Combining genomic analyses with tumour-derived slice cultures for the characterization of an EGFR-activating kinase mutation in a case of glioblastoma.

BACKGROUND: Epidermal growth factor receptor (EGFR) gene alterations and amplification are frequently reported in cases of glioblastoma (GBM). However, EGFR-activating mutations that confer proven sensitivity to tyrosine kinase inhibitors (TKIs) in lung cancer have not yet been reported in GBM. CASE PRESENTATION: Using next-generation sequencing, array comparative genomic hybridization and droplet digital PCR, we identified the p.L861Q EGFR mutation in a case of GBM for the first time. The mutation was associated with gene amplification. L861Q may be a clinically valuable mutation because it is known to sensitize non-small-cell lung cancers to treatment with the second-generation EGFR TKI afatinib in particular. Furthermore, we used slice culture of the patient's GBM explant to evaluate the tumour's sensitivity to various EGFR-targeting drugs. Our results suggested that the tumour was not intrinsically sensitive to these drugs. CONCLUSIONS: Our results highlight (i) the value of comprehensive genomic analyses for identifying patient-specific, targetable alterations, and (ii) the need to combine genomic analyses with functional assays, such as tumour-derived slice cultures.

The Conserved YPX3L Motif in the BK Polyomavirus VP1 Protein Is Important for Viral Particle Assembly but Not for Its Secretion into Extracellular Vesicles.

The BK polyomavirus (BKPyV) is a small DNA non-enveloped virus whose infection is asymptomatic in most of the world's adult population. However, in cases of immunosuppression, the reactivation of the virus can cause various complications, and in particular, nephropathies in kidney transplant recipients or hemorrhagic cystitis in bone marrow transplant recipients. Recently, it was demonstrated that BKPyV virions can use extracellular vesicles to collectively traffic in and out of cells, thus exiting producing cells without cell lysis and entering target cells by diversified entry routes. By a comparison to other naked viruses, we investigated the possibility that BKPyV virions recruit the Endosomal-Sorting Complexes Required for Transport (ESCRT) machinery through late domains in order to hijack extracellular vesicles. We identified a single potential late domain in the BKPyV structural proteins, a YPX3L motif in the VP1 protein, and used pseudovirions to study the effect of point mutations found in a BKPyV clinical isolate or known to ablate the interaction of such a domain with the ESCRT machinery. Our results suggest that this domain is not involved in BKPyV association with extracellular vesicles but is crucial for capsomere interaction and thus viral particle assembly.

PIEZO1 is essential for the survival and proliferation of acute myeloid leukemia cells.

INTRODUCTION: Leukemogenesis is a complex process that interconnects tumoral cells with their microenvironment, but the effect of mechanosensing in acute myeloid leukemia (AML) blasts is poorly known. PIEZO1 perceives and transmits the constraints of the environment to human cells by acting as a non-selective calcium channel, but very little is known about its role in leukemogenesis. RESULTS: For the first time, we show that PIEZO1 is preferentially expressed in healthy hematopoietic stem and progenitor cells in human hematopoiesis, and globally overexpressed in AML cells. In AML subtypes, PIEZO1 expression associates with favorable outcomes as better overall (OS) and disease-free survival (DFS). If PIEZO1 is expressed and functional in THP1 leukemic myeloid cell line, its chemical activation doesn't impact the proliferation, differentiation, nor survival of cells. However, the downregulation of PIEZO1 expression dramatically reduces the proliferation and the survival of THP1 cells. We show that PIEZO1 knock-down blocks the cell cycle in G0/G1 phases of AML cells, impairs the DNA damage response pathways, and critically increases cell death by triggering extrinsic apoptosis pathways. CONCLUSIONS: Altogether, our results reveal a new role for PIEZO1 mechanosensing in the survival and proliferation of leukemic blasts, which could pave the way for new therapeutic strategies to target AML cells.

STIM2 is involved in the regulation of apoptosis and the cell cycle in normal and malignant monocytic cells.

Calcium is a ubiquitous messenger that regulates a wide range of cellular functions, but its involvement in the pathophysiology of acute myeloid leukemia (AML) is not widely investigated. Here, we identified, from an analysis of The Cancer Genome Atlas and genotype-tissue expression databases, stromal interaction molecule 2 (STIM2) as being highly expressed in AML with monocytic differentiation and negatively correlated with overall survival. This was confirmed on a validation cohort of 407 AML patients. We then investigated the role of STIM2 in cell proliferation, differentiation, and survival in two leukemic cell lines with monocytic potential and in normal hematopoietic stem cells. STIM2 expression increased at the RNA and protein levels upon monocyte differentiation. Phenotypically, STIM2 knockdown drastically inhibited cell proliferation and induced genomic stress with DNA double-strand breaks, as shown by increased levels of phosphorylate histone H2AXγ (p-H2AXγ), followed by activation of the cellular tumor antigen p53 pathway, decreased expression of cell cycle regulators such as cyclin-dependent kinase 1 (CDK1)-cyclin B1 and M-phase inducer phosphatase 3 (CDC25c), and a decreased apoptosis threshold with a low antiapoptotic/proapoptotic protein ratio. Our study reports STIM2 as a new actor regulating genomic stability and p53 response in terms of cell cycle and apoptosis of human normal and malignant monocytic cells.

