Biomarker screen for efficacy of oncolytic virotherapy in patient-derived pancreatic cancer cultures.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a tumour entity with unmet medical need. To assess the therapeutic potential of oncolytic virotherapy (OVT) against PDAC, different oncolytic viruses (OVs) are currently investigated in clinical trials. However, systematic comparisons of these different OVs in terms of efficacy against PDAC and biomarkers predicting therapeutic response are lacking. METHODS: We screened fourteen patient-derived PDAC cultures which reflect the intra- and intertumoural heterogeneity of PDAC for their sensitivity to five clinically relevant OVs, namely serotype 5 adenovirus Ad5-hTERT, herpes virus T-VEC, measles vaccine strain MV-NIS, reovirus jin-3, and protoparvovirus H-1PV. Live cell analysis, quantification of viral genome/gene expression, cell viability as well as cytotoxicity assays and titration of viral progeny were conducted. Transcriptome profiling was employed to identify potential predictive biomarkers for response to OV treatment. FINDINGS: Patient-derived PDAC cultures showed individual response patterns to OV treatment. Twelve of fourteen cultures were responsive to at least one OV, with no single OV proving superior or inferior across all cultures. Known host factors for distinct viruses were retrieved as potential biomarkers. Compared to the classical molecular subtype, the quasi-mesenchymal or basal-like subtype of PDAC was found to be more sensitive to H-1PV, jin-3, and T-VEC. Generally, expression of viral entry receptors did not correlate with sensitivity to OV treatment, with one exception: Expression of Galectin-1 (LGALS1), a factor involved in H-1PV entry, positively correlated with H-1PV induced cell killing. Rather, cellular pathways controlling immunological, metabolic and proliferative signaling appeared to determine outcome. For instance, high baseline expression of interferon-stimulated genes (ISGs) correlated with relative resistance to oncolytic measles virus, whereas low cyclic GMP-AMP synthase (cGAS) expression was associated with exceptional response. Combination treatment of MV-NIS with a cGAS inhibitor improved tumour cell killing in several PDAC cultures and cells overexpressing cGAS were found to be less sensitive to MV oncolysis. INTERPRETATION: Considering the heterogeneity of PDAC and the complexity of biological therapies such as OVs, no single biomarker can explain the spectrum of response patterns. For selection of a particular OV, PDAC molecular subtype, ISG expression as well as activation of distinct signaling and metabolic pathways should be considered. Combination therapies can overcome resistance in specific constellations. Overall, oncolytic virotherapy is a viable treatment option for PDAC, which warrants further development. This study highlights the need for personalised treatment in OVT. By providing all primary data, this study provides a rich source and guidance for ongoing developments. FUNDING: German National Science Foundation (Deutsche Forschungsgemeinschaft, DFG), German Cancer Aid (Deutsche Krebshilfe), German National Academic Scholarship Foundation (Studienstiftung des deutschen Volkes), Survival with Pancreatic Cancer Foundation.

OpenPBTA: The Open Pediatric Brain Tumor Atlas.

Pediatric brain and spinal cancers are collectively the leading disease-related cause of death in children; thus, we urgently need curative therapeutic strategies for these tumors. To accelerate such discoveries, the Children's Brain Tumor Network (CBTN) and Pacific Pediatric Neuro-Oncology Consortium (PNOC) created a systematic process for tumor biobanking, model generation, and sequencing with immediate access to harmonized data. We leverage these data to establish OpenPBTA, an open collaborative project with over 40 scalable analysis modules that genomically characterize 1,074 pediatric brain tumors. Transcriptomic classification reveals universal TP53 dysregulation in mismatch repair-deficient hypermutant high-grade gliomas and TP53 loss as a significant marker for poor overall survival in ependymomas and H3 K28-mutant diffuse midline gliomas. Already being actively applied to other pediatric cancers and PNOC molecular tumor board decision-making, OpenPBTA is an invaluable resource to the pediatric oncology community.

Evaluation of circulating Dickkopf-1 as a prognostic biomarker in ovarian cancer patients.

