Inactivation of CREBBP expands the germinal center B cell compartment, down-regulates MHCII expression and promotes DLBCL growth

The genes encoding the histone acetyl-transferases (HATs) CREB binding protein (CREBBP) and EP300 are recurrently mutated in the activated B cell-like and germinal center (GC) B cell-like subtypes of diffuse large B cell lymphoma (DLBCL). Here, we introduced a patient mutation into a human DLBCL cell line using CRISPR and deleted Crebbp and Ep300 in the GC B cell compartment of mice. CREBBP-mutant DLBCL clones exhibited reduced histone H3 acetylation, expressed significantly less MHCII, and grew faster than wild-type clones in s.c. and orthotopic xenograft models. Mice lacking Crebbp in GC B cells exhibited hyperproliferation of their GC compartment upon immunization, had reduced MHCII surface expression on GC cells, and developed accelerated MYC-driven lymphomas. Ep300 inactivation reproduced some, but not all, consequences of Crebbp inactivation. MHCII deficiency phenocopied the effects of CREBBP loss in spontaneous and serial transplantation models of MYC-driven lymphomagenesis, supporting the idea that the mutational inactivation of CREBBP promotes immune evasion. Indeed, the depletion of CD4(+) T cells greatly facilitated the engraftment of lymphoma cells in serial transplantation models. In summary, we provide evidence that both HATs are bona fide tumor suppressors that control MHCII expression and promote tumor immune control; mutational inactivation of CREBBP, but not of EP300, has additional cell-intrinsic engraftment and growth-promoting effects

Impact of renal impairment on atrial fibrillation: ESC-EHRA EORP-AF Long-Term General Registry

Background: Atrial fibrillation (AF) and renal impairment share a bidirectional relationship with important pathophysiological interactions. We evaluated the impact of renal impairment in a contemporary cohort of patients with AF. Methods: We utilised the ESC-EHRA EORP-AF Long-Term General Registry. Outcomes were analysed according to renal function by CKD-EPI equation. The primary endpoint was a composite of thromboembolism, major bleeding, acute coronary syndrome and all-cause death. Secondary endpoints were each of these separately including ischaemic stroke, haemorrhagic event, intracranial haemorrhage, cardiovascular death and hospital admission. Results: A total of 9306 patients were included. The distribution of patients with no, mild, moderate and severe renal impairment at baseline were 16.9%, 49.3%, 30% and 3.8%, respectively. AF patients with impaired renal function were older, more likely to be females, had worse cardiac imaging parameters and multiple comorbidities. Among patients with an indication for anticoagulation, prescription of these agents was reduced in those with severe renal impairment, p <.001. Over 24 months, impaired renal function was associated with significantly greater incidence of the primary composite outcome and all secondary outcomes. Multivariable Cox regression analysis demonstrated an inverse relationship between eGFR and the primary outcome (HR 1.07 [95% CI, 1.01–1.14] per 10 ml/min/1.73 m2 decrease), that was most notable in patients with eGFR <30 ml/min/1.73 m2 (HR 2.21 [95% CI, 1.23–3.99] compared to eGFR ≥90 ml/min/1.73 m2). Conclusion: A significant proportion of patients with AF suffer from concomitant renal impairment which impacts their overall management. Furthermore, renal impairment is an independent predictor of major adverse events including thromboembolism, major bleeding, acute coronary syndrome and all-cause death in patients with AF

Clinical complexity and impact of the ABC (Atrial fibrillation Better Care) pathway in patients with atrial fibrillation: a report from the ESC-EHRA EURObservational Research Programme in AF General Long-Term Registry

