ALBERT

Description: In this issue of Blood, Huang and colleagues examine the role of BACH2, a transcription factor known to be expressed highly in germinal center B cells.1-3

Description: High throughput sequencing is a revolutionary technology for the definition of the genomic features of tumors. This talk will provide a review of the relevant methodologies for non-experts in the field. The presentation will include a discussion of how high throughput sequencing is performed, its relative strengths and weaknesses, and how it is applicable to formalin-fixed and fresh/frozen tissue samples. The talk will also describe future directions in the genomic analysis of tumors. Disclosures No relevant conflicts of interest to declare.

Description: Background: Diffuse Large B Cell Lymphoma (DLBCL) is the most common form of lymphoma in adults. Gene expression profiling has demonstrated that DLBCL can be classified into two distinct subgroups – activated B-cell-like (ABC) and germinal center B-cell-like (GCB) DLBCL. These subgroups arise through distinct normal cells of origin, activate different oncogenic pathways and display markedly different clinical outcomes. Deregulation of the transcriptome is believed to play a key role in the malignant transformation of B cells that culminates in the development of either ABC or GCB DLBCL. Here we describe global differences in RNA expression, mutation and splicing in relation to the pathogenesis of these subgroups of DLBCL. Methods: RNA sequencing (RNAseq) has emerged as a powerful tool for defining the cancer transcriptome. While mRNA sequencing is the most widely applied method for RNAseq, it overlooks non-coding RNAs, requires high-quality RNA and lacks strand-specificity. To overcome these limitations, we developed a method for strand-specific total RNA sequencing (ssRNAseq) to characterize the transcriptomes of 112 DLBCL tumors. Results: Through this work, we defined the entire spectrum of coding and non-coding RNAs expressed in DLBCLs including hundreds of lincRNAs, snoRNAs and microRNAs in addition to mRNAs. We found that the strand-specificity of our method was greater than 95% in all cases. This strand-specific sequencing strategy allowed us to maintain the orientation of the transcript to enable more accurate transcript annotation and better prediction of novel transcripts. Furthermore, we showed that our method had equal efficacy on frozen and FFPE tumor specimens from the sample patient in 24 cases. In addition, through simultaneous measurement of expression of diverse RNA types combined with mutations in MYD88, GNA13, EZH2, and BCL2, we demonstrated that we could distinguish the clinically important subgroups of DLBCL. Finally, we applied ssRNAseq to distinct training and validation sets of DLBCL cases (N=86 and N=112) to define alternative splicing events in DLBCL and found 1,021 genes that were preferentially spliced in a subgroup-specific manner. These alternatively spliced genes were selectively enriched in a number of different pathways important in lymphomas including those related to immune function, cell cycle progression and focal adhesion pathways, suggesting that alternative splicing regulates a number of important oncogenic processes in DLCBL. Conclusions: Strand-specific total RNA sequencing is a powerful method for defining the transcriptome and alternative splicing events in DLBCL. Here we define a complete coding and non-coding transcriptome of DLBCL and report the first characterization of subgroup-specific alternative splicing in DLBCL using high throughput sequencing. Our data demonstrate the power of our ssRNAseq method in defining the molecular patterns underlying DLBCLs and provide a starting point for defining the role of alternative splicing in this complex and heterogeneous disease. Disclosures Mann: Quiagen: Research Funding.

