Iron-triggered signaling via ETS1 and the p38/JNK MAPK pathway regulates Bmp6 expression.

BMP6 is an iron-sensing cytokine whose transcription in liver sinusoidal endothelial cells (LSECs) is enhanced by high iron levels, a step that precedes the induction of the iron-regulatory hormone hepcidin. While several reports suggested a cell-autonomous induction of Bmp6 by iron-triggered signals, likely via sensing of oxidative stress by the transcription factor NRF2, other studies proposed the dominant role of a paracrine yet unidentified signal released by iron-loaded hepatocytes. To further explore the mechanisms of Bmp6 transcriptional regulation, we used female mice aged 10-11 months, which are characterized by hepatocytic but not LSEC iron accumulation, and no evidence of systemic iron overload. We found that LSECs of aged mice exhibit increased Bmp6 mRNA levels as compared to young controls, but do not show a transcriptional signature characteristic of activated NFR2-mediated signaling in FACS-sorted LSECs. We further observed that primary murine LSECs derived from both wild-type and NRF2 knock-out mice induce Bmp6 expression in response to iron exposure. By analyzing transcriptomic data of FACS-sorted LSECs from aged versus young mice, as well as early after iron citrate injections, we identified ETS1 as a candidate transcription factor involved in Bmp6 transcriptional regulation. By performing siRNA-mediated knockdown, small-molecule treatments, and chromatin immunoprecipitation in primary LSECs, we show that Bmp6 transcription is regulated by iron via ETS1 and p38/JNK MAP kinase-mediated signaling, at least in part independently of NRF2. Thereby, these findings identify the new components of LSEC iron sensing machinery broadly associated with cellular stress responses.

The Risk Function of Breast and Ovarian Cancers in the Avrami-Dobrzynski Cellular Phase-Transition Model.

Specifying the role of genetic mutations in cancer development is crucial for effective screening or targeted treatments for people with hereditary cancer predispositions. Our goal here is to find the relationship between a number of cancerogenic mutations and the probability of cancer induction over the lifetime of cancer patients. We believe that the Avrami-Dobrzynski biophysical model can be used to describe this mechanism. Therefore, clinical data from breast and ovarian cancer patients were used to validate this model of cancer induction, which is based on a purely physical concept of the phase-transition process with an analogy to the neoplastic transformation. The obtained values of model parameters established using clinical data confirm the hypothesis that the carcinogenic process strongly follows fractal dynamics. We found that the model's theoretical prediction and population clinical data slightly differed for patients with the age below 30 years old, and that might point to the existence of an ancillary protection mechanism against cancer development. Additionally, we reveal that the existing clinical data predict breast or ovarian cancers onset two years earlier for patients with BRCA1/2 mutations.

Microbial and Metabolic Gut Profiling across Seven Malignancies Identifies Fecal Faecalibacillus intestinalis and Formic Acid as Commonly Altered in Cancer Patients.

The key association between gut dysbiosis and cancer is already known. Here, we used whole-genome shotgun sequencing (WGS) and gas chromatography/mass spectrometry (GC/MS) to conduct metagenomic and metabolomic analyses to identify common and distinct taxonomic configurations among 40, 45, 71, 34, 50, 60, and 40 patients with colorectal cancer, stomach cancer, breast cancer, lung cancer, melanoma, lymphoid neoplasms and acute myeloid leukemia (AML), respectively, and compared the data with those from sex- and age-matched healthy controls (HC). α-diversity differed only between the lymphoid neoplasm and AML groups and their respective HC, while ß-diversity differed between all groups and their HC. Of 203 unique species, 179 and 24 were under- and over-represented, respectively, in the case groups compared with HC. Of these, Faecalibacillus intestinalis was under-represented in each of the seven groups studied, Anaerostipes hadrus was under-represented in all but the stomach cancer group, and 22 species were under-represented in the remaining five case groups. There was a marked reduction in the gut microbiome cancer index in all case groups except the AML group. Of the short-chain fatty acids and amino acids tested, the relative concentration of formic acid was significantly higher in each of the case groups than in HC, and the abundance of seven species of Faecalibacterium correlated negatively with most amino acids and formic acid, and positively with the levels of acetic, propanoic, and butanoic acid. We found more differences than similarities between the studied malignancy groups, with large variations in diversity, taxonomic/metabolomic profiles, and functional assignments. While the results obtained may demonstrate trends rather than objective differences that correlate with different types of malignancy, the newly developed gut microbiota cancer index did distinguish most of the cancer cases from HC. We believe that these data are a promising step forward in the search for new diagnostic and predictive tests to assess intestinal dysbiosis among cancer patients.

