APC/PIK3CA mutations and ß-catenin status predict tankyrase inhibitor sensitivity of patient-derived colorectal cancer cells.

BACKGROUND: Aberrant WNT/ß-catenin signaling drives carcinogenesis. Tankyrases poly(ADP-ribosyl)ate and destabilize AXINs, ß-catenin repressors. Tankyrase inhibitors block WNT/ß-catenin signaling and colorectal cancer (CRC) growth. We previously reported that 'short' APC mutations, lacking all seven ß-catenin-binding 20-amino acid repeats (20-AARs), are potential predictive biomarkers for CRC cell sensitivity to tankyrase inhibitors. Meanwhile, 'Long' APC mutations, which possess more than one 20-AAR, do not predict inhibitor-resistant cells. Thus, additional biomarkers are needed to precisely predict the inhibitor sensitivity. METHODS: Using 47 CRC patient-derived cells (PDCs), we examined correlations between the sensitivity to tankyrase inhibitors (G007-LK and RK-582), driver mutations, and the expressions of signaling factors. NOD.CB17-Prkdcscid/J and BALB/c-nu/nu xenograft mice were treated with RK-582. RESULTS: Short APC mutant CRC cells exhibited high/intermediate sensitivities to tankyrase inhibitors in vitro and in vivo. Active ß-catenin levels correlated with inhibitor sensitivity in both short and long APC mutant PDCs. PIK3CA mutations, but not KRAS/BRAF mutations, were more frequent in inhibitor-resistant PDCs. Some wild-type APC PDCs showed inhibitor sensitivity in a ß-catenin-independent manner. CONCLUSIONS: APC/PIK3CA mutations and ß-catenin predict the sensitivity of APC-mutated CRC PDCs to tankyrase inhibitors. These observations may help inform the strategy of patient selection in future clinical trials of tankyrase inhibitors.

Skeleton phylogeny reconstructed with transcriptomes for the tribe Drosophilini (Diptera: Drosophilidae).

The family Drosophilidae is one of the most important model systems in evolutionary biology. Thanks to advances in high-throughput sequencing technology, a number of molecular phylogenetic analyses have been undertaken by using large data sets of many genes and many species sampled across this family. Especially, recent analyses using genome sequences have depicted the family-wide skeleton phylogeny with high confidence. However, the taxon sampling is still insufficient for minor lineages and non-Drosophila genera. In this study, we carried out phylogenetic analyses using a large number of transcriptome-based nucleotide sequences, focusing on the largest, core tribe Drosophilini in the Drosophilidae. In our analyses, some noise factors against phylogenetic reconstruction were taken into account by removing putative paralogy from the datasets and examining the effects of missing data, i.e. gene occupancy and site coverage, and incomplete lineage sorting. The inferred phylogeny has newly resolved the following phylogenetic positions/relationships at the genomic scale: (i) the monophyly of the subgenus Siphlodora including Zaprionus flavofasciatus to be transferred therein; (ii) the paraphyly of the robusta and melanica species groups within a clade comprised of the robusta, melanica and quadrisetata groups and Z. flavofasciatus; (iii) Drosophila curviceps (representing the curviceps group), D. annulipes (the quadrilineata subgroup of the immigrans group) and D. maculinotata clustered into a clade sister to the Idiomyia + Scaptomyza clade, forming together the expanded Hawaiian drosophilid lineage; (iv) Dichaetophora tenuicauda (representing the lineage comprised of the Zygothrica genus group and Dichaetophora) placed as the sister to the clade of the expanded Hawaiian drosophilid lineage and Siphlodora; and (v) relationships of the subgenus Drosophila and the genus Zaprionus as follows: (Zaprionus, (the quadrilineata subgroup, ((D. sternopleuralis, the immigrans group proper), (the quinaria radiation, the tripunctata radiation)))). These results are to be incorporated into the so-far published phylogenomic tree as a backbone (constraint) tree for grafting much more species based on sequences of a limited number of genes. Such a comprehensive, highly confident phylogenetic tree with extensive and dense taxon sampling will provide an essential framework for comparative studies of the Drosophilidae.

