Direct infiltration of clonal plasma cells causes renal insufficiency in patients with monoclonal gammopathy of undetermined significance.

Monoclonal gammopathy of undetermined significance (MGUS) is a benign but precancerous condition that can progress to multiple myeloma. Patients with MGUS are typically monitored closely for signs of disease progression, but in some cases, they may also develop renal insufficiency, a condition known as monoclonal gammopathy of renal significance (MGRS). In MGRS, M-protein secreted by a nonmalignant or premalignant cell clone triggers renal damage by definition. Herein, we report a case of a 66-year-old Asian male with MGUS complicated by renal insufficiency. A kidney biopsy showed no evidence of renal injury mediated by M-protein; instead, the direct infiltration of clonal cells into renal tissues was observed. Although five similar cases have been previously reported, our case is unique in that the involvement of clonal cells was directly confirmed by fluorescence in situ hybridization. Our findings suggest the need to consider a novel disease concept, as this phenomenon appears to be reproduced.

Caspase 3-specific cleavage of MEK1 suppresses ERK signaling and sensitizes cells to stress-induced apoptosis.

Proper regulation of apoptotic cell death is crucial for normal development and homeostasis in multicellular organisms and is achieved by the balance between pro-apoptotic processes, such as caspase activation, and pro-survival signaling, such as extracellular signal-regulated kinase (ERK) activation. However, the functional interplay between these opposing signaling pathways remains incompletely understood. Here, we identified MAPK/ERK kinase (MEK) 1, a central component of the ERK pathway, as a specific substrate for the executioner caspase-3. During apoptosis, MEK1 is cleaved at an evolutionarily conserved Asp282 residue in the kinase domain, thereby losing its catalytic activity. Gene knockout experiments showed that MEK1 cleavage was mediated by caspase-3, but not by the other executioner caspases, caspase-6 or -7. Following exposure of cells to osmotic stress, elevated ERK activity gradually decreased, and this was accompanied by increased cleavage of MEK1. In contrast, the expression of a caspase-uncleavable MEK1(D282N) mutant in cells maintained stress-induced ERK activity and thereby attenuated apoptotic cell death. Thus, caspase-3-mediated, proteolytic inhibition of MEK1 sensitizes cells to apoptosis by suppressing pro-survival ERK signaling. Furthermore, we found that a RASopathy-associated MEK1(Y130C) mutation prevented this caspase-3-mediated proteolytic inactivation of MEK1 and efficiently protected cells from stress-induced apoptosis. Our data reveal the functional crosstalk between ERK-mediated cell survival and caspase-mediated cell death pathways and suggest that its dysregulation by a disease-associated MEK1 mutation is at least partly involved in the pathophysiology of congenital RASopathies.

Refractory immune thrombocytopenic purpura associated with IgM monoclonal gammopathy of undetermined significance: Successful treatment with tirabrutinib plus conventional therapies.

When immune thrombocytopenia (ITP) is secondary to malignant diseases, chemotherapy is expected to improve the platelet count (PC) as well. Herein, we report a case of a 72-year-old man with ITP refractory to standard therapies. IgM monoclonal gammopathy of undetermined significance (MGUS) was determined as an underlying disease. After bendamustine and rituximab (BR) therapy was found inadequately effective, tirabrutinib, a novel Bruton's tyrosine kinase inhibitor, was initiated, and the PC normalised subsequently. Surveillance of underlying diseases with which effective therapies are available may help manage refractory ITP, and IgM-MGUS is potentially a targetable underlying disease with this newly available drug.

Qualitative differences in disease-associated MEK mutants reveal molecular signatures and aberrant signaling-crosstalk in cancer.

Point-mutations of MEK1, a central component of ERK signaling, are present in cancer and RASopathies, but their precise biological effects remain obscure. Here, we report a mutant MEK1 structure that uncovers the mechanisms underlying abnormal activities of cancer- and RASopathy-associated MEK1 mutants. These two classes of MEK1 mutations differentially impact on spatiotemporal dynamics of ERK signaling, cellular transcriptional programs, gene expression profiles, and consequent biological outcomes. By making use of such distinct characteristics of the MEK1 mutants, we identified cancer- and RASopathy-signature genes that may serve as diagnostic markers or therapeutic targets for these diseases. In particular, two AKT-inhibitor molecules, PHLDA1 and 2, are simultaneously upregulated by oncogenic ERK signaling, and mediate cancer-specific ERK-AKT crosstalk. The combined expression of PHLDA1/2 is critical to confer resistance to ERK pathway-targeted therapeutics on cancer cells. Finally, we propose a therapeutic strategy to overcome this drug resistance. Our data provide vital insights into the etiology, diagnosis, and therapeutic strategy of cancers and RASopathies.

