Relapse Timing Is Associated With Distinct Evolutionary Dynamics in Diffuse Large B-Cell Lymphoma.

PURPOSE: Diffuse large B-cell lymphoma (DLBCL) is cured in more than 60% of patients, but outcomes remain poor for patients experiencing disease progression or relapse (refractory or relapsed DLBCL [rrDLBCL]), particularly if these events occur early. Although previous studies examining cohorts of rrDLBCL have identified features that are enriched at relapse, few have directly compared serial biopsies to uncover biological and evolutionary dynamics driving rrDLBCL. Here, we sought to confirm the relationship between relapse timing and outcomes after second-line (immuno)chemotherapy and determine the evolutionary dynamics that underpin that relationship. PATIENTS AND METHODS: Outcomes were examined in a population-based cohort of 221 patients with DLBCL who experienced progression/relapse after frontline treatment and were treated with second-line (immuno)chemotherapy with an intention-to-treat with autologous stem-cell transplantation (ASCT). Serial DLBCL biopsies from a partially overlapping cohort of 129 patients underwent molecular characterization, including whole-genome or whole-exome sequencing in 73 patients. RESULTS: Outcomes to second-line therapy and ASCT are superior for late relapse (>2 years postdiagnosis) versus primary refractory (<9 months) or early relapse (9-24 months). Diagnostic and relapse biopsies were mostly concordant for cell-of-origin classification and genetics-based subgroup. Despite this concordance, the number of mutations exclusive to each biopsy increased with time since diagnosis, and late relapses shared few mutations with their diagnostic counterpart, demonstrating a branching evolution pattern. In patients with highly divergent tumors, many of the same genes acquired new mutations independently in each tumor, suggesting that the earliest mutations in a shared precursor cell constrain tumor evolution toward the same genetics-based subgroups at both diagnosis and relapse. CONCLUSION: These results suggest that late relapses commonly represent genetically distinct and chemotherapy-naïve disease and have implications for optimal patient management.

Genetic subdivisions of follicular lymphoma defined by distinct coding and noncoding mutation patterns.

Follicular lymphoma (FL) accounts for ∼20% of all new lymphoma cases. Increases in cytological grade are a feature of the clinical progression of this malignancy, and eventual histologic transformation (HT) to the aggressive diffuse large B-cell lymphoma (DLBCL) occurs in up to 15% of patients. Clinical or genetic features to predict the risk and timing of HT have not been described comprehensively. In this study, we analyzed whole-genome sequencing data from 423 patients to compare the protein coding and noncoding mutation landscapes of untransformed FL, transformed FL, and de novo DLBCL. This revealed 2 genetically distinct subgroups of FL, which we have named DLBCL-like (dFL) and constrained FL (cFL). Each subgroup has distinguishing mutational patterns, aberrant somatic hypermutation rates, and biological and clinical characteristics. We implemented a machine learning-derived classification approach to stratify patients with FL into cFL and dFL subgroups based on their genomic features. Using separate validation cohorts, we demonstrate that cFL status, whether assigned with this full classifier or a single-gene approximation, is associated with a reduced rate of HT. This implies distinct biological features of cFL that constrain its evolution, and we highlight the potential for this classification to predict HT from genetic features present at diagnosis.

Relapse timing is associated with distinct evolutionary dynamics in DLBCL.

Diffuse large B-cell lymphoma (DLBCL) is cured in over 60% of patients, but outcomes are poor for patients with relapsed or refractory disease (rrDLBCL). Here, we performed whole genome/exome sequencing (WGS/WES) on tumors from 73 serially-biopsied patients with rrDLBCL. Based on the observation that outcomes to salvage therapy/autologous stem cell transplantation are related to time-to-relapse, we stratified patients into groups according to relapse timing to explore the relationship to genetic divergence and sensitivity to salvage immunochemotherapy. The degree of mutational divergence increased with time between biopsies, yet tumor pairs were mostly concordant for cell-of-origin, oncogene rearrangement status and genetics-based subgroup. In patients with highly divergent tumors, several genes acquired exclusive mutations independently in each tumor, which, along with concordance of genetics-based subgroups, suggests that the earliest mutations in a shared precursor cell constrain tumor evolution. These results suggest that late relapses commonly represent genetically distinct and chemotherapy-naïve disease.

