Altered enhancer-promoter interaction leads to MNX1 expression in pediatric acute myeloid leukemia with t(7;12)(q36;p13).

ABSTRACT: Acute myeloid leukemia (AML) with the t(7;12)(q36;p13) translocation occurs only in very young children and has a poor clinical outcome. The expected oncofusion between break point partners (motor neuron and pancreas homeobox 1 [MNX1] and ETS variant transcription factor 6 [ETV6]) has only been reported in a subset of cases. However, a universal feature is the strong transcript and protein expression of MNX1, a homeobox transcription factor that is normally not expressed in hematopoietic cells. Here, we map the translocation break points on chromosomes 7 and 12 in affected patients to a region proximal to MNX1 and either introns 1 or 2 of ETV6. The frequency of MNX1 overexpression in pediatric AML is 2.4% and occurs predominantly in t(7;12)(q36;p13) AML. Chromatin interaction assays in a t(7;12)(q36;p13) induced pluripotent stem cell line model unravel an enhancer-hijacking event that explains MNX1 overexpression in hematopoietic cells. Our data suggest that enhancer hijacking may be a more widespread consequence of translocations in which no oncofusion product was identified, including t(1;3) or t(4;12) AML.

Neutrophil-specific expression of JAK2-V617F or CALRmut induces distinct inflammatory profiles in myeloproliferative neoplasia.

BACKGROUND: Neutrophils play a crucial role in inflammation and in the increased thrombotic risk in myeloproliferative neoplasms (MPNs). We have investigated how neutrophil-specific expression of JAK2-V617F or CALRdel re-programs the functions of neutrophils. METHODS: Ly6G-Cre JAK2-V617F and Ly6G-Cre CALRdel mice were generated. MPN parameters as blood counts, splenomegaly and bone marrow histology were compared to wild-type mice. Megakaryocyte differentiation was investigated using lineage-negative bone marrow cells upon in vitro incubation with TPO/IL-1ß. Cytokine concentrations in serum of mice were determined by Mouse Cytokine Array. IL-1α expression in various hematopoietic cell populations was determined by intracellular FACS analysis. RNA-seq to analyse gene expression of inflammatory cytokines was performed in isolated neutrophils from JAK2-V617F and CALR-mutated mice and patients. Bioenergetics of neutrophils were recorded on a Seahorse extracellular flux analyzer. Cell motility of neutrophils was monitored in vitro (time lapse microscopy), and in vivo (two-photon microscopy) upon creating an inflammatory environment. Cell adhesion to integrins, E-selectin and P-selection was investigated in-vitro. Statistical analysis was carried out using GraphPad Prism. Data are shown as mean ± SEM. Unpaired, two-tailed t-tests were applied. RESULTS: Strikingly, neutrophil-specific expression of JAK2-V617F, but not CALRdel, was sufficient to induce pro-inflammatory cytokines including IL-1 in serum of mice. RNA-seq analysis in neutrophils from JAK2-V617F mice and patients revealed a distinct inflammatory chemokine signature which was not expressed in CALR-mutant neutrophils. In addition, IL-1 response genes were significantly enriched in neutrophils of JAK2-V617F patients as compared to CALR-mutant patients. Thus, JAK2-V617F positive neutrophils, but not CALR-mutant neutrophils, are pathogenic drivers of inflammation in MPN. In line with this, expression of JAK2-V617F or CALRdel elicited a significant difference in the metabolic phenotype of neutrophils, suggesting a stronger inflammatory activity of JAK2-V617F cells. Furthermore, JAK2-V617F, but not CALRdel, induced a VLA4 integrin-mediated adhesive phenotype in neutrophils. This resulted in reduced neutrophil migration in vitro and in an inflamed vessel. This mechanism may contribute to the increased thrombotic risk of JAK2-V617F patients compared to CALR-mutant individuals. CONCLUSIONS: Taken together, our findings highlight genotype-specific differences in MPN-neutrophils that have implications for the differential pathophysiology of JAK2-V617F versus CALR-mutant disease.