Fusarium mindanaoense sp. nov., a New Fusarium Wilt Pathogen of Cavendish Banana from the Philippines Belonging to the F. fujikuroi Species Complex.

The pathogen causing Fusarium wilt in banana is reported to be Fusarium oxysporum f. sp. cubense (FOC). In 2019, wilt symptoms in banana plants (cultivar: Cavendish) in the Philippines were detected, i.e., the yellowing of the leaves and discoloration of the pseudostem and vascular tissue. The fungus isolated from the vascular tissue was found to be pathogenic to Cavendish bananas and was identified as a new species, F. mindanaoense, belonging to the F. fujikuroi species complex (FFSC); species classification was assessed using molecular phylogenetic analyses based on the tef1, tub2, cmdA, rpb1, and rpb2 genes and morphological analyses. A reciprocal blast search using genomic data revealed that this fungus exclusively included the Secreted in Xylem 6 (SIX6) gene among the SIX homologs related to pathogenicity; it exhibited a highly conserved amino acid sequence compared with that of species in the FFSC, but not with that of FOC. This was the first report of Fusarium wilt in Cavendish bananas caused by a species of the genus Fusarium other than those in the F. oxysporum species complex.

Surface modification and adhesive-free adhesion of polytetrafluoroethylene (PTFE) and silicone gel containing oleophilic SiO2 powder by plasma treatment.

Polytetrafluoroethylene (PTFE) has high-frequency characteristics and low transmission loss, and is expected to be used as a substrate material of printed wiring board for high-frequency applications. Meanwhile, silicone gel has superior properties such as attaching/detaching, weather resistance, and human safety. If the PTFE and silicone gel can be strongly adhered to, they can be applied to internet of things (IoT) devices that can be attached and detached freely. However, adhesion between PTFE, which has poor adhesion, and silicone gel, which has low mechanical strength, is difficult and has not been reported. In this study, PTFE was modified with heat-assisted plasma treatment, and silicone gel was treated with oleophilic SiO2 powder to improve elastic modulus and modified with plasma jet treatment, and then bonded without adhesive. The adhesion strength of PTFE/silicone gel assembly was 1.13 N mm-1 when treated moderately, but only 0.01 N mm-1 when untreated and treated excessively. To investigate the factors causing the difference in the adhesion strength, the surface of silicone gel was evaluated by water contact angle measurement, Fourier transform infrared spectroscopy, and confocal laser scanning microscopy. When treated moderately, hydrophilic functional groups and cross-linking were most frequently increased. Furthermore, when treated excessively, surface degradation was observed, which was expected to lower the adhesion strength. The adhesive-free bonding between PTFE and silicone gel can open a new path for developing IoT devices that can be freely attached and detached.

Flexible selection of the functional-group ratio on a polytetrafluoroethylene (PTFE) surface using a single-gas plasma treatment.

During plasma treatment of polymers, etching occurs and functional groups are introduced on their surface. We assumed that controlling the etching rate would enable plasma treatment using a single gas to control the ratio of functional groups generated on a polymer's surface, although previous studies have indicated that several different types of functional groups are formed when the gaseous species are varied. In this study, we selected the base pressure (BP) as a parameter for controlling the etching rate and subjected polytetrafluoroethylene (PTFE) to plasma treatments using only He gas at various BPs. The chemical composition of the surface of the plasma-treated PTFE samples was evaluated by X-ray photoelectron spectroscopy (XPS), and the ratios of fluorine (CF3, CF2, C-F), oxygen (O-C[double bond, length as m-dash]O, C[double bond, length as m-dash]O, C-O), and carbon (C-C, C[double bond, length as m-dash]C) groups were quantified from the C 1s-XPS spectra. The fluorine-group ratio decreased and the oxygen- and carbon-group ratios increased with decreasing BP. The results demonstrated that plasma treatment using a single gas enabled flexible selection of the ratio of functional groups generated on PTFE via control of the BP.