Genetic subgroups inform on pathobiology in adult and pediatric Burkitt lymphoma.

Burkitt lymphoma (BL) accounts for most pediatric non-Hodgkin lymphomas, being less common but significantly more lethal when diagnosed in adults. Much of the knowledge of the genetics of BL thus far has originated from the study of pediatric BL (pBL), leaving its relationship to adult BL (aBL) and other adult lymphomas not fully explored. We sought to more thoroughly identify the somatic changes that underlie lymphomagenesis in aBL and any molecular features that associate with clinical disparities within and between pBL and aBL. Through comprehensive whole-genome sequencing of 230 BL and 295 diffuse large B-cell lymphoma (DLBCL) tumors, we identified additional significantly mutated genes, including more genetic features that associate with tumor Epstein-Barr virus status, and unraveled new distinct subgroupings within BL and DLBCL with 3 predominantly comprising BLs: DGG-BL (DDX3X, GNA13, and GNAI2), IC-BL (ID3 and CCND3), and Q53-BL (quiet TP53). Each BL subgroup is characterized by combinations of common driver and noncoding mutations caused by aberrant somatic hypermutation. The largest subgroups of BL cases, IC-BL and DGG-BL, are further characterized by distinct biological and gene expression differences. IC-BL and DGG-BL and their prototypical genetic features (ID3 and TP53) had significant associations with patient outcomes that were different among aBL and pBL cohorts. These findings highlight shared pathogenesis between aBL and pBL, and establish genetic subtypes within BL that serve to delineate tumors with distinct molecular features, providing a new framework for epidemiologic, diagnostic, and therapeutic strategies.

Minimal information for reporting a genomics experiment.

Exome and genome sequencing has facilitated the identification of hundreds of genes and other regions that are recurrently mutated in hematologic neoplasms. The data sets from these studies theoretically provide opportunities. Quality differences between data sets can confound secondary analyses. We explore the consequences of these on the conclusions from some recent studies of B-cell lymphomas. We highlight the need for a minimum reporting standard to increase transparency in genomic research.

Super-enhancer hypermutation alters oncogene expression in B cell lymphoma.

Diffuse large B cell lymphoma (DLBCL) is the most common B cell non-Hodgkin lymphoma and remains incurable in around 40% of patients. Efforts to sequence the coding genome identified several genes and pathways that are altered in this disease, including potential therapeutic targets1-5. However, the non-coding genome of DLBCL remains largely unexplored. Here we show that active super-enhancers are highly and specifically hypermutated in 92% of samples from individuals with DLBCL, display signatures of activation-induced cytidine deaminase activity, and are linked to genes that encode B cell developmental regulators and oncogenes. As evidence of oncogenic relevance, we show that the hypermutated super-enhancers linked to the BCL6, BCL2 and CXCR4 proto-oncogenes prevent the binding and transcriptional downregulation of the corresponding target gene by transcriptional repressors, including BLIMP1 (targeting BCL6) and the steroid receptor NR3C1 (targeting BCL2 and CXCR4). Genetic correction of selected mutations restored repressor DNA binding, downregulated target gene expression and led to the counter-selection of cells containing corrected alleles, indicating an oncogenic dependency on the super-enhancer mutations. This pervasive super-enhancer mutational mechanism reveals a major set of genetic lesions deregulating gene expression, which expands the involvement of known oncogenes in DLBCL pathogenesis and identifies new deregulated gene targets of therapeutic relevance.

Analyzing the Interaction of RBPJ with Mitotic Chromatin and Its Impact on Transcription Reactivation upon Mitotic Exit.

The sequence-specific transcription factor RBPJ, also known as CSL (CBF1, Su(H), Lag1), is an evolutionarily conserved protein that mediates Notch signaling to guide cell fates. When cells enter mitosis, DNA is condensed and most transcription factors dissociate from chromatin; however, a few, select transcription factors, termed bookmarking factors, remain associated. These mitotic chromatin-bound factors are believed to play important roles in maintaining cell fates through cell division. RBPJ is one such factor that remains mitotic chromatin associated and therefore could function as a bookmarking factor. Here, we describe how to obtain highly purified mitotic cells from the mouse embryonal carcinoma cell line F9, perform chromatin immunoprecipitation with mitotic cells, and measure the first run of RNA synthesis upon mitotic exit. These methods serve as basis to understand the roles of mitotic bookmarking by RBPJ in propagating Notch signals through cell division.