CryoGrid-PIXUL-RNA: high throughput RNA isolation platform for tissue transcript analysis.

BACKGROUND: Disease molecular complexity requires high throughput workflows to map disease pathways through analysis of vast tissue repositories. Great progress has been made in tissue multiomics analytical technologies. To match the high throughput of these advanced analytical platforms, we have previously developed a multipurpose 96-well microplate sonicator, PIXUL, that can be used in multiple workflows to extract analytes from cultured cells and tissue fragments for various downstream molecular assays. And yet, the sample preparation devices, such as PIXUL, along with the downstream multiomics analytical capabilities have not been fully exploited to interrogate tissues because storing and sampling of such biospecimens remain, in comparison, inefficient. RESULTS: To mitigate this tissue interrogation bottleneck, we have developed a low-cost user-friendly system, CryoGrid, to catalog, cryostore and sample tissue fragments. TRIzol is widely used to isolate RNA but it is labor-intensive, hazardous, requires fume-hoods, and is an expensive reagent. Columns are also commonly used to extract RNA but they involve many steps, are prone to human errors, and are also expensive. Both TRIzol and column protocols use test tubes. We developed a microplate PIXUL-based TRIzol-free and column-free RNA isolation protocol that uses a buffer containing proteinase K (PK buffer). We have integrated the CryoGrid system with PIXUL-based PK buffer, TRIzol, and PureLink column methods to isolate RNA for gene-specific qPCR and genome-wide transcript analyses. CryoGrid-PIXUL, when integrated with either PK buffer, TRIzol or PureLink column RNA isolation protocols, yielded similar transcript profiles in frozen organs (brain, heart, kidney and liver) from a mouse model of sepsis. CONCLUSIONS: RNA isolation using the CryoGrid-PIXUL system combined with the 96-well microplate PK buffer method offers an inexpensive user-friendly high throughput workflow to study transcriptional responses in tissues in health and disease as well as in therapeutic interventions.

Concurrent depletion of Vps37 proteins evokes ESCRT-I destabilization and profound cellular stress responses.

Molecular details of how endocytosis contributes to oncogenesis remain elusive. Our in silico analysis of colorectal cancer (CRC) patients revealed stage-dependent alterations in the expression of 112 endocytosis-related genes. Among them, transcription of the endosomal sorting complex required for transport (ESCRT)-I component VPS37B was decreased in the advanced stages of CRC. Expression of other ESCRT-I core subunits remained unchanged in the investigated dataset. We analyzed an independent cohort of CRC patients, which also showed reduced VPS37A mRNA and protein abundance. Transcriptomic profiling of CRC cells revealed non-redundant functions of Vps37 proteins. Knockdown of VPS37A and VPS37B triggered p21 (CDKN1A)-mediated inhibition of cell proliferation and sterile inflammatory response driven by the nuclear factor (NF)-κB transcription factor and associated with mitogen-activated protein kinase signaling. Co-silencing of VPS37C further potentiated activation of these independently induced processes. The type and magnitude of transcriptional alterations correlated with the differential ESCRT-I stability upon individual and concurrent Vps37 depletion. Our study provides novel insights into cancer cell biology by describing cellular stress responses that are associated with ESCRT-I destabilization.

MEETING HIGHLIGHTS: THE THIRD MARIE SKLODOWSKA-CURIE SYMPOSIUM ON CANCER RESEARCH AND CARE AT ROSWELL PARK COMPREHENSIVE CANCER CENTER, BUFFALO, NY, SEPTEMBER 20-22, 2023.