OBJECTIVES: Dickkopf-1 (DKK1) is a secreted protein, known for suppressing the differentiation and activity of bone-building osteoblasts by acting as an inhibitor of Wnt-signalling. Soluble DKK1 (sDKK1) has been proposed as prognostic biomarker for a wide range of malignancies, however, clinical relevance of sDKK1 as potential blood-based marker for ovarian cancer is unknown. METHODS: sDKK1 levels were quantified in a cohort of 150 clinically documented ovarian cancer patients by a commercially available DKK1 ELISA (Biomedica, Vienna, Austria). RESULTS: Median sDKK1 level was significantly elevated at primary diagnosis of ovarian cancer compared to healthy controls (estimated difference (ED) of 7.75 ng/mL (95% CI: 3.01-12.30 ng/mL, p=0.001)). Higher levels of sDKK1 at diagnosis indicated an increased volume of intraoperative malignant ascites (ED 7.08 pmol/L, 95% CI: 1.46-13.05, p=0.02) and predicted suboptimal debulking surgery (ED 6.88 pmol/L, 95% CI: 1.73-11.87, p=0.01). sDKK1 did not correlate with CA125 and higher sDKK1 levels predicted a higher risk of recurrence and poor survival (PFS: HR=0.507, 95% CI: 0.317-0.809; p=0.004; OS: HR=0.561, 95% CI: 0.320-0.986; p=0.044). Prognostic relevance of sDKK1 was partly sustained in wtBRCA patients (PFS: HR=0.507, 95% CI: 0.317-0.809; p=0.004). CONCLUSIONS: This is the first study demonstrating the prognostic relevance of sDKK1 in ovarian cancer patients, including those with wtBRCA1/2 status. Our data encourage further evaluation of sDKK1 in ovarian cancer patients, possibly in terms of a therapy monitoring marker or a response predictor for sDKK1-directed targeted therapies.

Phylogenetic analysis of SARS-CoV-2 lineage development across the first and second waves in Eastern Germany in 2020: insights into the cause of the second wave.

In Germany, Eastern regions had a mild first wave of coronavirus disease 2019 (COVID-19) from March to May 2020, but were badly hit by a second wave later in autumn and winter. It is unknown how the second wave was initiated and developed in Eastern Germany where the number of COVID-19 cases was close to zero in June and July 2020. We used genomic epidemiology to investigate the dynamic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineage development across the first and second waves in Eastern Germany. With detailed phylogenetic analyses we could show that SARS-CoV-2 lineages prevalent in the first and second waves in Eastern Germany were different, with several new variants including four predominant lineages in the second wave, having been introduced into Eastern Germany between August and October 2020. The results indicate that the major driving force behind the second wave was the introduction of new variants.

Deep Learning Improves Pancreatic Cancer Diagnosis Using RNA-Based Variants.

For optimal pancreatic cancer treatment, early and accurate diagnosis is vital. Blood-derived biomarkers and genetic predispositions can contribute to early diagnosis, but they often have limited accuracy or applicability. Here, we seek to exploit the synergy between them by combining the biomarker CA19-9 with RNA-based variants. We use deep sequencing and deep learning to improve differentiating pancreatic cancer and chronic pancreatitis. We obtained samples of nucleated cells found in peripheral blood from 268 patients suffering from resectable, non-resectable pancreatic cancer, and chronic pancreatitis. We sequenced RNA with high coverage and obtained millions of variants. The high-quality variants served as input together with CA19-9 values to deep learning models. Our model achieved an area under the curve (AUC) of 96% in differentiating resectable cancer from pancreatitis using a test cohort. Moreover, we identified variants to estimate survival in resectable cancer. We show that the blood transcriptome harbours variants, which can substantially improve noninvasive clinical diagnosis.

AHNAK controls 53BP1-mediated p53 response by restraining 53BP1 oligomerization and phase separation.

p53-binding protein 1 (53BP1) regulates both the DNA damage response and p53 signaling. Although 53BP1's function is well established in DNA double-strand break repair, how its role in p53 signaling is modulated remains poorly understood. Here, we identify the scaffolding protein AHNAK as a G1 phase-enriched interactor of 53BP1. We demonstrate that AHNAK binds to the 53BP1 oligomerization domain and controls its multimerization potential. Loss of AHNAK results in hyper-accumulation of 53BP1 on chromatin and enhanced phase separation, culminating in an elevated p53 response, compromising cell survival in cancer cells but leading to senescence in non-transformed cells. Cancer transcriptome analyses indicate that AHNAK-53BP1 cooperation contributes to the suppression of p53 target gene networks in tumors and that loss of AHNAK sensitizes cells to combinatorial cancer treatments. These findings highlight AHNAK as a rheostat of 53BP1 function, which surveys cell proliferation by preventing an excessive p53 response.

