reporter assays revealed that BCL6 repressed LITAF expression by binding to its promoter. Accordingly, BCL6 silencing with siRNAs or after exposure to a BCL6-inhibitor peptide increased LITAF expression, indicating that LITAF is transcriptionally repressed by BCL6 in GC B lymphocytes and in B-cell lymphoma cells. To initially elucidate the function of LITAF in B cells, gain-and-loss of function experiments were performed in different cellular models. LITAF expression was not related to TNFa secretion after LPS exposure, nor modulated cell proliferation or apoptosis in B cells. However, sustained expression of LITAF in B-cell lymphoma cells increased cell size, lysosome content and mitochondrial mass. Gene expression microarray studies defined a LITAF-related transcriptional signature containing genes involved in the regulation of endomembranes, vesicle trafficking and protein transport. Accordingly, immunofluorescence analysis co-localized LITAF with lysosomes and with autophagosomes expressing LC3, the mammalian homolog of yeast autophagy-related protein (Atg8), as well as with the lysosomal sorting-associated proteins NEDD4 and TSG101, both in normal CD19+ B lymphocytes and in B-cell lymphoma cells. In addition, LITAF expression induced autophagic activity in B cells, shown by an increase in the FL1/FL3 ratio after acridine orange staining and by converting LC3-I to LC3-II, which were more evident upon cell starvation. Together, these data suggest that LITAF may play a role in the processing of proteins in autophagosomes through regulating autophagy. To investigate LITAF function in vivo, we generated mice with targeted deletion of the Litaf gene in B lymphocytes by using the Cre-loxP system. Litaf -mb1-Cre (Litaf−/− ) mice developed healthy and showed normal distribution of hematopoietic cell subpopulations. However, Litaf−/− mice were unable to develop full T-cell dependent immune responses, presenting PNA-stained, Litaf-negative GCs that were absent or had marked reduction in size and number. Accordingly, reduced amounts of IgM, IgG1 and IgG3 antibodies as a consequence of abnormal class switch recombination (CSR) were detected in immunized mice. However, in experiments testing CSR in vitro, in which B cells are artificially activated in the absence of T cells, the amounts of IgM/IgG1/IgG3 did not differ between knock-out and control groups. Similarly, mouse immunization with a T-cell independent antigen did not induce differences in immunoglobulin production. Further studies of GCs in T-cell immunized Litaf−/− mice using an antibody for the Class II-associated invariant chain peptide (CLIP) revealed that the atrophic GCs in Litaf−/− mice showed strong CLIP expression in comparison to wild-type littermates. In normal immune responses, CLIP peptides bind to MHC class II molecules in endolysosomes, until they are displaced by the antigen, then releasing CLIP and allowing MHC II-antigen complexes to be transported to the cell membrane for T-cell presentation. The failure to develop appropriate immune responses together with the accumulation of CLIP peptides in Litaf -deficient mice indicate that Litaf is essential for adequate T-cell dependent immune responses in GC B lymphocytes, possibly through facilitating the presentation of the antigens to MHC II molecules in the endolysosomes. Once this process is assembled and the T-cell activated B lymphocytes enter the GCs, BCL6 represses LITAF to prevent additional interactions between B and T cells during BCR editing. Disclosures:No relevant conflicts of interest to declare.

Translocations activating IRF4 identify a subtype of germinal center-derived B-cell lymphoma affecting predominantly children and young adults

AbstractThe prognosis of germinal center–derived B-cell (GCB) lymphomas, including follicular lymphoma and diffuse large-B-cell lymphoma (DLBCL), strongly depends on age. Children have a more favorable outcome than adults. It is not known whether this is because of differences in host characteristics, treatment protocols, or tumor biology, including the presence of chromosomal alterations. By screening for novel IGH translocation partners in pediatric and adult lymphomas, we identified chromosomal translocations juxtaposing the IRF4 oncogene next to one of the immunoglobulin (IG) loci as a novel recurrent aberration in mature B-cell lymphoma. FISH revealed 20 of 427 lymphomas to carry an IG/IRF4-fusion. Those were predominantly GCB-type DLBCL or follicular lymphoma grade 3, shared strong expression of IRF4/MUM1 and BCL6, and lacked PRDM1/BLIMP1 expression and t(14;18)/BCL2 breaks. BCL6 aberrations were common. The gene expression profile of IG/IRF4-positive lymphomas differed from other subtypes of DLBCL. A classifier for IG/IRF4 positivity containing 27 genes allowed accurate prediction. IG/IRF4 positivity was associated with young age and a favorable outcome. Our results suggest IRF4 translocations to be primary alterations in a molecularly defined subset of GCB-derived lymphomas. The probability for this subtype of lymphoma significantly decreases with age, suggesting that diversity in tumor biology might contribute to the age-dependent differences in prognosis of lymphoma.