Background: Clinical complexity is increasingly prevalent among patients with atrial fibrillation (AF). The ‘Atrial fibrillation Better Care’ (ABC) pathway approach has been proposed to streamline a more holistic and integrated approach to AF care; however, there are limited data on its usefulness among clinically complex patients. We aim to determine the impact of ABC pathway in a contemporary cohort of clinically complex AF patients. Methods: From the ESC-EHRA EORP-AF General Long-Term Registry, we analysed clinically complex AF patients, defined as the presence of frailty, multimorbidity and/or polypharmacy. A K-medoids cluster analysis was performed to identify different groups of clinical complexity. The impact of an ABC-adherent approach on major outcomes was analysed through Cox-regression analyses and delay of event (DoE) analyses. Results: Among 9966 AF patients included, 8289 (83.1%) were clinically complex. Adherence to the ABC pathway in the clinically complex group reduced the risk of all-cause death (adjusted HR [aHR]: 0.72, 95%CI 0.58–0.91), major adverse cardiovascular events (MACEs; aHR: 0.68, 95%CI 0.52–0.87) and composite outcome (aHR: 0.70, 95%CI: 0.58–0.85). Adherence to the ABC pathway was associated with a significant reduction in the risk of death (aHR: 0.74, 95%CI 0.56–0.98) and composite outcome (aHR: 0.76, 95%CI 0.60–0.96) also in the high-complexity cluster; similar trends were observed for MACEs. In DoE analyses, an ABC-adherent approach resulted in significant gains in event-free survival for all the outcomes investigated in clinically complex patients. Based on absolute risk reduction at 1 year of follow-up, the number needed to treat for ABC pathway adherence was 24 for all-cause death, 31 for MACEs and 20 for the composite outcome. Conclusions: An ABC-adherent approach reduces the risk of major outcomes in clinically complex AF patients. Ensuring adherence to the ABC pathway is essential to improve clinical outcomes among clinically complex AF patients

Impact of clinical phenotypes on management and outcomes in European atrial fibrillation patients: a report from the ESC-EHRA EURObservational Research Programme in AF (EORP-AF) General Long-Term Registry

Background: Epidemiological studies in atrial fibrillation (AF) illustrate that clinical complexity increase the risk of major adverse outcomes. We aimed to describe European AF patients\u2019 clinical phenotypes and analyse the differential clinical course. Methods: We performed a hierarchical cluster analysis based on Ward\u2019s Method and Squared Euclidean Distance using 22 clinical binary variables, identifying the optimal number of clusters. We investigated differences in clinical management, use of healthcare resources and outcomes in a cohort of European AF patients from a Europe-wide observational registry. Results: A total of 9363 were available for this analysis. We identified three clusters: Cluster 1 (n = 3634; 38.8%) characterized by older patients and prevalent non-cardiac comorbidities; Cluster 2 (n = 2774; 29.6%) characterized by younger patients with low prevalence of comorbidities; Cluster 3 (n = 2955;31.6%) characterized by patients\u2019 prevalent cardiovascular risk factors/comorbidities. Over a mean follow-up of 22.5 months, Cluster 3 had the highest rate of cardiovascular events, all-cause death, and the composite outcome (combining the previous two) compared to Cluster 1 and Cluster 2 (all P <.001). An adjusted Cox regression showed that compared to Cluster 2, Cluster 3 (hazard ratio (HR) 2.87, 95% confidence interval (CI) 2.27\u20133.62; HR 3.42, 95%CI 2.72\u20134.31; HR 2.79, 95%CI 2.32\u20133.35), and Cluster 1 (HR 1.88, 95%CI 1.48\u20132.38; HR 2.50, 95%CI 1.98\u20133.15; HR 2.09, 95%CI 1.74\u20132.51) reported a higher risk for the three outcomes respectively. Conclusions: In European AF patients, three main clusters were identified, differentiated by differential presence of comorbidities. Both non-cardiac and cardiac comorbidities clusters were found to be associated with an increased risk of major adverse outcomes

Identification and Functional Analysis of Epigenetic Alterations in Diffuse Large B cell Lymphoma