RTEL1 Regulates G4/R-Loops to Avert Replication-Transcription Collisions.

Regulator of telomere length 1 (RTEL1) is an essential helicase that maintains telomere integrity and facilitates DNA replication. The source of replication stress in Rtel1-deficient cells remains unclear. Here, we report that loss of RTEL1 confers extensive transcriptional changes independent of its roles at telomeres. The majority of affected genes in Rtel1-/- cells possess G-quadruplex (G4)-DNA-forming sequences in their promoters and are similarly altered at a transcriptional level in wild-type cells treated with the G4-DNA stabilizer TMPyP4 (5,10,15,20-Tetrakis-(N-methyl-4-pyridyl)porphine). Failure to resolve G4-DNAs formed in the displaced strand of RNA-DNA hybrids in Rtel1-/- cells is suggested by increased R-loops and elevated transcription-replication collisions (TRCs). Moreover, removal of R-loops by RNaseH1 overexpression suppresses TRCs and alleviates the global replication defects observed in Rtel1-/- and Rtel1PIP_box knockin cells and in wild-type cells treated with TMPyP4. We propose that RTEL1 unwinds G4-DNA/R-loops to avert TRCs, which is important to prevent global deregulation in both transcription and DNA replication.

AP-Seq: A Method to Measure Apurinic Sites and Small Base Adducts Genome-Wide.

DNA is constantly challenged by chemical modification and spontaneous loss of its bases, which results in apurinic sites (AP-sites). In addition to the direct route, modified bases may be converted into AP-sites through enzymatic removal of the base as part of the base excision repair pathway. Here we present the method AP-seq, which allows enriching and sequencing AP-sites genome-wide. Quantification of DNA recovery (AP-quant) allows for relative quantification of global AP-sites, and AP-site pulldown followed by qPCR (AP-qPCR) allows for site-specific damage assessment. Taking advantage of glycosylases that specifically excise modified bases also in vitro, this method allows not only to address the genomic distribution of AP-sites but also to detect base modifications, e.g., 8-oxo-7,8-dihydroguanine (8-oxoG). AP-quant, AP-qPCR, and AP-seq can be applied to investigate quantitatively the relative amount and genome specificity of DNA damage and repair, effects of radiation, as well as multiple other questions around AP-sites and base modifications.

Detection and Monitoring of Lineage-Specific Chimerism by Digital Droplet PCR-Based Testing of Deletion/Insertion Polymorphisms.

Analysis of specific leukocyte subsets for post-transplantation monitoring of chimerism provides greater sensitivity and clinical informativeness on dynamic changes in donor- and recipient-derived cells. Limitations of the most commonly used approach to chimerism testing relying on PCR-based analysis of microsatellite markers prompted us to assess the applicability of digital droplet (dd) PCR amplification of deletion/insertion polymorphisms (DIPs) for lineage-specific chimerism testing in the related stem cell transplantation setting, where the identification of informative markers facilitating the discrimination between donor-derived and recipient-derived cells can be challenging. We analyzed 100 genetically related patient-donor pairs by ddPCR analysis using commercially available DIP kits including large sets of polymorphic markers. At least 1 informative marker was identified in all related pairs analyzed, and 2 or more discriminating markers were detected in the majority (82%) of instances. The achievable detection limit is dependent on the number of cells available for analysis and was as low as 0.1% in the presence of ≥20,000 leukocytes available for DNA extraction. Moreover, the reproducibility and accuracy of quantitative chimerism analysis compared favorably to highly optimized microsatellite assays. Thus, the use of ddPCR-based analysis of DIP markers is an attractive approach to lineage-specific monitoring of chimerism in any allogeneic transplantation setting.

The genomics of oxidative DNA damage, repair, and resulting mutagenesis.

