TMEM8C-mediated fusion is regionalized and regulated by NOTCH signalling during foetal myogenesis

The location and regulation of fusion events within skeletal muscles during development remain unknown. Using the fusion marker myomaker (Mymk), named TMEM8C in chicken, as a readout of fusion, we identified a co-segregation of TMEM8C-positive cells and MYOG-positive cells in single-cell RNA-sequencing datasets of limbs from chicken embryos. We found that TMEM8C transcripts, MYOG transcripts and the fusion-competent MYOG-positive cells were preferentially regionalized in central regions of foetal muscles. We also identified a similar regionalization for the NOTCH ligand JAGGED2 along with an absence of NOTCH activity in TMEM8C+ fusion-competent myocytes. NOTCH function in myoblast fusion had not been addressed so far. We analysed the consequences of NOTCH inhibition for TMEM8C expression and myoblast fusion during foetal myogenesis in chicken embryos. NOTCH inhibition increased myoblast fusion and TMEM8C expression and released the HEYL transcriptional repressor from the TMEM8C regulatory regions. These results identify a regionalization of TMEM8C-dependent fusion and a molecular mechanism underlying the fusion-inhibiting effect of NOTCH in foetal myogenesis. The modulation of NOTCH activity in the fusion zone could regulate the flux of fusion events.

Shooting at Moving and Hidden Targets—Tumour Cell Plasticity and the Notch Signalling Pathway in Head and Neck Squamous Cell Carcinomas

Head and Neck Squamous Cell Carcinoma (HNSCC) is often aggressive, with poor response to current therapies in approximately 40–50% of the patients. Current therapies are restricted to operation and irradiation, often combined with a small number of standard-of-care chemotherapeutic drugs, preferentially for advanced tumour patients. Only very recently, newer targeted therapies have entered the clinics, including Cetuximab, which targets the EGF receptor (EGFR), and several immune checkpoint inhibitors targeting the immune receptor PD-1 and its ligand PD-L1. HNSCC tumour tissues are characterized by a high degree of intra-tumour heterogeneity (ITH), and non-genetic alterations that may affect both non-transformed cells, such as cancer-associated fibroblasts (CAFs), and transformed carcinoma cells. This very high degree of heterogeneity likely contributes to acquired drug resistance, tumour dormancy, relapse, and distant or lymph node metastasis. ITH, in turn, is likely promoted by pronounced tumour cell plasticity, which manifests in highly dynamic and reversible phenomena such as of partial or hybrid forms of epithelial-to-mesenchymal transition (EMT), and enhanced tumour stemness. Stemness and tumour cell plasticity are strongly promoted by Notch signalling, which remains poorly understood especially in HNSCC. Here, we aim to elucidate how Notch signal may act both as a tumour suppressor and proto-oncogenic, probably during different stages of tumour cell initiation and progression. Notch signalling also interacts with numerous other signalling pathways, that may also have a decisive impact on tumour cell plasticity, acquired radio/chemoresistance, and metastatic progression of HNSCC. We outline the current stage of research related to Notch signalling, and how this pathway may be intricately interconnected with other, druggable targets and signalling mechanisms in HNSCC.

Three-Dimensional Culture Systems for Dissecting Notch Signalling in Health and Disease

Three-dimensional (3D) culture systems opened up new horizons in studying the biology of tissues and organs, modelling various diseases, and screening drugs. Producing accurate in vitro models increases the possibilities for studying molecular control of cell–cell and cell–microenvironment interactions in detail. The Notch signalling is linked to cell fate determination, tissue definition, and maintenance in both physiological and pathological conditions. Hence, 3D cultures provide new accessible platforms for studying activation and modulation of the Notch pathway. In this review, we provide an overview of the recent advances in different 3D culture systems, including spheroids, organoids, and “organ-on-a-chip” models, and their use in analysing the crucial role of Notch signalling in the maintenance of tissue homeostasis, pathology, and regeneration.