Adhesive-Free Adhesion between Plasma-Treated Glass-Cloth-Containing Polytetrafluoroethylene (GC-PTFE) and Stainless Steel: Comparison between GC-PTFE and Pure PTFE.

In this study, the effect of plasma treatment on glass-cloth-containing polytetrafluoroethylene (GC-PTFE) was investigated. Previous plasma studies investigated pure PTFE (which does not contain glass cloth) but not GC-PTFE. The effect of Ar + H2O plasma treatment on GC-PTFE was investigated. The Ar + H2O plasma-treated GC-PTFE sheets were thermally compressed to stainless steel (SUS304) foils without using adhesive, and the GC-PTFE/SUS304 adhesion strengths were measured using a 90° peel test. The adhesion strength increased with the increase in the plasma treatment time (0.8 and 1.0 N/mm at 20 s and 300 s, respectively). Thus, strong adhesion between GC-PTFE/SUS304 was achieved without adhesive. This improvement in the adhesion properties of GC-PTFE can be attributed to the generation of oxygen-containing functional groups and the decrease in the surface roughness of the samples. Thereafter, the adhesion properties of GC-PTFE and pure PTFE were compared. Because, unlike pure PTFE, GC-PTFE has no weak boundary layer, GC-PTFE exhibited better adhesion properties than pure PTFE under short plasma treatment times.

Soluble PD-L1 works as a decoy in lung cancer immunotherapy via alternative polyadenylation.

Immune checkpoint therapy targeting the PD-1/PD-L1 axis is a potentially novel development in anticancer therapy and has been applied to clinical medicine. However, there are still some problems, including a relatively low response rate, innate mechanisms of resistance against immune checkpoint blockades, and the absence of reliable biomarkers to predict responsiveness. In this study of in vitro and in vivo models, we demonstrate that PD-L1-vInt4, a splicing variant of PD-L1, plays a role as a decoy in anti-PD-L1 antibody treatment. First, we showed that PD-L1-vInt4 was detectable in clinical samples and that it was possible to visualize the secreting variants with IHC. By overexpressing the PD-L1-secreted splicing variant on MC38 cells, we observed that an immune-suppressing effect was not induced by their secretion alone. We then demonstrated that PD-L1-vInt4 secretion resisted anti-PD-L1 antibody treatment, compared with WT PD-L1, which was explicable by the PD-L1-vInt4's decoying of the anti-PD-L1 antibody. The decoying function of PD-L1 splicing variants may be one of the reasons for cancers being resistant to anti-PD-L1 therapy. Measuring serum PD-L1 levels might be helpful in deciding the therapeutic strategy.

Effects of He and Ar Heat-Assisted Plasma Treatments on the Adhesion Properties of Polytetrafluoroethylene (PTFE).

Heat-assisted plasma (HAP) treatment using He gas is known to improve the adhesive-bonding and adhesive-free adhesion properties of polytetrafluoroethylene (PTFE). In this study, we investigated the effects of He and Ar gaseous species on the HAP-treated PTFE surface. Epoxy (EP) adhesive-coated stainless steel (SUS304) and isobutylene-isoprene rubber (IIR) were used as adherents for the evaluation of the adhesive-bonding and adhesive-free adhesion properties of PTFE. In the case of adhesive bonding, the PTFE/EP-adhesive/SUS304 adhesion strength of the Ar-HAP-treated PTFE was the same as that of the He-HAP-treated PTFE. In the case of adhesive-free adhesion, the PTFE/IIR adhesion strength of the Ar-HAP-treated PTFE was seven times lower than that of the He-HAP-treated PTFE. The relation among gaseous species used in HAP treatment, adhesion properties, peroxy radical density ratio, surface chemical composition, surface modification depth, surface morphology, surface hardness, and the effect of irradiation with vacuum ultraviolet (VUV) and UV photons were investigated. The different adhesive-free adhesion properties obtained by the two treatments resulted from the changes in surface chemical composition, especially the ratios of oxygen-containing functional groups and C-C crosslinks.

