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Christoph Plass and colleagues investigate the transcriptomic and epigenomic changes induced by treatment with inhibitors of DNMT and HDAC in cancer cell lines. They observe large numbers of treatment-induced non-annotated TSSs (TINATs) encoded in long-terminal repeats that are normally repressed in most cell types. Several mechanisms of action have been proposed for DNA methyltransferase and histone deacetylase inhibitors (DNMTi and HDACi), primarily based on candidate-gene approaches. However, less is known about their genome-wide transcriptional and epigenomic consequences. By mapping global transcription start site (TSS) and chromatin dynamics, we observed the cryptic transcription of thousands of treatment-induced non-annotated TSSs (TINATs) following DNMTi and HDACi treatment. The resulting transcripts frequently splice into protein-coding exons and encode truncated or chimeric ORFs translated into products with predicted abnormal or immunogenic functions. TINAT transcription after DNMTi treatment coincided with DNA hypomethylation and gain of classical promoter histone marks, while HDACi specifically induced a subset of TINATs in association with H2AK9ac, H3K14ac, and H3K23ac. Despite this mechanistic difference, both inhibitors convergently induced transcription from identical sites, as we found TINATs to be encoded in solitary long terminal repeats of the ERV9/LTR12 family, which are epigenetically repressed in virtually all normal cells.

Christoph Plass, Christopher Oakes and colleagues study genome-wide DNA methylation dynamics during B cell maturation and the pathogenic role of transcription factor dysregulation in chronic lymphocytic leukemia (CLL). By comparing normal and malignant B cells, they find that tumors derive from a continuum of maturation states, which correlate with different clinical outcomes. Charting differences between tumors and normal tissue is a mainstay of cancer research. However, clonal tumor expansion from complex normal tissue architectures potentially obscures cancer-specific events, including divergent epigenetic patterns. Using whole-genome bisulfite sequencing of normal B cell subsets, we observed broad epigenetic programming of selective transcription factor binding sites coincident with the degree of B cell maturation. By comparing normal B cells to malignant B cells from 268 patients with chronic lymphocytic leukemia (CLL), we showed that tumors derive largely from a continuum of maturation states reflected in normal developmental stages. Epigenetic maturation in CLL was associated with an indolent gene expression pattern and increasingly favorable clinical outcomes. We further uncovered that most previously reported tumor-specific methylation events are normally present in non-malignant B cells. Instead, we identified a potential pathogenic role for transcription factor dysregulation in CLL, where excess programming by EGR and NFAT with reduced EBF and AP-1 programming imbalances the normal B cell epigenetic program.

Background The decline of hematopoietic stem cell (HSC) function upon aging contributes to aging-associated immune remodeling and leukemia pathogenesis. Aged HSCs show changes to their epigenome, such as alterations in DNA methylation and histone methylation and acetylation landscapes. We previously showed a correlation between high Cdc42 activity in aged HSCs and the loss of intranuclear epigenetic polarity, or epipolarity, as indicated by the specific distribution of H4K16ac. Results Here, we show that not all histone modifications display a polar localization and that a reduction in H4K16ac amount and loss of epipolarity are specific to aged HSCs. Increasing the levels of H4K16ac is not sufficient to restore polarity in aged HSCs and the restoration of HSC function. The changes in H4K16ac upon aging and rejuvenation of HSCs are correlated with a change in chromosome 11 architecture and alterations in nuclear volume and shape. Surprisingly, by taking advantage of knockout mouse models, we demonstrate that increased Cdc42 activity levels correlate with the repression of the nuclear envelope protein LaminA/C, which controls chromosome 11 distribution, H4K16ac polarity, and nuclear volume and shape in aged HSCs. Conclusions Collectively, our data show that chromatin architecture changes in aged stem cells are reversible by decreasing the levels of Cdc42 activity, revealing an unanticipated way to pharmacologically target LaminA/C expression and revert alterations of the epigenetic architecture in aged HSCs.