Multi-omic and functional analysis for classification and treatment of sarcomas with FUS-TFCP2 or EWSR1-TFCP2 fusions.

Linking clinical multi-omics with mechanistic studies may improve the understanding of rare cancers. We leverage two precision oncology programs to investigate rhabdomyosarcoma with FUS/EWSR1-TFCP2 fusions, an orphan malignancy without effective therapies. All tumors exhibit outlier ALK expression, partly accompanied by intragenic deletions and aberrant splicing resulting in ALK variants that are oncogenic and sensitive to ALK inhibitors. Additionally, recurrent CKDN2A/MTAP co-deletions provide a rationale for PRMT5-targeted therapies. Functional studies show that FUS-TFCP2 blocks myogenic differentiation, induces transcription of ALK and truncated TERT, and inhibits DNA repair. Unlike other fusion-driven sarcomas, TFCP2-rearranged tumors exhibit genomic instability and signs of defective homologous recombination. DNA methylation profiling demonstrates a close relationship with undifferentiated sarcomas. In two patients, sarcoma was preceded by benign lesions carrying FUS-TFCP2, indicating stepwise sarcomagenesis. This study illustrates the potential of linking precision oncology with preclinical research to gain insight into the classification, pathogenesis, and therapeutic vulnerabilities of rare cancers.

NCT/DKFZ MASTER handbook of interpreting whole-genome, transcriptome, and methylome data for precision oncology.

Analysis of selected cancer genes has become an important tool in precision oncology but cannot fully capture the molecular features and, most importantly, vulnerabilities of individual tumors. Observational and interventional studies have shown that decision-making based on comprehensive molecular characterization adds significant clinical value. However, the complexity and heterogeneity of the resulting data are major challenges for disciplines involved in interpretation and recommendations for individualized care, and limited information exists on how to approach multilayered tumor profiles in clinical routine. We report our experience with the practical use of data from whole-genome or exome and RNA sequencing and DNA methylation profiling within the MASTER (Molecularly Aided Stratification for Tumor Eradication Research) program of the National Center for Tumor Diseases (NCT) Heidelberg and Dresden and the German Cancer Research Center (DKFZ). We cover all relevant steps of an end-to-end precision oncology workflow, from sample collection, molecular analysis, and variant prioritization to assigning treatment recommendations and discussion in the molecular tumor board. To provide insight into our approach to multidimensional tumor profiles and guidance on interpreting their biological impact and diagnostic and therapeutic implications, we present case studies from the NCT/DKFZ molecular tumor board that illustrate our daily practice. This manual is intended to be useful for physicians, biologists, and bioinformaticians involved in the clinical interpretation of genome-wide molecular information.

DNA methylation landscape of tumor-associated macrophages reveals pathways, transcription factors and prognostic value relevant to triple-negative breast cancer patients.

The accumulation of myeloid cells, particularly tumor-associated macrophages (TAMs), characterizes the tumor microenvironment (TME) of many solid cancers, including breast cancer. Compared to healthy tissue-resident macrophages, TAMs acquire distinct transcriptomes and tumor-promoting functions by largely unknown mechanisms. Here, we hypothesize the involvement of TME signaling and subsequent epigenetic reprogramming of TAMs. Using the 4T1 mouse model of triple-negative breast cancer, we demonstrate that the presence of cancer cells significantly alters the DNA methylation landscape of macrophages and, to a lesser extent, bone marrow-derived monocytes (BMDMs). TAM methylomes, dissected into BMDM-originating and TAM-specific epigenetic programs, implicated transcription factors (TFs) and signaling pathways involved in TAM reprogramming, correlated with cancer-specific gene expression patterns. Utilizing published single-cell gene expression data, we linked microenvironmentally-derived signals to the cancer-specific DNA methylation landscape of TAMs. These integrative analyses highlighted the role of altered cytokine production in the TME (eg, TGF-ß, IFN-γ and CSF1) on the induction of specific TFs (eg, FOSL2, STAT1 and RUNX3) responsible for the epigenetic reprogramming of TAMs. DNA methylation deconvolution identified a TAM-specific signature associated with the identified signaling pathways and TFs, corresponding with severe tumor grade and poor prognosis of breast cancer patients. Similarly, immunosuppressive TAM functions were identified, such as induction of the immune inhibitory receptor-ligand PD-L1 by DNA hypomethylation of Cd274. Collectively, these results provide strong evidence that the epigenetic landscapes of macrophages and monocytes are perturbed by the presence of breast cancer, pointing to molecular mechanisms of TAM reprogramming, impacting patient outcomes.

