Exploration of Pharmacogenomic Biomarkers in Chronic Immune Diseases Using Single-Cell RNA Sequencing

Biological therapies have revolutionized management of the severe cases of Chronic Immune Diseases refractory to the standard therapies. However, many patients do not respond to the selected biological therapy, loose response over time, or develop adverse effects. A personalized approach to treatment of these patients, based on reliable biomarkers is thus clearly needed. Non-invasive approaches, such as use of the peripheral blood immune cells, are favored for novel biomarker discovery. However, the attention has shifted away from the bulk immune cells and towards specific immune cell sub-populations. Thus, the single-cell RNA sequencing (scRNA-seq) can prove highly valuable. By simultaneously capturing and profiling all the cells in a sample, scRNA-seq allows the analysis of cellular heterogeneity and gene expression in all immune cell sub-populations, targeted or adversely affected by the biological treatment. In our ongoing research, scRNA-seq was utilized to analyze samples from Inflammatory Bowel Disease and Childhood Asthma patients with varied response to the biological therapy. Confounding effects of disease conditions and (biological) therapies on marker genes were eliminated using computational integration in order to identify conserved marker genes across all states. It turned out, that a reliable identification of the different immune cell sub-populations in this setting is quite challenging due to subjective cell-landscape clustering resolution. Several resolutions and automated annotation approaches were subsequently tested and validated.A reference-based approach (Seurat-Azimuth) combined with manual cluster validation proved superior. Alas, manual cluster validation is time consuming. Annotation validation is important, especially to provide additional insights into unidentified clusters, which are essential for the identification of predictive biomarkers for personalized therapies in the vast heterogeneity of immune cell landscapes residing behind pathophysiology of chronic immune diseases.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

The human DEK oncogene regulates DNA damage response signaling and repair

The human DEK gene is frequently overexpressed and sometimes amplified in human cancer. Consistent with oncogenic functions, Dek knockout mice are partially resistant to chemically induced papilloma formation. Additionally, DEK knockdown in vitro sensitizes cancer cells to DNA damaging agents and induces cell death via p53-dependent and -independent mechanisms. Here we report that DEK is important for DNA double-strand break repair. DEK depletion in human cancer cell lines and xenografts was sufficient to induce a DNA damage response as assessed by detection of γH2AX and FANCD2. Phosphorylation of H2AX was accompanied by contrasting activation and suppression, respectively, of the ATM and DNA-PK pathways. Similar DNA damage responses were observed in primary Dek knockout mouse embryonic fibroblasts (MEFs), along with increased levels of DNA damage and exaggerated induction of senescence in response to genotoxic stress. Importantly, Dek knockout MEFs exhibited distinct defects in non-homologous end joining (NHEJ) when compared to their wild-type counterparts. Taken together, the data demonstrate new molecular links between DEK and DNA damage response signaling pathways, and suggest that DEK contributes to DNA repair

Pre-Treatment Biomarkers of Anti-Tumour Necrosis Factor Therapy Response in Crohn’s Disease—A Systematic Review and Gene Ontology Analysis

The most prominent treatment for the serious cases of Crohn’s disease (CD) are biological tumour necrosis factor (TNF) inhibitors. Unfortunately, therapy nonresponse is still a serious issue in ~1/3 of CD patients. Accurate prediction of responsiveness prior to therapy start would therefore be of great value. Clinical predictors have, however, proved insufficient. Here, we integrate genomic and expression data on potential pre-treatment biomarkers of anti-TNF nonresponse. We show that there is almost no overlap between genomic (annotated with tissue-specific expression quantitative trait loci data) and transcription (RNA and protein data) biomarkers. Furthermore, using interaction networks we demonstrate there is little direct interaction between the proposed biomarkers, though a majority do have common interactors connecting them into networks. Our gene ontology analysis shows that these networks have roles in apoptotic signalling, response to oxidative stress and inflammation pathways. We conclude that a more systematic approach with genome-wide search of genomic and expression biomarkers in the same patients is needed in future studies

Pre-Treatment Biomarkers of Anti-Tumour Necrosis Factor Therapy Response in Crohn’s Disease—A Systematic Review and Gene Ontology Analysis

The most prominent treatment for the serious cases of Crohn’s disease (CD) are biological tumour necrosis factor (TNF) inhibitors. Unfortunately, therapy nonresponse is still a serious issue in ~1/3 of CD patients. Accurate prediction of responsiveness prior to therapy start would therefore be of great value. Clinical predictors have, however, proved insufficient. Here, we integrate genomic and expression data on potential pre-treatment biomarkers of anti-TNF nonresponse. We show that there is almost no overlap between genomic (annotated with tissue-specific expression quantitative trait loci data) and transcription (RNA and protein data) biomarkers. Furthermore, using interaction networks we demonstrate there is little direct interaction between the proposed biomarkers, though a majority do have common interactors connecting them into networks. Our gene ontology analysis shows that these networks have roles in apoptotic signalling, response to oxidative stress and inflammation pathways. We conclude that a more systematic approach with genome-wide search of genomic and expression biomarkers in the same patients is needed in future studies

Cleavage-mediated regulation of Myd88 signaling by inflammasome-activated caspase-1

