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.

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.

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.

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.

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.

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.

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.

ZSWIM8 is a myogenic protein that partly prevents C2C12 differentiation.

Cell adhesion molecule-related/downregulated by oncogenes (Cdon) is a cell-surface receptor that mediates cell-cell interactions and positively regulates myogenesis. The cytoplasmic region of Cdon interacts with other proteins to form a Cdon/JLP/Bnip-2/CDC42 complex that activates p38 mitogen-activated protein kinase (MAPK) and induces myogenesis. However, Cdon complex may include other proteins during myogenesis. In this study, we found that Cullin 2-interacting protein zinc finger SWIM type containing 8 (ZSWIM8) ubiquitin ligase is induced during C2C12 differentiation and is included in the Cdon complex. We knocked-down Zswim8 in C2C12 cells to determine the effect of ZSWIM8 on differentiation. However, we detected neither ZSWIM8-dependent ubiquitination nor the degradation of Bnip2, Cdon, or JLP. In contrast, ZSWIM8 knockdown accelerated C2C12 differentiation. These results suggest that ZSWIM8 is a Cdon complex-included myogenic protein that prevents C2C12 differentiation without affecting the stability of Bnip2, Cdon, and JLP.

The efficacy of nintedanib in 158 patients with idiopathic pulmonary fibrosis in real-world settings: A multicenter retrospective study.

BACKGROUND: The INPULSIS trials revealed that nintedanib reduced the decline in lung function in patients with idiopathic pulmonary fibrosis. We aimed to evaluate the efficacy and safety of nintedanib in Japanese idiopathic pulmonary fibrosis patients in real-world settings. METHOD: Medical records of idiopathic pulmonary fibrosis patients, who received treatment with nintedanib in five institutions between July 2015 and June 2017, were reviewed. Patients with % forced vital capacity ⩾50% and % predicted diffusing capacity of the lung carbon monoxide ⩾30% were classified as the moderate group and those with more impaired lung functions as the severe group. RESULT: Among 158 patients analyzed, 132 (84.6%) were classified as the moderate group and 26 (15.4%) as the severe group. In the moderate group, changes in forced vital capacity in 12 months were significantly different between before and after nintedanib administration (-253 ± 163 vs -125 ± 235 mL; p = 0.0027). In contrast, changes in forced vital capacity in 12 months were not significantly changed by nintedanib treatment in the severe group (-353 ± 250 vs -112 ± 341 mL; p = 0.2374). Incidence of acute exacerbation was higher in the severe group than in the moderate group (30.8% vs 18.9%). The overall survival of the moderate and the severe groups was 17.2 and 10.1 months. CONCLUSION: In real-world practice, nintedanib showed comparable efficacy to those observed in previous trials. In the severe group, the efficacy of nintedanib might be limited.

Comparative analysis of sperm motility in liquid and seminal coagulum portions between Bornean orangutan (Pongo pygmaeus) and chimpanzee (Pan troglodytes).

Coagulum in the semen of some primates plays different roles, depending on the species. In the present study, we examined sperm motility in the coagulum and liquid portions of semen collected from captive individuals from two great ape species: two adult Bornean orangutans (Pongo pygmaeus) (n = 27) and three adult chimpanzees (Pan troglodytes) (n = 14). The results revealed that orangutan sperm remained motile for significantly longer in the coagulum than in the liquid portion (> 18 h). By contrast, chimpanzee sperm motility did not differ significantly over time between the two portions of the semen, although motility was slightly higher in the liquid portion than in the coagulum. The evolution of the seminal coagulum is thought to be related to postcopulatory sperm competition; however, functions of seminal coagulum have not been completely elucidated. Our data from the orangutan semen suggest that in this species, seminal coagulum may strengthen own-sperm survival. This report is the first to provide evidence for this distinctive function of the seminal coagulum. This unique property of orangutan seminal coagulum might be attributable to their reproductive traits, e.g., difficulty in predicting ovulation due to a lack of genital swelling during estrus. The orangutan is a Critically Endangered species, and captive breeding, including artificial insemination (AI), is expected. However, worldwide, only one case of orangutan AI has been successful. Our findings may contribute to an understanding of their basic semen characteristics and help improve the AI method.

A Phase I/II Study of Biweekly Carboplatin and Nab-paclitaxel With Concurrent Radiotherapy for Patients With Locally Advanced Unresectable Stage III Non-small-cell Lung Cancer.

