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Supplementary Data from New Molecular and Biological Mechanism of Antitumor Activities of KW-2478, a Novel Nonansamycin Heat Shock Protein 90 Inhibitor, in Multiple Myeloma Cells

Purpose: The heat shock protein 90 (Hsp90) plays an important role in chaperoning oncogenic client proteins in multiple myeloma (MM) cells, and several Hsp90 inhibitors have shown antitumor activities both in vitro and in vivo. However the precise mechanism of action of Hsp90 inhibitor in MM has not been fully elucidated.Experimental Design: We evaluated the antitumor activities of KW-2478, a nonansamycin Hsp90 inhibitor, in MM cells with various chromosomal translocations of immunoglobulin heavy chain (IgH) loci both in vitro and in vivo.Results: Our studies revealed that exposure of KW-2478 to MM cells resulted in growth inhibition and apoptosis, which were associated with degradation of well-known client proteins as well as a decrease in IgH translocation products (FGFR3, c-Maf, and cyclin D1), and FGFR3 was shown to be a new client protein of Hsp90 chaperon complex. In addition, KW-2478 depleted the Hsp90 client Cdk9, a transcriptional kinase, and the phosphorylated 4E-BP1, a translational inhibitor. Both inhibitory effects of KW-2478 on such transcriptional and translational pathways were shown to reduce c-Maf and cyclin D1 expression. In NCI-H929 s.c. inoculated model, KW-2478 showed a significant suppression of tumor growth and induced the degradation of client proteins in tumors. Furthermore, in a novel orthotopic MM model of i.v. inoculated OPM-2/green fluorescent protein, KW-2478 showed a significant reduction of both serum M protein and MM tumor burden in the bone marrow.Conclusions: These results suggest that targeting such diverse pathways by KW-2478 could be a promising strategy for the treatment of MM with various cytogenetic abnormalities. Clin Cancer Res; 16(10); 2792–802. ©2010 AACR.

DEK is a highly conserved nuclear factor that plays an important role in the regulation of multiple cellular processes.DEKwas discovered to be an oncogene as a fusion withNUP214gene, which results in producing DEK-NUP214 proteins, in a subset of patients with acute myeloid leukemia. Subsequently, DEK overexpression was reported in many cancers, thus DEK itself is considered to be an oncoprotein. DEK has been reported to play important roles in the progression of early and late stage squamous cell carcinoma (SCC) and is useful for early diagnosis of the disease. These findings have made DEK an attractive therapeutic target, especially for human papillomavirus (HPV)-associated SCC. However, the mechanism of DEK in SCC remains unclear. In this review, we discuss humanDEKoncogene-related SCC.

Phenotypic screening in drug discovery has been revived with the expectation of providing promising lead compounds and drug targets and improving the success rate of drug approval. However, target identification remains a major bottleneck in phenotype-based drug discovery. We identified the lead compounds K542 and K405 with a selective inhibition of cell viability against sphingosine-1-phosphate lyase 1 (SGPL1)-transduced ES-2 cells by phenotypic screening. We therefore performed an in vivo pharmacological examination and observed the antitumor activity of K542 in an HT-1080 tumor-bearing mouse xenograft model. SGPL1 was expected to be a therapeutic target in some cancers, suggesting that these lead molecules might be promising candidates; however, their mechanisms of action still remain unexplained. We therefore synthesized the affinity probe Ind-tag derived from K542 and identified the proteins binding to Ind-tag via a pull-down experiment. Proteomics and biochemical analyses revealed that the target molecule of these lead compounds was Nicotinamide phosphoribosyltransferase (NAMPT). We established K542-resistant DLD-1 and HT-1080 cells, and genetic analyses of these cells identified a missense mutation in the NAMPT-encoding gene. This enzymatic experiment clearly showed that K393 exerts enzymatic inhibition against NAMPT. These proteomics, genetics and biochemical analyses clarified that compounds K542 and K405 were NAMPT inhibitors.

Oral squamous cell carcinoma (OSCC) patients have a low 5-year survival rate and poor prognosis. To improve survival and prognosis, the causes and processes involved in lesion development should be evaluated. For this purpose, the use of OSCC mouse models, such as chemically induced mouse models, genetically modified mouse models, and transplanted (xenograft) models, is crucial. These OSCC models exhibit both advantages and disadvantages when studying OSCC development and progression. Until a model resembling human OSCC is developed, both the advantages and disadvantages of each model should be carefully considered. In this review, we discuss OSCC mouse models and their use in cancer research worldwide.