A Novel Flow Cytometric Assay to Identify Inhibitors of RBPJ-DNA Interactions.

Notch signaling is often involved in cancer cell initiation and proliferation. Aberrant Notch activation underlies more than 50% of T-cell acute lymphoblastic leukemia (T-ALL); accordingly, chemicals disrupting Notch signaling are of potential to treat Notch-dependent cancer. Here, we developed a flow cytometry-based high-throughput assay to identify compounds that disrupt the interactions of DNA and RBPJ, the major downstream effector of Notch signaling. From 1492 compounds, we identified 18 compounds that disrupt RBPJ-DNA interactions in a dose-dependent manner. Cell-based assays further revealed that auranofin downregulates Notch-dependent transcription and decreases RBPJ-chromatin interactions in cells. Most strikingly, T-ALL cells that depend on Notch signaling for proliferation are more sensitive to auranofin treatment, supporting the notion that auranofin downregulates Notch signaling by disrupting RBPJ-DNA interaction. These results validate the feasibility of our assay scheme to screen for additional Notch inhibitors and provide a rationale to further test the use of auranofin in treating Notch-dependent cancer.

Gene Expression and DNA Methylation Alterations During Non-alcoholic Steatohepatitis-Associated Liver Carcinogenesis.

Hepatocellular carcinoma (HCC) is one of the most aggressive human cancers. HCC is characterized by an acquisition of multiple abnormal phenotypes driven by genetic and epigenetic alterations, especially abnormal DNA methylation. Most of the existing clinical and experimental reports provide only a snapshot of abnormal DNA methylation patterns in HCC rather than their dynamic changes. This makes it difficult to elucidate the significance of these changes in the development of HCC. In the present study, we investigated hepatic gene expression and gene-specific DNA methylation alterations in mice using the Stelic Animal Model (STAM) of non-alcoholic steatohepatitis (NASH)-derived liver carcinogenesis. Analysis of the DNA methylation status in aberrantly expressed epigenetically regulated genes showed the accumulation of DNA methylation abnormalities during the development of HCC, with the greatest number of aberrantly methylated genes being found in full-fledged HCC. Among these genes, only one gene, tubulin, beta 2B class IIB (Tubb2b), was increasingly hypomethylated and over-expressed during the progression of the carcinogenic process. Furthermore, the TUBB2B gene was also over-expressed and hypomethylated in poorly differentiated human HepG2 cells as compared to well-differentiated HepaRG cells. The results of this study indicate that unique gene-expression alterations mediated by aberrant DNA methylation of selective genes may contribute to the development of HCC and may have diagnostic value as the disease-specific indicator.

HDAC1 negatively regulates selective mitotic chromatin binding of the Notch effector RBPJ in a KDM5A-dependent manner.

Faithful propagation of transcription programs through cell division underlies cell-identity maintenance. Transcriptional regulators selectively bound on mitotic chromatin are emerging critical elements for mitotic transcriptional memory; however, mechanisms governing their site-selective binding remain elusive. By studying how protein-protein interactions impact mitotic chromatin binding of RBPJ, the major downstream effector of the Notch signaling pathway, we found that histone modifying enzymes HDAC1 and KDM5A play critical, regulatory roles in this process. We found that HDAC1 knockdown or inactivation leads to increased RBPJ occupancy on mitotic chromatin in a site-specific manner, with a concomitant increase of KDM5A occupancy at these sites. Strikingly, the presence of KDM5A is essential for increased RBPJ occupancy. Our results uncover a regulatory mechanism in which HDAC1 negatively regulates RBPJ binding on mitotic chromatin in a KDM5A-dependent manner. We propose that relative chromatin affinity of a minimal regulatory complex, reflecting a specific transcription program, renders selective RBPJ binding on mitotic chromatin.

Quantitative comparison of in vitro genotoxicity between metabolically competent HepaRG cells and HepG2 cells using the high-throughput high-content CometChip assay.

