Analysis of off-tumour toxicities of T-cell-engaging bispecific antibodies via donor-matched intestinal organoids and tumouroids.

Predicting the toxicity of cancer immunotherapies preclinically is challenging because models of tumours and healthy organs do not typically fully recapitulate the expression of relevant human antigens. Here we show that patient-derived intestinal organoids and tumouroids supplemented with immune cells can be used to study the on-target off-tumour toxicities of T-cell-engaging bispecific antibodies (TCBs), and to capture clinical toxicities not predicted by conventional tissue-based models as well as inter-patient variabilities in TCB responses. We analysed the mechanisms of T-cell-mediated damage of neoplastic and donor-matched healthy epithelia at a single-cell resolution using multiplexed immunofluorescence. We found that TCBs that target the epithelial cell-adhesion molecule led to apoptosis in healthy organoids in accordance with clinical observations, and that apoptosis is associated with T-cell activation, cytokine release and intra-epithelial T-cell infiltration. Conversely, tumour organoids were more resistant to damage, probably owing to a reduced efficiency of T-cell infiltration within the epithelium. Patient-derived intestinal organoids can aid the study of immune-epithelial interactions as well as the preclinical and clinical development of cancer immunotherapies.

Corrigendum: VHL-Mediated Regulation of CHCHD4 and Mitochondrial Function.

[This corrects the article DOI: 10.3389/fonc.2018.00388.].

Genome-wide CRISPR/Cas9 deletion screen defines mitochondrial gene essentiality and identifies routes for tumour cell viability in hypoxia.

Mitochondria are typically essential for the viability of eukaryotic cells, and utilize oxygen and nutrients (e.g. glucose) to perform key metabolic functions that maintain energetic homeostasis and support proliferation. Here we provide a comprehensive functional annotation of mitochondrial genes that are essential for the viability of a large panel (625) of tumour cell lines. We perform genome-wide CRISPR/Cas9 deletion screening in normoxia-glucose, hypoxia-glucose and normoxia-galactose conditions, and identify both unique and overlapping genes whose loss influences tumour cell viability under these different metabolic conditions. We discover that loss of certain oxidative phosphorylation (OXPHOS) genes (e.g. SDHC) improves tumour cell growth in hypoxia-glucose, but reduces growth in normoxia, indicating a metabolic switch in OXPHOS gene function. Moreover, compared to normoxia-glucose, loss of genes involved in energy-consuming processes that are energetically demanding, such as translation and actin polymerization, improve cell viability under both hypoxia-glucose and normoxia-galactose. Collectively, our study defines mitochondrial gene essentiality in tumour cells, highlighting that essentiality is dependent on the metabolic environment, and identifies routes for regulating tumour cell viability in hypoxia.

The Tumor Profiler Study: integrated, multi-omic, functional tumor profiling for clinical decision support.

The application and integration of molecular profiling technologies create novel opportunities for personalized medicine. Here, we introduce the Tumor Profiler Study, an observational trial combining a prospective diagnostic approach to assess the relevance of in-depth tumor profiling to support clinical decision-making with an exploratory approach to improve the biological understanding of the disease.

CHCHD4 regulates tumour proliferation and EMT-related phenotypes, through respiratory chain-mediated metabolism.

BACKGROUND: Mitochondrial oxidative phosphorylation (OXPHOS) via the respiratory chain is required for the maintenance of tumour cell proliferation and regulation of epithelial to mesenchymal transition (EMT)-related phenotypes through mechanisms that are not fully understood. The essential mitochondrial import protein coiled-coil helix coiled-coil helix domain-containing protein 4 (CHCHD4) controls respiratory chain complex activity and oxygen consumption, and regulates the growth of tumours in vivo. In this study, we interrogate the importance of CHCHD4-regulated mitochondrial metabolism for tumour cell proliferation and EMT-related phenotypes, and elucidate key pathways involved. RESULTS: Using in silico analyses of 967 tumour cell lines, and tumours from different cancer patient cohorts, we show that CHCHD4 expression positively correlates with OXPHOS and proliferative pathways including the mTORC1 signalling pathway. We show that CHCHD4 expression significantly correlates with the doubling time of a range of tumour cell lines, and that CHCHD4-mediated tumour cell growth and mTORC1 signalling is coupled to respiratory chain complex I (CI) activity. Using global metabolomics analysis, we show that CHCHD4 regulates amino acid metabolism, and that CHCHD4-mediated tumour cell growth is dependent on glutamine. We show that CHCHD4-mediated tumour cell growth is linked to CI-regulated mTORC1 signalling and amino acid metabolism. Finally, we show that CHCHD4 expression in tumours is inversely correlated with EMT-related gene expression, and that increased CHCHD4 expression in tumour cells modulates EMT-related phenotypes. CONCLUSIONS: CHCHD4 drives tumour cell growth and activates mTORC1 signalling through its control of respiratory chain mediated metabolism and complex I biology, and also regulates EMT-related phenotypes of tumour cells.