// Ayumi Niwa 1, * , Seiya Kuwano 1, * , Hiroyuki Tomita 1 , Keita Kimura 1 , Yukiya Orihara 1 , Tomohiro Kanayama 1 , Kei Noguchi 1 , Kenji Hisamatsu 1 , Takayuki Nakashima 1 , Yuichiro Hatano 1 , Akihiro Hirata 2 , Tatsuhiko Miyazaki 3 , Kazuhiro Kaneko 4 , Takuji Tanaka 5 and Akira Hara 1 1 Department of Tumor Pathology, Gifu University Graduate School of Medicine, Gifu, Japan 2 Division of Animal Experiment, Life Science Research Center, Gifu University, Gifu, Japan 3 Division of Pathology, Gifu University Hospital, Gifu, Japan 4 Department of Gastroenterology, Endoscopy Division, National Cancer Center Hospital East, Kashiwa, Japan 5 Department of Diagnostic Pathology (DDP) and Research Center of Diagnostic Pathology (RC-DiP), Gifu Municipal Hospital, Gifu, Japan * These authors have contributed equally to this work Correspondence to: Hiroyuki Tomita, email: h_tomita@gifu-u.ac.jp Keywords: duodenal neoplasms, beta-catenin Received: October 24, 2016 Accepted: February 27, 2017 Published: April 12, 2017 ABSTRACT Non-ampullary duodenal adenoma with activation of Wnt/β-catenin signalling is common in familial adenomatous polyposis (FAP) patients, whereas sporadic non-ampullary adenoma is uncommon. The adenoma-carcinoma sequence similar to colon cancer is associated with duodenal tumors in FAP, but not always in sporadic tumors. We obtained 37 non-ampullary duodenal tumors, including 25 adenomas and 12 adenocarcinomas, were obtained from biopsies and endoscopic resections. We performed immunohistochemistry for β-catenin, the hallmark of Wnt activation, and aldehyde dehydrogenase 1 (ALDH1), a putative cancer stem cell marker. In non-ampullary lesions, abnormal nuclear localization of β-catenin was observed in 21 (84.0%) of 25 adenomas and 4 (33.3%) of 12 adenocarcinomas. In the proximal duodenum, nuclear β-catenin was less frequent in both adenomas and adenocarcinomas. Gastric duodenal metaplasia (GDM) was observed only in the proximal duodenum. All adenomas with GDM were the gastric foveolar and pyloric gland types, and showed only membranous β-catenin. The intestinal-type adenomas had nuclear β-catenin in the proximal and distal duodenum. ALDH1-positive cells were more frequent in adenocarcinomas than adenomas. Nuclear β-catenin accumulation frequently occurred in ALDH1-positive cells in adenoma, but not in adenocarcinoma. In the non-ampullary proximal duodenum, Wnt/β-catenin pathway activation was more closely associated with adenomas than adenocarcinomas, and while it might cooperate with ALDH1 in adenoma, it does not in adenocarcinoma. The pathogenesis thus may differ between sporadic adenoma and adenocarcinoma of non-ampullary duodenal lesions, especially in the proximal and distal duodenum.

Serrated lesions in the colorectum are currently predominantly classified as hyperplastic polyps (HPs), sessile serrated adenomas/polyps (SSA/Ps), and traditional serrated adenomas (TSAs) according to their morphology. However, the histological morphology and the molecular changes in the serrated lesions are still unclear. We performed immunohistochemistry for Ki67, p16INK4a, and WNT5A in human HPs (n=22), SSA/Ps (n=41), and TSAs (n=19). The distribution of Ki67 and p16INK4a positive cells in TSAs was different from that in HPs and SSA/Ps. Co-expression of Ki67 and P16INK4a was infrequent in HPs and SSA/Ps; p16INK4a-positive cells were found in the crypt cleft and stromal WNT5A-positive stromal cells were localized near the cleft in SSA/Ps, while intraepithelial lymphocytes (IELs) in SSA/Ps were more abundant than HPs. In conclusion, our study provides evidence that HPs branch because of the increase in and patchy distribution of senescent and proliferative cells, with increased and misdistributed stromal and inflammatory cells, which might contribute to creation of L- and/or T-shaped crypts, which are of distinctive shapes in SSA/Ps. Our findings may facilitate better understanding and therapy in the serrated lesions.