In vitro genotoxicity testing that employs metabolically active human cells may be better suited for evaluating human in vivo genotoxicity than current bacterial or non-metabolically active mammalian cell systems. In the current study, 28 compounds, known to have different genotoxicity and carcinogenicity modes of action (MoAs), were evaluated over a wide range of concentrations for the ability to induce DNA damage in human HepG2 and HepaRG cells. DNA damage dose-responses in both cell lines were quantified using a combination of high-throughput high-content (HTHC) CometChip technology and benchmark dose (BMD) quantitative approaches. Assays of metabolic activity indicated that differentiated HepaRG cells had much higher levels of cytochromes P450 activity than did HepG2 cells. DNA damage was observed for four and two out of five indirect-acting genotoxic carcinogens in HepaRG and HepG2 cells, respectively. Four out of seven direct-acting carcinogens were positive in both cell lines, with two of the three negatives being genotoxic mainly through aneugenicity. The four chemicals positive in both cell lines generated HTHC Comet data in HepaRG and HepG2 cells with comparable BMD values. All the non-genotoxic compounds, including six non-genotoxic carcinogens, were negative in HepaRG cells; five genotoxic non-carcinogens also were negative. Our results indicate that the HTHC CometChip assay detects a greater proportion of genotoxic carcinogens requiring metabolic activation (i.e., indirect carcinogens) when conducted with HepaRG cells than with HepG2 cells. In addition, BMD genotoxicity potency estimate is useful for quantitatively evaluating CometChip assay data in a scientifically rigorous manner.

Histopathological and Molecular Signatures of a Mouse Model of Acute-on-Chronic Alcoholic Liver Injury Demonstrate Concordance With Human Alcoholic Hepatitis.

Human alcoholic hepatitis (AH) carries a high mortality rate. AH is an acute-on-chronic form of liver injury characterized by hepatic steatosis, ballooned hepatocytes, neutrophil infiltration, and pericellular fibrosis. We aimed to study the pathogenesis of AH in an animal model which combines chronic hepatic fibrosis with intragastric alcohol administration. Adult male C57BL6/J mice were treated with CCl4 (0.2 ml/kg, 2×weekly by intraperitoneal injections for 6 weeks) to induce chronic liver fibrosis. Then, ethyl alcohol (up to 25 g/kg/day for 3 weeks) was administered continuously to mice via a gastric feeding tube, with or without one-half dose of CCl4. Liver and serum markers and liver transcriptome were evaluated to characterize acute-on-chronic-alcoholic liver disease in our model. CCl4 or alcohol treatment alone induced liver fibrosis or steatohepatitis, respectively, findings that were consistent with expected pathology. Combined treatment resulted in a marked exacerbation of liver injury, as evident by the development of inflammation, steatosis, and pericellular fibrosis, pathological features of human AH. E. coli and Candida were also detected in livers of mice cotreated with CCl4 and alcohol, indicating pathogen translocation from gut to liver, similar to human AH. Importantly, liver transcriptomic changes specific to combined treatment group demonstrated close concordance with pathways perturbed in patients with severe AH. Overall, mice treated with CCl4 and alcohol displayed key molecular and pathological characteristics of human AH-pericellular fibrosis, increased hepatic bacterial load, and dysregulation of the same molecular pathways. This model may be useful for developing therapeutics for AH.

Poly(ADP-ribose) polymerase 1 (PARP1) promotes oxidative stress-induced association of Cockayne syndrome group B protein with chromatin.