Leukaemia Cells Induced Metabolic Alterations in AML Associated Mesenchymal Stem Cells Via Notch Signalling

Introduction: Acute myeloid leukaemia (AML) is a haematological malignancy with a high relapse rate and poor prognosis. Leukaemia cell proliferation is dependent on its interaction with the bone marrow (BM) microenvironment. AML associated mesenchymal stem cells (AML-MSCs) supported the proliferation of leukaemia cells and contributed to disease progression. Stromal microenvironment promoted a metabolic switch but precise underlying molecular mechanisms are poorly understood. Previous studies have demonstrated transfer of functional mitochondria from AML-MSCs to AML blasts facilitating energy requirements. To further improve our understanding of the crosstalk between leukaemia and AML-MSCs, we sought to determine contribution of AML-MSCs and signalling cascades regulating metabolic processes. Methods: Sorted MSCs from non-leukaemic and MLL-AF9 leukaemic mice were isolated, and gene expression profiling was performed using RNA microarray. Additionally sorted MSCs from long-term cultures were cultured alone or with MLL-AF9 leukaemia cells and analysed by RNA-sequencing. Gene set enrichment analysis (GSEA) was used to identify the hallmark gene sets overrepresented in AML-MSCs. We further cocultured murine wild type BM-MSCs alone or together with murine AML cells (C1498 and MLL-AF9) or the control lineage negative cells (Lin -). Metabolic alterations, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were analysed by Agilent Seahorse XFe96 analyser. Additionally, glucose consumption, lactate secretion and mitochondrial DNA copy number were measured. Results: Microarray analysis in sorted MSCs from leukaemic and non-leukaemic mice have identified hallmark oxidative phosphorylation (p<0.01, NES=-1.6) and glycolysis (p<0.01, NES=-1.3) gene sets to be negatively enriched in AML-MSCs. Interestingly, both the gene sets were also negatively enriched in sorted AML-MSCs when cocultured with leukaemia but not control cells. To validate these findings, we analysed OCR and EACR in WT-MSCs in an identical setting. The oxidative phosphorylation was significantly decreased in MSCs cocultured with C1498 (p<0.0001) and MLL-AF9 (p<0.005) but not with Lin - cells. Interestingly, glycolysis rate, glucose consumption, lactate secretion were significantly decreased in MSCs cocultured with leukaemia cells. Mitochondrial DNA copy number were significantly decreased in MSCs cocultured with C1498 (p<0.001) or MLL-AF9 (p<0.005) but not with control cells. Recent evidence from the lab has demonstrated an essential role for Notch signalling in the leukaemia and AML-MSCs interaction. To functionally determine the crosstalk of leukaemia-MSC interaction and subsequent Notch signalling, we ectopically expressed the Notch intracellular domain (Notch-ICN1) to mimic Notch activation in a murine stromal cell line, MS-5. Confirming Notch activation, Hes1 mRNA expression (encoding a transcriptional target of Notch signalling) was significantly increased in these cells. Underscoring a role for Notch signalling and activation, Notch-ICN1 overexpression in MS-5 cells demonstrated less oxidative phosphorylation and glycolysis rates as compared to MS-5 cells transduced with empty vector. Conclusion: In line with our microarray and GSEA analysis, our findings confirmed that leukaemia cells indeed induced metabolic alterations decreasing oxidative phosphorylation and glycolysis, and thereby potentially altering AML-MSCs function. At the molecular level, Notch signalling (via upregulated Notch1 and 2 expressions and Notch-ICN) in AML-MSCs contributed to metabolic alterations. Therefore, therapeutically interfering this pathway could target the bidirectional interaction between leukaemia and AML-MSCs improving therapeutic efficacy of AML. Disclosures Khandanpour: GSK: Honoraria; Takeda: Honoraria; Janssen: Honoraria; AstraZeneca: Honoraria, Research Funding; Pfizer: Honoraria; Sanofi: Honoraria, Research Funding; BMS/Celgene: Honoraria.