DNA methylation biomarkers for noninvasive detection of triple-negative breast cancer using liquid biopsy.

Noninvasive detection of aberrant DNA methylation could provide invaluable biomarkers for earlier detection of triple-negative breast cancer (TNBC) which could help clinicians with easier and more efficient treatment options. We evaluated genome-wide DNA methylation data derived from TNBC and normal breast tissues, peripheral blood of TNBC cases and controls and reference samples of sorted blood and mammary cells. Differentially methylated regions (DMRs) between TNBC and normal breast tissues were stringently selected, verified and externally validated. A machine-learning algorithm was applied to select the top DMRs, which then were evaluated on plasma-derived circulating cell-free DNA (cfDNA) samples of TNBC patients and healthy controls. We identified 23 DMRs accounting for the methylation profile of blood cells and reference mammary cells and then selected six top DMRs for cfDNA analysis. We quantified un-/methylated copies of these DMRs by droplet digital PCR analysis in a plasma test set from TNBC patients and healthy controls and confirmed our findings obtained on tissues. Differential cfDNA methylation was confirmed in an independent validation set of plasma samples. A methylation score combining signatures of the top three DMRs overlapping with the SPAG6, LINC10606 and TBCD/ZNF750 genes had the best capability to discriminate TNBC patients from controls (AUC = 0.78 in the test set and AUC = 0.74 in validation set). Our findings demonstrate the usefulness of cfDNA-based methylation signatures as noninvasive liquid biopsy markers for the diagnosis of TNBC.

Dynamic DNA methylation reveals novel cis-regulatory elements in mouse hematopoiesis.

Differentiation of hematopoietic stem and progenitor cells to terminally differentiated immune cells is accompanied by large-scale remodeling of the DNA methylation landscape. Although significant insights into the molecular mechanisms of hematopoietic tissue regeneration were derived from mouse models, profiling of DNA methylation has been hampered by high cost or low resolution using available methods. The recent development of the Infinium Mouse Methylation BeadChip (MMBC) array facilitates methylation profiling of the mouse genome at a single CpG resolution at affordable cost. We extended the RnBeads package to provide a computational framework for the analysis of MMBC data. This framework was applied to a newly generated reference map of mouse hematopoiesis encompassing nine different cell types. Analysis of dynamically regulated CpG sites showed progressive and unidirectional DNA methylation changes from hematopoietic stem and progenitor cells to differentiated hematopoietic cells and allowed the identification of lineage- and cell type-specific DNA methylation programs. Comparison with previously published catalogs of cis-regulatory elements (CREs) revealed 12,856 novel putative CREs that were dynamically regulated by DNA methylation (mdCREs). These mdCREs were predominantly associated with patterns of cell type-specific DNA hypomethylation and could be identified as epigenetic control regions regulating the expression of key hematopoietic genes during differentiation. In summary, we established an analysis pipeline for MMBC data sets and provide a DNA methylation atlas of mouse hematopoiesis. This resource allowed us to identify novel putative CREs involved in hematopoiesis and will serve as a platform to study epigenetic regulation of normal and malignant hematopoiesis.

Usp22 is an intracellular regulator of systemic emergency hematopoiesis.