Cockayne syndrome protein B (CSB) is an ATP-dependent chromatin remodeler that relieves oxidative stress by regulating DNA repair and transcription. CSB is proposed to participate in base-excision repair (BER), the primary pathway for repairing oxidative DNA damage, but exactly how CSB participates in this process is unknown. It is also unclear whether CSB contributes to other repair pathways during oxidative stress. Here, using a patient-derived CS1AN-sv cell line, we examined how CSB is targeted to chromatin in response to menadione-induced oxidative stress, both globally and locus-specifically. We found that menadione-induced, global CSB-chromatin association does not require CSB's ATPase activity and is, therefore, mechanistically distinct from UV-induced CSB-chromatin association. Importantly, poly(ADP-ribose) polymerase 1 (PARP1) enhanced the kinetics of global menadione-induced CSB-chromatin association. We found that the major BER enzymes, 8-oxoguanine DNA glycosylase (OGG1) and apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1), do not influence this association. Additionally, the level of γ-H2A histone family member X (γ-H2AX), a marker for dsDNA breaks, was not increased in menadione-treated cells. Therefore, our results support a model whereby PARP1 localizes to ssDNA breaks and recruits CSB to participate in DNA repair. Furthermore, this global CSB-chromatin association occurred independently of RNA polymerase II-mediated transcription elongation. However, unlike global CSB-chromatin association, both PARP1 knockdown and inhibition of transcription elongation interfered with menadione-induced CSB recruitment to specific genomic regions. This observation supports the hypothesis that CSB is also targeted to specific genomic loci to participate in transcriptional regulation in response to oxidative stress.

Effect of aflatoxin B1, benzo[a]pyrene, and methapyrilene on transcriptomic and epigenetic alterations in human liver HepaRG cells.

The increasing number of man-made chemicals in the environment that may pose a carcinogenic risk highlights the need for developing reliable time- and cost-effective approaches for carcinogen detection and identification. To address this issue, we investigated the utility of high-throughput microarray gene expression and next-generation genome-wide DNA methylation sequencing for the in vitro identification of genotoxic and non-genotoxic carcinogens. Terminally differentiated and metabolically competent human liver HepaRG cells were treated at minimally cytotoxic concentrations of (i) the genotoxic human liver carcinogen aflatoxin B1 (AFB1) and its structural non-carcinogenic analog aflatoxin B2 (AFB2); (ii) the genotoxic human lung carcinogen benzo[a]pyrene (B[a]P) and its non-carcinogenic isomer benzo[e]pyrene (B[e]P); and (iii) the non-genotoxic liver carcinogen methapyrilene for 72 h and transcriptomic and DNA methylation profiles were examined. Treatment of HepaRG cells with the liver carcinogens AFB1 and methapyrilene generated distinct gene-expression profiles, whereas B[a]P had only a slight effect on gene expression. In contrast to transcriptomic alterations, treatment of HepaRG cells with the carcinogenic and non-carcinogenic chemicals resulted in profound changes in the DNA methylation footprint; however, the correlation between gene-specific DNA methylation and gene expression changes was minimal. Among the carcinogen-altered genes, transferrin (TF) emerged as sensitive marker for an initial screening of chemicals for their potential liver carcinogenicity. Potential liver carcinogens (i.e., chemicals causing altered TF gene expression) could then be subjected to gene-expression analyses to differentiate genotoxic from non-genotoxic liver carcinogens. This approach may substantially enhance the identification and assessment of potential liver carcinogens.

Cellular and Molecular Effects of Prolonged Low-Level Sodium Arsenite Exposure on Human Hepatic HepaRG Cells.

Inorganic arsenic is a human carcinogen associated with several types of cancers, including liver cancer. Inorganic arsenic has been postulated to target stem cells, causing their oncogenic transformation. This is proposed to be one of the key events in arsenic-associated carcinogenesis; however, the underlying mechanisms for this process remain largely unknown. To address this question, human hepatic HepaRG cells, at progenitor and differentiated states, were continuously treated with a noncytotoxic concentration of 1 µM sodium arsenite (NaAsO2). The HepaRG cells demonstrated active intracellular arsenite metabolism that shared important characteristic with primary human hepatocytes. Treatment of proliferating progenitor-like HepaRG cells with NaAsO2 inhibited their differentiation into mature hepatocyte-like cells, up-regulated genes involved in cell growth, proliferation, and survival, and down-regulated genes involved in cell death. In contrast, treatment of differentiated hepatocyte-like HepaRG cells with NaAsO2 resulted in enhanced cell death of mature hepatocyte-like cells, overexpression of cell death-related genes, and down-regulation of genes in the cell proliferation pathway, while biliary-like cells remained largely unaffected. Mechanistically, the cytotoxic effect of arsenic on mature hepatocyte-like HepaRG cells may be attributed to arsenic-induced dysregulation of cellular iron metabolism. The inhibitory effect of NaAsO2 on the differentiation of progenitor cells, the resistance of biliary-like cells to cell death, and the enhanced cell death of functional hepatocyte-like cells resulted in stem-cell activation. These effects favored the proliferation of liver progenitor cells that can serve as a source of initiation and driving force of arsenic-mediated liver carcinogenesis.

