ARID1A orchestrates SWI/SNF-mediated sequential binding of transcription factors with ARID1A loss driving pre-memory B cell fate and lymphomagenesis.

ARID1A, a subunit of the canonical BAF nucleosome remodeling complex, is commonly mutated in lymphomas. We show that ARID1A orchestrates B cell fate during the germinal center (GC) response, facilitating cooperative and sequential binding of PU.1 and NF-kB at crucial genes for cytokine and CD40 signaling. The absence of ARID1A tilts GC cell fate toward immature IgM+CD80-PD-L2- memory B cells, known for their potential to re-enter new GCs. When combined with BCL2 oncogene, ARID1A haploinsufficiency hastens the progression of aggressive follicular lymphomas (FLs) in mice. Patients with FL with ARID1A-inactivating mutations preferentially display an immature memory B cell-like state with increased transformation risk to aggressive disease. These observations offer mechanistic understanding into the emergence of both indolent and aggressive ARID1A-mutant lymphomas through the formation of immature memory-like clonal precursors. Lastly, we demonstrate that ARID1A mutation induces synthetic lethality to SMARCA2/4 inhibition, paving the way for potential precision therapy for high-risk patients.

Lysine demethylase LSD1 is associated with stemness in EBV-positive B cell lymphoma.

EBV-infected lymphoma has a poor prognosis and various treatment strategies are being explored. Reports suggesting that B cell lymphoma can be induced by epigenetic regulation have piqued interest in studying mechanisms targeting epigenetic regulation. Here, we set out to identify an epigenetic regulator drug that acts synergistically with doxorubicin in EBV-positive lymphoma. We expressed the major EBV protein, LMP1, in B-cell lymphoma cell lines and used them to screen 100 epigenetic modifiers in combination with doxorubicin. The screening results identified TCP, which is an inhibitor of LSD1. Further analyses revealed that LMP1 increased the activity of LSD1 to enhance stemness ability under doxorubicin treatment, as evidenced by colony-forming and ALDEFLUOR activity assays. Quantseq 3' mRNA sequencing analysis of potential targets regulated by LSD1 in modulating stemness revealed that the LMP1-induced upregulation of CHAC2 was decreased when LSD1 was inhibited by TCP or downregulated by siRNA. We further observed that SOX2 expression was altered in response to CHAC2 expression, suggesting that stemness is regulated. Collectively, these findings suggest that LSD1 inhibitors could serve as promising therapeutic candidates for EBV-positive lymphoma, potentially reducing stemness activity when combined with conventional drugs to offer an effective treatment approach.

Alterations in Genome Organization in Lymphoma Cell Nuclei due to the Presence of the t(14;18) Translocation.

Chromosomal rearrangements have been shown to alter genome organization, consequently having an impact on gene expression. Studies on certain types of leukemia have shown that gene expression can be exacerbated by the altered nuclear positioning of fusion genes arising from chromosomal translocations. However, studies on lymphoma have been, so far, very limited. The scope of this study was to explore genome organization in lymphoma cells carrying the t(14;18)(q32;q21) rearrangement known to results in over-expression of the BCL2 gene. In order to achieve this aim, we used fluorescence in situ hybridization to carefully map the positioning of whole chromosome territories and individual genes involved in translocation in the lymphoma-derived cell line Pfeiffer. Our data show that, although there is no obvious alteration in the positioning of the whole chromosome territories, the translocated genes may take the nuclear positioning of either of the wild-type genes. Furthermore, the BCL2 gene was looping out in a proportion of nuclei with the t(14;18) translocation but not in control nuclei without the translocation, indicating that chromosome looping may be an essential mechanism for BCL2 expression in lymphoma cells.

E3 ubiquitin ligase casitas B-lineage lymphoma-b modulates T-cell anergic resistance via phosphoinositide 3-kinase signaling in patients with immune thrombocytopenia.