Emergency hematopoiesis is a concerted response aimed toward enhanced protection from infection, involving multiple cell types and developmental stages across the immune system. Despite its importance, the underlying molecular regulation remains poorly understood. The deubiquitinase USP22 regulates the levels of monoubiquitinated histone H2B (H2Bub1), which is associated with activation of interferon responses upon viral infection. Here, we show that in the absence of infection or inflammation, mice lacking Usp22 in all hematopoietic cells display profound systemic emergency hematopoiesis, evident by increased hematopoietic stem cell proliferation, myeloid bias, and extramedullary hematopoiesis. Functionally, loss of Usp22 results in elevated phagocytosis by neutrophilic granulocytes and enhanced innate protection against Listeria monocytogenes infection. At the molecular level, we found this state of emergency hematopoiesis associated with transcriptional signatures of myeloid priming, enhanced mitochondrial respiration, and innate and adaptive immunity and inflammation. Augmented expression of many inflammatory genes was linked to elevated locus-specific H2Bub1 levels. Collectively, these results demonstrate the existence of a tunable epigenetic state that promotes systemic emergency hematopoiesis in a cell-autonomous manner to enhance innate protection, identifying potential paths toward immune enhancement.

Comprehensive genomic and epigenomic analysis in cancer of unknown primary guides molecularly-informed therapies despite heterogeneity.

The benefit of molecularly-informed therapies in cancer of unknown primary (CUP) is unclear. Here, we use comprehensive molecular characterization by whole genome/exome, transcriptome and methylome analysis in 70 CUP patients to reveal substantial mutational heterogeneity with TP53, MUC16, KRAS, LRP1B and CSMD3 being the most frequently mutated known cancer-related genes. The most common fusion partner is FGFR2, the most common focal homozygous deletion affects CDKN2A. 56/70 (80%) patients receive genomics-based treatment recommendations which are applied in 20/56 (36%) cases. Transcriptome and methylome data provide evidence for the underlying entity in 62/70 (89%) cases. Germline analysis reveals five (likely) pathogenic mutations in five patients. Recommended off-label therapies translate into a mean PFS ratio of 3.6 with a median PFS1 of 2.9 months (17 patients) and a median PFS2 of 7.8 months (20 patients). Our data emphasize the clinical value of molecular analysis and underline the need for innovative, mechanism-based clinical trials.

The antileukemic activity of decitabine upon PML/RARA-negative AML blasts is supported by all-trans retinoic acid: in vitro and in vivo evidence for cooperation.

The prognosis of AML patients with adverse genetics, such as a complex, monosomal karyotype and TP53 lesions, is still dismal even with standard chemotherapy. DNA-hypomethylating agent monotherapy induces an encouraging response rate in these patients. When combined with decitabine (DAC), all-trans retinoic acid (ATRA) resulted in an improved response rate and longer overall survival in a randomized phase II trial (DECIDER; NCT00867672). The molecular mechanisms governing this in vivo synergism are unclear. We now demonstrate cooperative antileukemic effects of DAC and ATRA on AML cell lines U937 and MOLM-13. By RNA-sequencing, derepression of >1200 commonly regulated transcripts following the dual treatment was observed. Overall chromatin accessibility (interrogated by ATAC-seq) and, in particular, at motifs of retinoic acid response elements were affected by both single-agent DAC and ATRA, and enhanced by the dual treatment. Cooperativity regarding transcriptional induction and chromatin remodeling was demonstrated by interrogating the HIC1, CYP26A1, GBP4, and LYZ genes, in vivo gene derepression by expression studies on peripheral blood blasts from AML patients receiving DAC + ATRA. The two drugs also cooperated in derepression of transposable elements, more effectively in U937 (mutated TP53) than MOLM-13 (intact TP53), resulting in a "viral mimicry" response. In conclusion, we demonstrate that in vitro and in vivo, the antileukemic and gene-derepressive epigenetic activity of DAC is enhanced by ATRA.