Tranilast [N-(3′,4′-dimethoxycinnamoyl)-anthranilic acid], initially developed as an antiallergic drug, also exhibits a growth inhibitory effect on various types of cancer. Osteosarcoma is treated mainly with high-dose methotrexate, doxorubicin, cisplatin and ifosfamide; however, 20–30 % of patients cannot be cured of metastatic disease. We investigated whether tranilast enhances the anticancer effects of chemotherapeutic drugs and analyzed its mechanism of action in osteosarcomas. Tranilast inhibited proliferation of HOS, 143B, U2OS and MG-63 osteosarcoma cells in a dose-dependent manner, as well as enhancing the effects of cisplatin and doxorubicin. The average combination index at effect levels for tranilast in combination with cisplatin was 0.57 in HOS, 0.4 in 143B, 0.39 in U2OS and 0.51 in MG-63 cells. Tranilast and cisplatin synergistically inhibited the viability of osteosarcoma cells. In flow cytometric analysis, although tranilast alone did not induce significant apoptosis, the combination of tranilast and cisplatin induced early and late apoptotic cell death. Expression of cleaved caspase-3, cleaved poly(ADP-ribose) polymerase and p-H2AX was enhanced by tranilast in combination with cisplatin. Tranilast alone increased expression of p21 and Bim protein in a dose-dependent manner. Cell cycle analysis using flow cytometry demonstrated that the combination of tranilast and cisplatin increased the number of cells in the G2/M phase. Compared with cisplatin alone, the combination increased levels of phospho-cyclin-dependent kinase 1 (Y15). In the 143B xenograft model, tumor growth was significantly inhibited by combined tranilast and cisplatin compared with the controls, whereas cisplatin alone did not significantly inhibit tumor growth. In conclusion, tranilast has a cytostatic effect on osteosarcoma cells and enhances the effect of anticancer drugs, especially cisplatin. Enhanced sensitivity to cisplatin was mediated by increased apoptosis through G2/M arrest. Since tranilast has been clinically approved and has few adverse effects, clinical trials of osteosarcoma chemotherapy in combination with tranilast are expected.

Various silicon-containing six-membered-ring molecules were synthesized by Pd-catalyzed coupling reaction of 1,2-bis(dimethylsilyl)arenes with 1,2-diiodoarenes in moderate yields. Their optical properties were investigated via UV–vis absorption, fluorescence and phosphorescence spectroscopies, which indicated that the σ*–π* conjugated system expanded owing to the silicon atoms. Some of the compounds exhibited enhanced luminescence, larger Stokes shift, and phosphorescence in comparison with 9,9,10,10-tetramethyl-9,10-dihydroanthracene. Density functional theory (DFT) studies were also performed to investigate the photophysical properties of the compounds.

Aldehyde dehydrogenase 1A1 (ALDH1A1) is considered to be a cancer stem cell marker in several human malignancies. However, the role of ALDH1A1 in hepatocellular carcinoma (HCC) has not been well elucidated. In this study, we investigated the relationship between ALDH1A1 and clinicopathological findings and examined whether ALDH1A1 deserves to be a cancer stem cell marker in HCC. Sixty HCC samples obtained from surgical resection were collected for immunohistochemical (IHC) staining. Of these 60 samples, 47 samples of HCC tumorous and non-tumorous tissues were evaluated with qRT-PCR. There was no significant difference in the ALDH1A1-mRNA level between tumorous and non-tumorous tissues. Tumorous ALDH1A1-mRNA level had no relationship with the clinicopathological features. Immunoreactivity of ALDH1A1 was classified into two groups based on the percentage of ALDH1A1-overexpressing cells. The ALDH1A1-high group was significantly associated with low serum levels of α-fetoprotein, small tumor diameter, very little lymphovascular invasion, more differentiated pathology and good stage. The ALDH1A1-high group showed more favorable prognosis for recurrence-free survival. In double-staining IHC, ALDH1A1 was not co-expressed with BMI1, EpCAM, CD13, CD24, CD90 and CD133, which reported as cancer stem cell markers in HCC. In conclusion, ALDH1A1-overexpressing cells could appear to be differentiated cells rather than cancer stem cells in HCC.