Coordination among multiple signaling pathways ensures an appropriate immune response, where a signaling pathway may impair or augment another signaling pathway. Here, we report a negative feedback regulation of signaling through the key innate immune mediator MyD88 by inflammasome-activated caspase-1. NLRP3 inflammasome activation impaired agonist- or infection-induced TLR signaling and cytokine production through the proteolytic cleavage of MyD88 by caspase-1. Site-specific mutagenesis was used to identify caspase-1 cleavage site within MyD88 intermediary segment. Different cleavage site location within MyD88 defined the functional consequences of MyD88 cleavage between mouse and human cells. LPS/monosodium urate–induced mouse inflammation model corroborated the physiological role of this mechanism of regulation, that could be reversed by chemical inhibition of NLRP3. While Toll/interleukin-1 receptor (TIR) domain released by MyD88 cleavage additionally contributed to the inhibition of signaling, Waldenström’s macroglobulinemia associated MyD88L265P mutation is able to evade the caspase-1-mediated inhibition of MyD88 signaling through the ability of its TIRL265P domain to recruit full length MyD88 and facilitate signaling. The characterization of this mechanism reveals an additional layer of innate immunity regulation

Dual effect of combined metformin and 2-deoxy-D-glucose treatment on mitochondrial biogenesis and PD-L1 expression in triple-negative breast cancer cells

Metformin and 2-deoxy-D-glucose (2DG) exhibit multiple metabolic and immunomodulatory anti-cancer effects, such as suppressed proliferation or PD-L1 expression. Their combination or 2DG alone induce triple-negative breast cancer (TNBC) cell detachment, but their effects on mitochondria, crucial for anchorage-independent growth and metastasis formation, have not yet been evaluated. In the present study, we explored the effects of metformin, 2DG and their combination (metformin + 2DG) on TNBC cell mitochondria in vitro. Metformin + 2DG increased mitochondrial mass in TNBC cells. This was associated with an increased size but not number of morphologically normal mitochondria and driven by the induction of mitochondrial biogenesis rather than suppressed mitophagy. 2DG and metformin + 2DG strongly induced the unfolded protein response by inhibiting protein N-glycosylation. Together with adequate energy stress, this was one of the possible triggers of mitochondrial enlargement. Suppressed N-glycosylation by 2DG or metformin + 2DG also caused PD-L1 deglycosylation and reduced surface expression in MDA-MB-231 cells. PD-L1 was increased in low glucose and normalized by both drugs. 2DG and metformin + 2DG reduced PD-1 expression in Jurkat cells beyond the effects on activation, while cytokine secretion was mostly preserved. Despite increasing mitochondrial mass in TNBC cells, metformin and 2DG could therefore potentially be used as an adjunct therapy to improve anti-tumor immunity in TNBC

Endonuclease G promotes mitochondrial genome cleavage and replication

CERVOxyInternational audienceEndonuclease G (EndoG) is a nuclear-encoded endonuclease, mostly localised in mitochondria. In the nucleus EndoG participates in site-specific cleavage during replication stress and genome-wide DNA degradation during apoptosis. However, the impact of EndoG on mitochondrial DNA (mtDNA) metabolism is poorly understood. Here, we investigated whether EndoG is involved in the regulation of mtDNA replication and removal of aberrant copies. We applied the single-cell mitochondrial Transcription and Replication Imaging Protocol (mTRIP) and PCR-based strategies on human cells after knockdown/knockout and re-expression of EndoG. Our analysis revealed that EndoG stimulates both mtDNA replication initiation and mtDNA depletion, the two events being interlinked and dependent on EndoG's nuclease activity. Stimulation of mtDNA replication by EndoG was independent of 7S DNA processing at the replication origin. Importantly, both mtDNA-directed activities of EndoG were promoted by oxidative stress. Inhibition of base excision repair (BER) that repairs oxidative stress-induced DNA damage unveiled a pronounced effect of EndoG on mtDNA removal, reminiscent of recently discovered links between EndoG and BER in the nucleus. Altogether with the downstream effects on mitochondrial transcription, protein expression, redox status and morphology, this study demonstrates that removal of damaged mtDNA by EndoG and compensatory replication play a critical role in mitochondria homeostasis

Mesenchymal Stem Cells Isolated from Paediatric Paravertebral Adipose Tissue Show Strong Osteogenic Potential

Mesenchymal stem cells (MSCs) represent the basis of novel clinical concepts in cellular therapy and tissue regeneration. Therefore, the isolation of MSCs from various tissues has become an important endeavour for stem cell biobanking and the development of regenerative therapies. Paravertebral adipose tissue is readily exposed during spinal procedures in children and could be a viable source of stem cells for therapeutic applications. Here, we describe the first case of MSCs isolated from paravertebral adipose tissue (PV-ADMSCs), obtained during a routine spinal surgery on a child. Using quantitative real-time PCR and flow cytometry, we show that PV-ADMSCs have different levels of stem marker expression compared to the MSCs from other sources while having the highest proliferation rate. Furthermore, we evaluate the multipotency of PV-ADMSCs by the three-lineage (adipogenic, osteogenic and chondrogenic) differentiation and compare it to the multipotency of MSCs from other sources. It was found that the PV-ADMSCs have a strong osteogenic potential in particular. Taken together, our data indicate that PV-ADMSCs meet the criteria for successful cell therapy, defined by the International Society for Cellular Therapy (ISCT), and thus, could provide a source of MSCs that is relatively easy to isolate and expand in culture. Due to their strong osteogenic potential, these cells provide a promising basis, especially for orthopaedic applications