Description: Background: HDAC inhibitors (HDACi) are being investigated as treatment for relapsed/refractory non Hodgkin lymphoma (NHL) and other cancers. However, the mechanisms underlying sensitivity and resistance to HDAC inhibition in lymphomas have not been fully characterized. We probed the cellular and molecular response to HDACi in vitro and in vivo in order to determine factors that dictate the response to HDACi and to enable design of approaches to incorporate HDACi into novel combination therapeutics. Methods: High-throughput cytotoxicity screening was performed using two different HDAC inhibitors, LBH589 (panobinostat) and SAHA (vorinostat) in 52 lymphoid cell lines characterized through RNA-seq and microarray gene expression profiling. This screen revealed a greater than 50-fold range in concentration needed to induce cytotoxicity for the 2 different HDAC inhibitors and there was moderate correlation between the 2 compounds in this panel (Pearson correlation r = 0.76, p 〈 0.01). By pairing this chemosensitivity data with gene expression profiles of the screened cell lines, we developed a gene expression classifier for LBH589 that identified resistant and sensitive cell line groups. This predictor was applied to B-cell NHL cell lines tested with LBH589 in the Cancer Cell Line Encyclopedia (CCLE) and we found that the sensitive and resistant cell line groups distinguished by this method differed more than 5-fold in IC50 (0.021 vs. 1.24 nM, P 〈 0.01 by Wilcoxon rank sum), thus validating the ability of this approach to distinguish HDACi resistant cell lines. We further initiated a clinical trial of LBH589 in relapsed/refractory diffuse large B cell lymphoma patients combined with RNAseq profiling of their tumors prior to embarking on treatment. We treated nine patients with LBH589, and application of our response predictor to scaled RNAseq gene expression data revealed 4 predicted responders and 5 predicted non-responders. Two of the predicted responders had a clinical response to LBH589, whereas none of the predicted non-responders had a clinical response, thus our classifier was able to identify all of the LBH589-responsive patients from this cohort (P = 0.08 by Fisher's exact test). Analysis of differentially expressed molecular pathways in HDACi sensitive and resistant samples by gene set enrichment revealed the JAK-STAT pathway as the most differentially expressed pathway associated with HDACi resistance (at P 〈 0.001 and FDR 〈 0.20). We further identified a number of distinct mutations including STAT3, SOCS1 and JAK1 that were associated with activation of the JAK-STAT pathway by gene expression signatures and the LBH589 response signature in DLBCL cell lines and patient samples by analysis of RNA-seq data. Phosphoprotein analysis by Western blot and Sis-inducible-element (SIE) luciferase reporter assays were used to confirm JAK-STAT activation in these samples and we found that overexpression of STAT3 Src-homology domain mutations activated JAK-STAT3 signaling in isogenic cell lines and fostered resistance to LBH589 in vitro. Conversely, using in vivo DLBCL xenograft models, we found that combining JAK-STAT and HDAC inhibition by treatment with LBH589 and ruxolitinib resulted in synergistic reduction of tumor cell viability and tumor growth with tolerable toxicity in mice. Conclusions: Sustained JAK-STAT activation appears to mediate resistance to HDAC inhibition in DLBCL and other NHLs and several recurrent genetic lesions drive JAK-STAT activation in these diseases. This process can be overcome by JAK 1/2 inhibition with ruxolitinib and these findings demonstrate a role for combination therapy with HDAC inhibitors and small molecules targeting the JAK-STAT pathway in lymphoid malignancies. Disclosures No relevant conflicts of interest to declare.

Description: Gene expression profiling of diffuse large B-cell lymphoma (DLBCL) has revealed distinct molecular subtypes that include germinal center B cell–like (GCB) and activated B cell–like (ABC) DLBCL. ABC DLBCL has a worse survival after upfront chemotherapy and is characterized by constitutive activation of the antiapoptotic nuclear factor–kappa B (NF-κB) pathway, which can inhibit chemotherapy. We hypothesized that inhibition of NF-κB might sensitize ABC but not GCB DLBCL to chemotherapy and improve outcome. As the proteasome inhibitor bortezomib can inhibit NF-κB through blocking IκBα degradation, we investigated bortezomib alone followed by bortezomib and doxorubicin-based chemotherapy in recurrent DLBCL. Tumor tissue was analyzed by gene expression profiling and/or immunohistochemistry to identify molecular DLBCL subtypes. As a control, we showed that relapsed/refractory ABC and GCB DLBCL have equally poor survivals after upfront chemotherapy. Bortezomib alone had no activity in DLBCL, but when combined with chemotherapy, it demonstrated a significantly higher response (83% vs 13%; P 〈 .001) and median overall survival (10.8 vs 3.4 months; P = .003) in ABC compared with GCB DLBCL, respectively. These results suggest bortezomib enhances the activity of chemotherapy in ABC but not GCB DLBCL, and provide a rational therapeutic approach based on genetically distinct DLBCL subtypes. This trial is registered with http://www.ClinicalTrials.gov under identifier NCT00057902.