CHCHD4 confers metabolic vulnerabilities to tumour cells through its control of the mitochondrial respiratory chain.

Background: Tumour cells rely on glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) to survive. Thus, mitochondrial OXPHOS has become an increasingly attractive area for therapeutic exploitation in cancer. However, mitochondria are required for intracellular oxygenation and normal physiological processes, and it remains unclear which mitochondrial molecular mechanisms might provide therapeutic benefit. Previously, we discovered that coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4) is critical for regulating intracellular oxygenation and required for the cellular response to hypoxia (low oxygenation) in tumour cells through molecular mechanisms that we do not yet fully understand. Overexpression of CHCHD4 in human cancers correlates with increased tumour progression and poor patient survival. Results: Here, we show that elevated CHCHD4 expression provides a proliferative and metabolic advantage to tumour cells in normoxia and hypoxia. Using stable isotope labelling with amino acids in cell culture (SILAC) and analysis of the whole mitochondrial proteome, we show that CHCHD4 dynamically affects the expression of a broad range of mitochondrial respiratory chain subunits from complex I-V, including multiple subunits of complex I (CI) required for complex assembly that are essential for cell survival. We found that loss of CHCHD4 protects tumour cells from respiratory chain inhibition at CI, while elevated CHCHD4 expression in tumour cells leads to significantly increased sensitivity to CI inhibition, in part through the production of mitochondrial reactive oxygen species (ROS). Conclusions: Our study highlights an important role for CHCHD4 in regulating tumour cell metabolism and reveals that CHCHD4 confers metabolic vulnerabilities to tumour cells through its control of the mitochondrial respiratory chain and CI biology.

VHL-Mediated Regulation of CHCHD4 and Mitochondrial Function.

Dysregulated mitochondrial function is associated with the pathology of a wide range of diseases including renal disease and cancer. Thus, investigating regulators of mitochondrial function is of particular interest. Previous work has shown that the von Hippel-Lindau tumor suppressor protein (pVHL) regulates mitochondrial biogenesis and respiratory chain function. pVHL is best known as an E3-ubiquitin ligase for the α-subunit of the hypoxia inducible factor (HIF) family of dimeric transcription factors. In normoxia, pVHL recognizes and binds hydroxylated HIF-α (HIF-1α and HIF-2α), targeting it for ubiquitination and proteasomal degradation. In this way, HIF transcriptional activity is tightly controlled at the level of HIF-α protein stability. At least 80% of clear cell renal carcinomas exhibit inactivation of the VHL gene, which leads to HIF-α protein stabilization and constitutive HIF activation. Constitutive HIF activation in renal carcinoma drives tumor progression and metastasis. Reconstitution of wild-type VHL protein (pVHL) in pVHL-defective renal carcinoma cells not only suppresses HIF activation and tumor growth, but also enhances mitochondrial respiratory chain function via mechanisms that are not fully elucidated. Here, we show that pVHL regulates mitochondrial function when re-expressed in pVHL-defective 786O and RCC10 renal carcinoma cells distinct from its regulation of HIF-α. Expression of CHCHD4, a key component of the disulphide relay system (DRS) involved in mitochondrial protein import within the intermembrane space (IMS) was elevated by pVHL re-expression alongside enhanced expression of respiratory chain subunits of complex I (NDUFB10) and complex IV (mtCO-2 and COX IV). These changes correlated with increased oxygen consumption rate (OCR) and dynamic changes in glucose and glutamine metabolism. Knockdown of HIF-2α also led to increased OCR, and elevated expression of CHCHD4, NDUFB10, and COXIV in 786O cells. Expression of pVHL mutant proteins (R200W, N78S, D126N, and S183L) that constitutively stabilize HIF-α but differentially promote glycolytic metabolism, were also found to differentially promote the pVHL-mediated mitochondrial phenotype. Parallel changes in mitochondrial morphology and the mitochondrial network were observed. Our study reveals a new role for pVHL in regulating CHCHD4 and mitochondrial function in renal carcinoma cells.