Shikonin (SHK), a natural small agent (MW 288.3), reportedly induces cell death in various tumor cells. We have found that SHK also exerts potent cytocidal effects on human multiple myeloma (MM) cells, but its anticancer mechanism in MM cells remains to be elucidated. SHK at 2.5–5 μM induced apoptosis in seven MM cell lines, including the bortezomib-resistant cell line KMS11/BTZ. The IC50 value of SHK against KMS11/BTZ was comparable to that of a parental cell line KMS11 (1.1 and 1.56 μM, respectively). SHK induces accumulation of ubiquitinated proteins and activates XBP-1 in MM cells, suggesting that SHK functions as a proteasome inhibitor, eventually inducing ER stress-associated apoptosis. SHK increases levels of HSP70/72, which protects cells from apoptosis, and exerts greater cytocidal effects in combination with the HSP70/72 inhibitor VER-155008. At higher concentrations (10–20 μM), SHK induced cell death, which was completely inhibited by a necroptosis inhibitor, necrostatin-1 (Nec-1), while the cytocidal activity was unaffected by Z-VAD-FMK, strongly suggesting that cell death is induced by SHK at high concentrations through necroptosis. The present data show for the first time that SHK induces cell death in MM cells. SHK efficiently induces apoptosis and combination of heat shock protein inhibitor with low dose SHK enhances apoptosis, while high dose SHK induces necroptosis in MM cells. These findings together support the use of SHK as a potential therapeutic agent for MM.

Oral squamous cell carcinoma (OSCC) develops through a multistep carcinogenic process involving field cancerization. The DEK gene is a proto‐oncogene with functions in genetic and epigenetic modifications, and has oncogenic functions, including cellular proliferation, differentiation, and senescence. DEK overexpression is associated with malignancies; however, the functional roles of DEK overexpression are unclear. We demonstrated that DEK‐expressing cells were significantly increased in human dysplasia/carcinoma in situ and OSCC. Furthermore, we generated ubiquitous and squamous cell‐specific doxycycline (DOX)‐inducible Dek mice (iDek and iDek‐e mice respectively). Both DOX+ iDek and iDek‐e mice did not show differences in the oral mucosa compared with DOX‐ mice. In the environment exposed to carcinogen, DOX‐treated (DOX+) iDek mice showed field cancerization and OSCC development. Microarray analysis revealed that DEK overexpression was mediated by the upregulation of DNA replication‐ and cell cycle‐related genes, particularly those related to the G 1/S transition. Tongue tumors overexpressing DEK showed increased proliferating cell nuclear antigen and elongator complex protein 3 expression. Our data suggest that DEK overexpression enhanced carcinogenesis, including field cancerization, in OSCC by stimulating the G 1/S phase transition and promoting DNA replication, providing important insights into the potential applications of DEK as a target in the treatment and prevention of OSCC.

Serum IgG, IgE and IgM have been shown to enhance the primary antibody responses upon exposure to the soluble antigens recognized by those antibodies. However, how IgA affects these responses remains unknown.We investigated the effects of intravenously administered monoclonal IgA on the immune responses in mice.DBA/1J mice were immunized with ovalbumin in the presence or absence of anti-ovalbumin monoclonal IgA. The Th1 and Th2 immune responses to ovalbumin and the anaphylaxis induced by re-exposure to ovalbumin were measured.IgA complexed with antigen attenuated the primary antibody responses to the antigen in mice, in contrast to IgG2b and IgE. The primary antibody responses, i.e. the de novo synthesis of anti-ovalbumin IgG2a, IgG1 and IgE in the serum, and the subsequent anaphylaxis induced with re-exposure to ovalbumin were reduced by the co-injection of anti-ovalbumin monoclonal IgA at ovalbumin immunization. The Th1, Th2 and Tr1 cytokines interferon-γ, interleukin-4 and interleukin-10, respectively, released from ovalbumin-restimulated cultured splenocytes collected from allergic mice were also reduced by the treatment. The induction of interferon-γ and interleukin-4 secretion by splenocytes from ovalbumin-immunized mice stimulated in vitro with ovalbumin was also significantly reduced by the antigen complexed with anti-ovalbumin IgA.These data suggest that the direct inhibition of Th1 and Th2 activation by anti-ovalbumin monoclonal IgA participates in the inhibition of the primary antibody responses. IgA plays important immunosuppressive roles under physiological and pathological conditions and is a promising candidate drug for the treatment of immune disorders.