Novel EGFRvIII-CAR transgenic mice for rigorous preclinical studies in syngeneic mice.

BACKGROUND: Rigorous preclinical studies of chimeric antigen receptor (CAR) immunotherapy will require large quantities of consistent and high-quality CAR-transduced T (CART) cells that can be used in syngeneic mouse glioblastoma (GBM) models. To this end, we developed a novel transgenic (Tg) mouse strain with a fully murinized CAR targeting epidermal growth factor receptor variant III (EGFRvIII). METHODS: We first established the murinized version of EGFRvIII-CAR and validated its function using a retroviral vector (RV) in C57BL/6J mice bearing syngeneic SB28 GBM expressing EGFRvIII. Next, we created C57BL/6J-background Tg mice carrying the anti-EGFRvIII-CAR downstream of a Lox-Stop-Lox cassette in the Rosa26 locus. We bred these mice with CD4-Cre Tg mice to allow CAR expression on T cells and evaluated the function of the CART cells both in vitro and in vivo. To inhibit immunosuppressive myeloid cells within SB28 GBM, we also evaluated a combination approach of CART and an anti-EP4 compound (ONO-AE3-208). RESULTS: Both RV- and Tg-CART cells demonstrated specific cytotoxic activities against SB28-EGFRvIII cells. A single intravenous infusion of EGFRvIII-CART cells prolonged the survival of glioma-bearing mice when preceded by a lymphodepletion regimen with recurrent tumors displaying profound EGFRvIII loss. The addition of ONO-AE3-208 resulted in long-term survival in a fraction of CART-treated mice and those survivors demonstrated delayed growth of subcutaneously re-challenged both EGFRvIII+ and parental EGFRvIII- SB28. CONCLUSION: Our new syngeneic CAR Tg mouse model can serve as a useful tool to address clinically relevant questions and develop future immunotherapeutic strategies.

Nylon mesh-based 3D scaffolds for the adherent culture of neural stem/progenitor cells.

We developed novel scaffolds for the adherent culture of neural stem/progenitor cells on the woven mesh. Nylon mesh (NM) is an inert material for cell adhesion. We prepared polyacrylic acid-grafted nylon mesh (PAA-NM) by graft polymerization method using gamma-irradiation. Matrigel was covalently immobilized to the carboxyl groups in PAA-NM by chemical conjugation using 1-ethyl-3-(3-dimethylamino propyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to prepare the Matrigel-immobilized PAA-grafted nylon mesh (M-PAA-NM). Cell adhesion property of mouse neural stem/progenitor cells (NSPCs) between the NM, PAA-NM, and M-PAA-NM was different from each other. The neurosphere-like clusters of NSPCs were weakly bound to NM and PAA-NM without spreading. The NSPCs were firmly adhered to, spread, and covered the surface of M-PAA-NM. We evaluated the state of differentiation by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immnocytochemistry. A neuronal marker ß III tubulin, a glial marker glial fibrillary acidic protein (GFAP) and a mature glial marker S100ß were expressed at a low level in the cultured cells while immature NSPCs marker Nestin and Sox2 were slightly lower without significant statistical difference. We concluded that the M-PAA-NM is a good substrate for adherent culture of NSPCs without triggering their cell differentiation, and also provides the maintenance of their growth with fewer passages in comparison with the conventional suspension culture of NSPCs in neurospheres.

Suppression of P-glycoprotein by cigarette smoke extract in human lung-derived A549/P-gp cells.

Effect of long-term treatment with cigarette smoke extract (CSE) on the function and expression of P-glycoprotein (P-gp) in lung alveolar epithelial cells was examined using A549/P-gp cell line expressing P-gp. CSE treatment suppressed P-gp activity in a concentration- and treatment time-dependent manner. The suppression of P-gp activity by CSE was irreversible for at least 96 h after removal of CSE. In addition, CSE treatment suppressed the expression of P-gp mRNA and protein. In order to understand the mechanisms underlying P-gp suppression by CSE, the role of reactive oxygen species (ROS) was examined. CSE treatment increased intracellular ROS level, and suppressed catalase activity. α-Tocopherol suppressed ROS production by CSE, and ameliorated the suppression of P-gp activity by CSE, suggesting that ROS is involved in CSE-induced suppression of P-gp. The role of intracellular signaling pathways such as the nuclear factor κB and mitogen-activated protein kinase pathways was also examined. Among these pathways, the involvement of extracellular signal-regulated kinase (ERK) pathway was suggested. Taken together, long-term CSE treatment may suppress P-gp via modulation of ROS level and ERK pathway in alveolar epithelial cells.