Model soups improve performance of dermoscopic skin cancer classifiers.

BACKGROUND: Image-based cancer classifiers suffer from a variety of problems which negatively affect their performance. For example, variation in image brightness or different cameras can already suffice to diminish performance. Ensemble solutions, where multiple model predictions are combined into one, can improve these problems. However, ensembles are computationally intensive and less transparent to practitioners than single model solutions. Constructing model soups, by averaging the weights of multiple models into a single model, could circumvent these limitations while still improving performance. OBJECTIVE: To investigate the performance of model soups for a dermoscopic melanoma-nevus skin cancer classification task with respect to (1) generalisation to images from other clinics, (2) robustness against small image changes and (3) calibration such that the confidences correspond closely to the actual predictive uncertainties. METHODS: We construct model soups by fine-tuning pre-trained models on seven different image resolutions and subsequently averaging their weights. Performance is evaluated on a multi-source dataset including holdout and external components. RESULTS: We find that model soups improve generalisation and calibration on the external component while maintaining performance on the holdout component. For robustness, we observe performance improvements for pertubated test images, while the performance on corrupted test images remains on par. CONCLUSIONS: Overall, souping for skin cancer classifiers has a positive effect on generalisation, robustness and calibration. It is easy for practitioners to implement and by combining multiple models into a single model, complexity is reduced. This could be an important factor in achieving clinical applicability, as less complexity generally means more transparency.

Inflammatory exposure drives long-lived impairment of hematopoietic stem cell self-renewal activity and accelerated aging.

Hematopoietic stem cells (HSCs) mediate regeneration of the hematopoietic system following injury, such as following infection or inflammation. These challenges impair HSC function, but whether this functional impairment extends beyond the duration of inflammatory exposure is unknown. Unexpectedly, we observed an irreversible depletion of functional HSCs following challenge with inflammation or bacterial infection, with no evidence of any recovery up to 1 year afterward. HSCs from challenged mice demonstrated multiple cellular and molecular features of accelerated aging and developed clinically relevant blood and bone marrow phenotypes not normally observed in aged laboratory mice but commonly seen in elderly humans. In vivo HSC self-renewal divisions were absent or extremely rare during both challenge and recovery periods. The progressive, irreversible attrition of HSC function demonstrates that temporally discrete inflammatory events elicit a cumulative inhibitory effect on HSCs. This work positions early/mid-life inflammation as a mediator of lifelong defects in tissue maintenance and regeneration.

Gene expression-based prediction of pazopanib efficacy in sarcoma.

BACKGROUND: The multi-receptor tyrosine kinase inhibitor pazopanib is approved for the treatment of advanced soft-tissue sarcoma and has also shown activity in other sarcoma subtypes. However, its clinical efficacy is highly variable, and no reliable predictors exist to select patients who are likely to benefit from this drug. PATIENTS AND METHODS: We analysed the molecular profiles and clinical outcomes of patients with pazopanib-treated sarcoma enrolled in a prospective observational study by the German Cancer Consortium, DKTK MASTER, that employs whole-genome/exome sequencing and transcriptome sequencing to inform the care of young adults with advanced cancer across histology and patients with rare cancers. RESULTS: Among 109 patients with available whole-genome/exome sequencing data, there was no correlation between clinical parameters, specific genetic alterations or mutational signatures and clinical outcome. In contrast, the analysis of a subcohort of 62 patients who underwent molecular analysis before pazopanib treatment and had transcriptome sequencing data available showed that mRNA levels of NTRK3 (hazard ratio [HR] = 0.53, p = 0.021), IGF1R (HR = 1.82, p = 0.027) and KDR (HR = 0.50, p = 0.011) were independently associated with progression-free survival (PFS). Based on the expression of these receptor tyrosine kinase genes, i.e. the features NTRK3-high, IGF1R-low and KDR-high, we developed a pazopanib efficacy predictor that stratified patients into three groups with significantly different PFS (p < 0.0001). Application of the pazopanib efficacy predictor to an independent cohort of patients with pazopanib-treated sarcoma from DKTK MASTER (n = 43) confirmed its potential to separate patient groups with significantly different PFS (p = 0.02), whereas no such association was observed in patients with sarcoma from DKTK MASTER (n = 97) or The Cancer Genome Atlas sarcoma cohort (n = 256) who were not treated with pazopanib. CONCLUSION: A score based on the combined expression of NTRK3, IGF1R and KDR allows the identification of patients with sarcoma and with good, intermediate and poor outcome following pazopanib therapy and warrants prospective investigation as a predictive tool to optimise the use of this drug in the clinic.