Description: Background: MicroRNAs (miRNAs) are small non coding RNAs that have been shown to play a regulatory role in a number of different settings including development, hematopoiesis and lineage-selection. The expression patterns of miRNAs in various cellular processes and in various normal and malignant tissues are an area of active exploration. Bioinformatic analyses of the genome suggest that there might be thousands of miRNAs encoded in the genome. However, thus far only about 600 unique miRNAs have been identified in humans. The role of microRNAs in B cell malignancies is poorly understood. Mature B cells comprise naive, germinal center, memory and plasma cells. These B cell stages comprise the majority of leukemias and lymphomas. We have previously demonstrated a role for microRNAs in the regulation of key transcription factors and oncogenes including PRDM1, LMO2 and MYBL1. We hypothesized that microRNAs play a role in the pathogenesis of lymphomas and have applied high throughput sequencing to understand the pattern and function of microRNA expression in normal B cells and their malignant counterparts. Methods: CD19+ mature B cells were obtained from normal patients undergoing tonsillectomy and sorted using flow cytometry into naive, germinal center, memory and plasma cells. We also obtained cells from tumor cell lines derived from mantle cell lymphoma (Mino, JVM2), Burkitt lymphoma (Ramos, BL41) and multiple myeloma (H929, U266), as well as patient tumor samples derived from Burkitt lymphoma and diffuse large B cell lymphomas. From these cells, we purified small RNAs (18-25 nucleotides) and ligated sequencing adapters to these small RNAs and subjected them to 15 cycles of PCR amplification. The constructs were then subjected to massively parallel high throughput sequencing (Illumina) in picoliter wells to identify millions of sequences per sample. Sequences thus identified were matched to the genome and microRNAs were identified based on their characteristic stem-loop secondary structure, thermodynamic stability, and evidence of processing with the microRNA-related enzymes drosha and dicer. Results: Using massively parallel high-throughput sequencing of small RNAs isolated from these B cell subsets, we analyzed a total of 62,293,147 sequences (〉 1.6 billion bases). We found that 261 known microRNAs are expressed in normal and malignant B cells, a number that is three times higher than previously recognized. Our work also identified the expression of 86 novel miRNAs in normal and malignant B cells, many of which appear to target genes important in B cell differentiation including BCL6, NLK, EBF, as well as oncogenes including MYC, LMO2, and CCND1. We found no evidence of decreased expression of microRNAs in B cell malignancies, in contrast to the described down-regulation of microRNAs in tumors from other lineages. On the other hand, there were striking differences between the microRNA expression patterns in normal and malignant B cells, although B cell malignancies still frequently express miRNAs that are characteristic of their normal B cell counterpart. Each malignancy had a characteristic pattern of microRNA expression that was distinct from other malignancies and normal B cells. Conclusion: Through high throughput sequencing, we have discovered novel microRNAs that target important oncogenes including BCL6, MYC, LMO2, and CCND1, suggesting a role for microRNAs in B cell lymphomas.

Description: Subjects with X-linked hyper-IgM syndrome (X-HIgM) have a markedly reduced frequency of CD27+ memory B cells, and their Ig genes have a low level of somatic hypermutation (SHM). To analyze the nature of SHM in X-HIgM, we sequenced 209 nonproductive and 926 productive Ig heavy chain genes. In nonproductive rearrangements that were not subjected to selection, as well as productive rearrangements, most of the mutations were within targeted RGYW, WRCY, WA, or TW motifs (R = purine, Y = pyrimidine, and W = A or T). However, there was significantly decreased targeting of the hypermutable G in RGYW motifs. Moreover, the ratio of transitions to transversions was markedly increased compared with normal. Microarray analysis documented that specific genes involved in SHM, including activation-induced cytidine deaminase (AICDA) and uracil-DNA glycosylase (UNG2), were up-regulated in normal germinal center (GC) B cells, but not induced by CD40 ligation. Similar results were obtained from light chain rearrangements. These results indicate that in the absence of CD40-CD154 interactions, there is a marked reduction in SHM and, specifically, mutations of AICDA-targeted G residues in RGYW motifs along with a decrease in transversions normally related to UNG2 activity.