BACKGROUND: The E3 ubiquitin ligase casitas B-lineage lymphoma-b (CBLB) is a newly identified component of the ubiquitin-dependent protein degradation system and is considered an important negative regulator of immune cells. CBLB is essential for establishing a threshold of T-cell activation and regulating peripheral T-cell tolerance through various mechanisms. However, the involvement of CBLB in the pathogenesis of immune thrombocytopenia (ITP) is unknown. OBJECTIVES: We aimed to investigate the expression and role of CBLB in CD4+ T cells obtained from patients with ITP through quantitative proteomics analyses. METHODS: CD4+ T cells were transfected with adenoviral vectors overexpressing CBLB to clarify the effect of CBLB on anergic induction of T cells in patients with ITP. DNA methylation levels of the CBLB promoter and 5' untranslated region (UTR) in patient-derived CD4+ T cells were detected via MassARRAY EpiTYPER assay (Agena Bioscience). RESULTS: CD4+ T cells from patients with ITP showed resistance to anergic induction, highly activated phosphoinositide 3-kinase-protein kinase B (AKT) signaling, decreased CBLB expression, and 5' UTR hypermethylation of CBLB. CBLB overexpression in T cells effectively attenuated the elevated phosphorylated protein kinase B level and resistance to anergy. Low-dose decitabine treatment led to significantly elevated levels of CBLB expression in CD4+ T cells from 7 patients showing a partial or complete response. CONCLUSION: These results indicate that the 5' UTR hypermethylation of CBLB in CD4+ T cells induces resistance to T-cell anergy in ITP. Thus, the upregulation of CBLB expression by low-dose decitabine treatment may represent a potential therapeutic approach to ITP.

Predictive and prognostic molecular biomarkers in lymphomas.

Recent advances in molecular diagnostics have markedly expanded our understanding of the genetic underpinnings of lymphomas and catalysed a transformation in not just how we classify lymphomas, but also how we treat, target, and monitor affected patients. Reflecting these advances, the World Health Organization Classification, International Consensus Classification, and National Comprehensive Cancer Network guidelines were recently updated to better integrate these molecular insights into clinical practice. We summarise here the molecular biomarkers of lymphomas with an emphasis on biomarkers that have well-supported prognostic and predictive utility, as well as emerging biomarkers that show promise for clinical practice. These biomarkers include: (1) diagnostic entity-defining genetic abnormalities [e.g., B-cell acute lymphoblastic leukaemia (B-ALL) with KMT2A rearrangement]; (2) molecular alterations that guide patients' prognoses (e.g., TP53 loss frequently conferring worse prognosis); (3) mutations that serve as the targets of, and often a source of acquired resistance to, small molecular inhibitors (e.g., ABL1 tyrosine kinase inhibitors for B-ALL BCR::ABL1, hindered by ABL1 kinase domain resistance mutations); (4) the growing incorporation of molecular measurable residual disease (MRD) in the management of lymphoma patients (e.g., molecular complete response and sequencing MRD-negative criteria in multiple myeloma). Altogether, our review spans the spectrum of lymphoma types, from the genetically defined subclasses of precursor B-cell lymphomas to the highly heterogeneous categories of small and large cell mature B-cell lymphomas, Hodgkin lymphomas, plasma cell neoplasms, and T/NK-cell lymphomas, and provides an expansive summary of our current understanding of their molecular pathology.

Cell-Free DNA Extraction of Vitreous and Aqueous Humor Specimens for Diagnosis and Monitoring of Vitreoretinal Lymphoma.

Vitreoretinal lymphoma (VRL) represents an aggressive lymphoma, often categorized as primary central nervous system diffuse large B-cell lymphoma. To diagnose VRL, specimens such as vitreous humor and, more recently, aqueous humor are collected. Diagnostic testing for VRL on these specimens includes cytology, flow cytometry, and molecular testing. However, both cytopathology and flow cytometry, along with molecular testing using cellular DNA, necessitate intact whole cells. The challenge lies in the fact that vitreous and aqueous humor typically have low cellularity, and many cells get destroyed during collection, storage, and processing. Moreover, these specimens pose additional difficulties for molecular testing due to the high viscosity of vitreous humor and the low volume of both vitreous and aqueous humor. This study proposes a method for extracting cell-free DNA from vitreous and aqueous specimens. This approach complements the extraction of cellular DNA or allows the cellular component of these specimens to be utilized for other diagnostic methods, including cytology and flow cytometry.