Platelet-derived lysophosphatidic acid mediated LPAR1 activation as a therapeutic target for osteosarcoma metastasis.

Osteosarcoma is the most common primary malignant bone cancer, with high rates of pulmonary metastasis. Osteosarcoma patients with pulmonary metastasis have worse prognosis than those with localized disease, leading to dramatically reduced survival rates. Therefore, understanding the biological characteristics of metastatic osteosarcoma and the molecular mechanisms of invasion and metastasis of osteosarcoma cells will lead to the development of innovative therapeutic intervention for advanced osteosarcoma. Here, we identified that osteosarcoma cells commonly exhibit high platelet activation-inducing characteristics, and molecules released from activated platelets promote the invasiveness of osteosarcoma cells. Given that heat-denatured platelet releasate maintained the ability to promote osteosarcoma invasion, we focused on heat-tolerant molecules, such as lipid mediators in the platelet releasate. Osteosarcoma-induced platelet activation leads to abundant lysophosphatidic acid (LPA) release. Exposure to LPA or platelet releasate induced morphological changes and increased invasiveness of osteosarcoma cells. By analyzing publicly available transcriptome datasets and our in-house osteosarcoma patient-derived xenograft tumors, we found that LPA receptor 1 (LPAR1) is notably upregulated in osteosarcoma. LPAR1 gene KO in osteosarcoma cells abolished the platelet-mediated osteosarcoma invasion in vitro and the formation of early pulmonary metastatic foci in experimental pulmonary metastasis models. Of note, the pharmacological inhibition of LPAR1 by the orally available LPAR1 antagonist, ONO-7300243, prevented pulmonary metastasis of osteosarcoma in the mouse models. These results indicate that the LPA-LPAR1 axis is essential for the osteosarcoma invasion and metastasis, and targeting LPAR1 would be a promising therapeutic intervention for advanced osteosarcoma.

Chromatin structure-dependent histone incorporation revealed by a genome-wide deposition assay.

In eukaryotes, histone variant distribution within the genome is the key epigenetic feature. To understand how each histone variant is targeted to the genome, we developed a new method, the RhIP (Reconstituted histone complex Incorporation into chromatin of Permeabilized cell) assay, in which epitope-tagged histone complexes are introduced into permeabilized cells and incorporated into their chromatin. Using this method, we found that H3.1 and H3.3 were incorporated into chromatin in replication-dependent and -independent manners, respectively. We further found that the incorporation of histones H2A and H2A.Z mainly occurred at less condensed chromatin (open), suggesting that condensed chromatin (closed) is a barrier for histone incorporation. To overcome this barrier, H2A, but not H2A.Z, uses a replication-coupled deposition mechanism. Our study revealed that the combination of chromatin structure and DNA replication dictates the differential histone deposition to maintain the epigenetic chromatin states.

Microsecond-timescale MD simulation of EGFR minor mutation predicts the structural flexibility of EGFR kinase core that reflects EGFR inhibitor sensitivity.

Approximately 15-30% of patients with lung cancer harbor mutations in the EGFR gene. Major EGFR mutations (>90% of EGFR-mutated lung cancer) are highly sensitive to EGFR tyrosine kinase inhibitors (TKIs). Many uncommon EGFR mutations have been identified, but little is known regarding their characteristics, activation, and sensitivity to various EGFR-TKIs, including allosteric inhibitors. We encountered a case harboring an EGFR-L747P mutation, originally misdiagnosed with EGFR-del19 mutation using a routine diagnostic EGFR mutation test, which was resistant to EGFR-TKI gefitinib. Using this minor mutation and common EGFR-activating mutations, we performed the binding free energy calculations and microsecond-timescale molecular dynamic (MD) simulations, revealing that the L747P mutation considerably stabilizes the active conformation through a salt-bridge formation between K745 and E762. We further revealed why several EGFR inhibitors, including the allosteric inhibitor, were ineffective. Our computational structural analysis strategy would be beneficial for future drug development targeting the EGFR minor mutations.