Description: Nonhodgkin Lymphoma (NHL) is among the most common cancer subtypes, with approximately 〉350,000 new cases diagnosed annually worldwide. The vast majority of NHLs arise from germinal center (GC) B cells. We and others have identified GNA13 as one of the most frequently mutated genes in GC-derived lymphomas, including ~30% of Burkitt Lymphoma and ~25% of Germinal Center B Cell-like (GCB) Diffuse Large B cell Lymphoma. Despite this association, the role of GNA13 in lymphomagenesis remains elusive. In human breast and prostate cancer, GNA13 behaves as an oncogene, with increased expression linked to cellular invasion and metastasis. Intriguingly, GNA13 mutations in GCB DLBCL and Burkitt Lymphoma are frequently inactivating, possessing a high number of nonsense and missense mutations in conserved domains. This suggests that GNA13 may function as a tumor suppressor in the context of lymphoma, in contrast to its role in solid tumors. The purpose of this study is to define the role of GNA13 in GC B cells and to clarify how GNA13 loss may contribute to lymphoma within the germinal center niche. We first investigated the expression pattern of GNA13 in lymphocyte populations from normal human tonsil. Our data demonstrated that GNA13 is enriched in GC B cells by quantitative PCR and immunohistochemistry. To determine the effect of GNA13 abundance on global mRNA expression patterns, we performed RNA sequencing on lymphoma derived cell lines. Using this method, we found that GNA13 knockdown and overexpression was highly correlated with GC dark and light zone gene signatures, respectively. We next devised a proteomics approach to identify potential GNA13 binding partners in GCs. Lysates from lymphoma-derived cell lines overexpressing FLAG-tagged GNA13 were subjected to immunoprecipitation with M2-antibody bound magnetic beads, followed by LC-MS/MS. Our results demonstrated an enrichment of proteins involved in focal adhesion, consistent with the known involvement of GNA13 in processes of cytoskeletal reorganization and cell migration. We next explored the role of GNA13 in vivo. Since GNA13 mutations are a unique feature of GC-derived lymphomas, we developed mouse models that would allow us study GNA13 exclusively in the germinal center context. We generated B cell and GC specific GNA13 knockout mice by crossing GNA13fl/fl mice with MB1-Cre and AID-Cre strains. After immunization with sheep red blood cells, both B cell and GC specific GNA13 deficient mice possessed normal levels of B, T and GC cells within secondary lymphoid sites including Peyer’s patches and spleen, suggesting that GNA13 is not essential for GC formation. GC B cells from both GNA13 deficient strains demonstrated enhanced cellular motility toward GC directed chemokines CXCL12, CXCL13 and S1P using in vitro transwell migration assays. Furthermore, B cells isolated from GNA13 deficient animals showed enhanced RhoA activity. These data suggested that GNA13 inhibits GC B cell migration and RhoA mediated cell motility in normal conditions. Loss of GNA13 may then deregulate normal chemokine gradient signaling, resulting in global increases in GC migration. We also demonstrated that GNA13 deficient B cells possess elevated levels of phosphorylated AKT, an effect potentiated by the addition of CXCL12 and S1P. AKT signaling is known to promote cell survival in a variety of cell types, which may further promote oncogenesis. In this study, we have synthesized the complementary approaches of next generation sequencing, proteomics and genetic mouse models to gain novel insight into the biological function of GNA13, a gene that is mutated in a high proportion of GC-derived lymphomas. As a whole, our work suggests that GNA13 serves as a tumor suppressor during the germinal center reaction. The acquisition of inactivating GNA13 mutations may promote lymphoma by allowing cells to physically escape the germinal center niche and evade apoptosis while continuing to express GC signature genes. Affected cells may be subjected to persistent somatic hypermutation, which, over time, could result in the accumulation of additional oncogenic mutations, culminating in development of GC-derived lymphoma. Disclosures No relevant conflicts of interest to declare.