Diffuse large B cell lymphoma (DLBCL) is the most common type of non- Hodgkin lymphoma accounting for around 30% of newly diagnosed cases per year. It is a very heterogeneous and aggressive disease, which arises from mature B-cells at different stages of differentiation and is characterized by a diffuse growth of neoplastic large B lymphocytes. Over the past years, enormous progress has been made in identifying key alterations associated with DLBCL, which have largely improved our understanding of the underlying molecular pathogenesis. Having said this, the majority of work has focused on genetic mechanisms and mutational events leading to malignant transformation, the contribution of epigenetic processes such as deregulated microRNA expression or aberrant DNA methylation to lymphomagenesis remains poorly understood. My doctoral studies have therefore been dedicated to elucidate the functional contribution of epigenetic alterations to the pathogenesis of DLBCL. Previous work in our group has focused on the analysis of aberrantly regulated microRNAs in DLBCL. In the scope of these studies one important tumor suppressor microRNA, miR-34a, was identified and characterized in vitro. In order to investigate the translational potential of the knowledge gained from these in vitro experiments, pre-clinical DLBCL xenograft studies were conducted. Strikingly, growth of subcutaneous tumor grafts was significantly reduced in mice treated locally or systemically with miR-34a mimetics in comparison to control treated mice. These findings suggest that miR-34a replacement therapy constitutes a valid therapeutic approach, especially for treatment of DLBCL patients with low miR-34a expression. Another major epigenetic event that gained increasing attention in the context of cancer research in recent years is DNA methylation. Aberrant DNA methylation of promoter regions is associated with the silencing of the respective gene and constitutes an important mechanism for cancer cells to silence tumor suppressor genes. The genes targeted by this mechanism in B cell lymphomas and their direct functional contribution to pathogenesis however remain to be elucidated. To address this issue, I set out to identify and functionally characterize hypermethylated genes in DLBCL. Whole genome DNA methylation analysis of low-grade marginal zone lymphoma of MALT (mucosa-associated lymphoid tissue) type, gastric and nodal DLBCL as well as control tonsil samples revealed a characteristic pattern of aberrant promoter-specific DNA hypermethylation, which is shared between all lymphoma entities analyzed. This suggests that global methylation changes represent an early event in lymphomagenesis and that these alterations are mediated by a shared mechanism operating irrespective of lymphoma localization. Further integration of this methylation data with data obtained from RNA sequencing after pharmacological demethylation of DLBCL cell lines allowed us to identify candidate tumor suppressor genes, which we subsequently validated in a series of functional screening experiments. One gene that emerged as a particularly interesting tumor suppressor candidate was the dual specificity phosphatase DUSP4, a negative regulator of mitogen-activated protein kinases. We could show that DUSP4 is either epigenetically silenced or deleted in the majority of nodal and extranodal DLBCL cases and that lack of DUSP4 negatively correlates with overall survival probability of DLBCL patients. Furthermore, we could demonstrate that DUSP4 dephosphorylates and thereby inhibits c-JUN N-terminal kinase (JNK) resulting in increased apoptosis in DLBCL cells. Pharmacological inhibition of JNK phenocopies these tumor suppressive effects in vitro and in vivo, reinforcing the importance of active JNK signaling for DLBCL cell survival and providing a mechanistic rationale for targeted JNK therapy as novel treatment approach. In conclusion, the work performed in the scope of this thesis contributes another valuable puzzle piece to understanding the complex epigenetic and genetic determinants of DLBCL and proposes novel therapeutic approaches for clinical consideration in the future

The role of microRNAs in the pathogenesis and treatment of hematopoietic malignancies

microRNAs (miRNAs) comprise a recently discovered class of non-coding RNAs with regulatory functions in post-transcriptional gene expression control. Many miRNAs are located in genomic regions that are frequently deleted in cancer, or are subject to epigenetic and transcriptional deregulation in cancer cells. The miRNA transcriptome of cancer cells is very different from that of their normal cell counterparts. miRNAs can exhibit oncogenic or tumor suppressive or even both properties depending on the specific targets and cellular context. It is becoming increasingly clear that miRNAs not only serve as useful tumor biomarkers with implications for diagnosis, prognosis and the prediction of treatment responses, but may also be used for targeted cancer treatment and even as therapeutics. In this review, we provide an overview of recent advances in our understanding of the tumor suppressor miRNAs and oncomiRs involved in the pathogenesis of leukemias and lymphomas, and their target transcripts in cancer signaling networks. In particular, we focus on the role of miRNAs in chronic lymphocytic and acute lymphoblastic leukemia and in B-cell lymphomas. In the second part, we review the various alternative strategies of targeting miRNAs in cancer therapy. Methods of oncomiR antagonization by antagomiRs or locked nucleid acids are contrasted with strategies that harness the tumor suppressive properties of certain miRNAs for cancer treatment. Preclinical progress, also with regard to delivery strategies, possible side effects and other pharmacological aspects, is presented along with results from the first human trials assessing the safety and efficacy of miRNA-targeting therapeutics

DUSP4 deficiency caused by promoter hypermethylation drives JNK signaling and tumor cell survival in diffuse large B cell lymphoma