Whole-genome CRISPR screening identifies molecular mechanisms of PD-L1 expression in adult T-cell leukemia/lymphoma.

ABSTRACT: Adult T-cell leukemia/lymphoma (ATLL) is an aggressive T-cell malignancy with a poor prognosis and limited treatment options. Programmed cell death ligand 1(PD-L1) is recognized to be involved in the pathobiology of ATLL. However, what molecules control PD-L1 expression and whether genetic or pharmacological intervention might modify PD-L1 expression in ATLL cells are still unknown. To comprehend the regulatory mechanisms of PD-L1 expression in ATLL cells, we performed unbiased genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening in this work. In ATLL cells, we discovered that the neddylation-associated genes NEDD8, NAE1, UBA3, and CUL3 negatively regulated PD-L1 expression, whereas STAT3 positively did so. We verified, in line with the genetic results, that treatment with the JAK1/2 inhibitor ruxolitinib or the neddylation pathway inhibitor pevonedistat resulted in a decrease in PD-L1 expression in ATLL cells or an increase in it, respectively. It is significant that these results held true regardless of whether ATLL cells had the PD-L1 3' structural variant, a known genetic anomaly that promotes PD-L1 overexpression in certain patients with primary ATLL. Pevonedistat alone showed cytotoxicity for ATLL cells, but compared with each single modality, pevonedistat improved the cytotoxic effects of the anti-PD-L1 monoclonal antibody avelumab and chimeric antigen receptor (CAR) T cells targeting PD-L1 in vitro. As a result, our work provided insight into a portion of the complex regulatory mechanisms governing PD-L1 expression in ATLL cells and demonstrated the in vitro preliminary preclinical efficacy of PD-L1-directed immunotherapies by using pevonedistat to upregulate PD-L1 in ATLL cells.

Comparative evaluation of 11C-methionine and 18F-fluorodeoxyglucose positron emission tomography for distinguishing between primary central nervous system lymphoma and isocitrate dehydrogenase-wildtype glioblastoma.

PURPOSE: Distinguishing between primary central nervous system lymphoma (PCNSL) and isocitrate dehydrogenase (IDH)-wildtype glioblastoma is important for therapeutic decision-making. This study aimed to compare the performance of 11C-methionine (MET) and 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) for distinguishing between these two major malignant brain tumors. METHODS: We retrospectively conducted qualitative and semiquantitative analyses of pre-treatment MET and FDG PET/computed tomography (CT) images of 22 patients with PCNSL and 64 patients with IDH-wildtype glioblastoma. For semiquantitative analysis, we calculated the tumor-to-normal tissue (T/N) ratio by dividing the maximum standardized uptake value (SUV) for the tumor (T) by the average SUV for the normal tissue (N). For performance evaluation, we employed receiver operating characteristic curve analysis and calculated the areas under the curve (AUC) values. RESULTS: In the qualitative analysis, all PCNSLs and IDH-wildtype glioblastomas were MET-positive, while 95% and 84% of PCNSLs and IDH-wildtype glioblastomas, respectively, were FDG-positive. Eleven patients were excluded from the FDG PET/CT semiquantitative analysis because of hyperglycemia. There was no difference in MET T/N ratio between PCNSL and IDH-wildtype glioblastoma (p = 0.37). FDG T/N ratio was significantly higher in PCNSL than in IDH-wildtype glioblastoma (p < 0.001). The AUC value for distinguishing PCNSL from IDH-wildtype glioblastoma was significantly higher for the FDG T/N ratio (0.871) than for the MET T/N ratio (0.565) (p = 0.0027). CONCLUSION: MET PET could detect both PCNSL and IDH-wildtype glioblastoma, but unlike FDG PET, it could not distinguish between these two major malignant brain tumors.

Noncoding mutations cause super-enhancer retargeting resulting in protein synthesis dysregulation during B cell lymphoma progression.

