Deregulation and epigenetic modification of BCL2-family genes cause resistance to venetoclax in hematologic malignancies.

The BCL2 inhibitor venetoclax has been approved to treat different hematological malignancies. Because there is no common genetic alteration causing resistance to venetoclax in chronic lymphocytic leukemia (CLL) and B-cell lymphoma, we asked if epigenetic events might be involved in venetoclax resistance. Therefore, we employed whole-exome sequencing, methylated DNA immunoprecipitation sequencing, and genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 screening to investigate venetoclax resistance in aggressive lymphoma and high-risk CLL patients. We identified a regulatory CpG island within the PUMA promoter that is methylated upon venetoclax treatment, mediating PUMA downregulation on transcript and protein level. PUMA expression and sensitivity toward venetoclax can be restored by inhibition of methyltransferases. We can demonstrate that loss of PUMA results in metabolic reprogramming with higher oxidative phosphorylation and adenosine triphosphate production, resembling the metabolic phenotype that is seen upon venetoclax resistance. Although PUMA loss is specific for acquired venetoclax resistance but not for acquired MCL1 resistance and is not seen in CLL patients after chemotherapy-resistance, BAX is essential for sensitivity toward both venetoclax and MCL1 inhibition. As we found loss of BAX in Richter's syndrome patients after venetoclax failure, we defined BAX-mediated apoptosis to be critical for drug resistance but not for disease progression of CLL into aggressive diffuse large B-cell lymphoma in vivo. A compound screen revealed TRAIL-mediated apoptosis as a target to overcome BAX deficiency. Furthermore, antibody or CAR T cells eliminated venetoclax resistant lymphoma cells, paving a clinically applicable way to overcome venetoclax resistance.

[High-throughput sequencing of frozen and paraffin-embedded tumor and normal tissue]. / Hochdurchsatz-Sequenzierung von gefrorenem und in Paraffin eingebettetem Tumor- und Normalgewebe.

Until now high-throughput sequencing of tumor samples relied on DNA isolated from fresh frozen tissues, the preparation of which, however, is relatively laborious. The use of preserved material, i.e. from tissue banks, could help to avoid this limitation and would enable the reanalysis of diverse clinical trials. So far we have shown that formalin-fixed paraffin-embedded (FFPE) tissue samples can be used for genomic re-sequencing processes. FFPE samples are amply available from surgical tumor resections and histopathological diagnosis, and comprise tissue from precursor lesions, primary tumors, and lymphogenic and/or hematogenic metastases. To generate models which predict the response to therapy, FFPE tissue also has the advantage that it is available from a variety of clinical trials. Second generation sequencing techniques are not only applicable to snap frozen and FFPE tissues for whole genome analyses but also for targeted resequencing approaches. In addition, the detection of copy number variations and mutations in FFPE tissues can be obtained within one sequencing run. The possibility of using genome-wide technologies irrespective of the mode of storage facilitates the retrieval of useful material and is a prerequisite for subsequent computational modelling approaches.

Campylobacter jejuni impairs sodium transport and epithelial barrier function via cytokine release in human colon.

This corrects the article DOI: 10.1038/mi.2017.66.

Campylobacter jejuni impairs sodium transport and epithelial barrier function via cytokine release in human colon.

Campylobacter jejuni is the most prevalent cause of foodborne bacterial enteritis worldwide. Patients present with diarrhea and immune responses lead to complications like arthritis and irritable bowel syndrome. Although studies exist in animal and cell models, we aimed at a functional and structural characterization of intestinal dysfunction and the involved regulatory mechanisms in human colon. First, in patients' colonic biopsies, sodium malabsorption was identified as an important diarrheal mechanism resulting from hampered epithelial ion transport via impaired epithelial sodium channel (ENaC) ß- and γ-subunit. In addition, barrier dysfunction from disrupted epithelial tight junction proteins (claudin-1, -3, -4, -5, and -8), epithelial apoptosis, and appearance of lesions was detected, which cause leak-flux diarrhea and can perpetuate immune responses. Importantly, these effects in human biopsies either represent direct action of Campylobacter jejuni (ENaC impairment) or are caused by proinflammatory signaling (barrier dysfunction). This was revealed by regulator analysis from RNA-sequencing (cytometric bead array-checked) and confirmed in cell models, which identified interferon-γ, TNFα, IL-13, and IL-1ß. Finally, bioinformatics' predictions yielded additional information on protective influences like vitamin D, which was confirmed in cell models. Thus, these are candidates for intervention strategies against C. jejuni infection and post-infectious sequelae, which result from the permissive barrier defect along the leaky gut.

The bromodomain protein BRD4 regulates the KEAP1/NRF2-dependent oxidative stress response.

The epigenetic sensor BRD4 (bromodomain protein 4) is a potent target for anti-cancer therapies. To study the transcriptional impact of BRD4 in cancer, we generated an expression signature of BRD4 knockdown cells and found oxidative stress response genes significantly enriched. We integrated the RNA-Seq results with DNA-binding sites of BRD4 generated by chromatin immunoprecipitations, correlated these with gene expressions from human prostate cancers and identified 21 top BRD4 candidate genes among which the oxidative stress pathway genes KEAP1, SESN3 and HDAC6 are represented. Knock down of BRD4 or treatment with the BRD4 inhibitor JQ1 resulted in decreased reactive oxygen species (ROS) production and increased cell viability under H2O2 exposure. Consistently, a deregulation of BRD4 diminished the KEAP1/NRF2 axis and led to a disturbed regulation of the inducible heme oxygenase 1 (HMOX1). Without exogenous stress induction, we also found BRD4 directly targeting the HMOX1 promoter over the SP1-binding sites. Our findings provide insight into the transcriptional regulatory network of BRD4 and highlight BRD4 as signal transducer of the cellular response to oxidative stress.

The Interlaboratory RObustness of Next-generation sequencing (IRON) study: a deep sequencing investigation of TET2, CBL and KRAS mutations by an international consortium involving 10 laboratories.

Massively parallel pyrosequencing allows sensitive deep sequencing to detect molecular aberrations. Thus far, data are limited on the technical performance in a clinical diagnostic setting. Here, we investigated as an international consortium the robustness, precision and reproducibility of amplicon next-generation deep sequencing across 10 laboratories in eight countries. In a cohort of 18 chronic myelomonocytic leukemia patients, mutational analyses were performed on TET2, a frequently mutated gene in myeloproliferative neoplasms. Additionally, hotspot regions of CBL and KRAS were investigated. The study was executed using GS FLX sequencing instruments and the small volume 454 Life Sciences Titanium emulsion PCR setup. We report a high concordance in mutation detection across all laboratories, including a robust detection of novel variants, which were undetected by standard Sanger sequencing. The sensitivity to detect low-level variants present with as low as 1-2% frequency, compared with the 20% threshold for Sanger-based sequencing is increased. Together with the output of high-quality long reads and fast run time, we demonstrate the utility of deep sequencing in clinical applications. In conclusion, this multicenter analysis demonstrated that amplicon-based deep sequencing is technically feasible, achieves high concordance across multiple laboratories and allows a broad and in-depth molecular characterization of cancer specimens with high diagnostic sensitivity.