Phase 2 trial of crizotinib in Japanese patients with advanced NSCLC harboring a MET gene alteration: a Co-MET study.

BACKGROUND: MET exon 14 skipping mutations occur in 3-4% and MET high amplifications occur in < 1% of patients with non-small-cell lung cancer (NSCLC). Crizotinib, a selective ATP-competitive small-molecule inhibitor of c-Met, ALK, and ROS1 tyrosine kinases, has shown activity in cancer models with various types of MET activation. METHODS: The Co-MET study is a single-arm phase 2 trial to assess the safety and efficacy of crizotinib in MET inhibitor-naïve patients with advanced NSCLC harboring MET exon 14 skipping mutation (cohort 1) or high MET gene copy number of ≥ 7 (cohort 2). The primary endpoint was the objective response rate (ORR) per RECIST v1.1 by independent radiology review in cohort 1. The key secondary endpoints were the duration of response (DoR), progression-free survival (PFS), overall survival (OS), and safety. RESULTS: A total of 28 patients (23 in cohort 1 and 5 in cohort 2) were enrolled between March 2018 and February 2020. The primary endpoint was met as the ORR (90% confidence interval: CI) in cohort 1 was 38.1% (20.6-58.3). Median DoR, PFS, and OS (95% CI) were 7.6 (1.9-NE), 5.7 (2.1-11.3), 9.1 (4.0-19.9) months, respectively, in cohort 1. ORR in cohort 2 was 40.0% (18.9-92.4). The safety signals were generally consistent with the known safety profile of crizotinib. CONCLUSIONS: Crizotinib showed a clinical activity similar to that of tepotinib and capmatinib in patients with NSCLC harboring MET exon 14 skipping mutations. CLINICAL TRIAL INFORMATION: UMIN000031623.

The Ubiquitin Ligase SCF(Ucc1) Acts as a Metabolic Switch for the Glyoxylate Cycle.

Despite the crucial role played by the glyoxylate cycle in the virulence of pathogens, seed germination in plants, and sexual development in fungi, we still have much to learn about its regulation. Here, we show that a previously uncharacterized SCF(Ucc1) ubiquitin ligase mediates proteasomal degradation of citrate synthase in the glyoxylate cycle to maintain metabolic homeostasis in glucose-grown cells. Conversely, transcription of the F box subunit Ucc1 is downregulated in C2-compound-grown cells, which require increased metabolic flux for gluconeogenesis. Moreover, in vitro analysis demonstrates that oxaloacetate regenerated through the glyoxylate cycle induces a conformational change in citrate synthase and inhibits its recognition and ubiquitination by SCF(Ucc1), suggesting the existence of an oxaloacetate-dependent positive feedback loop that stabilizes citrate synthase. We propose that SCF(Ucc1)-mediated regulation of citrate synthase acts as a metabolic switch for the glyoxylate cycle in response to changes in carbon source, thereby ensuring metabolic versatility and flexibility.

Triacylglycerol lipase Tgl4 is a stable protein and its dephosphorylation is regulated in a cell cycle-dependent manner in Saccharomyces cerevisiae.

Triacylglycerols (TGs) serve as reservoirs for diacylglycerols and fatty acids, which play important roles in synthesizing energy and membrane lipids that are required for cell cycle progression. In the yeast, Saccharomyces cerevisiae, Tgl4, the functional ortholog of murine adipose triacylglycerol lipase (ATGL), is activated by Cdk1/Cdc28-mediated phosphorylation and facilitates the G1/S transition. However, little is known about how Tgl4 is inactivated during the cell cycle. To monitor the phosphorylation status and the stability of endogenous Tgl4, we raised a specific antibody against Tgl4. We found that in contrast to the previous suggestion, Tgl4 was a stable protein throughout the cell cycle. We also showed that Tgl4 was dephosphorylated upon entry into G1 phase. These results suggest that Tgl4 is a stable protein and is inactivated during G1 phase by dephosphorylation.

Curcumin partly prevents ISG15 activation via ubiquitin-activating enzyme E1-like protein and decreases ISGylation.