Changes in active chromatin marks in old activated satellite cells lead to Hoxa9 activation, which induces the expression of developmental pathway genes with a known inhibitory effect on satellite cell function and muscle regeneration in aged mice. Hox genes and muscle stem-cell decline Satellite cells ensure muscle regeneration following injury, but their capacity for repair declines with age. Hox genes are regulators of organ formation during embryogenesis but so far, they have not been linked to ageing. Lenhard Rudolph and colleagues have found that changes in active chromatin marks and in old satellite cells lead to activation of Hoxa9 , which in turn induces the expression genes normally involved in development but with a known inhibitory effect on satellite cell function or important for induction of senescence. Preventing this activation in old mice rescues muscle regeneration, and inducing Hoxa9 expression in young mice impairs satellite cells function and muscle repair. The functionality of stem cells declines during ageing, and this decline contributes to ageing-associated impairments in tissue regeneration and function 1 . Alterations in developmental pathways have been associated with declines in stem-cell function during ageing 2 , 3 , 4 , 5 , 6 , but the nature of this process remains poorly understood. Hox genes are key regulators of stem cells and tissue patterning during embryogenesis with an unknown role in ageing 7 , 8 . Here we show that the epigenetic stress response in muscle stem cells (also known as satellite cells) differs between aged and young mice. The alteration includes aberrant global and site-specific induction of active chromatin marks in activated satellite cells from aged mice, resulting in the specific induction of Hoxa9 but not other Hox genes. Hoxa9 in turn activates several developmental pathways and represents a decisive factor that separates satellite cell gene expression in aged mice from that in young mice. The activated pathways include most of the currently known inhibitors of satellite cell function in ageing muscle, including Wnt, TGFβ, JAK/STAT and senescence signalling 2 , 3 , 4 , 6 . Inhibition of aberrant chromatin activation or deletion of Hoxa9 improves satellite cell function and muscle regeneration in aged mice, whereas overexpression of Hoxa9 mimics ageing-associated defects in satellite cells from young mice, which can be rescued by the inhibition of Hoxa9-targeted developmental pathways. Together, these data delineate an altered epigenetic stress response in activated satellite cells from aged mice, which limits satellite cell function and muscle regeneration by Hoxa9-dependent activation of developmental pathways.

Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative disorder of childhood caused by mutations in the Ras pathway. Outcomes in JMML vary markedly from spontaneous resolution to rapid relapse after hematopoietic stem cell transplantation. Here, we hypothesized that DNA methylation patterns would help predict disease outcome and therefore performed genome-wide DNA methylation profiling in a cohort of 39 patients. Unsupervised hierarchical clustering identifies three clusters of patients. Importantly, these clusters differ significantly in terms of 4-year event-free survival, with the lowest methylation cluster having the highest rates of survival. These findings were validated in an independent cohort of 40 patients. Notably, all but one of 14 patients experiencing spontaneous resolution cluster together and closer to 22 healthy controls than to other JMML cases. Thus, we show that DNA methylation patterns in JMML are predictive of outcome and can identify the patients most likely to experience spontaneous resolution. Juvenile myelomonocytic leukemia (JMML) is an aggressive disease with limited options for treatment. Here, the authors utilize DNA methylation based subgroups in JMML to predict clinical outcome.

Recent years have been witnessing a substantial improvement in the accuracy of skin cancer classification using convolutional neural networks (CNNs). CNNs perform on par with or better than dermatologists with respect to the classification tasks of single images. However, in clinical practice, dermatologists also use other patient data beyond the visual aspects present in a digitized image, further increasing their diagnostic accuracy. Several pilot studies have recently investigated the effects of integrating different subtypes of patient data into CNN-based skin cancer classifiers. This systematic review focuses on the current research investigating the impact of merging information from image features and patient data on the performance of CNN-based skin cancer image classification. This study aims to explore the potential in this field of research by evaluating the types of patient data used, the ways in which the nonimage data are encoded and merged with the image features, and the impact of the integration on the classifier performance. Google Scholar, PubMed, MEDLINE, and ScienceDirect were screened for peer-reviewed studies published in English that dealt with the integration of patient data within a CNN-based skin cancer classification. The search terms skin cancer classification, convolutional neural network(s), deep learning, lesions, melanoma, metadata, clinical information, and patient data were combined. A total of 11 publications fulfilled the inclusion criteria. All of them reported an overall improvement in different skin lesion classification tasks with patient data integration. The most commonly used patient data were age, sex, and lesion location. The patient data were mostly one-hot encoded. There were differences in the complexity that the encoded patient data were processed with regarding deep learning methods before and after fusing them with the image features for a combined classifier. This study indicates the potential benefits of integrating patient data into CNN-based diagnostic algorithms. However, how exactly the individual patient data enhance classification performance, especially in the case of multiclass classification problems, is still unclear. Moreover, a substantial fraction of patient data used by dermatologists remains to be analyzed in the context of CNN-based skin cancer classification. Further exploratory analyses in this promising field may optimize patient data integration into CNN-based skin cancer diagnostics for patients' benefits.

Precision cancer medicine aims to improve patient outcomes by providing individually tailored recommendations for clinical management based on the evaluation of biological disease profiles in multidisciplinary molecular tumor boards (MTBs). The quality of MTB decisions depends on the comprehensive, reliable, and reproducible interpretation of increasingly complex molecular data. We developed and implemented, as part of a multicenter precision oncology program, the Knowledge Connector (KC), a decision support system that integrates individual patients’ molecular and clinical data with world knowledge to generate and document MTB recommendations. The KC supports data curation, database integration, and discussion based on multiomics data and provides an interface for creating a cross-institutional knowledge base. Furthermore, it extracts relevant biomarker-drug associations and increases the efficacy of data interpretation in a clinically relevant manner by reducing reliance on external sources and optimizing inter-curator concordance. Our results demonstrate that the KC is a versatile tool that supports medical decision-making in MTBs, thus enabling the scalability of precision cancer medicine. Integrating complex multi-omics data for individual patient decision making can be challenging. Here, the authors develop Knowledge Connector as a decision support system to generate and document Molecular Tumor Board recommendations and support medical decision-making.