MGMT inactivation as a new biomarker in patients with advanced biliary tract cancers.

Biliary tract cancers (BTCs) have poor prognosis and limited therapeutic options. The impact of O6 -methylguanine-DNA methyltransferase (MGMT) inactivation in advanced BTC patients is not established. We investigated the prevalence, prognostic, and predictive impact of MGMT inactivation in two multicenter cohorts. MGMT inactivation was assessed through PCR and immunohistochemistry (IHC) in an Italian cohort; the results were then externally validated using RNA sequencing (RNA-seq) data from the BTC subcohort of the Molecularly Aided Stratification for Tumor Eradication Research (MASTER) precision oncology program of the National Center for Tumor Diseases Heidelberg and the German Cancer Consortium. Among 164 Italian cases, 18% presented MGMT promoter hypermethylation (> 14%) and 73% had negative MGMT protein expression. Both were associated with worse overall survival (OS; HR 2.31; P < 0.001 and HR 1.99, P = 0.012, respectively). In the MASTER cohort, patients with lower MGMT mRNA expression showed significantly poorer OS (median OS [mOS] 20.4 vs 31.7 months, unadjusted HR 1.89; P = 0.043). Our results suggest that MGMT inactivation is a frequent epigenetic alteration in BTC, with a significant prognostic impact, and provide the rationale to explore DNA-damaging agents in MGMT-inactivated BTCs.

Explainable artificial intelligence in skin cancer recognition: A systematic review.

BACKGROUND: Due to their ability to solve complex problems, deep neural networks (DNNs) are becoming increasingly popular in medical applications. However, decision-making by such algorithms is essentially a black-box process that renders it difficult for physicians to judge whether the decisions are reliable. The use of explainable artificial intelligence (XAI) is often suggested as a solution to this problem. We investigate how XAI is used for skin cancer detection: how is it used during the development of new DNNs? What kinds of visualisations are commonly used? Are there systematic evaluations of XAI with dermatologists or dermatopathologists? METHODS: Google Scholar, PubMed, IEEE Explore, Science Direct and Scopus were searched for peer-reviewed studies published between January 2017 and October 2021 applying XAI to dermatological images: the search terms histopathological image, whole-slide image, clinical image, dermoscopic image, skin, dermatology, explainable, interpretable and XAI were used in various combinations. Only studies concerned with skin cancer were included. RESULTS: 37 publications fulfilled our inclusion criteria. Most studies (19/37) simply applied existing XAI methods to their classifier to interpret its decision-making. Some studies (4/37) proposed new XAI methods or improved upon existing techniques. 14/37 studies addressed specific questions such as bias detection and impact of XAI on man-machine-interactions. However, only three of them evaluated the performance and confidence of humans using CAD systems with XAI. CONCLUSION: XAI is commonly applied during the development of DNNs for skin cancer detection. However, a systematic and rigorous evaluation of its usefulness in this scenario is lacking.

DNMT1 Deficiency Impacts on Plasmacytoid Dendritic Cells in Homeostasis and Autoimmune Disease.