Whole-genome sequencing of longitudinal tumor pairs representing transformation of follicular lymphoma to high-grade B cell lymphoma with MYC and BCL2 rearrangements (double-hit lymphoma) identified coding and noncoding genomic alterations acquired during lymphoma progression. Many of these transformation-associated alterations recurrently and focally occur at topologically associating domain resident regulatory DNA elements, including H3K4me3 promoter marks located within H3K27ac super-enhancer clusters in B cell non-Hodgkin lymphoma. One region found to undergo recurrent alteration upon transformation overlaps a super-enhancer affecting the expression of the PAX5/ZCCHC7 gene pair. ZCCHC7 encodes a subunit of the Trf4/5-Air1/2-Mtr4 polyadenylation-like complex and demonstrated copy number gain, chromosomal translocation and enhancer retargeting-mediated transcriptional upregulation upon lymphoma transformation. Consequently, lymphoma cells demonstrate nucleolar dysregulation via altered noncoding 5.8S ribosomal RNA processing. We find that a noncoding mutation acquired during lymphoma progression affects noncoding rRNA processing, thereby rewiring protein synthesis leading to oncogenic changes in the lymphoma proteome.

Cytogenomics of B-cell non-Hodgkin lymphomas: The "old" meets the "new".

For the routine diagnosis of haematological neoplasms an integrative approach is used considering the morphology, and the immunophenotypic, and molecular features of the tumor sample, along with clinical information. The identification and characterization of recurrent chromosomal aberrations mainly detected by conventional and molecular cytogenetics in the tumor cells has a major impact on the classification of lymphoid neoplasms. Some of the B-cell non-Hodgkin lymphomas are characterized by particular chromosomal aberrations, highlighting the relevance of conventional and molecular cytogenetic studies in their diagnosis and prognosis. In the current genomics era, next generation sequencing provides relevant information as the mutational profiles of haematological malignancies, improving their classification and also the clinical management of the patients. In addition, other new technologies have emerged recently, such as the optical genome mapping, which can overcome some of the limitations of conventional and molecular cytogenetics and may become more widely used in the cytogenetic laboratories in the upcoming years. Moreover, epigenetic alterations may complement genetic changes for a deeper understanding of the pathogenesis underlying B-cell neoplasms and a more precise risk-based patient stratification. Overall, here we describe the current state of the genomic data integrating chromosomal rearrangements, copy number alterations, and somatic variants, as well as a succinct overview of epigenomic changes, which altogether constitute a comprehensive diagnostic approach in B-cell non-Hodgkin lymphomas.

Mechanisms of ferroptosis and targeted therapeutic approaches in lymphoma.

Lymphoma is the sixth most common type of cancer worldwide. Under the current treatment standards, patients with lymphoma often fail to respond to treatment or relapse early and require further therapy. Hence, novel therapeutic strategies need to be explored and our understanding of the molecular underpinnings of lymphomas should be expanded. Ferroptosis, a non-apoptotic regulated cell death, is characterized by increased reactive oxygen species and lipid peroxidation due to metabolic dysfunction. Excessive or lack of ferroptosis has been implicated in tumor development. Current preclinical evidences suggest that ferroptosis participates in tumorigenesis, progression, and drug resistance of lymphoma, identifying a potential biomarker and an attractive molecular target. Our review summarizes the core mechanisms and regulatory networks of ferroptosis and discusses existing evidences of ferroptosis induction for the treatment of lymphoma, with intent to provide a framework for understanding the role of ferroptosis in lymphomagenesis and a new perspective of lymphoma treatment.

TCR Pathway Mutations in Mature T Cell Lymphomas.

Mature T cell lymphomas are heterogeneous neoplasms that are aggressive and resistant to treatment. Many of these cancers retain immunological properties of their cell of origin. They express cytokines, cytotoxic enzymes, and cell surface ligands normally induced by TCR signaling in untransformed T cells. Until recently, their molecular mechanisms were unclear. Recently, high-dimensional studies have transformed our understanding of their cellular and genetic characteristics. Somatic mutations in the TCR signaling pathway drive lymphomagenesis by disrupting autoinhibitory domains, increasing affinity to ligands, and/or inducing TCR-independent signaling. Collectively, most of these mutations augment signaling pathways downstream of the TCR. Emerging data suggest that these mutations not only drive proliferation but also determine lymphoma immunophenotypes. For example, RHOA mutations are sufficient to induce disease-relevant CD4+ T follicular helper cell phenotypes. In this review, we describe how mutations in the TCR signaling pathway elucidate lymphoma pathophysiology but also provide insights into broader T cell biology.