Marie Sklodowska-Curie Symposia on Cancer Research and Care (MSCS-CRC) promote collaborations between cancer researchers and care providers in the United States, Canada and Central and Eastern European Countries (CEEC), to accelerate the development of new cancer therapies, advance early detection and prevention, increase cancer awareness, and improve cancer care and the quality of life of patients and their families. The third edition of MSCS-CRC, held at Roswell Park Comprehensive Cancer Center, Buffalo, NY, in September 2023, brought together 137 participants from 20 academic institutions in the US, Poland, Ukraine, Lithuania, Croatia and Hungary, together with 16 biotech and pharma entities. The key areas of collaborative opportunity identified during the meeting are a) creating of a database of available collaborative projects in the areas of early-phase clinical trials, preclinical development, and identification of early biomarkers; b) promoting awareness of cancer risks and efforts at cancer prevention; c) laboratory and clinical training; and d) sharing experience in cost-effective delivery of cancer care and improving the quality of life of cancer patients and their families. Examples of ongoing international collaborations in the above areas were discussed. Participation of the representatives of the Warsaw-based Medical Research Agency, National Cancer Institute (NCI) of the United States, National Cancer Research Institutes of Poland and Lithuania, New York State Empire State Development, Ministry of Health of Ukraine and Translational Research Cancer Center Consortium of 13 cancer centers from the US and Canada, facilitated the discussion of available governmental and non-governmental funding initiatives in the above areas.

Prediction of fetal blood group antigens from maternal plasma using Ion AmpliSeq HD technology.

BACKGROUND: Fetal blood group (BG) and platelet (HPA) antigens may trigger maternal immunization, causing a fetal disease. Noninvasive prenatal diagnostics (NIPT) predicts fetal genotype, identifying pregnancies with no risk. All current techniques detect fetal antigen alleles with unspecific background and without estimation of fetal fraction, thus new protocols for detection of fetal BG/HPA alleles with ultrahigh sensitivity still need to be tested to improve NIPT. AIM: To design NIPT of clinically important antigens using Ion AmpliSeq HD technology. METHODS: Plasma DNA from 36 pregnant women (9-33 week of gestation, 24 immunized with anti-HPA-1a,-3b,-15a, -K, or -D+C+S), with known BG/HPA genotypes of their neonates/partners, was tested on Ion S5 System using the Ion AmpliSeq HD designer custom gene panel. NGS contained 25 rs-targets encoding relevant BG/HPA antigens and 10 markers. RESULTS: Using the NGS protocol, 76 out of 85 differences in fetal/maternal BG/HPA genotypes were determined in concentration above 2% fetal paternally inherited allele chimerism. The level of unspecific reads for BG/HPA alleles was below 0.87%. In 24 immunized women NGS revealed feto-maternal incompatibility in 11 cases (from 2.44% to 7.41%) and excluded in 10 (<0.05%), three cases had inconclusive results (1.79%, 0.19%, 0.11%). The presence of fetal DNA was confirmed in each case by detecting markers with at least 2% chimerism. CONCLUSION: The use of Ion AmpliSeq HD technology improves the prediction of feto-maternal incompatibility, increasing the sensitivity of BG/HPA NIPT and serving confirmation of the fetal DNA at the same workflow.

Genetic associations with lymphomas in Polish patients: A pooled-DNA genome-wide association analysis.

Several single nucleotide polymorphisms (SNPs) associated with susceptibility to Hodgkin lymphoma (HL) and diffuse large B-cell lymphoma (DLBCL) have been identified. The aim of this study was to identify susceptibility loci for HL and DLBCL in Polish patients. Altogether, DLBCL (n = 218 and HL patients (n = 224) and healthy individuals (n = 1181) were recruited. Lymphoma diagnosis was based on standard criteria. Genome-wide association study (GWAS) was performed using pooled-DNA samples on llumina Infinium Omni2.5 Exome-8 v1.3, and selected loci were replicated by TaqMan SNP genotyping of individuals. GWAS detected thirteen and seven SNPs associated with DLBCL and HL, respectively. In the replication study, six and seven SNPs reached significance after correction for multiple testing in the DLBCL and HL cohorts, respectively. One and four SNPs associated with DLBCL and HL, respectively, were localized within, and two SNPs-near the major histocompatibility complex (MHC) region. In conclusion, the majority of loci associated with HL and DLBCL aetiology in previous studies have potential roles in immune function. Our pooled-DNA GWAS enabled the identification of several susceptibility loci for DLBCL and HL in the Polish population; some of them were mapped within or adjacent to the MHC, and other associated SNPs were located outside the MHC.