Effect of cigarette smoke extract on P-glycoprotein function in primary cultured and newly developed alveolar epithelial cells.

The effect of cigarette smoke extract (CSE) on P-glycoprotein (P-gp) function in the distal lung is unclear. In this study, we first examined the expression and function of P-gp and the effect of CSE in rat primary cultured alveolar epithelial cells. The expression of P-gp protein was observed in type I-like cells, but not in type II cells. In type I-like cells, rhodamine 123 (Rho123) accumulation was enhanced by various P-gp inhibitors such as verapamil and cyclosporine A. In addition, the expression of P-gp mRNAs, mdr1a and mdr1b, as well as P-gp activity increased along with the transdifferentiation. When type I-like cells were co-incubated with CSE, P-gp activity was suppressed. Next, we attempted to clarify the effect of CSE on P-gp function in human-derived cultured alveolar epithelial cells. For this purpose, we isolated an A549 clone (A549/P-gp) expressing P-gp, because P-gp expression in native A549 cells was negligible. In A549/P-gp cells, P-gp was functionally expressed, and the inhibitory effect of CSE on P-gp was observed. These results suggested that smoking would directly suppress P-gp activity, and that A549/P-gp cell line should be a useful model to further study the effect of xenobiotics on P-gp function in the alveolar epithelial cells.

Accumulation of free complex-type N-glycans in MKN7 and MKN45 stomach cancer cells.

During the N-glycosylation reaction, it has been shown that 'free' N-glycans are generated either from lipid-linked oligosaccharides or from misfolded glycoproteins. In both cases, occurrence of high mannose-type free glycans is well-documented, and the molecular mechanism for their catabolism in the cytosol has been studied. On the other hand, little, if anything, is known with regard to the accumulation of more processed, complex-type free oligosaccharides in the cytosol of mammalian cells. During the course of comprehensive analysis of N-glycans in cancer cell membrane fractions [Naka et al. (2006) J. Proteome Res. 5, 88-97], we found that a significant amount of unusual, complex-type free N-glycans were accumulated in the stomach cancer-derived cell lines, MKN7 and MKN45. The most abundant and characteristic glycan found in these cells was determined to be NeuAcalpha2-6Galbeta1-4GlcNAcbeta1-2Manalpha1-3Manbeta1-4GlcNAc. Biochemical analyses indicated that those glycans found were cytosolic glycans derived from lysosomes due to low integrity of the lysosomal membrane. Since the accumulation of these free N-glycans was specific to only two cell lines among the various cancer cell lines examined, these cytosolic N-glycans may serve as a specific biomarker for diagnosis of specific tumours. A cytosolic sialidase, Neu2, was shown to be involved in the degradation of these sialoglycans, indicating that the cytosol of mammalian cells might be equipped for metabolism of complex-type glycans.

Analysis of total N-glycans in cell membrane fractions of cancer cells using a combination of serotonin affinity chromatography and normal phase chromatography.

Cell surface glycans and recognition molecules of these glycans play important roles in cellular recognition and trafficking, such as in the inflammation response by sialyl LewisX oligosaccharides. Malignant cells also utilize a similar mechanism during colonization and establishment of tumor tissues in the host. These considerations prompt us to develop a screening method for comprehensive analysis of N-glycans derived from membrane fractions of cancer cells. The method involves two step separations. Initially, N-glycans released from cell membrane fractions with N-glycoamidase F were labeled with 2-aminobenzoic acid and separated based on the number of sialic acid residues attached to the oligosaccharides using affinity chromatography on a serotonin-immobilized stationary phase. Each of the nonretarded fractions containing asialo- and high-mannose type oligosaccharides and mono-, di-, tri-, and tetra-sialooligosaccharide fractions which were desialylated with neuraminidase was analyzed by a combination of HPLC using an Amide-80 column as the stationary phase and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We analyzed total N-glycan pools of membrane fractions obtained from some cancer cells, and found that U937 cells (Histocytic lymphoma cells) expressed a large amount of oligosaccharides having polylactosamine residues and MKN45 cells (Gastric adenocarcinoma cells) contained hyper-fucosylated oligosaccharides which contained multiple fucose residues. The method described here will be a powerful technique for glycomics studies in cell surface glycoproteins, and will enable one to search marker oligosaccharides characteristically observed in various diseases such as cancer, inflammation, and congenital disorder.