Various silicon-containing six-membered-ring molecules were synthesized by Pd-catalyzed coupling reaction of 1,2-bis(dimethylsilyl)arenes with 1,2-diiodoarenes in moderate yields. Their optical properties were investigated via UV–vis absorption, fluorescence and phosphorescence spectroscopies, which indicated that the σ*–π* conjugated system expanded owing to the silicon atoms. Some of the compounds exhibited enhanced luminescence, larger Stokes shift, and phosphorescence in comparison with 9,9,10,10-tetramethyl-9,10-dihydroanthracene. Density functional theory (DFT) studies were also performed to investigate the photophysical properties of the compounds.

軟部肉腫は通常それ自体は疼痛を伴わず無痛性腫瘤を主訴として診断されることが多い．ゆえに深部発生で腫瘤を触知しない症例に対しては疼痛の存在のみで肉腫を疑うことは困難である．今回我々は，鼠径部痛を主訴に医療機関を受診し，保存的加療を行われた後に腫瘤が明らかとなった肉腫の2症例を経験したので報告した．鼠径部痛をきたす疾患は多く，一般整形外科医にとってはその発生頻度の低さからも，鼠径部痛のみで肉腫を疑うことは困難と思われる．しかし迅速かつ的確に軟部肉腫を診断し，早期に治療を開始する事は患者の予後を左右する．大腿から鼠径部は成人軟部肉腫の好発部位であることを念頭におき，保存的加療に抵抗性の場合にはMRIなどを早期に行うことが望ましいと考えられた．

小児孤立性骨嚢腫（Simple Bone Cyst：以下SBC）のactive phaseに，骨穿孔術を施行した症例の治療成績を検討した．2001年1月以降に当院で加療したSBC患者35例のうち，小児SBCのactive phaseで骨穿孔術を施行した7例（男5例，女2例，平均年齢9歳4ケ月）を対象とした．観察期間は平均67.6ケ月，発生部位は，上腕骨近位4例，大腿骨近位3例であった．7例中3例に初回手術後の再発を認め再発率は43％であった．再発を認めた3例には全例に再度骨穿孔術を施行し，最終観察時には7例中6例（85.7％）が良好な結果であった．骨穿孔術は低侵襲であり，特にactive phaseに対しては骨端線損傷の危険性を回避できる有効な治療法であると考えられる．しかし自験例においても再発率が43％と高率であり，低侵襲でかつより再発率が低い方法の開発が望まれる．

Bcl-2 is an intracellular membrane protein that prevents cells from undergoing apoptosis in response to various cell-death signals. It negatively regulates mitochondrial outer membrane permeabilization, which is responsible for the release of apoptogenic factors and the subsequent activation of caspases. A microbial metabolite, aranorosin, was identified as an inhibitor of the anti-apoptotic function of Bcl-2. Based on its structure, a more potent derivative, K050, was synthesized. Apoptosis could be induced in a cell line that overexpressed Bcl-2 when cells were treated with an anti-Fas antibody in addition to K050, at sub-micromolar concentrations. Furthermore, K050 inhibited anti-apoptotic functions regulated by Bcl-2, resulting in a Fas-triggered mitochondrial transmembrane potential loss, the activation of caspase-9, and a morphological change to apoptosis. Inhibition of cell-based function of Bcl-2 and its anti-apoptotic effects could serve as useful pharmacological effects. Thus, a novel aranorosin derivative, K050, could be a potent therapeutic agent against Bcl-2-overexpressing human malignancies.

Intraosseous epidermoid cysts are exceedingly rare. Known as pseudotumors, not true neoplasms, intraosseous epidermoid cysts usually involve the phalanges, the skull, and the toes. Intraosseous epidermoid cysts typically present as destructive osteolytic lesions on X-ray, mimicking malignant bone tumors. Here, we present two cases of an intraosseous epidermoid cyst in the distal phalanx treated with curettage and synthetic bone graft, followed by a review of the relevant literature. In both cases, the patient presented with a painful enlargement of the fingertip following a minor trauma. Magnetic resonance imaging demonstrated lesions involving the distal phalanx that had a low signal on T1-weighted imaging (WI) and a high intensity on T2-WI. In both cases, the lesions were not enhanced by gadolinium. Good remodeling and functional recoveries were obtained. For physically active patients with substantial bone defects, synthetic bone graft may be recommended.