BACKGROUND: Concurrent chemoradiotherapy (CCRT) is the standard treatment for patients with locally advanced non-small-cell lung cell cancer (LA-NSCLC). We conducted a phase I/II study of biweekly carboplatin and nab-paclitaxel (nab-PTX) with radiotherapy (RT). MATERIALS AND METHODS: In the phase I part, patients with inoperable stage IIIA/IIIB NSCLC were treated with carboplatin (area under the time-concentration curve, 4) and nab-PTX (60-100 mg/m2) on days 1, 15, and 29. Thoracic RT was administered from day 1 to a total dose of 60 Gy in 30 fractions. In the phase II part, patients were administered carboplatin and nab-PTX on days 1, 15, and 29 at the recommended dose (RD). The primary endpoint of the phase I part was to determine the maximum tolerated dose and the RD. In the phase II part, the primary endpoint was 2-year overall survival (OS) rate, and secondary endpoints were the objective response rate, progression-free survival, OS, and safety profile. RESULTS: In the phase I part, although maximum tolerated dose was not obtained, the RD was carboplatin (area under the time-concentration curve, 4) and nab-PTX (100 mg/m2). Of the evaluable 28 patients, the rate of 2-year OS was 67.8% (95% confidence interval, 49.3%-82.1%). The objective response rate was 96.4%, and the median follow-up time was 33.2 months. The median progression-free survival was 18.2 months (95% confidence interval, 13.1 months to not reached). The most common toxicities of grade 3 or higher were neutropenia (60.5%), anemia (14.2%), thrombocytopenia (7.2%), and pneumonitis (3.6%). CONCLUSIONS: This study achieved the primary endpoint. Biweekly carboplatin and nab-PTX with concurrent RT was well-tolerated and exerted promising antitumor activity.

A pilot study of adjuvant chemotherapy with carboplatin and oral S-1 for patients with completely resected stage II to IIIA non-small cell lung cancer.

BACKGROUND: Adjuvant chemotherapy with platinum-based regimens for completely resected early-stage non-small cell lung cancer (NSCLC) provides overall survival benefit in several clinical trials. OBJECTIVES: We conducted this prospective study to evaluate the efficacy and safety of adjuvant chemotherapy with carboplatin and S-1 for patients with completely resected stage II to IIIA NSCLC. METHODS: Patients with completely resected stage IIA to IIIA NSCLC were treated with four cycles of carboplatin with area under the concentration time curve of 5 mg/mL/min on day 1 plus S-1 at 80-120 mg/bodyweight per day for two weeks, followed by one-week rest as adjuvant chemotherapy. The primary endpoint was the completion rate of three cycles of the treatment. The secondary endpoints were safety and two-year survival rate. RESULTS: A total of 19 patients were enrolled, until the study was terminated prematurely because of fatal pulmonary embolism in two patients. The median number of treatment cycles was three (range: 1-4). The completion rate of three cycles was 78.9% (95% confidence interval [CI]: 56.6-91.4%). Two-year disease-free survival rate was 57.8%. Grade 3 or 4 hematological toxicities included neutropenia (26.2%), anemia (5.2%), and thrombocytopenia (15.7%). Grade 3 or 4 nonhematological toxicities were anorexia (10.5%) and nausea (10.5%). Febrile neutropenia developed in 5.2%. In two patients (10.5%), grade five pulmonary embolism was observed, and the causal relationship with treatment could not be denied. CONCLUSIONS: Carboplatin and oral S-1 had modest survival benefit, but this regimen was not tolerable in an adjuvant setting because fatal pulmonary embolism occurred in two patients. KEY POINTS: Carboplatin and oral S-1 had modest survival benefit but this regimen was not tolerable. Fatal pulmonary embolism occurred in this regimen.

Cul5-type Ubiquitin Ligase KLHDC1 Contributes to the Elimination of Truncated SELENOS Produced by Failed UGA/Sec Decoding.

The UGA codon signals protein translation termination, but it can also be translated into selenocysteine (Sec, U) to produce selenocysteine-containing proteins (selenoproteins) by dedicated machinery. As Sec incorporation can fail, Sec-containing longer and Sec-lacking shorter proteins co-exist. Cul2-type ubiquitin ligases were recently shown to destabilize such truncated proteins; however, which ubiquitin ligase targets truncated proteins for degradation remained unclear. We report that the Cul5-type ubiquitin ligase KLHDC1 targets truncated SELENOS, a selenoprotein, for proteasomal degradation. SELENOS is involved in endoplasmic reticulum (ER)-associated degradation, which is linked to reactive oxygen species (ROS) production, and the knockdown of KLHDC1 in U2OS cells decreased ER stress-induced cell death. Knockdown of SELENOS increased the cell population with lower ROS levels. Our findings reveal that, in addition to Cul2-type ubiquitin ligases, KLHDC1 is involved in the elimination of truncated oxidoreductase-inactive SELENOS, which would be crucial for maintaining ROS levels and preventing cancer development.

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.

The HECT-type ubiquitin ligase Tom1 contributes to the turnover of Spo12, a component of the FEAR network, in G2/M phase.

The ubiquitin-proteasome system plays a crucial role in cell cycle progression. A previous study suggested that Spo12, a component of the Cdc14 early anaphase release (FEAR) network, is targeted for degradation by the APC/CCdh1 complex in G1 phase. In the present study, we demonstrate that the Hect-type ubiquitin ligase Tom1 contributes to the turnover of Spo12 in G2/M phase. Coimmunoprecipitation analysis confirmed that Tom1 and Spo12 interact. Overexpression of Spo12 is cytotoxic in the absence of Tom1. Notably, Spo12 is degraded in S phase even in the absence of Tom1 and Cdh1, suggesting that an additional E3 ligase(s) also mediates Spo12 degradation. Together, we propose that several distinct degradation pathways control the level of Spo12 during the cell cycle.