Reactive oxygen species are a constant threat to DNA as they modify bases with the risk of disrupting genome function, inducing genome instability and mutation. Such risks are due to primary oxidative DNA damage and also mediated by the repair process. This leads to a delicate decision process for the cell as to whether to repair a damaged base at a specific genomic location or better leave it unrepaired. Persistent DNA damage can disrupt genome function, but on the other hand it can also contribute to gene regulation by serving as an epigenetic mark. When such processes are out of balance, pathophysiological conditions could get accelerated, because oxidative DNA damage and resulting mutagenic processes are tightly linked to ageing, inflammation, and the development of multiple age-related diseases, such as cancer and neurodegenerative disorders. Recent technological advancements and novel data analysis strategies have revealed that oxidative DNA damage, its repair, and related mutations distribute heterogeneously over the genome at multiple levels of resolution. The involved mechanisms act in the context of genome sequence, in interaction with genome function and chromatin. This review addresses what we currently know about the genome distribution of oxidative DNA damage, repair intermediates, and mutations. It will specifically focus on the various methodologies to measure oxidative DNA damage distribution and discuss the mechanistic conclusions derived from the different approaches. It will also address the consequences of oxidative DNA damage, specifically how it gives rise to mutations, genome instability, and how it can act as an epigenetic mark.

Target-Specific Precision of CRISPR-Mediated Genome Editing.

The CRISPR-Cas9 system has successfully been adapted to edit the genome of various organisms. However, our ability to predict the editing outcome at specific sites is limited. Here, we examined indel profiles at over 1,000 genomic sites in human cells and uncovered general principles guiding CRISPR-mediated DNA editing. We find that precision of DNA editing (i.e., recurrence of a specific indel) varies considerably among sites, with some targets showing one highly preferred indel and others displaying numerous infrequent indels. Editing precision correlates with editing efficiency and a preference for single-nucleotide homologous insertions. Precise targets and editing outcome can be predicted based on simple rules that mainly depend on the fourth nucleotide upstream of the protospacer adjacent motif (PAM). Indel profiles are robust, but they can be influenced by chromatin features. Our findings have important implications for clinical applications of CRISPR technology and reveal general patterns of broken end joining that can provide insights into DNA repair mechanisms.

Genomic landscape of oxidative DNA damage and repair reveals regioselective protection from mutagenesis.

BACKGROUND: DNA is subject to constant chemical modification and damage, which eventually results in variable mutation rates throughout the genome. Although detailed molecular mechanisms of DNA damage and repair are well understood, damage impact and execution of repair across a genome remain poorly defined. RESULTS: To bridge the gap between our understanding of DNA repair and mutation distributions, we developed a novel method, AP-seq, capable of mapping apurinic sites and 8-oxo-7,8-dihydroguanine bases at approximately 250-bp resolution on a genome-wide scale. We directly demonstrate that the accumulation rate of apurinic sites varies widely across the genome, with hot spots acquiring many times more damage than cold spots. Unlike single nucleotide variants (SNVs) in cancers, damage burden correlates with marks for open chromatin notably H3K9ac and H3K4me2. Apurinic sites and oxidative damage are also highly enriched in transposable elements and other repetitive sequences. In contrast, we observe a reduction at chromatin loop anchors with increased damage load towards inactive compartments. Less damage is found at promoters, exons, and termination sites, but not introns, in a seemingly transcription-independent but GC content-dependent manner. Leveraging cancer genomic data, we also find locally reduced SNV rates in promoters, coding sequence, and other functional elements. CONCLUSIONS: Our study reveals that oxidative DNA damage accumulation and repair differ strongly across the genome, but culminate in a previously unappreciated mechanism that safeguards the regulatory and coding regions of genes from mutations.

Inactivation of the ATMIN/ATM pathway protects against glioblastoma formation.

Glioblastoma multiforme (GBM) is the most aggressive human primary brain cancer. Using a Trp53-deficient mouse model of GBM, we show that genetic inactivation of the Atm cofactor Atmin, which is dispensable for embryonic and adult neural development, strongly suppresses GBM formation. Mechanistically, expression of several GBM-associated genes, including Pdgfra, was normalized by Atmin deletion in the Trp53-null background. Pharmacological ATM inhibition also reduced Pdgfra expression, and reduced the proliferation of Trp53-deficient primary glioma cells from murine and human tumors, while normal neural stem cells were unaffected. Analysis of GBM datasets showed that PDGFRA expression is also significantly increased in human TP53-mutant compared with TP53-wild-type tumors. Moreover, combined treatment with ATM and PDGFRA inhibitors efficiently killed TP53-mutant primary human GBM cells, but not untransformed neural stem cells. These results reveal a new requirement for ATMIN-dependent ATM signaling in TP53-deficient GBM, indicating a pro-tumorigenic role for ATM in the context of these tumors.