Dendritic cells (DCs) are heterogeneous immune regulators involved in autoimmune diseases. Epigenomic mechanisms orchestrating DC development and DC subset diversification remain insufficiently understood but could be important to modulate DC fate for clinical purposes. By combining whole-genome methylation assessment with the analysis of mice expressing reduced DNA methyltransferase 1 levels, we show that distinct DNA methylation levels and patterns are required for the development of plasmacytoid DC and conventional DC subsets. We provide clonal in vivo evidence for DC lineage establishment at the stem cell level, and we show that a high DNA methylation threshold level is essential for Flt3-dependent survival of DC precursors. Importantly, reducing methylation predominantly depletes plasmacytoid DC and alleviates systemic lupus erythematosus in an autoimmunity mouse model. This study shows how DNA methylation regulates the production of DC subsets and provides a potential rationale for targeting autoimmune disease using hypomethylating agents.

Epigenomic technologies for precision oncology.

Epigenetic patterns in a cell control the expression of genes and consequently determine the phenotype of a cell. Cancer cells possess altered epigenomes which include aberrant patterns of DNA methylation, histone tail modifications, nucleosome positioning and of the three-dimensional chromatin organization within a nucleus. These altered epigenetic patterns are potential useful biomarkers to detect cancer cells and to classify tumor types. In addition, the cancer epigenome dictates the response of a cancer cell to therapeutic intervention and, therefore its knowledge, will allow to predict response to different therapeutic approaches. Here we review the current state-of-the-art technologies that have been developed to decipher epigenetic patterns on the genomic level and discuss how these methods are potentially useful for precision oncology.

Control of APOBEC3B induction and cccDNA decay by NF-κB and miR-138-5p.

BACKGROUND & AIMS: Immune-mediated induction of cytidine deaminase APOBEC3B (A3B) expression leads to HBV covalently closed circular DNA (cccDNA) decay. Here, we aimed to decipher the signalling pathway(s) and regulatory mechanism(s) involved in A3B induction and related HBV control. METHODS: Differentiated HepaRG cells (dHepaRG) knocked-down for NF-κB signalling components, transfected with siRNA or micro RNAs (miRNA), and primary human hepatocytes ± HBV or HBVΔX or HBV-RFP, were treated with lymphotoxin beta receptor (LTßR)-agonist (BS1). The biological outcomes were analysed by reverse transcriptase-qPCR, immunoblotting, luciferase activity, chromatin immune precipitation, electrophoretic mobility-shift assay, targeted-bisulfite-, miRNA-, RNA-, genome-sequencing, and mass-spectrometry. RESULTS: We found that canonical and non-canonical NF-κB signalling pathways are mandatory for A3B induction and anti-HBV effects. The degree of immune-mediated A3B production is independent of A3B promoter demethylation but is controlled post-transcriptionally by the miRNA 138-5p expression (hsa-miR-138-5p), promoting A3B mRNA decay. Hsa-miR-138-5p over-expression reduced A3B levels and its antiviral effects. Of note, established infection inhibited BS1-induced A3B expression through epigenetic modulation of A3B promoter. Twelve days of treatment with a LTßR-specific agonist BS1 is sufficient to reduce the cccDNA pool by 80% without inducing significant damages to a subset of cancer-related host genes. Interestingly, the A3B-mediated effect on HBV is independent of the transcriptional activity of cccDNA as well as on rcDNA synthesis. CONCLUSIONS: Altogether, A3B represents the only described enzyme to target both transcriptionally active and inactive cccDNA. Thus, inhibiting hsa-miR-138-5p expression should be considered in the combinatorial design of new therapies against HBV, especially in the context of immune-mediated A3B induction. LAY SUMMARY: Immune-mediated induction of cytidine deaminase APOBEC3B is transcriptionally regulated by NF-κB signalling and post-transcriptionally downregulated by hsa-miR-138-5p expression, leading to cccDNA decay. Timely controlled APOBEC3B-mediated cccDNA decay occurs independently of cccDNA transcriptional activity and without damage to a subset of cancer-related genes. Thus, APOBEC3B-mediated cccDNA decay could offer an efficient therapeutic alternative to target hepatitis B virus chronic infection.