The expression of the ubiquitin-like molecule interferon-stimulated gene 15 kDa (ISG15) and post-translational protein modification by ISG15 (ISGylation) are strongly activated by interferons or pathogen infection, suggesting that ISG15 and ISGylation play an important role in innate immune responses. More than 400 proteins have been found to be ISGylated. ISG15 is removed from substrates by interferon-induced ubiquitin-specific peptidase 18 or severe acute respiratory syndrome coronavirus 2‒derived papain-like protease. Therefore, maintaining strong ISGylation may help prevent the spread of coronavirus disease 2019 (COVID-19). However, it is unknown whether nutrients or chemicals affect ISGylation level. Curcumin is the major constituent of turmeric and functions as an immunomodulator. Here, we investigated the effect of curcumin on ISGylation. MCF10A and A549 cells were treated with interferon α and curcumin after which the expression levels of various proteins were determined. The effect of curcumin on ubiquitylation was also determined. Curcumin treatment was found to reduce ISGylation in a dose-dependent manner. The findings suggested that curcumin partly prevents disulfide bond-mediated ISG15 dimerization directly or indirectly, thereby increasing monomer ISG15 levels. Reduced ISGylation may also occur via the prevention of ISG15 activation by ubiquitin-activating enzyme E1-like protein. In conclusion, curcumin treatment was found to reduce ISGylation, suggesting that it may contribute to severe COVID-19. This is the first study to report a relationship between ISGylation and a food component.

Insulin-like growth factor 1 receptor stabilizes the ETV6-NTRK3 chimeric oncoprotein by blocking its KPC1/Rnf123-mediated proteasomal degradation.

Many oncogenes, including chimeric oncoproteins, require insulin-like growth factor 1 receptor (IGF1R) for promoting cell transformation. The ETS variant 6 (ETV6)-neurotrophic receptor tyrosine kinase 3 (NTRK3) (EN) chimeric tyrosine kinase is expressed in mesenchymal, epithelial, and hematopoietic cancers and requires the IGF1R axis for transformation. However, current models of IGF1R-mediated EN activation are lacking mechanistic detail. We demonstrate here that IGF-mediated IGF1R stimulation enhances EN tyrosine phosphorylation and that blocking IGF1R activity or decreasing protein levels of the adaptor protein insulin receptor substrate 1/2 (IRS1/2) results in rapid EN degradation. This was observed both in vitro and in vivo in fibroblast and breast epithelial cell line models and in MO91, an EN-expressing human leukemia cell line. Stable isotope labeling with amino acids in cell culture (SILAC)-based MS analysis identified the E3 ligase RING-finger protein 123 (Rnf123, more commonly known as KPC1) as an EN interactor upon IGF1R/insulin receptor (INSR) inhibitor treatment. KPC1/Rnf123 ubiquitylated EN in vitro, and its overexpression decreased EN protein levels. In contrast, KPC1/Rnf123 knockdown rendered EN resistant to IGF1R inhibitor-mediated degradation. These results support a critical function for IGF1R in protecting EN from KPC1/Rnf123-mediated proteasomal degradation. Attempts to therapeutically target oncogenic chimeric tyrosine kinases have traditionally focused on blocking kinase activity to restrict downstream activation of essential signaling pathways. In this study, we demonstrate that IGF1R inhibition results in rapid ubiquitylation and degradation of the EN oncoprotein through a proteasome-dependent mechanism that is reversible, highlighting a potential strategy for targeting chimeric tyrosine kinases in cancer.

Proteolytic regulation of metabolic enzymes by E3 ubiquitin ligase complexes: lessons from yeast.

