Multiomic analysis of malignant pleural mesothelioma identifies molecular axes and specialized tumor profiles driving intertumor heterogeneity.

Malignant pleural mesothelioma (MPM) is an aggressive cancer with rising incidence and challenging clinical management. Through a large series of whole-genome sequencing data, integrated with transcriptomic and epigenomic data using multiomics factor analysis, we demonstrate that the current World Health Organization classification only accounts for up to 10% of interpatient molecular differences. Instead, the MESOMICS project paves the way for a morphomolecular classification of MPM based on four dimensions: ploidy, tumor cell morphology, adaptive immune response and CpG island methylator profile. We show that these four dimensions are complementary, capture major interpatient molecular differences and are delimited by extreme phenotypes that-in the case of the interdependent tumor cell morphology and adapted immune response-reflect tumor specialization. These findings unearth the interplay between MPM functional biology and its genomic history, and provide insights into the variations observed in the clinical behavior of patients with MPM.

Redefining malignant pleural mesothelioma types as a continuum uncovers immune-vascular interactions.

BACKGROUND: Malignant Pleural Mesothelioma (MPM) is an aggressive disease related to asbestos exposure, with no effective therapeutic options. METHODS: We undertook unsupervised analyses of RNA-sequencing data of 284 MPMs, with no assumption of discreteness. Using immunohistochemistry, we performed an orthogonal validation on a subset of 103 samples and a biological replication in an independent series of 77 samples. FINDINGS: A continuum of molecular profiles explained the prognosis of the disease better than any discrete model. The immune and vascular pathways were the major sources of molecular variation, with strong differences in the expression of immune checkpoints and pro-angiogenic genes; the extrema of this continuum had specific molecular profiles: a "hot" bad-prognosis profile, with high lymphocyte infiltration and high expression of immune checkpoints and pro-angiogenic genes; a "cold" bad-prognosis profile, with low lymphocyte infiltration and high expression of pro-angiogenic genes; and a "VEGFR2+/VISTA+" better-prognosis profile, with high expression of immune checkpoint VISTA and pro-angiogenic gene VEGFR2. We validated the gene expression levels at the protein level for a subset of five selected genes belonging to the immune and vascular pathways (CD8A, PDL1, VEGFR3, VEGFR2, and VISTA), in the validation series, and replicated the molecular profiles as well as their prognostic value in the replication series. INTERPRETATION: The prognosis of MPM is best explained by a continuous model, which extremes show specific expression patterns of genes involved in angiogenesis and immune response.

MsCYS1, a developmentally-regulated cystatin from alfalfa.

Several roles have been attributed to cystatins in plants, ranging from the regulation of host [endogenous] cysteine proteases to the inhibition of herbivorous pest [exogenous] proteases. We report here the cloning, expression and functional characterization of a novel cystatin from alfalfa, Medicago sativa L. The new sequence, isolated from a cDNA expression library prepared from young leaves, encodes a protein, MsCYS1, with the typical inhibitory motifs of cystatins, namely the central signature motif QxVxG, a GG doublet in the N-terminal trunk, and a W residue in the C-terminal region, about 30 amino acids distant from the central inhibitory motif. As shown by a protein-based phylogenetic reconstruction, MsCYS1 is a close relative of other cystatins from Fabaceae presumably involved in the regulation of endogenous proteases. This cystatin is developmentally regulated in stems and leaves, and not induced by stress signals including methyl jasmonate, known to activate cystatins involved in plant defense. A recombinant form of MsCYS1 expressed in Escherichia coli was shown to strongly inhibit alfalfa leaf cysteine proteases while showing weak affinity for the digestive cysteine proteases of different herbivorous pests. Overall, these observations suggest an endogenous protease regulatory role for MsCYS1, possibly associated with the early development of stems and leaves.

A multicomponent, elicitor-inducible cystatin complex in tomato, Solanum lycopersicum.

We assessed the ability of the fungal elicitor arachidonic acid to induce cystatin genes in tomato (Solanum lycopersicum), using a cDNA expression library from arachidonate-treated leaves. The cDNAs of two novel cystatins were isolated, coding for an approx. 11-kDa protein, SlCYS10; and for a 23.6-kDa protein, SlCYS9, bearing an N-terminal signal peptide and a long, 11.5-kDa extension at the C terminus. Both genes were induced by arachidonate but not by methyl jasmonate, an inducer of the 88-kDa eight-unit cystatin, multicystatin, accumulated in the cytosol of leaf cells upon herbivory. A truncated form of SlCYS9, tSlCYS9, was produced by deletion of the C-terminal extension to assess the influence of this structural element on the cystatin moiety. As shown by kinetic and stability assays with recombinant variants expressed in Escherichia coli, deleting the extension influenced both the overall stability and inhibitory potency of SlCYS9 against cysteine proteases of herbivorous organisms. These findings provide evidence for a multicomponent elicitor-inducible cystatin complex in tomato, including at least 10 cystatin units produced via two metabolic routes.

A hybrid, broad-spectrum inhibitor of Colorado potato beetle aspartate and cysteine digestive proteinases.

Protein engineering approaches are currently being devised to improve the inhibitory properties of plant proteinase inhibitors against digestive proteinases of herbivorous insects. Here we engineered a potent hybrid inhibitor of aspartate and cysteine digestive proteinases found in the Colorado potato beetle, Leptinotarsa decemlineata Say. Three cathepsin D inhibitors (CDIs) from stressed potato and tomato were first compared in their potency to inhibit digestive cathepsin D-like activity of the insect. After showing the high inhibitory potency of tomato CDI (M(r) approximately 21 kDa), an approximately 33-kDa hybrid inhibitor was generated by fusing this inhibitor to the N terminus of corn cystatin II (CCII), a potent inhibitor of cysteine proteinases. Inhibitory assays with recombinant forms of CDI, CCII, and CDI-CCII expressed in Escherichia coli showed the CDI-CCII fusion to exhibit a dual inhibitory effect against cystatin-sensitive and cathepsin D-like enzymes of the potato beetle, resulting in detrimental effects against 3rd-instar larvae fed the hybrid inhibitor. The inhibitory potency of CDI and CCII was not altered after their fusion, as suggested by IC(50) values for the interaction of CDI-CCII with target proteinases similar to those measured for each inhibitor. These observations suggest the potential of plant CDIs and cystatins as functional inhibitory modules for the design of effective broad-spectrum, hybrid inhibitors of herbivorous insect cysteine and aspartate digestive proteinases.