RASA4 undergoes DNA hypermethylation in resistant juvenile myelomonocytic leukemia.

Aberrant DNA methylation at specific genetic loci is a key molecular feature of juvenile myelomonocytic leukemia (JMML) with poor prognosis. Using quantitative high-resolution mass spectrometry, we identified RASA4 isoform 2, which maps to chromosome 7 and encodes a member of the GAP1 family of GTPase-activating proteins for small G proteins, as a recurrent target of isoform-specific DNA hypermethylation in JMML (51% of 125 patients analyzed). RASA4 isoform 2 promoter methylation correlated with clinical parameters predicting poor prognosis (older age, elevated fetal hemoglobin), with higher risk of relapse after hematopoietic stem cell transplantation, and with PTPN11 mutation. The level of isoform 2 methylation increased in relapsed cases after transplantation. Interestingly, most JMML cases with monosomy 7 exhibited hypermethylation on the remaining RASA4 allele. The results corroborate the significance of epigenetic modifications in the phenotype of aggressive JMML.

Parg deficiency confers radio-sensitization through enhanced cell death in mouse ES cells exposed to various forms of ionizing radiation.

Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme involved in poly(ADP-ribose) degradation. Here, the effects of Parg deficiency on sensitivity to low and high linear-energy-transfer (LET) radiation were investigated in mouse embryonic stem (ES) cells. Mouse Parg(-/-) and poly(ADP-ribose) polymerase-1 deficient (Parp-1(-/-)) ES cells were used and responses to low and high LET radiation were assessed by clonogenic survival and biochemical and biological analysis methods. Parg(-/-) cells were more sensitive to γ-irradiation than Parp-1(-/-) cells. Transient accumulation of poly(ADP-ribose) was enhanced in Parg(-/-) cells. Augmented levels of phosphorylated H2AX (γ-H2AX) from early phase were observed in Parg(-/-) ES cells. The induction level of p53 phophorylation at ser18 was similar in wild-type and Parp-1(-/-) cells and apoptotic cell death process was mainly observed in the both genotypes. These results suggested that the enhanced sensitivity of Parg(-/-) ES cells to γ-irradiation involved defective repair of DNA double strand breaks. The effects of Parg and Parp-1 deficiency on the ES cell response to carbon-ion irradiation (LET13 and 70 keV/µm) and Fe-ion irradiation (200 keV/µm) were also examined. Parg(-/-) cells were more sensitive to LET 70 keV/µm carbon-ion irradiation than Parp-1(-/-) cells. Enhanced apoptotic cell death also accompanied augmented levels of γ-H2AX in a biphasic manner peaked at 1 and 24h. The induction level of p53 phophorylation at ser18 was not different between wild-type and Parg(-/-) cells. The augmented level of poly(ADP-ribose) accumulation was noted after carbon-ion irradiation compared to γ-irradiation even in the wild-type cells. An enhanced poly(ADP-ribose) accumulation was further observed in Parg(-/-) cells. Both Parg(-/-) cells and Parp-1(-/-) cells did not show sensitization to Fe-ion irradiation. Parg deficiency sensitizes mouse ES cells to a wide therapeutic range of LET radiation through the effects on DNA double strand break repair responses and enhanced cell death.

Quantitative analyses of DAPK1 methylation in AML and MDS.

Aberrant DNA methylation and concomitant transcriptional silencing of death-associated protein kinase 1 (DAPK1) have been demonstrated to be key pathogenic events in chronic lymphocytic leukemia (CLL). In acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), however, the presence of elevated DNA methylation levels has been a matter of continued controversy. Several studies demonstrated highly variable frequencies of DAPK1 promoter methylation by the use of methylation-specific PCR (MSP). By quantitative high-resolution assessment, we demonstrate that aberrant DNA methylation is an extremely rare event in this region. We observed elevated levels just in one out of 246 (0.4%) AML patients, all 42 MDS patients were unmethylated. In conclusion, we present a refined DAPK1 methylation analysis in a large representative patient cohort of AML and MDS patients proofing almost complete absence of elevated DNA methylation. Our results highlight the importance of quantitative measurements for translational research questions on primary patient specimens, particularly.