Transcriptional reprogramming by mutated IRF4 in lymphoma.

Disease-causing mutations in genes encoding transcription factors (TFs) can affect TF interactions with their cognate DNA-binding motifs. Whether and how TF mutations impact upon the binding to TF composite elements (CE) and the interaction with other TFs is unclear. Here, we report a distinct mechanism of TF alteration in human lymphomas with perturbed B cell identity, in particular classic Hodgkin lymphoma. It is caused by a recurrent somatic missense mutation c.295 T > C (p.Cys99Arg; p.C99R) targeting the center of the DNA-binding domain of Interferon Regulatory Factor 4 (IRF4), a key TF in immune cells. IRF4-C99R fundamentally alters IRF4 DNA-binding, with loss-of-binding to canonical IRF motifs and neomorphic gain-of-binding to canonical and non-canonical IRF CEs. IRF4-C99R thoroughly modifies IRF4 function by blocking IRF4-dependent plasma cell induction, and up-regulates disease-specific genes in a non-canonical Activator Protein-1 (AP-1)-IRF-CE (AICE)-dependent manner. Our data explain how a single mutation causes a complex switch of TF specificity and gene regulation and open the perspective to specifically block the neomorphic DNA-binding activities of a mutant TF.

Correlation study of malignant lymphoma and breast Cancer in different gender European populations: mendelian randomization analysis.

BACKGROUND: Previous research has already indicated an elevated risk of breast cancer (BC) among survivors of malignant lymphoma, but the underlying reasons remain unknown. Our objective is to elucidate the causal relationship between malignant lymphoma and BC through Mendelian randomization (MR). Genome-wide association studies (GWAS) data from 181,125 Hodgkin lymphoma (HL) patients and 181,289 non-Hodgkin lymphoma (NHL) patients from the FinnGen Consortium were utilized as exposure. We selected single nucleotide polymorphisms (SNPs) strongly associated with the exposure as instrumental variables to investigate their relationship with BC in a cohort of 107,722 participants. Subsequently, we obtained data from the UK Biobank containing gender-stratified information on HL, NHL, and BC. We validated the findings from our analysis and explored the impact of gender. The Inverse-Variance Weighted (IVW) method served as the primary reference for the two-sample MR, accompanied by tests for heterogeneity and pleiotropy. RESULTS: The analysis results from the FinnGen consortium indicate that there is no causal relationship between HL and NHL with BC. HL (OR = 1.01, 95% CI = 0.98-1.04, p = 0.29), NHL (OR = 1.01, 95% CI = 0.96-1.05, p = 0.64). When utilizing GWAS data from the UK Biobank that includes different gender cohorts, the lack of association between HL, NHL, and BC remains consistent. HL (OR = 1.08, 95% CI = 0.74-1.56, p = 0.69), HL-Female (OR = 0.84, 95% CI = 0.59-1.19, p = 0.33), NHL (OR = 0.89, 95% CI = 0.66-1.19, p = 0.44), and NHL-Female (OR = 0.81, 95% CI = 0.58-1.11, p = 0.18). CONCLUSIONS: The two-sample MR analysis indicates that there is no significant causal relationship between malignant lymphoma (HL and NHL) and BC. The association between malignant lymphoma and breast cancer requires further in-depth research and exploration.

circ_SPEF2 Regulates the Balance of Treg Cells by Regulating miR-16-5p/BACH2 in Lymphoma and Participates in the Immune Response.