Tetrocarcin A was recently identified as an inhibitor of the anti-apoptotic function of Bcl-2. We synthesized novel tetrocarcin derivatives in order to increase their selective inhibitory activity against Bcl-2. It was found that 21-acetoxy-9-glycosyloxy derivatives had potent Bcl-2 inhibitory activity without significant antimicrobial activity.

Administration of the influenza vaccination to patients with an egg allergy is major health concern. Contaminating egg antigens occasionally induce severe anaphylactic shock in these patients following administration of the vaccination; therefore, the development of a safer vaccination is needed. In the present study, we investigated whether a mixture of four newly and previously generated anti-ovalbumin (OVA) IgA monoclonal antibodies (mAbs) could inhibit both anaphylactic shock upon a subcutaneous OVA challenge and subsequent further sensitization against OVA in passively anti-OVA IgE-sensitized mice and actively sensitized mice with an injection of OVA. The prevention of anaphylaxis by anti-OVA IgA mAbs was suggested to be mediated through the inhibition of OVA binding to allergenic antibodies such as anti-OVA IgE on mast cells and deceleration of the rate of OVA penetration from the injected site into the systemic circulation. Anti-OVA IgA mAbs inhibited further sensitization against OVA in mice actively sensitized with OVA, but did not affect sensitization against the unrelated antigen, phosphorylcholine-keyhole limpet hemocyanin co-injected with OVA. Our findings indicate that adding the anti-egg antigen IgA to the influenza vaccine should reduce not only the risk of inducing anaphylactic shock, but also undesired further sensitization against egg antigens following the vaccination without affecting the intended beneficial effect of the vaccine, namely the upregulation of immune responses to influenza viruses.

Aromatic groups were incorporated into 9H-9-silafluorene units at the 9-position (mono-9H-silafluorenes) and 9,9′-positions (di-9H-9-silafluorenes). The aryl substituents showed weak conjugation to the 9H-9-silafluorene for 9-aryl substituted ones 1–7 and a 9,9′-phenylene substituted one (compound 8) and they exhibited similar absorption and emission spectra. The 9H-9-silafluorene 10 containing a 5,5′-(2,2′-bithiophenyl) group showed a significantly red-shifted absorption and fluorescence maxima in the solid-state. Single-crystal X-ray diffraction studies found J-type aggregated structures formed by intermolecular CH–π interactions (ca. 2.6–2.7 Å). Density functional theory (DFT), time-dependent DFT (TD-DFT), and configuration interaction single (CIS) calculations were conducted to explain the observed optical properties.

Heat shock protein 90 (Hsp90) is a promising target for anti-tumor therapy. We previously reported the anti-tumor activity of a novel Hsp90 inhibitor, KW-2478, in multiple myeloma (MM) as a single agent. In this study, we examined the combinational effect of KW-2478 and bortezomib, a proteasome inhibitor, in vitro and in vivo. In vitro, KW-2478 enhanced bortezomib-induced cell growth inhibition, both in MM cell lines and primary patient MM cells. The combination of KW-2478 and bortezomib also induced caspase activation in MM cell lines. Interestingly, the combination synergistically enhanced the expression of Hsp70B, a homolog of Hsp70, in human MM cells and peripheral blood mononuclear cells, indicating Hsp70B could be a surrogate biomarker for the combination of Hsp90 and proteasome inhibitors. In vivo, the combination of KW-2478 with bortezomib showed synergistic anti-tumor activity without significant body weight loss in a subcutaneously inoculated human myeloma model. Furthermore, the combination also showed synergistic reduction of tumor burden in bone marrow in an orthotopic myeloma model. Our results strongly suggest that combination of KW-2478 with bortezomib could exhibit enhanced anti-tumor activity against human myeloma.

Protein synthesis and autophagic degradation are regulated in an opposite manner by mammalian target of rapamycin (mTOR), whereas under certain conditions it would be beneficial if they occurred in unison to handle rapid protein turnover. We observed a distinct cellular compartment at the trans side of the Golgi apparatus, the TOR-autophagy spatial coupling compartment (TASCC), where (auto)lysosomes and mTOR accumulated during Ras-induced senescence. mTOR recruitment to the TASCC was amino acid- and Rag guanosine triphosphatase-dependent, and disruption of mTOR localization to the TASCC suppressed interleukin-6/8 synthesis. TASCC formation was observed during macrophage differentiation and in glomerular podocytes; both displayed increased protein secretion. The spatial coupling of cells' catabolic and anabolic machinery could augment their respective functions and facilitate the mass synthesis of secretory proteins.