Multiple microRNAs function as self-protective modules in acetaminophen-induced hepatotoxicity in humans.

Acetaminophen (APAP) overdose is the leading cause of acute liver failure. Yet the mechanisms underlying adaptive tolerance toward APAP-induced liver injury are not fully understood. To better understand molecular mechanisms contributing to adaptive tolerance to APAP is an underpinning foundation for APAP-related precision medicine. In the current study, the mRNA and microRNA (miRNA) expression profiles derived from next generation sequencing data for APAP-treated (5 and 10 mM) HepaRG cells and controls were analyzed systematically. Putative miRNAs targeting key dysregulated genes involved in APAP hepatotoxicity were selected using in silico prediction algorithms, un-biased gene ontology, and network analyses. Luciferase reporter assays, RNA electrophoresis mobility shift assays, and miRNA pull-down assays were performed to investigate the role of miRNAs affecting the expression of dysregulated genes. Levels of selected miRNAs were measured in serum samples obtained from children with APAP overdose (58.6-559.4 mg/kg) and from healthy controls. As results, 2758 differentially expressed genes and 47 miRNAs were identified. Four of these miRNAs (hsa-miR-224-5p, hsa-miR-320a, hsa-miR-449a, and hsa-miR-877-5p) suppressed drug metabolizing enzyme (DME) levels involved in APAP-induced liver injury by downregulating HNF1A, HNF4A and NR1I2 expression. Exogenous transfection of these miRNAs into HepaRG cells effectively rescued them from APAP toxicity, as indicated by decreased alanine aminotransferase levels. Importantly, hsa-miR-320a and hsa-miR-877-5p levels were significantly elevated in serum samples obtained from children with APAP overdose compared to health controls. Collectively, these data indicate that hsa-miR-224-5p, hsa-miR-320a, hsa-miR-449a, and hsa-miR-877-5p suppress DME expression involved in APAP-induced hepatotoxicity and they contribute to an adaptive response in hepatocytes.

Epigenetically mediated inhibition of S-adenosylhomocysteine hydrolase and the associated dysregulation of 1-carbon metabolism in nonalcoholic steatohepatitis and hepatocellular carcinoma.

The substantial rise in the prevalence of nonalcoholic steatohepatitis (NASH), an advanced form of nonalcoholic fatty liver disease, and the strong association between NASH and the development of hepatocellular carcinoma indicate the urgent need for a better understanding of the underlying mechanisms. In the present study, by using the Stelic animal model of NASH and NASH-derived liver carcinogenesis, we investigated the role of the folate-dependent 1-carbon metabolism in the pathogenesis of NASH. We demonstrated that advanced NASH and NASH-related liver carcinogenesis are characterized by a significant dysregulation of 1-carbon homeostasis, with diminished expression of key 1-carbon metabolism genes, especially a marked inhibition of the S-adenosylhomocysteine hydrolase ( Ahcy) gene and an increased level of S-adenosyl-l-homocysteine (SAH). The reduction in Ahcy expression was associated with gene-specific cytosine DNA hypermethylation and enrichment of the gene promoter by trimethylated histone H3 lysine 27 and deacetylated histone H4 lysine 16, 2 main transcription-inhibiting markers. These results indicate that epigenetically mediated inhibition of Ahcy expression may be a driving force in causing SAH elevation and subsequent downstream disturbances in transsulfuration and transmethylation pathways during the development and progression of NASH.-Pogribny, I. P., Dreval, K., Kindrat, I., Melnyk, S., Jimenez, L., de Conti, A., Tryndyak, V., Pogribna, M., Ortega, J. F., James, S. J., Rusyn, I., Beland, F. A. Epigenetically mediated inhibition of S-adenosylhomocysteine hydrolase and the associated dysregulation of 1-carbon metabolism in nonalcoholic steatohepatitis and hepatocellular carcinoma.