Transcriptional regulation by DNA methylation.

Epigenetic mechanisms synergize with genetic alterations in modulating gene expression patterns in cancer cells. While epigenetic alterations are reversible genetic modifications are not. This has raised the attention of many groups to focus on a better understanding of the molecular mechanisms that underlie the establishment of altered DNA methylation, histone modifications patterns and miRNA expression. The improved understanding of these mechanisms we will in turn allow us to improve the strategies that can be used for epigenetic therapies. In this review we will discuss and summarize briefly our current knowledge of epigenetic alterations in leukemias and will turn our attention to a concrete example of epigenetic deregulation of CCAAT/enhancer-binding protein alpha (C/EBPα), a key regulator for granulocytic differentiation of common myeloid progenitor cells in order to highlight the cooperativity of genetic and epigenetic mechanisms acting on this gene during the process of leukemogenesis.

Aberrant DNA methylation characterizes juvenile myelomonocytic leukemia with poor outcome.

Aberrant DNA methylation contributes to the malignant phenotype in virtually all types of cancer, including myeloid leukemia. We hypothesized that CpG island hypermethylation also occurs in juvenile myelomonocytic leukemia (JMML) and investigated whether it is associated with clinical, hematologic, or prognostic features. Based on quantitative measurements of DNA methylation in 127 JMML cases using mass spectrometry (MassARRAY), we identified 4 gene CpG islands with frequent hypermethylation: BMP4 (36% of patients), CALCA (54%), CDKN2B (22%), and RARB (13%). Hypermethylation was significantly associated with poor prognosis: when the methylation data were transformed into prognostic scores using a LASSO Cox regression model, the 5-year overall survival was 0.41 for patients in the top tertile of scores versus 0.72 in the lowest score tertile (P = .002). Among patients given allogeneic hematopoietic stem cell transplantation, the 5-year cumulative incidence of relapse was 0.52 in the highest versus 0.10 in the lowest score tertile (P = .007). In multivariate models, DNA methylation retained prognostic value independently of other clinical risk factors. Longitudinal analyses indicated that some cases acquired a more extensively methylated phenotype at relapse. In conclusion, our data suggest that a high-methylation phenotype characterizes an aggressive biologic variant of JMML and is an important molecular predictor of outcome.

Insulin-like growth factor-II methylation status in lymphocyte DNA and colon cancer risk in the Northern Sweden Health and Disease cohort.

Loss of imprinting (LOI) of the insulin-like growth factor II (IGFII) gene is a frequent phenomenon in colorectal tumor tissues. Previous reports indicated that subjects with colorectal neoplasias show LOI of IGFII in circulating lymphocytes. Furthermore, LOI of IGFII is strongly related to the methylation of a differentially methylated region (DMR) in intron 2 of IGFII, suggesting that the methylation status could serve as a biomarker for early detection. Thus, hypermethylation of this DMR, even at a systemic level, e.g., in lymphocyte DNA, could be used for screening for colon cancer. To validate this, we performed a case-control study of 97 colon cancer cases and 190 age-matched and gender-matched controls, nested within the prospective Northern Sweden Health and Disease Study cohort. Methylation levels of the IGFII-DMR in lymphocyte DNA were measured at two specific CpG sites of the IGFII-DMR using a mass-spectrometric method called short oligonucleotide mass analysis, the measurements of which showed high reproducibility between replicate measurements for the two CpG sites combined and showed almost perfect validity when performed on variable mixtures of methylated and unmethylated standards. Mean fractions of CpG methylation, for the two CpG sites combined, were identical for cases and controls (0.47 and 0.46, respectively; P(difference) = 0.75), and logistic regression analyses showed no relationship between colon cancer risk and quartile levels of CpG methylation. The results from this study population do not support the hypothesis that colon cancer can be predicted from the different degrees of methylation of DMR in the IGFII gene from lymphocyte DNA.