Eukaryotic organisms use diverse mechanisms to control metabolic rates in response to changes in the internal and/or external environment. Fine metabolic control is a highly responsive, energy-saving process that is mediated by allosteric inhibition/activation and/or reversible modification of preexisting metabolic enzymes. In contrast, coarse metabolic control is a relatively long-term and expensive process that involves modulating the level of metabolic enzymes. Coarse metabolic control can be achieved through the degradation of metabolic enzymes by the ubiquitin-proteasome system (UPS), in which substrates are specifically ubiquitinated by an E3 ubiquitin ligase and targeted for proteasomal degradation. Here, we review select multi-protein E3 ligase complexes that directly regulate metabolic enzymes in Saccharomyces cerevisiae. The first part of the review focuses on the endoplasmic reticulum (ER) membrane-associated Hrd1 and Doa10 E3 ligase complexes. In addition to their primary roles in the ER-associated degradation pathway that eliminates misfolded proteins, recent quantitative proteomic analyses identified native substrates of Hrd1 and Doa10 in the sterol synthesis pathway. The second part focuses on the SCF (Skp1-Cul1-F-box protein) complex, an abundant prototypical multi-protein E3 ligase complex. While the best-known roles of the SCF complex are in the regulation of the cell cycle and transcription, accumulating evidence indicates that the SCF complex also modulates carbon metabolism pathways. The increasing number of metabolic enzymes whose stability is directly regulated by the UPS underscores the importance of the proteolytic regulation of metabolic processes for the acclimation of cells to environmental changes.

The nutrient stress-induced small GTPase Rab5 contributes to the activation of vesicle trafficking and vacuolar activity.

Rab family small GTPases regulate membrane trafficking by spatiotemporal recruitment of various effectors. However, it remains largely unclear how the expression and functions of Rab proteins are regulated in response to extracellular or intracellular stimuli. Here we show that Ypt53, one isoform of Rab5 in Saccharomyces cerevisiae, is up-regulated significantly under nutrient stress. Under non-stress conditions, Vps21, a constitutively expressed Rab5 isoform, is crucial to Golgi-vacuole trafficking and to vacuolar hydrolase activity. However, when cells are exposed to nutrient stress for an extended period of time, the up-regulated Ypt53 and the constitutive Vps21 function redundantly to maintain these activities, which, in turn, prevent the accumulation of reactive oxygen species and maintain mitochondrial respiration. Together, our results clarify the relative roles of these constitutive and nutrient stress-inducible Rab5 proteins that ensure adaptable vesicle trafficking and vacuolar hydrolase activity, thereby allowing cells to adapt to environmental changes.

Activation of double-stranded RNA-activated protein kinase (PKR) by interferon-stimulated gene 15 (ISG15) modification down-regulates protein translation.

The ubiquitin-like molecule ISG15 (UCRP) and protein modification by ISG15 (ISGylation) are strongly induced by interferon, genotoxic stress, and pathogen infection, suggesting that ISG15 plays an important role in innate immune responses. However, how ISGylation contributes to innate immune responses is not clear. The dsRNA-dependent protein kinase (PKR) inhibits translation by phosphorylating eIF2α to exert its anti-viral effect. ISG15 and PKR are induced by interferon, suggesting that a relationship exists between ISGylation and translational regulation. Here, we report that PKR is ISGylated at lysines 69 and 159. ISG15-modified PKR is active in the absence of virus infection and phosphorylates eIF2α to down-regulate protein translation. The present study describes a novel pathway for the activation of PKR and the regulation of protein translation.

TRIM6 interacts with Myc and maintains the pluripotency of mouse embryonic stem cells.

The proto-oncogene product Myc is a master regulator of cell proliferation through its specific binding to the E-box motif in genomic DNA. It has been reported that Myc has an important role in the proliferation and maintenance of the pluripotency of embryonic stem (ES) cells and that the transcriptional activity of Myc is regulated by several post-translational modifications, including ubiquitination. In this study, we showed that tripartite motif containing 6 (TRIM6), one of the TRIM family ubiquitin ligases, was selectively expressed in ES cells and interacted with Myc followed by attenuation of the transcriptional activity of Myc. Knockdown of TRIM6 in ES cells enhanced the transcriptional activity of Myc and repressed expression of NANOG, resulting in the promotion of ES cell differentiation. These findings indicate that TRIM6 regulates the transcriptional activity of Myc during the maintenance of ES cell pluripotency, suggesting that TRIM6 functions as a novel regulator for Myc-mediated transcription in ES cells.

Role of the San1 ubiquitin ligase in the heat stress-induced degradation of nonnative Nup1 in the nuclear pore complex.