Higher genome variability within metabolism genes associates with recurrent Clostridium difficile infection.

BACKGROUND: Clostridium difficile (C. difficile) is a major source of healthcare-associated infection with a high risk of recurrence, attributable to many factors such as usage of antibiotics, older age and immunocompromised status of the patients. C. difficile has also a highly diverse genome, which may contribute to its high virulence. Herein we examined whether the genome conservation, measured as non-synonymous to synonymous mutations ratio (dN/dS) in core genes, presence of single genes, plasmids and prophages increased the risk of reinfection in a subset of 134 C. difficile isolates from our previous study in a singly hemato-oncology ward. METHODS: C. difficile isolates were subjected to whole-genome sequencing (WGS) on Ion Torrent PGM sequencer. Genomes were assembled with MIRA5 and annotated with prokka and VRprofile. Logistic regression was used to asses the relationship between single gene presence and the odds of infection recurrence. DN/dS ratios were computed with codeml. Functional annotation was conducted with eggNOG-Mapper. RESULTS: We have found that the presence of certain genes, associated with carbon metabolism and oxidative phosphorylation, increased the odds of infection recurrence. More core genes were under positive selective pressure in recurrent disease isolates - they were mostly associated with the metabolism of aminoacids. Finally, prophage elements were more prevalent in single infection isolates and plasmids did not influence the odds of recurrence. CONCLUSIONS: Our findings suggest higher genetic plasticity in isolates causing recurrent infection, associated mainly with metabolism. On the other hand, the presence of prophages seems to reduce the isolates' virulence.

Integrative genomics reveal a role for MCPIP1 in adipogenesis and adipocyte metabolism.

Obesity is considered a serious chronic disease, associated with an increased risk of developing cardiovascular diseases, non-alcoholic fatty liver disease and type 2 diabetes. Monocyte chemoattractant protein-1-induced protein-1 (MCPIP1) is an RNase decreasing stability of transcripts coding for inflammation-related proteins. In addition, MCPIP1 plays an important role in the regulation of adipogenesis in vitro by reducing the expression of key transcription factors, including C/EBPß. To elucidate the role of MCPIP1 in adipocyte biology, we performed RNA-Seq and proteome analysis in 3T3-L1 adipocytes overexpressing wild-type (WTMCPIP1) and the mutant form of MCPIP1 protein (D141NMCPIP1). Our RNA-Seq analysis followed by confirmatory Q-RT-PCR revealed that elevated MCPIP1 levels in 3T3-L1 adipocytes upregulated transcripts encoding proteins involved in signal transmission and cellular remodeling and downregulated transcripts of factors involved in metabolism. These data are consistent with our proteomic analysis, which showed that MCPIP1 expressing adipocytes exhibit upregulation of proteins involved in cellular organization and movement and decreased levels of proteins involved in lipid and carbohydrate metabolism. Moreover, MCPIP1 adipocytes are characterized by decreased level of insulin receptor, reduced insulin-induced Akt phosphorylation, as well as depleted Glut4 level and impaired glucose uptake. Overexpression of Glut4 in 3T3-L1 cells expressed WTMCPIP1 rescued adipogenesis. Interestingly, we found decreased level of MCPIP1 along with an increase in body mass index in subcutaneous adipose tissue. The presented data show a novel role of MCPIP1 in modulating insulin sensitivity in adipocytes. Overall, our findings demonstrate that MCPIP1 is an important regulator of adipogenesis and adipocyte metabolism.

Cytotoxic Efficacy and Resistance Mechanism of a TRAIL and VEGFA-Peptide Fusion Protein in Colorectal Cancer Models.