Rescuing SLAMF3 Expression Restores Sorafenib Response in Hepatocellular Carcinoma Cells through the Induction of Mesenchymal-to-Epithelial Transition.

BACKGROUND: Acquired resistance to sorafenib in hepatocellular carcinoma (HCC) patients results in poor prognosis. Epithelial-to-mesenchymal transition (EMT) is the major mechanism implicated in the resistance to sorafenib. We have reported the tumor suppressor role of SLAMF3 (signaling lymphocytic activation molecules family 3) in HCC progression and highlighted its implication in controlling the MRP-1 transporter activity. These data suggest the implication of SLAMF3 in sorafenib resistance mechanisms. METHODS: We evaluated the resistance to sorafenib in Huh-7 cells treated with progressive doses (Res cells). We investigated the link between acquired resistance to sorafenib and SLAMF3 expression by flow cytometry and Western blot methods. Furthermore, we analyzed the EMT and the stem cell potential of cells resistant to sorafenib. RESULTS: Sorafenib resistance was confirmed in Res cells by analyzing the cell viability in the presence of sorafenib. The mesenchymal transition, in Res cells, was confirmed by high migratory index and the expression of EMT antigens. Interestingly, we found that loss of SLAMF3 expression corresponded to sorafenib-resistant phenotypes. The overexpression of SLAMF3 reversed EMT, decreased metastatic potential and inhibited mTOR/ERK1/2 in Res cells. CONCLUSIONS: We propose that rescuing SLAMF3 expression in resistant cells could represent a potential therapeutic strategy to enhance sorafenib efficacy in HCC patients.

An Alginate-Based Hydrogel with a High Angiogenic Capacity and a High Osteogenic Potential.

In bone tissue engineering, autologous cells are combined with osteoconductive scaffolds and implanted into bone defects. The major challenge is the lack of post-implantation vascular growth into biomaterial. The objective of the present study was to develop a new alginate-based hydrogel that enhances the regeneration of bone defects after surgery. The viability of human bone marrow-derived mesenchymal stem cells (BM-MSCs) or human endothelial cells (ECs) cultured alone or together on the hydrogel was analyzed for 24 and 96 h. After seeding, the cells self-assembled and aggregated to form clusters. For functional validation, empty or cellularized hydrogel matrices were implanted ectopically at subcutaneous sites in nude mice. After 2 months, the matrices were explanted. Transplanted human cells were present, and we observed vessels expressing human von Willebrand factor (resulting from the incorporation of transplanted ECs into neovessels and/or the differentiation of BM-MSCs into ECs). The addition of BM-MSCs improved host vascularization and neovessel formation from human cells, relative to ECs alone. Although we did not observe bone formation, the transplanted BM-MSCs were able to differentiate into osteoblasts. This new biomaterial provided an appropriate three-dimensional environment for transplanted cells and has a high angiogenic capacity and an osteogenic potential.

The combination of a poly-caprolactone/nano-hydroxyapatite honeycomb scaffold and mesenchymal stem cells promotes bone regeneration in rat calvarial defects.

Bone tissue engineering goes beyond the limitations of conventional methods of treating bone loss, such as autograft-induced morbidity and a lack of integration for large grafts. Novel biomimicry approaches (using three-dimensional [3D] electrospinning and printing techniques) have been designed to offer the most appropriate environment for cells and thus promote bone regeneration. In the present study, we assessed the bone regeneration properties of a composite 3D honeycomb structure from the electrostatic template-assisted deposition process by an alternate deposition of electrospun polycaprolactone (PCL) nanofibers and electrosprayed hydroxyapatite nanoparticles (nHA) on a honeycomb micropatterned substrate. We first confirmed the cytocompatibility of this honeycomb PCL-nHA scaffold in culture with bone marrow-derived mesenchymal stem cells (BM-MSCs). The scaffold was then implanted (alone or with seeded MSCs) for 2 months in a rat critical-sized calvarial defect model. The observation of new bone synthesis in situ (monitored using microcomputed tomography every 2 weeks and a histological assessment upon extraction) demonstrated that the honeycomb PCL-nHA scaffold was osteoconductive. Moreover, the combination of the scaffold with BM-MSCs was associated with significantly greater bone volume and mineralized regeneration during the 2-month experiment. The combination of the biomimetic honeycomb PCL-nHA scaffold with patient mesenchymal stem cells might therefore have great potential for clinical applications and specifically in maxillofacial surgery.