Overcoming resistance by ALK compound mutation (I1171S + G1269A) after sequential treatment of multiple ALK inhibitors in non-small cell lung cancer.

BACKGROUND: Anaplastic lymphoma kinase (ALK) fusion genes are found in 3%-5% of non-small cell lung cancers (NSCLCs). ALK inhibitors show a very high response rate to ALK-positive NSCLCs. However, the emergence of acquired resistance is inevitable. In this study, we investigated the drugs for overcoming resistance especially compound mutations after sequential treatment with crizotinib, alectinib, and lorlatinib. METHOD: Next-generation sequencing (NGS) and Sanger sequencing were performed on a liver biopsy tissue obtained from a clinical case. Ba/F3 cells in which mutant EML4-ALK were overexpressed were prepared, and cell viability assay and immunoblotting were performed to check the sensitivity of five independent ALK inhibitors. RESULTS: I1171S + G1269A double mutation was identified by NGS and Sanger sequencing on a liver biopsy tissue from a patient who relapsed on lorlatinib treatment. Ceritinib and brigatinib-but not other ALK inhibitors-were active against the compound mutations in the cell line model. CONCLUSIONS: With the sequential ALK inhibitors treatment, cancer cells accumulate new mutations in addition to mutations acquired previously. The identified compound mutation (I1171S + G1269A) was found to be sensitive to ceritinib and brigatinib, and indeed the patient's tumor partially responded to ceritinib. KEY POINTS: ALK compound mutation was found in a clinical sample that was resistant to lorlatinib after sequential ALK-tyrosine kinase inhibitor (TKI) treatment. Ceritinib and brigatinib are potential overcoming drugs against ALK I1171S + G1269A double mutation.

The new-generation selective ROS1/NTRK inhibitor DS-6051b overcomes crizotinib resistant ROS1-G2032R mutation in preclinical models.

ROS1 gene rearrangement was observed in around 1-2 % of NSCLC patients and in several other cancers such as cholangiocarcinoma, glioblastoma, or colorectal cancer. Crizotinib, an ALK/ROS1/MET inhibitor, is highly effective against ROS1-rearranged lung cancer and is used in clinic. However, crizotinib resistance is an emerging issue, and several resistance mechanisms, such as secondary kinase-domain mutations (e.g., ROS1-G2032R) have been identified in crizotinib-refractory patients. Here we characterize a new selective ROS1/NTRK inhibitor, DS-6051b, in preclinical models of ROS1- or NTRK-rearranged cancers. DS-6051b induces dramatic growth inhibition of both wild type and G2032R mutant ROS1-rearranged cancers or NTRK-rearranged cancers in vitro and in vivo. Here we report that DS-6051b is effective in treating ROS1- or NTRK-rearranged cancer in preclinical models, including crizotinib-resistant ROS1 positive cancer with secondary kinase domain mutations especially G2032R mutation which is highly resistant to crizotinib as well as lorlatinib and entrectinib, next generation ROS1 inhibitors.

Mini-review an insect-specific system for terrestrialization: Laccase-mediated cuticle formation.

Insects are often regarded as the most successful group of animals in the terrestrial environment. Their success can be represented by their huge biomass and large impact on ecosystems. Among the factors suggested to be responsible for their success, we focus on the possibility that the cuticle might have affected the process of insects' evolution. The cuticle of insects, like that of other arthropods, is composed mainly of chitin and structural cuticle proteins. However, insects seem to have evolved a specific system for cuticle formation. Oxidation reaction of catecholamines catalyzed by a copper enzyme, laccase, is the key step in the metabolic pathway for hardening of the insect cuticle. Molecular phylogenetic analysis indicates that laccase functioning in cuticle sclerotization has evolved only in insects. In this review, we discuss a theory on how the insect-specific "laccase" function has been advantageous for establishing their current ecological position as terrestrial animals.