Purpose: The heat shock protein 90 (Hsp90) plays an important role in chaperoning oncogenic client proteins in multiple myeloma (MM) cells, and several Hsp90 inhibitors have shown antitumor activities both in vitro and in vivo. However the precise mechanism of action of Hsp90 inhibitor in MM has not been fully elucidated. Experimental Design: We evaluated the antitumor activities of KW-2478, a nonansamycin Hsp90 inhibitor, in MM cells with various chromosomal translocations of immunoglobulin heavy chain (IgH) loci both in vitro and in vivo. Results: Our studies revealed that exposure of KW-2478 to MM cells resulted in growth inhibition and apoptosis, which were associated with degradation of well-known client proteins as well as a decrease in IgH translocation products (FGFR3, c-Maf, and cyclin D1), and FGFR3 was shown to be a new client protein of Hsp90 chaperon complex. In addition, KW-2478 depleted the Hsp90 client Cdk9, a transcriptional kinase, and the phosphorylated 4E-BP1, a translational inhibitor. Both inhibitory effects of KW-2478 on such transcriptional and translational pathways were shown to reduce c-Maf and cyclin D1 expression. In NCI-H929 s.c. inoculated model, KW-2478 showed a significant suppression of tumor growth and induced the degradation of client proteins in tumors. Furthermore, in a novel orthotopic MM model of i.v. inoculated OPM-2/green fluorescent protein, KW-2478 showed a significant reduction of both serum M protein and MM tumor burden in the bone marrow. Conclusions: These results suggest that targeting such diverse pathways by KW-2478 could be a promising strategy for the treatment of MM with various cytogenetic abnormalities. Clin Cancer Res; 16(10); 2792–802. ©2010 AACR.

Hsp90 is an attractive chemotherapeutic target because it is essential to maturation of multiple oncogenes. We describe the conformational significance of EH21A1–A4, phenolic derivatives of geldanamycin isolated from Streptomyces sp. Their native free structures are similar to the active form of geldanamycin bound to Hsp90 protein. Their conformational character is a probable reason for their high-affinity binding. Lack of toxic benzoquinone in EH21A1–A4 also adds to their potential as lead compounds for anti-tumor drugs.

今回，我々は極めて稀な軸椎骨折を伴った環軸関節回旋脱臼骨折の一例を経験したので，報告する．症例は38歳男性で，平成21年3月，飲酒後誤って階段より転落し頭部を強打して受傷．以後，頚部痛，斜頚を認め翌日近医整形外科受診．神経症状みられず，頚椎牽引行うも症状改善しなかったため，受傷後1週で当院紹介となる．画像上，右側環軸椎関節が回旋位に脱臼しており軸椎上関節突起が一部骨折していた．頸椎牽引の量を増やし加療していたが整復位を得ることができず，全身麻酔下で透視下に徒手整復術を施行し，整復位を得ることができた．整復後画像診断にて，椎骨動脈の異常を認めなかった．整復後カラーにて外固定を継続し，術後2カ月で骨癒合を得ることができ，また不安定性もみられなかった．骨折を伴った環軸関節回旋脱臼は，ほとんど報告がなく極めて稀な症例といえる．今回，我々は観血的治療を行わず，良好な骨癒合を得ることができた．

Hepatocyte nuclear factor 4 (HNF-4) is a liver-enriched transcription factor involved in the expression of many liver-specific genes. In the preceding communication (Hata, S., Tsukamoto, T. and Osumi, T. (1992) Biochim. Biophys. Acta 1131, 211–213), we reported the presence of two isoforms of mRNA for HNF-4 in rat liver and kidney. The longer isoform contained a segment of 30 bases which was not present in the shorter one. As an initial step to determine whether or not other mammals have these mRNA isoforms, we isolated a cDNA for mouse HNF-4 using the rat HNF-4 gene as a probe. The cDNA had an open reading frame for a 465 amino acid polypeptide. The deduced amino acid sequence was remarkably conserved between mouse HNF-4 and rat HNF-4 (99.6% identical). Moreover, like the cDNA for the larger rat isoform, the mouse cDNA contained an extra segment of 30 bp in the coding region near the C-terminus. Blotting analyses showed that the mRNA is about 3.7 kb in size and that a single copy of the gene is present in the mouse genome. Next we carried out the polymerase chain reaction (PCR) using primers located just upstream and downstream of the extra segment. Two PCR products were amplified from a mouse liver cDNA library. Determination of their nucleotide sequences proved that they exactly corresponded to the two rat isoforms. Finally, we amplified a DNA fragment (1.1 kb in size) from mouse genomic DNA using the same PCR primers as above. Its nucleotide sequence unequivocally confirmed that different splice donor sites were used to generate the two isoforms.