Ubiquitin ligase SPSB4 diminishes cell repulsive responses mediated by EphB2.

Eph receptor tyrosine kinases and their ephrin ligands are overexpressed in various human cancers, including colorectal malignancies, suggesting important roles in many aspects of cancer development and progression as well as in cellular repulsive responses. The ectodomain of EphB2 receptor is cleaved by metalloproteinases (MMPs) MMP-2/MMP-9 and released into the extracellular space after stimulation by its ligand. The remaining membrane-associated fragment is further cleaved by the presenilin-dependent γ-secretase and releases an intracellular peptide that has tyrosine kinase activity. Although the cytoplasmic fragment is degraded by the proteasome, the responsible ubiquitin ligase has not been identified. Here, we show that SOCS box-containing protein SPSB4 polyubiquitinates EphB2 cytoplasmic fragment and that SPSB4 knockdown stabilizes the cytoplasmic fragment. Importantly, SPSB4 down-regulation enhances cell repulsive responses mediated by EphB2 stimulation. Altogether, we propose that SPSB4 is a previously unidentified ubiquitin ligase regulating EphB2-dependent cell repulsive responses.

Hypoxia-inducible factor-2α stabilizes the von Hippel-Lindau (VHL) disease suppressor, Myb-related protein 2.

Ubiquitin ligase von Hippel-Lindau tumor suppressor (pVHL) negatively regulates protein levels of hypoxia-inducible factor-α (HIF-α). Loss of pVHL causes HIF-α accumulation, which contributes to the pathogenesis of von Hippel-Lindau (VHL) disease. In contrast, v-Myb avian myeloblastosis viral oncogene homolog-like 2 (MYBL2; B-Myb), a transcription factor, prevents VHL pathogenesis by regulating gene expression of HIF-independent pathways. Both HIF-α and B-Myb are targets of pVHL-mediated polyubiquitination and proteasomal degradation. Here, we show that knockdown of HIF-2α induces downregulation of B-Myb in 786-O cells, which are deficient in pVHL, and this downregulation is prevented by proteasome inhibition. In the presence of pVHL and under hypoxia-like conditions, B-Myb and HIF-2α are both upregulated, and the upregulation of B-Myb requires expression of HIF-2α. We also show that HIF-2α and B-Myb interact in the nucleus, and this interaction is mediated by the central region of HIF-2α and the C-terminal region of B-Myb. These data indicate that oncogenic HIF-2α stabilizes B-Myb to suppress VHL pathogenesis.

ASB7 regulates spindle dynamics and genome integrity by targeting DDA3 for proteasomal degradation.

Proper dynamic regulation of the spindle is essential for successful cell division. However, the molecular mechanisms that regulate spindle dynamics in mitosis are not fully understood. In this study, we show that Cullin 5-interacting suppressor of cytokine signaling box protein ASB7 ubiquitinates DDA3, a regulator of spindle dynamics, thereby targeting it for proteasomal degradation. The presence of microtubules (MTs) prevented the ASB7-DDA3 interaction, thus stabilizing DDA3. Knockdown of ASB7 decreased MT polymerization and increased the proportion of cells with unaligned chromosomes, and this phenotype was rescued by deletion of DDA3. Collectively, these data indicate that ASB7 plays a crucial role in regulating spindle dynamics and genome integrity by controlling the expression of DDA3.

Parallel Regulation of von Hippel-Lindau Disease by pVHL-Mediated Degradation of B-Myb and Hypoxia-Inducible Factor α.

pVHL, the protein product of the von Hippel-Lindau (VHL) tumor suppressor gene, is a ubiquitin ligase that targets hypoxia-inducible factor α (HIF-α) for proteasomal degradation. Although HIF-α activation is necessary for VHL disease pathogenesis, constitutive activation of HIF-α alone did not induce renal clear cell carcinomas and pheochromocytomas in mice, suggesting the involvement of an HIF-α-independent pathway in VHL pathogenesis. Here, we show that the transcription factor B-Myb is a pVHL substrate that is degraded via the ubiquitin-proteasome pathway and that vascular endothelial growth factor (VEGF)- and/or platelet-derived growth factor (PDGF)-dependent tyrosine 15 phosphorylation of B-Myb prevents its degradation. Mice injected with B-Myb knockdown 786-O cells developed dramatically larger tumors than those bearing control cell tumors. Microarray screening of B-Myb-regulated genes showed that the expression of HIF-α-dependent genes was not affected by B-Myb knockdown, indicating that B-Myb prevents HIF-α-dependent tumorigenesis through an HIF-α-independent pathway. These data indicate that the regulation of B-Myb by pVHL plays a critical role in VHL disease.