The Wide Distribution and Change of Target Specificity of R2 Non-LTR Retrotransposons in Animals.

Transposons, or transposable elements, are the major components of genomes in most eukaryotes. Some groups of transposons have developed target specificity that limits the integration sites to a specific nonessential sequence or a genomic region to avoid gene disruption caused by insertion into an essential gene. R2 is one of the most intensively investigated groups of sequence-specific non-LTR retrotransposons and is inserted at a specific site inside of 28S ribosomal RNA (rRNA) genes. R2 is known to be distributed among at least six animal phyla even though its occurrence is reported to be patchy. Here, in order to obtain a more detailed picture of the distribution of R2, we surveyed R2 using both in silico screening and degenerate PCR, particularly focusing on actinopterygian fish. We found two families of the R2C lineage from vertebrates, although it has previously only been found in platyhelminthes. We also revealed the apparent movement of insertion sites of a lineage of actinopterygian R2, which was likely concurrent with the acquisition of a 28S rRNA-derived sequence in their 3' UTR. Outside of actinopterygian fish, we revealed the maintenance of a single R2 lineage in birds; the co-existence of four lineages of R2 in the leafcutter bee Megachile rotundata; the first examples of R2 in Ctenophora, Mollusca, and Hemichordata; and two families of R2 showing no target specificity. These findings indicate that R2 is relatively stable and universal, while differences in the distribution and maintenance of R2 lineages probably reflect characteristics of some combination of both R2 lineages and host organisms.

Re-examination of a α-chymotrypsin-solubilized laccase in the pupal cuticle of the silkworm, Bombyx mori: Insights into the regulation system for laccase activation during the ecdysis process.

The laccase in the pupal cuticle of the silkworm, Bombyx mori, is thought to accumulate as an inactive precursor that can be activated stage-dependently. In this study we isolated an 81-kDa laccase from cuticular extract of B. mori that was prepared by digestion of the pupal cuticles with α-chymotrypsin. The mass spectrometric analysis of the purified protein indicates that this 81-kDa laccase is a product of the Bombyx laccase2 gene. The purified 81-kDa laccase (α-chymotrypsin-solubilized Bombyx laccase2: Bm-clac2) has an N-terminal sequence of RNPADS that corresponds to Arg146 to Ser151 of the deduced protein sequence of Bmlaccase2 cDNA, indicating that Bm-clac2 lacks the N-terminal part upstream from residue Arg146. Bm-clac2 shows enzymatic activity, but its specific activity is increased around 17-fold after treatment with trypsin, which involves cleavage of peptide bonds at the C-terminal region. We also found that the activity of Bm-clac2 is increased in the presence of isopropanol. In previous reports, proteolytic processing has been hypothesized as a system for laccase activation in vivo, but the present result implies that this type of processing is not the only way to convert Bm-clac2 to the high-activity enzyme.

Extensive differences in antifungal immune response in two Drosophila species revealed by comparative transcriptome analysis.

The innate immune system of Drosophila is activated by ingestion of microorganisms. D. melanogaster breeds on fruits fermented by Saccharomyces cerevisiae, whereas D. virilis breeds on slime flux and decaying bark of tree housing a variety of bacteria, yeasts, and molds. In this study, it is shown that D. virilis has a higher resistance to oral infection of a species of filamentous fungi belonging to the genus Penicillium compared to D. melanogaster. In response to the fungal infection, a transcriptome profile of immune-related genes was considerably different between D. melanogaster and D. virilis: the genes encoding antifungal peptides, Drosomycin and Metchnikowin, were highly expressed in D. melanogaster whereas, the genes encoding Diptericin and Defensin were highly expressed in D. virilis. On the other hand, the immune-induced molecule (IM) genes showed contrary expression patterns between the two species: they were induced by the fungal infection in D. melanogaster but tended to be suppressed in D. virilis. Our transcriptome analysis also showed newly predicted immune-related genes in D. virilis. These results suggest that the innate immune system has been extensively differentiated during the evolution of these Drosophila species.