TNF-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane protein capable of selectively inducing apoptosis in cancer cells by binding to its cognate receptors. Here, we examined the anticancer efficacy of a recently developed chimeric AD-O51.4 protein, a TRAIL fused to the VEGFA-originating peptide. We tested AD-O51.4 protein activity against human colorectal cancer (CRC) models and investigated the resistance mechanism in the non-responsive CRC models. The quantitative comparison of apoptotic activity between AD-O51.4 and the native TRAIL in nine human colorectal cancer cell lines revealed dose-dependent toxicity in seven of them; the immunofluorescence-captured receptor abundance correlated with the extent of apoptosis. AD-O51.4 reduced the growth of CRC patient-derived xenografts (PDXs) with good efficacy. Cell lines that acquired AD-O51.4 resistance showed a significant decrease in surface TRAIL receptor expression and apoptosis-related proteins, including Caspase-8, HSP60, and p53. These results demonstrate the effectiveness of AD-O51.4 protein in CRC preclinical models and identify the potential mechanism underlying acquired resistance. Progression of AD-O51.4 to clinical trials is expected.

Selective Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) Inhibition by the SCH772984 Compound Attenuates In Vitro and In Vivo Inflammatory Responses and Prolongs Survival in Murine Sepsis Models.

Sepsis is the leading cause of death in intensive care units worldwide. Current treatments of sepsis are largely supportive and clinical trials using specific pharmacotherapy for sepsis have failed to improve outcomes. Here, we used the lipopolysaccharide (LPS)-stimulated mouse RAW264.7 cell line and AlphaLisa assay for TNFa as a readout to perform a supervised drug repurposing screen for sepsis treatment with compounds targeting epigenetic enzymes, including kinases. We identified the SCH772984 compound, an extracellular signal-regulated kinase (ERK) 1/2 inhibitor, as an effective blocker of TNFa production in vitro. RNA-Seq of the SCH772984-treated RAW264.7 cells at 1, 4, and 24 h time points of LPS challenge followed by functional annotation of differentially expressed genes highlighted the suppression of cellular pathways related to the immune system. SCH772984 treatment improved survival in the LPS-induced lethal endotoxemia and cecal ligation and puncture (CLP) mouse models of sepsis, and reduced plasma levels of Ccl2/Mcp1. Functional analyses of RNA-seq datasets for kidney, lung, liver, and heart tissues from SCH772984-treated animals collected at 6 h and 12 h post-CLP revealed a significant downregulation of pathways related to the immune response and platelets activation but upregulation of the extracellular matrix organization and retinoic acid signaling pathways. Thus, this study defined transcriptome signatures of SCH772984 action in vitro and in vivo, an agent that has the potential to improve sepsis outcome.

Mutational landscape of primary and recurrent Ewing sarcoma.

INTRODUCTION: Ewing sarcoma (ES) is a highly aggressive malignancy of bone and soft tissues characterized by the presence of a genetic fusion involving the EWSR1 gene. More than one-third of patients develop distant metastases, which are associated with unfavorable prognosis. Knowledge about the disease's genetic landscape may help foster progress in using targeted therapies in the treatment of ES. AIM OF THE STUDY: The objective is to assess the mutational landscape of ES in pretreatment samples, tumor samples after neoadjuvant chemotherapy, and in metastatic/recurrent tumors in children and adults. MATERIAL AND METHODS: DNA from 39 formalin-fixed paraffin-embedded tumor samples of 22 patients (17 adults, 5 children) were analyzed by targeted next generation sequencing (NGS) using the Oncomine Comprehensive Assay v3gene panel. Additional functional analyses were performed between patient subgroups. RESULTS: All samples were characterized by low tumor mutation burden (< 10 mut/Mb). The most commonly mutated genes were PIK3R1 (59%) and POLE (50%). The most widely detected variants in biopsy samples were PIK3R1 T369I (50%), FGFR1 E159K, and TP53 at codon 72 (both in 27.3%). Additionally, the ATR,BRCA1, RAD50,ATM,CHEK1, and NBN genes showed a significantly higher number of mutations in ES. Mutations in PIK3R1 were significantly more frequent in adults, while mutations in the pathways responsible for cell cycle control, DNA repair, and transcriptional regulation were more frequent in children. CONCLUSIONS: Besides EWSR1 fusion, ES is characterized by numerous point mutations that are potential targets for precision medicine. There is high genomic heterogeneity that may explain differences in outcomes between patient subgroups.