The epigenetic dysregulation of tumor suppressor genes is an important driver of human carcinogenesis. We have combined genome-wide DNA methylation analyses and gene expression profiling after pharmacological DNA demethylation with functional screening to identify novel tumor suppressors in diffuse large B cell lymphoma (DLBCL). We find that a CpG island in the promoter of the dual-specificity phosphatase DUSP4 is aberrantly methylated in nodal and extranodal DLBCL, irrespective of ABC or GCB subtype, resulting in loss of DUSP4 expression in 75% of >200 examined cases. The DUSP4 genomic locus is further deleted in up to 13% of aggressive B cell lymphomas, and the lack of DUSP4 is a negative prognostic factor in three independent cohorts of DLBCL patients. Ectopic expression of wild-type DUSP4, but not of a phosphatase-deficient mutant, dephosphorylates c-JUN N-terminal kinase (JNK) and induces apoptosis in DLBCL cells. Pharmacological or dominant-negative JNK inhibition restricts DLBCL survival in vitro and in vivo and synergizes strongly with the Bruton's tyrosine kinase inhibitor ibrutinib. Our results indicate that DLBCL cells depend on JNK signaling for survival. This finding provides a mechanistic basis for the clinical development of JNK inhibitors in DLBCL, ideally in synthetic lethal combinations with inhibitors of chronic active B cell receptor signaling

The hematopoietic oncoprotein FOXP1 promotes tumor cell survival in diffuse large B-cell lymphoma by repressing S1PR2 signaling

Aberrant expression of the oncogenic transcription factor FOXP1 is a common feature of diffuse large B-cell lymphoma (DLBCL). We have combined chromatin immunoprecipitation and gene expression profiling after FOXP1 depletion with functional screening to identify targets of FOXP1 contributing to tumor cell survival. We find that the sphingosine-1-phosphate receptor 2 (S1PR2) is repressed by FOXP1 in activated B-cell (ABC) and germinal center B-cell (GCB) DLBCL cell lines with aberrantly high FOXP1 levels; S1PR2 expression is further inversely correlated with FOXP1 expression in three patient cohorts. Ectopic expression of wild type S1PR2, but not of a point mutant incapable of activating downstream signaling pathways, induces apoptosis in DLBCL cells and restricts tumor growth in subcutaneous and orthotopic models of the disease. The pro-apoptotic effects of S1PR2 are phenocopied by ectopic expression of the small G-protein Gα13, but are independent of AKT signaling. We further show that low S1PR2 expression is a strong negative prognosticator of patient survival, alone and especially in combination with high FOXP1 expression. The S1PR2 locus has previously been demonstrated to be recurrently mutated in GCB DLBCL; the transcriptional silencing of S1PR2 by FOXP1 represents an alternative mechanism leading to inactivation of this important hematopoietic tumor suppressor

Inactivation of CREBBP expands the germinal center B cell compartment, down-regulates MHCII expression and promotes DLBCL growth

The genes encoding the histone acetyl-transferases (HATs) CREB binding protein (CREBBP) and EP300 are recurrently mutated in the activated B cell-like and germinal center (GC) B cell-like subtypes of diffuse large B cell lymphoma (DLBCL). Here, we introduced a patient mutation into a human DLBCL cell line using CRISPR and deleted Crebbp and Ep300 in the GC B cell compartment of mice. CREBBP-mutant DLBCL clones exhibited reduced histone H3 acetylation, expressed significantly less MHCII, and grew faster than wild-type clones in s.c. and orthotopic xenograft models. Mice lacking Crebbp in GC B cells exhibited hyperproliferation of their GC compartment upon immunization, had reduced MHCII surface expression on GC cells, and developed accelerated MYC-driven lymphomas. Ep300 inactivation reproduced some, but not all, consequences of Crebbp inactivation. MHCII deficiency phenocopied the effects of CREBBP loss in spontaneous and serial transplantation models of MYC-driven lymphomagenesis, supporting the idea that the mutational inactivation of CREBBP promotes immune evasion. Indeed, the depletion of CD4(+) T cells greatly facilitated the engraftment of lymphoma cells in serial transplantation models. In summary, we provide evidence that both HATs are bona fide tumor suppressors that control MHCII expression and promote tumor immune control; mutational inactivation of CREBBP, but not of EP300, has additional cell-intrinsic engraftment and growth-promoting effects