BACKGROUND: This study aims to explore the potential mechanism of action of the newly discovered hsa_circ_0128899 (circSPEF2) in diffuse large B-cell lymphoma (DLBCL). METHODS: circSPEF2, miR-16-5p and BTB and CNC homologue 2 (BACH2) expression patterns in DLBCL patients and cell lines were studied by RT-qPCR. The biological function of circSPEF2 in vitro and in vivo was investigated by function acquisition experiments. The proliferation activity of lymphoma cells was detected by MTT. Bax, Caspase-3, and Bcl-2 were determined by Western Blot. Apoptosis and the ratio of CD4 to Treg of immune cells in the co-culture system were analyzed by flow cytometry. The mechanism of action of circSPEF2 in DLBCL progression was further investigated by RIP and dual luciferase reporter experiments. RESULTS: circSPEF2 was a circRNA with abnormally down-regulated expression in DLBCL. Increasing circSPEF2 expression inhibited the proliferative activity and induced apoptosis of lymphoma cells in vitro and in vivo, as well as increased CD4+T cells and decreased Treg cell proportion of immune cells in the tumor microenvironment. Mechanically, circSPEF2 was bound to miR-16-5p expression, while BACH2 was targeted by miR-16-5p. circSPEF2 overexpression-mediated effects on lymphoma progression were reversible by upregulating miR-16-5p or downregulating BACH2. CONCLUSIONS: circSPEF2 can influence DLBCL progression by managing cellular proliferation and apoptosis and the proportion of immune cells Treg and CD4 through the miR-16-5p/BACH2 axis.

Aging and Age-Related Epigenetic Drift in the Pathogenesis of Leukemia and Lymphomas: New Therapeutic Targets.

One of the traits of cancer cells is abnormal DNA methylation patterns. The idea that age-related epigenetic changes may partially explain the increased risk of cancer in the elderly is based on the observation that aging is also accompanied by comparable changes in epigenetic patterns. Lineage bias and decreased stem cell function are signs of hematopoietic stem cell compartment aging. Additionally, aging in the hematopoietic system and the stem cell niche have a role in hematopoietic stem cell phenotypes linked with age, such as leukemia and lymphoma. Understanding these changes will open up promising pathways for therapies against age-related disorders because epigenetic mechanisms are reversible. Additionally, the development of high-throughput epigenome mapping technologies will make it possible to identify the "epigenomic identity card" of every hematological disease as well as every patient, opening up the possibility of finding novel molecular biomarkers that can be used for diagnosis, prediction, and prognosis.

Circadian disruption does not alter tumorigenesis in a mouse model of lymphoma.

Background: Disruption of natural light cycles, as experienced by shift workers, is linked to enhanced cancer incidence. Several mouse models of cancer develop more severe disease when exposed to irregular light/dark cycles, supporting the connection between circadian disruption and increased cancer risk. Cryptochrome 2 (CRY2), a repressive component of the molecular circadian clock, facilitates turnover of the oncoprotein c-MYC, one mechanism that may link the molecular clock to tumorigenesis. In Eµ-MYC mice, which express transgenic c-MYC in B cells and develop aggressive lymphomas and leukemia, global Cry2 deletion reduces survival and enhances tumor formation. Lighting conditions that mimic the disruption experienced by shift workers dampen Cry2 transcripts in peripheral tissues of C57BL/6J mice. Although it is milder than homozygous deletion of Cry2, we hypothesized that reduced Cry2 rhythmicity could alter MYC protein accumulation and contribute to enhanced cancer risk caused by circadian disruption. We tested this hypothesis in MYC-driven lymphoma. Methods: We housed Eµ-MYC mice in light-tight boxes set to either control (continuous cycles of 12-hours of light followed by 12-hours of dark, LD12:12) or chronic jetlag (eight-hour light phase advances every two to three days, CJL) lighting conditions and assessed the impact of disrupted light cycles on survival and tumor formation in Eµ-MYC mice. Results: Environmental disruption of circadian rhythms did not alter tumor location, tumor growth, or survival in Eµ-MYC mice. Conclusions: Dampened rhythms of Cry2 following disruption of circadian light exposures is milder than deletion of Cry2. The lack of phenotype caused by altered circadian gene expression in contrast to enhanced tumorigenesis caused by homozygous deletion of Cry2 suggests that CRY2 dosage impacts this model. Importantly, these findings indicate that increased cancer risk associated with circadian disruption arises from one or more mechanisms that are not recapitulated here, and may be different in distinct tumor types.