Mitochondrial amidoxime-reducing component 2 (MARC2) has a significant role in N-reductive activity and energy metabolism.

The mitochondrial amidoxime-reducing component (MARC) is a mammalian molybdenum-containing enzyme. All annotated mammalian genomes harbor two MARC genes, MARC1 and MARC2, which share a high degree of sequence similarity. Both molybdoenzymes reduce a variety of N-hydroxylated compounds. Besides their role in N-reductive drug metabolism, only little is known about their physiological functions. In this study, we characterized an existing KO mouse model lacking the functional MARC2 gene and fed a high-fat diet and also performed in vivo and in vitro experiments to characterize reductase activity toward known MARC substrates. MARC2 KO significantly decreased reductase activity toward several N-oxygenated substrates, and for typical MARC substrates, only small residual reductive activity was still detectable in MARC2 KO mice. The residual detected reductase activity in MARC2 KO mice could be explained by MARC1 expression that was hardly unaffected by KO, and we found no evidence of significant activity of other reductase enzymes. These results clearly indicate that MARC2 is mainly responsible for N-reductive biotransformation in mice. Striking phenotypical features of MARC2 KO mice were lower body weight, increased body temperature, decreased levels of total cholesterol, and increased glucose levels, supporting previous findings that MARC2 affects energy pathways. Of note, the MARC2 KO mice were resistant to high-fat diet-induced obesity. We propose that the MARC2 KO mouse model could be a powerful tool for predicting MARC-mediated drug metabolism and further investigating MARC's roles in energy homeostasis.

Mutational Mosaics of Cell-Free DNA from Pancreatic Cyst Fluids.

BACKGROUND: Pancreatic cyst fluids (PCFs) enriched in tumor-derived DNA are a potential source of new biomarkers. The study aimed to analyze germinal variants and mutational profiles of cell-free (cf)DNA shed into the cavity of pancreatic cysts. METHODS: The study cohort consisted of 71 patients who underwent endoscopic ultrasound fine-needle aspiration of PCF. Five malignant cysts, 19 intraductal papillary mucinous neoplasms (IPMNs), 11 mucinous cystic neoplasms (MCNs), eight serous cystic neoplasms (SCNs), and 28 pseudocysts were identified. The sequencing of 409 genes included in Comprehensive Cancer Panel was performed using Ion Proton System. The mutation rate of the KRAS and GNAS canonical loci was additionally determined using digital PCR. RESULTS: The number of mutations detected with NGS varied from 0 to 22 per gene, and genes with the most mutations were: TP53, KRAS, PIK3CA, GNAS, ADGRA2, and APC. The frequencies of the majority of mutations did not differ between non-malignant cystic neoplasms and pseudocysts. NGS detected KRAS mutations in malignant cysts (60%), IPMNs (32%), MCNs (64%), SCNs (13%), and pseudocysts (14%), with GNAS mutations in 20%, 26%, 27%, 13%, and 21% of samples, respectively. Digital PCR-based testing increased KRAS (68%) and GNAS (52%) mutations detection level in IPMNs, but not other cyst types. CONCLUSIONS: We demonstrate relatively high rates of somatic mutations of cancer-related genes, including KRAS and GNAS, in cfDNA isolated from PCFs irrespectively of the pancreatic cyst type. Further studies on molecular mechanisms of pancreatic cysts malignant transformation in relation to their mutational profiles are required.

PARP Inhibition Increases the Reliance on ATR/CHK1 Checkpoint Signaling Leading to Synthetic Lethality-An Alternative Treatment Strategy for Epithelial Ovarian Cancer Cells Independent from HR Effectiveness.