How Phosphorylation and ATPase Activity Regulate Anion Flux though the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).

The cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), mutations of which cause cystic fibrosis, belongs to the ATP-binding cassette (ABC) transporter family and works as a channel for small anions, such as chloride and bicarbonate. Anion channel activity is known to depend on phosphorylation by cAMP-dependent protein kinase A (PKA) and CFTR-ATPase activity. Whereas anion channel activity has been extensively investigated, phosphorylation and CFTR-ATPase activity are still poorly understood. Here, we show that the two processes can be measured in a label-free and non-invasive manner in real time in live cells, stably transfected with CFTR. This study reveals three key findings. (i) The major contribution (≥90%) to the total CFTR-related ATP hydrolysis rate is due to phosphorylation by PKA and the minor contribution (≤10%) to CFTR-ATPase activity. (ii) The mutant CFTR-E1371S that is still conductive, but defective in ATP hydrolysis, is not phosphorylated, suggesting that phosphorylation requires a functional nucleotide binding domain and occurs in the post-hydrolysis transition state. (iii) CFTR-ATPase activity is inversely related to CFTR anion flux. The present data are consistent with a model in which CFTR is in a closed conformation with two ATPs bound. The open conformation is induced by ATP hydrolysis and corresponds to the post-hydrolysis transition state that is stabilized by phosphorylation and binding of chloride channel potentiators.

Lipid bilayer domain fluctuations as a probe of membrane viscosity.

We argue that membrane viscosity, η(m), plays a prominent role in the thermal fluctuation dynamics of micron-scale lipid domains. A theoretical expression is presented for the timescales of domain shape relaxation, which reduces to the well-known η(m) = 0 result of Stone and McConnell in the limit of large domain sizes. Experimental measurements of domain dynamics on the surface of ternary phospholipid and cholesterol vesicles confirm the theoretical results and suggest domain flicker spectroscopy as a convenient means to simultaneously measure both the line tension, σ, and the membrane viscosity, η(m), governing the behavior of individual lipid domains.

Dynamic sorting of lipids and proteins in membrane tubes with a moving phase boundary.

Cellular organelle membranes maintain their integrity, global shape, and composition despite vigorous exchange among compartments of lipids and proteins during trafficking and signaling. Organelle homeostasis involves dynamic molecular sorting mechanisms that are far from being understood. In contrast, equilibrium thermodynamics of membrane mixing and sorting, particularly the phase behavior of binary and ternary model membrane mixtures and its coupling to membrane mechanics, is relatively well characterized. Elucidating the continuous turnover of live cell membranes, however, calls for experimental and theoretical membrane models enabling manipulation and investigation of directional mass transport. Here we introduce the phenomenon of curvature-induced domain nucleation and growth in membrane mixtures with fluid phase coexistence. Membrane domains were consistently observed to nucleate precisely at the junction between a strongly curved cylindrical (tube) membrane and a pipette-aspirated giant unilamellar vesicle. This experimental geometry mimics intracellular sorting compartments, because they often show tubular-vesicular membrane regions. Nucleated domains at tube necks were observed to present diffusion barriers to the transport of lipids and proteins. We find that curvature-nucleated domains grow with characteristic parabolic time dependence that is strongly curvature-dependent. We derive an analytical model that reflects the observed growth dynamics. Numerically calculated membrane shapes furthermore allow us to elucidate mechanical details underlying curvature-dependent directed lipid transport. Our observations suggest a novel dynamic membrane sorting principle that may contribute to intracellular protein and lipid sorting and trafficking.

Membrane charge dependent states of the beta-amyloid fragment Abeta (16-35) with differently charged micelle aggregates.