The nuclear pore complex (NPC) mediates the selective exchange of macromolecules between the nucleus and the cytoplasm. Neurodegenerative diseases such as amyotrophic lateral sclerosis are characterized by mislocalization of nucleoporins (Nups), transport receptors, and Ras-related nuclear proteins into nucleoplasmic or cytosolic aggregates, underscoring the importance of precise assembly of the NPC. The assembly state of large protein complexes is strictly monitored by the protein quality control system. The ubiquitin-proteasome system may eliminate aberrant, misfolded, and/or orphan components; however, the involvement of the ubiquitin-proteasome system in the degradation of nonnative Nups in the NPC remains unclear. Here, we show that in Saccharomyces cerevisiae, although Nup1 (the FG-Nup component of the central core of the NPC) was stable, C-terminally green fluorescent protein-tagged Nup1, which had been incorporated into the NPC, was degraded by the proteasome especially under heat stress conditions. The degradation was dependent on the San1 ubiquitin ligase and Cdc48/p97, as well as its cofactor Doa1. We also demonstrate that San1 weakly but certainly contributes to the degradation of nontagged endogenous Nup1 in cells defective in NPC biogenesis by the deletion of NUP120. In addition, the overexpression of SAN1 exacerbated the growth defect phenotype of nup120Δ cells, which may be caused by excess degradation of defective Nups due to the deletion of NUP120. These biochemical and genetic data suggest that San1 is involved in the degradation of nonnative Nups generated by genetic mutation or when NPC biogenesis is impaired.

TRIM67 protein negatively regulates Ras activity through degradation of 80K-H and induces neuritogenesis.

Tripartite motif (TRIM)-containing proteins, which are defined by the presence of a common domain structure composed of a RING finger, one or two B-box motifs and a coiled-coil motif, are involved in many biological processes including innate immunity, viral infection, carcinogenesis, and development. Here we show that TRIM67, which has a TRIM motif, an FN3 domain and a SPRY domain, is highly expressed in the cerebellum and that TRIM67 interacts with PRG-1 and 80K-H, which is involved in the Ras-mediated signaling pathway. Ectopic expression of TRIM67 results in degradation of endogenous 80K-H and attenuation of cell proliferation and enhances neuritogenesis in the neuroblastoma cell line N1E-115. Furthermore, morphological and biological changes caused by knockdown of 80K-H are similar to those observed by overexpression of TRIM67. These findings suggest that TRIM67 regulates Ras signaling via degradation of 80K-H, leading to neural differentiation including neuritogenesis.

TRIM32 promotes neural differentiation through retinoic acid receptor-mediated transcription.

Retinoic acid (RA), a metabolite of vitamin A, plays versatile roles in development, differentiation, cell cycles and regulation of apoptosis by regulating gene transcription through nuclear receptor activation. Ubiquitinylation, which is one of the post-translational modifications, appears to be involved in the transcriptional activity of intranuclear receptors including retinoic acid receptor α (RARα). Mutations in the tripartite motif-containing protein 32 gene (TRIM32; also known as E3 ubiquitin-protein ligase) have been reported to be responsible for limb-girdle muscular dystrophy type 2H in humans, and its encoded protein has been shown to interact with several other important proteins. In this study, we found that TRIM32 interacts with RARα and enhances its transcriptional activity in the presence of RA. We also found that overexpression of TRIM32 in mouse neuroblastoma cells and embryonal carcinoma cells promoted stability of RARα, resulting in enhancement of neural differentiation. These findings suggest that TRIM32 functions as one of the co-activators for RARα-mediated transcription, and thereby TRIM32 is a potential therapeutic target for developmental disorders and RA-dependent leukemias.

Granulomatosis with Polyangiitis following COVID-19 in an Adolescent.

Granulomatosis with polyangiitis (GPA) is a systemic disease that causes vasculitis in various organs. Although the mechanism of pathogenesis remains unclear, infection has been reported to be a causative factor. We herein report a case of GPA that developed following coronavirus disease 2019 (COVID-19) in an adolescent girl. One month after contracting mild COVID-19, the patient had facial allodynia, a fever, and weight loss and was admitted for multiple nodular shadows on a chest roentgenogram. GPA was diagnosed based on pathological findings of the lung and nasal mucosal biopsies. She received methylprednisolone and rituximab, and her symptoms and radiological findings improved.