Poly (ADP-ribose) polymerase inhibitor (PARPi, olaparib) impairs the repair of DNA single-strand breaks (SSBs), resulting in double-strand breaks (DSBs) that cannot be repaired efficiently in homologous recombination repair (HRR)-deficient cancers such as BRCA1/2-mutant cancers, leading to synthetic lethality. Despite the efficacy of olaparib in the treatment of BRCA1/2 deficient tumors, PARPi resistance is common. We hypothesized that the combination of olaparib with anticancer agents that disrupt HRR by targeting ataxia telangiectasia and Rad3-related protein (ATR) or checkpoint kinase 1 (CHK1) may be an effective strategy to reverse ovarian cancer resistance to olaparib. Here, we evaluated the effect of olaparib, the ATR inhibitor AZD6738, and the CHK1 inhibitor MK8776 alone and in combination on cell survival, colony formation, replication stress response (RSR) protein expression, DNA damage, and apoptotic changes in BRCA2 mutated (PEO-1) and HRR-proficient BRCA wild-type (SKOV-3 and OV-90) cells. Combined treatment caused the accumulation of DNA DSBs. PARP expression was associated with sensitivity to olaparib or inhibitors of RSR. Synergistic effects were weaker when olaparib was combined with CHK1i and occurred regardless of the BRCA2 status of tumor cells. Because PARPi increases the reliance on ATR/CHK1 for genome stability, the combination of PARPi with ATR inhibition suppressed ovarian cancer cell growth independently of the efficacy of HRR. The present results were obtained at sub-lethal doses, suggesting the potential of these inhibitors as monotherapy as well as in combination with olaparib.

Differences between Well-Differentiated Neuroendocrine Tumors and Ductal Adenocarcinomas of the Pancreas Assessed by Multi-Omics Profiling.

Most pancreatic neuroendocrine tumors (PNETs) are indolent, while pancreatic ductal adenocarcinomas (PDACs) are particularly aggressive. To elucidate the basis for this difference and to establish the biomarkers, by using the deep sequencing, we analyzed somatic variants across coding regions of 409 cancer genes and measured mRNA/miRNA expression in nine PNETs, eight PDACs, and four intestinal neuroendocrine tumors (INETs). There were 153 unique somatic variants considered pathogenic or likely pathogenic, found in 50, 57, and 24 genes in PDACs, PNETs, and INETs, respectively. Ten and 11 genes contained a pathogenic mutation in at least one sample of all tumor types and in PDACs and PNETs, respectively, while 28, 34, and 11 genes were found to be mutated exclusively in PDACs, PNETs, and INETs, respectively. The mRNA and miRNA transcriptomes of PDACs and NETs were distinct: from 54 to 1659 differentially expressed mRNAs and from 117 to 250 differentially expressed miRNAs exhibited high discrimination ability and resulted in models with an area under the receiver operating characteristics curve (AUC-ROC) >0.9 for both miRNA and mRNA. Given the miRNAs high stability, we proposed exploring that class of RNA as new pancreatic tumor biomarkers.

Ectopic overexpression of MCPIP1 impairs adipogenesis by modulating microRNAs.

Adipogenesis is a process of preadipocyte differentiation that requires action of numerous factors. Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) possesses the N-terminus of the PilT protein (PilT N-terminus or PIN domain) that has RNase properties. This protein degrades transcripts coding for inflammation and differentiation - related proteins. Moreover, MCPIP1 is a broad suppressor of the miRNA biogenesis. We previously found that MCPIP1 degrades transcript encoding CCAAT/Enhancer Binding Protein Beta (C/EBPß) and influences adipogenesis. Subsequently, we aimed to determine adipocyte miRNA expression profile in differentiating mouse preadipocytes, 3T3-L1, by overexpressing MCPIP1. Using Next-Generation Sequencing (NSG) we showed that MCPIP1 overexpression results in modulated levels of 58 miRNAs in adipocytes on day 2 of differentiation. Among them, 30 miRNAs showed significantly reduced levels and 28 showed increased levels in comparison to control. Approximately one third of the modulated miRNAs were not previously reported to be involved in adipocytes differentiation. Our analysis revealed that 24 down-regulated and 23 up-regulated miRNAs (at least 1.5-fold) influence 19 signaling pathways that are important for adipogenesis. Furthermore, reduced miRNA levels result in the up-regulation of their targets. By using luciferase reporter assay, we demonstrated that miR-32-5p and miR-9-3p directly target the 3'UTR region of Mapk8 and Tiam1, respectively. In addition, activation of MAP kinases pathway (JNK and p38), proposed as being regulated by down-regulated miRNAs, was higher in WTMCPIP1 than in D141NMCPIP1 or control 3T3-L1 adipocytes. Our results indicate a considerable impact of MCPIP1 on miRNAs levels and its significance in adipogenesis.