Abeta (16-35) is the hydrophobic central core of beta-amyloid peptide, the main component of plaques found in the brain tissue of Alzheimer's disease patients. Depending on the conditions present, beta-amyloid peptides undergo a conformational transition from random coil or alpha-helical monomers, to highly toxic beta-sheet oligomers and aggregate fibrils. The behavior of beta-amyloid peptide at plasma membrane level has been extensively investigated, and membrane charge has been proved to be a key factor modulating its conformational properties. In the present work we probed the conformational behavior of Abeta (16-35) in response to negative charge modifications of the micelle surface. CD and NMR conformational analyses were performed in negatively charged pure SDS micelles and in zwitterionic DPC micelles "doped" with small amounts of SDS. To analyze the tendency of Abeta (16-35) to interact with these micellar systems, we performed EPR experiments on three spin-labeled analogues of Abeta (16-35), bearing the methyl 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl) methanethiolsulfonate spin label at the N-terminus, in the middle of the sequence and at the C-terminus, respectively. Our conformational data show that, by varying the negative charge of the membrane, Abeta (16-35) undergoes a conformational transition from a soluble helical-kink-helical structure, to a U-turn shaped conformation that resembles protofibril models.

Bending stiffness depends on curvature of ternary lipid mixture tubular membranes.

Lipid and protein sorting and trafficking in intracellular pathways maintain cellular function and contribute to organelle homeostasis. Biophysical aspects of membrane shape coupled to sorting have recently received increasing attention. Here we determine membrane tube bending stiffness through measurements of tube radii, and demonstrate that the stiffness of ternary lipid mixtures depends on membrane curvature for a large range of lipid compositions. This observation indicates amplification by curvature of cooperative lipid demixing. We show that curvature-induced demixing increases upon approaching the critical region of a ternary lipid mixture, with qualitative differences along two roughly orthogonal compositional trajectories. Adapting a thermodynamic theory earlier developed by M. Kozlov, we derive an expression that shows the renormalized bending stiffness of an amphiphile mixture membrane tube in contact with a flat reservoir to be a quadratic function of curvature. In this analytical model, the degree of sorting is determined by the ratio of two thermodynamic derivatives. These derivatives are individually interpreted as a driving force and a resistance to curvature sorting. We experimentally show this ratio to vary with composition, and compare the model to sorting by spontaneous curvature. Our results are likely to be relevant to the molecular sorting of membrane components in vivo.

Structures and micelle locations of the nonlipidated and lipidated C-terminal membrane anchor of 2',3'-cyclic nucleotide-3'-phosphodiesterase.

2,3'-Cyclic nucleotide-3'-phosphodiesterase (CNP) is a myelin-associated protein, an enzyme abundantly present in the central nervous system of mammals and some vertebrates. In vitro, CNP specifically catalyzes the hydrolysis of 2',3'-cyclic nucleotides to produce 2'-nucleotides, but the physiologically relevant in vivo substrate is still unknown. Recently, it was found that CNP is a possible linker protein between microtubules and the plasma membranes. Since CNP is modified post-translationally by an isoprenylation process at its C terminus, the prenylation is hypothesized to be a requisite process, which permanently anchors CNP to the plasma membrane. This study investigates the molecular mechanism of the interaction between CNP and the plasma membrane, proposing a general model to interpret the structural bases of prenylated proteins binding to the membrane. A 13 residue, C-terminal CNP fragment, C13, was demonstrated to be directly responsible for CNP membrane anchoring. C13 and its lipidated derivative (LIPO-C13) were subjected to conformational analysis in membrane mimetic environments, by means of CD and NMR spectroscopies. The orientation of C13 in relation to the membrane was investigated by NMR and EPR spin labeling studies. Our structural investigation shows that the presence of the lipidic tail is essential for the peptide to be folded and correctly positioned on the membrane surface. A general model is proposed in which the post-translational lipidation is an important biomolecular trick to enlarge the hydrophobic surface and to enable the contact of the protein with membrane.

Obestatin conformational features: a strategy to unveil obestatin's biological role?