Defective import of mitochondrial metabolic enzyme elicits ectopic metabolic stress.

Deficiencies in mitochondrial protein import are associated with a number of diseases. However, although nonimported mitochondrial proteins are at great risk of aggregation, it remains largely unclear how their accumulation causes cell dysfunction. Here, we show that nonimported citrate synthase is targeted for proteasomal degradation by the ubiquitin ligase SCFUcc1. Unexpectedly, our structural and genetic analyses revealed that nonimported citrate synthase appears to form an enzymatically active conformation in the cytosol. Its excess accumulation caused ectopic citrate synthesis, which, in turn, led to an imbalance in carbon flux of sugar, a reduction of the pool of amino acids and nucleotides, and a growth defect. Under these conditions, translation repression is induced and acts as a protective mechanism that mitigates the growth defect. We propose that the consequence of mitochondrial import failure is not limited to proteotoxic insults, but that the accumulation of a nonimported metabolic enzyme elicits ectopic metabolic stress.

TRIM8 regulates Nanog via Hsp90ß-mediated nuclear translocation of STAT3 in embryonic stem cells.

TRIM8 is a member of a protein family defined by the presence of a common domain structure composed of a tripartite motif including a RING-finger, one or two B-box domains and a coiled-coil motif. Here, we show that TRIM8 interacts with Hsp90ß, which interacts with STAT3 and selectively downregulates transcription of Nanog in embryonic stem cells. Knock-down of TRIM8 increased phosphorylated STAT3 in the nucleus and also enhanced transcription of Nanog. These findings suggest that TRIM8 modulates translocation of phosphorylated STAT3 into the nucleus through interaction with Hsp90ß and consequently regulates transcription of Nanog in embryonic stem cells.

TRIM8 modulates STAT3 activity through negative regulation of PIAS3.

TRIM8 is a member of the protein family defined by the presence of a common domain structure composed of a tripartite motif: a RING-finger, one or two B-box domains and a coiled-coil motif. Here, we show that TRIM8 interacts with protein inhibitor of activated STAT3 (PIAS3), which inhibits IL-6-dependent activation of STAT3. Ectopic expression of TRIM8 cancels the negative effect of PIAS3 on STAT3, either by degradation of PIAS3 through the ubiquitin-proteasome pathway or exclusion of PIAS3 from the nucleus. Furthermore, expression of TRIM8 in NIH3T3 cells enhances Src-dependent tumorigenesis. These findings indicate that TRIM8 enhances the STAT3-dependent signal pathway by inhibiting the function of PIAS3.

TRIM32 promotes retinoic acid receptor α-mediated differentiation in human promyelogenous leukemic cell line HL60.

Ubiquitination, one of the posttranslational modifications, appears to be involved in the transcriptional activity of nuclear receptors including retinoic acid receptor α (RARα). We previously reported that an E3 ubiquitin ligase, TRIM32, interacts with several important proteins including RARα and enhances transcriptional activity of RARα in mouse neuroblastoma cells and embryonal carcinoma cells. Retinoic acid (RA), which acts as a ligand to nuclear receptors including RARα, plays crucial roles in development, differentiation, cell cycles and apoptosis. In this study, we found that TRIM32 enhances RARα-mediated transcriptional activity even in the absence of RA and stabilizes RARα in the human promyelogenous leukemic cell line HL60. Moreover, we found that overexpression of TRIM32 in HL60 cells suppresses cellular proliferation and induces granulocytic differentiation even in the absence of RA. These findings suggest that TRIM32 functions as one of the coactivators for RARα-mediated transcription in acute promyelogenous leukemia (APL) cells, and thus TRIM32 may become a potentially therapeutic target for APL.

Dot6/Tod6 degradation fine-tunes the repression of ribosome biogenesis under nutrient-limited conditions.