Prediction of fetal blood group and platelet antigens from maternal plasma using next-generation sequencing.

BACKGROUND: Fetuses whose mothers have produced antibodies to red blood cell (RBC) or platelet antigens are at risk of being affected by hemolytic disease or alloimmune thrombocytopenia, respectively, only if they inherit the incompatible antigen. Noninvasive diagnosis of the fetal antigen is employed for management of immunized pregnancies, but the specific detection of SNPs, encoding the majority of antigens, in maternal plasma is still a challenge. We applied targeted next-generation sequencing (NGS) to predict the fetal antigen based on the detection of fetomaternal chimerism. METHODS AND MATERIALS: The DNA of 13 pregnant women (with anti-K [3] anti-k [1], anti-Fya [1], anti-D + C + Jka [1], anti-D + E + K [1], anti-HPA-1a [1], anti-HPA-3b [1], anti-HPA-5b [1], and nonimmunized [3]) was sequenced using primers for regions encoding RhD, RhC, Rhc, RhE/e, K/k, Fya/b, Jka/b, MN, Ss, and HPA-1, 2, 3, 5, 15, 4 X-polymorphisms on the Ion Torrent Personal Genome Machine (PGM) System (Thermo Fisher Scientific, Inc., Waltham, MA, USA). RESULTS: NGS results were in agreement with the phenotype/genotype of women and their neonates (except for the unsuccessful detection of MN and RhC). NGS determined fetal allele chimerism for K, k, Fya, Fyb, Jka, Jkb, S, RhE (from 0.42% to 6.08%); RhD, Rhc (100%); HPA-1a, -2b, -3a, 3b, -5b, -15a, 15b (from 0.23% to 4.11%). NGS revealed fetal chimerism for incompatible antigens (from 0.7% to 4.8%) in 7 immunized cases, excluded in 3 (with anti-K, anti-Fya , anti-HPA-3b). CONCLUSION: The designed NGS predicts the fetal RBC and platelet antigen status universally in cases with various clinically significant antibodies as well as providing confirmation of the presence of fetal DNA. However, some improvement of the unsuccessful primers is required.

Whole-exome sequencing identifies novel pathogenic variants across the ATP7B gene and some modifiers of Wilson's disease phenotype.

BACKGROUND & AIMS: Wilson's disease (WD) is an autosomal recessive disorder associated with disease-causing alterations across the ATP7B gene, with highly variable symptoms and age of onset. We aimed to assess whether the clinical variability of WD relates to modifier genes. METHODS: A total of 248 WD patients were included, of whom 148 were diagnosed after age of 17. Human exome libraries were constructed using AmpliSeq technology and sequenced using the IonProton platform. RESULTS: ATP7B p.His1069Gln mutation was present in 215 patients, with 112 homozygotes and 103 heterozygotes. Three other mutations: p.Gln1351Ter, p.Trp779Ter and c.3402delC were identified in >10 patients. Among patients, 117 had a homozygous mutation, 101 were compound heterozygotes, 27 had one heterozygous mutation, and 3 other patients had no identifiable pathogenic variant of ATP7B. Sixteen mutations were novel, found as part of a compound mutation or as a sole, homozygous mutation. For disease phenotype prediction, age at diagnosis was a deciding factor, while frameshift allelic variants of ATP7B and being male increased the odds of developing a neurological phenotype. Rare allelic variants in ESD and INO80 increased and decreased chances for the neurological phenotype, respectively, while rare variants in APOE and MBD6 decreased the chances of WD early manifestation. Compound mutations contributed to earlier age of onset. CONCLUSIONS: In a Polish population, genetic screening for WD may help genotype for four variants (p.His1069Gln, p.Gln1351Ter, p.Trp779Ter and c.3402delC), with direct sequencing of all ATP7B amplicons as a second diagnostic step. We also identified some allelic variants that may modify a WD phenotype.