Obestatin and its derivative Ob(11-23) are recently discovered peptides produced in the rat stomach. They have proven to be involved in the regulation of energy balance, inhibiting feeding, causing reductions in food intake, body weight and jejunal contraction in rodents. The G-protein coupled receptor, GPR39, was originally proposed as being an obestatin target receptor, but this remains controversial. As such, the molecular mechanism for obestatin's effects in vivo is still uncertain. Here we report the CD and NMR conformational analysis of obestatin and Ob(11-23). Both peptides assume a regular secondary structure in the C-terminal region of the molecule. In this region, structural elements similar to other GPCR binding neuropeptides support the identity of obestatin as a new and functionally autonomous GPCR ligand. Conversely sequence and conformational specificity point to a new farmacoforic structure, on which innovative derivatives with a potential role in the treatment of obesity can be designed and synthetized.

Flicker spectroscopy of thermal lipid bilayer domain boundary fluctuations.

This contribution describes measurements of lipid bilayer domain line tension based on two-dimensional thermal undulations of membranes with liquid ordered/liquid disordered phase coexistence and near-critical composition at room temperature. Lateral inhomogeneity of lipid and protein composition is currently a subject of avid research aimed at determining both fundamental properties and biological relevance of membrane domains. Line tension at fluid lipid bilayer membrane domain boundaries controls the kinetics of domain growth and therefore regulates the size of compositional heterogeneities. High line tension promotes membrane domain budding and fission. Line tension could therefore be an important control parameter regulating functional aspects of biological membranes. Here the established method of fluid domain flicker spectroscopy is applied to examine thermal domain wall fluctuations of phase-separated bilayer membranes. We find a Gaussian probability distribution for the first few excited mode amplitudes, which permits an analysis by means of appropriately specialized capillary wave theory. Time autocorrelation functions are found to decay exponentially, and relaxation times are fitted by means of a hydrodynamic theory relating line tensions and excited mode relaxation kinetics. Line tensions below 1 pN are obtained, with these two approaches yielding similar results. We examine experimental artifacts that perturb the Fourier spectrum of domain traces and discuss ways to identify the number of modes that yield reliable line tension information.

Antibodies generated in cats by a lipopeptide reproducing the membrane-proximal external region of the feline immunodeficiency virus transmembrane enhance virus infectivity.

The immunogenicity of a lipoylated peptide (lipo-P59) reproducing the membrane-proximal external region (MPER) of the transmembrane glycoprotein of feline immunodeficiency virus (FIV) was investigated with cats. In the attempt to mimic the context in which MPER is located within intact virions, lipo-P59 was administered in association with membrane-like micelles. Analyses showed that in this milieu, lipo-P59 had a remarkable propensity to be positioned at the membrane interface, displayed a large number of ordered structures folded in turn helices, and was as active as lipo-P59 alone at inhibiting FIV infectivity in vitro. The antibodies developed differed from the ones previously obtained by immunizing cats with the nonlipoylated version of the peptide (G. Freer, S. Giannecchini, A. Tissot, M. F. Bachmann, P. Rovero, P. F. Serres, and M. Bendinelli, Virology 322:360-369, 2004) in epitope specificity and in the fact that they bound FIV virions. However, they too lacked virus-neutralizing activity and actually enhanced FIV infectivity for lymphoid cell cultures. It is concluded that the use of MPER-reproducing oligopeptides is not a viable approach for vaccinating against FIV.

Driving forces in the delivery of penetratin conjugated G protein fragment.

A42 is a chimera peptide consisting of Galphas(374-394)C379A--the 21-mer C terminus of the Galphas protein, able of adenosine inhibitory activity--and penetratin--the 16 residue fragment, derived from the homeodomain of the Drosophila transcription factor Antennapedia. A42 is able to cross cell membranes and to inhibit A2A and A2B adenosine and beta-adrenergic receptor stimulated camps (D'Ursi et al. Mol. Pharmacol. 2006, 69, 727-36). Here we present an extensive biophysical study of A42 in different membrane mimetics, with the objective to evaluate the molecular mechanisms which promote the membrane permeation. Fluorescence, CD, and NMR data were acquired in the presence of negatively charged and zwitterionic sodium dodecyl sulfate and dodecylphosphocholine surfactants. To validate the spectroscopic results in a larger scale, fluorescence microscopy experiments were performed on negatively charged and zwitterionic dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylcholine vesicles. Our results show that the internalization of A42 is mainly driven by electrostatic interactions, hydrophobic interactions playing only a secondary, sinergistic role. The distribution of the charges along the molecule has an important role, highlighting that internalization is a process which requires a specific matching of peptide and membrane properties.