Ribosome biogenesis (Ribi) is a complex and energy-consuming process, and should therefore be repressed under nutrient-limited conditions to minimize unnecessary cellular energy consumption. In yeast, the transcriptional repressors Dot6 and Tod6 are phosphorylated and inactivated by the TORC1 pathway under nutrient-rich conditions, but are activated and repress ∼200 Ribi genes under nutrient-limited conditions. However, we show that in the presence of rapamycin or under nitrogen starvation conditions, Dot6 and Tod6 were readily degraded by the proteasome in a SCFGrr1 and Tom1 ubiquitin ligase-dependent manner, respectively. Moreover, promiscuous accumulation of Dot6 and Tod6 excessively repressed Ribi gene expression as well as translation activity and caused a growth defect in the presence of rapamycin. Thus, we propose that degradation of Dot6 and Tod6 is a novel mechanism to ensure an appropriate level of Ribi gene expression and thereby fine-tune the repression of Ribi and translation activity for cell survival under nutrient-limited conditions.

TRIM40 promotes neddylation of IKKγ and is downregulated in gastrointestinal cancers.

Gastrointestinal neoplasia seems to be a common consequence of chronic inflammation in the gastrointestinal epithelium. Nuclear factor-kappaB (NF-κB) is an important transcription factor for carcinogenesis in chronic inflammatory diseases and plays a key role in promoting inflammation-associated carcinoma in the gastrointestinal tract. Activation of NF-κB is regulated by several posttranslational modifications including phosphorylation, ubiquitination and neddylation. In this study, we showed that tripartite motif (TRIM) 40 is highly expressed in the gastrointestinal tract and that TRIM40 physically binds to Nedd8, which is conjugated to target proteins by neddylation. We also found that TRIM40 promotes the neddylation of inhibitor of nuclear factor kappaB kinase subunit gamma, which is a crucial regulator for NF-κB activation, and consequently causes inhibition of NF-κB activity, whereas a dominant-negative mutant of TRIM40 lacking the RING domain does not inhibit NF-κB activity. Knockdown of TRIM40 in the small intestinal epithelial cell line IEC-6 caused NF-κB activation followed by increased cell growth. In addition, we found that TRIM40 is highly expressed in normal gastrointestinal epithelia but that TRIM40 is downregulated in gastrointestinal carcinomas and chronic inflammatory lesions of the gastrointestinal tract. These findings suggest that TRIM40 inhibits NF-κB activity via neddylation of inhibitor of nuclear factor kappaB kinase subunit gamma and that TRIM40 prevents inflammation-associated carcinogenesis in the gastrointestinal tract.

Plasma gelsolin facilitates interaction between ß2 glycoprotein I and α5ß1 integrin.

Antiphospholipid syndrome (APS) is characterized by thrombosis and the presence of antiphospholipid antibodies (aPL) that directly recognizes plasma ß(2)-glycoprotein I (ß(2) GPI). Tissue factor (TF), the major initiator of the extrinsic coagulation system, is induced on monocytes by aPL in vitro, explaining in part the pathophysiology in APS. We previously reported that the mitogen-activated protein kinase (MAPK) pathway plays an important role in aPL-induced TF expression on monocytes. In this study, we identified plasma gelsolin as a protein associated with ß(2) GPI by using immunoaffinity chromatography and mass spectrometric analysis. An in vivo binding assay showed that endogenous ß(2) GPI interacts with plasma gelsolin, which binds to integrin a(5) ß(1) through fibronectin. The tethering of ß(2) GPI to monoclonal anti-ß(2) GPI autoantibody on the cell surface was enhanced in the presence of plasma gelsolin. Immunoblot analysis demonstrated that p38 MAPK protein was phosphorylated by monoclonal anti-ß(2) GPI antibody treatment, and its phosphorylation was attenuated in the presence of anti-integrin a(5) ß(1) antibody. Furthermore, focal adhesion kinase, a downstream molecule of the fibronectin-integrin signalling pathway, was phosphorylated by anti-ß(2) GPI antibody treatment. These results indicate that molecules including gelsolin and integrin are involved in the anti-ß(2) GPI antibody-induced MAPK pathway on monocytes and that integrin is a possible therapeutic target to modify a prothrombotic state in patients with APS.