Background The JAK2-V617F mutation is the most frequent driver mutation in a group of malignant hematopoietic disorders called myeloproliferative neoplasms (MPN). JAK2-V617F is a somatic mutation originating in a hematopoietic stem cell and results in constitutively activated JAK-STAT signaling. High levels of pro-inflammatory cytokines in the blood are a hallmark of MPN patients and are a key factor in the severe clinical symptoms seen in these patients. The molecular mechanisms underlying the up-regulation of inflammatory cytokines in JAK2-V617F mutated hematopoietic cells remain to be elucidated. Methods 32D myeloid progenitor cells expressing JAK2-wildtype (WT) and JAK2-V617F, respectively were employed. In addition, primary hematopoietic cells from the JAK2-V617F knock-in MPN mouse model were investigated. Integrin outside-in signaling upon binding of cells to the adhesion molecules VCAM-1/ICAM-1 was characterized by Western blotting of phosphorylated FAK, STAT3, p65, SYK and JNK. Regulation of mRNA and protein expression of IL-1α, IL-1β, IL-6, TNF and CXCL10 was measured by qPCR and ELISA. RNAseq and DNA methylation analysis in primary mouse JAK2-V617F granulocytes was performed. In JAK2-V617F knock-in mice, anti-integrin treatment was applied to evaluate the impact of activated integrin signaling on IL-1 blood levels in vivo. Results Integrin stimulation via the adhesion molecules VCAM-1/ICAM-1 activated integrin outside-in signaling including FAK, SYK, NFκB, and JNK. This induced strong mRNA expression of IL-1α, IL-1β, IL-6, TNF and CXCL10. In 32D cells, the presence of the JAK2-V617F mutation further increased VCAM-1/ICAM-1-induced mRNA and protein levels of IL-1α and IL-1β, and active caspase 1 expression. In primary granulocytes, integrin stimulation resulted in an activated mRNA signature of inflammatory cytokines. Consistent with the mRNA results, adhesion to VCAM-1/ICAM-1 induced an increase in intracellular IL-1α and IL-1β protein levels in 32D cells. However, in primary hematopoietic cells, up-regulation of inflammatory cytokines was not observed at the protein level in vitro, whereas, in vivo, blocking of integrin binding to VCAM-1/ICAM-1 was sufficient to reduce elevated IL-1α levels in the blood of JAK2-V617F mice. Conclusions We conclude that integrin stimulation via the adhesion molecules VCAM-1/ICAM-1 activates integrin outside-in signaling, leading to the up-regulation of pro-inflammatory cytokines in both JAK2-mutated and non-mutated mouse hematopoietic cells.

Lung cancer, with its most prevalent form non-small-cell lung carcinoma (NSCLC), is one of the leading causes of cancer-related deaths worldwide, and is commonly treated with chemotherapeutic drugs such as cisplatin. Lung cancer patients frequently suffer from chemotherapy-induced anemia, which can be treated with erythropoietin (EPO). However, studies have indicated that EPO not only promotes erythropoiesis in hematopoietic cells, but may also enhance survival of NSCLC cells. Here, we verified that the NSCLC cell line H838 expresses functional erythropoietin receptors (EPOR) and that treatment with EPO reduces cisplatin-induced apoptosis. To pinpoint differences in EPO-induced survival signaling in erythroid progenitor cells (CFU-E, colony forming unit-erythroid) and H838 cells, we combined mathematical modeling with a method for feature selection, the L1 regularization. Utilizing an example model and simulated data, we demonstrated that this approach enables the accurate identification and quantification of cell type-specific parameters. We applied our strategy to quantitative time-resolved data of EPO-induced JAK/STAT signaling generated by quantitative immunoblotting, mass spectrometry and quantitative real-time PCR (qRT-PCR) in CFU-E and H838 cells as well as H838 cells overexpressing human EPOR (H838-HA-hEPOR). The established parsimonious mathematical model was able to simultaneously describe the data sets of CFU-E, H838 and H838-HA-hEPOR cells. Seven cell type-specific parameters were identified that included for example parameters for nuclear translocation of STAT5 and target gene induction. Cell type-specific differences in target gene induction were experimentally validated by qRT-PCR experiments. The systematic identification of pathway differences and sensitivities of EPOR signaling in CFU-E and H838 cells revealed potential targets for intervention to selectively inhibit EPO-induced signaling in the tumor cells but leave the responses in erythroid progenitor cells unaffected. Thus, the proposed modeling strategy can be employed as a general procedure to identify cell type-specific parameters and to recommend treatment strategies for the selective targeting of specific cell types.

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.