KW-2478 is a novel Hsp90 inhibitor with a new chemical structure and is under clinical investigation in multiple myeloma patients. Our previous study revealed that KW-2478 bind to N-terminal ATP/ADP pocket and thus inducing apoptosis in myeloma cells. Others have reported that bortezomib, a proteasome inhibitor, induces apoptosis of myeloma cells through increase of ER stress mediator protein such as Noxa. Since Hsp90 inhibition can also causes cellular stress, combining Hsp90 inhibitor and bortezomib may be of therapeutic benefit. In this study, we assessed the combined effect of KW-2478 with bortezomib using human myeloma cell lines. In OPM2/GFP cells, KW-2478 enhanced caspase activation of bortezomib in vitro and this effect was revealed to be mediated through synergistic induction of Noxa. Interestingly KW-2478 reduced bortezomib-induced level of another ER stress mediator, Puma in the same cells. To see if this in vitro effect is translated into in vivo setting, we evaluated combined anti-tumor activity of KW-2478 with bortezomib in s.c. inoculated NCI-H929 human myeloma model. In this model, twice weekly treatment of KW-2478 at the dose of 50 mg/kg (1/4 of MTD) showed synergistic anti-tumor effect with 1 mg/kg bortezomib, without significant body weight loss. Our previous studies showed that KW-2478 showed anti-tumor activity in bone marrow environment, the combined effect of KW-278 with bortezomib was further evaluated using an orthotopic myeloma model in which OPM2/GFP cells were i.v. inoculated. The combination of KW-2478 with bortezomib showed synergistic reduction of myeloma cells in bone marrow and this effect was reflected in a significant decrease in serum M protein level. Our results strongly suggested that combination of KW-2478 with bortezomib may exhibit the enhanced anti-tumor activity against human myeloma than bortezomib alone. Clinical study for combination of KW-2478 with bortzomib in myeloma patients is warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2636.

Abstract 3772 Poster Board III-708 [Purpose] Bortezomib (BTZ), a proteasome inhibitor, has been introduced into the treatment of multiple myeloma (MM). It shows remarkable response against both relapsed/refractory and newly diagnosed MM. However, it is often encountered that BTZ treatment achieves very short duration of response and permits early drug resistance. Therefore, understanding the mechanisms underlying this drug resistance is necessary to develop novel treatments to overcome this problem. [Materials & Methods] We established two stable BTZ-resistant MM cell lines, KMS-11/BTZ and OPM-2/BTZ, whose IC50 values were respectively 24.7- and 16.6-fold higher than their parental cell lines, under continuous exposure to BTZ. Using these resistant cells, we investigated on their proteasome activity, the alteration of proteasome β5 subunit (PSMB5) gene, misfolded protein accumulation, endoplasmic reticulum (ER) stress, and apoptosis signals including BH3 only proteins in comparison with their parental cells at clinically achievable concentration of BTZ treatment. [Results & Discussion] No activation of caspase -3,-8, and -9 and BH3 only protein, Noxa, which were initially up-regulated in BTZ-treated cells, were noted in BTZ-resistant cells even in the presence of BTZ. These results indicate avoidance of fatal intracellular stress may block transcriptional activation of Noxa in resistant cells at an early phase after BTZ exposure. In gel shift assay detecting NF-kB-DNA complexes, BTZ-resistant cells maintained constitutive NF-kB activation, whereas their parental cells lost its activity in the presence of BTZ. In addition, cellular proteasome activities including chymotrypsin-like and caspase-like activity were markedly inhibited by BTZ treatment in parental cells, and moderately also in BTZ-resistant cells, when detected by fluorogenic substrates specific for each proteasome activity. While time-dependent accumulation of ubiquitinated proteins was prevented only in BTZ-resistant cells, but not in their parental cells after BTZ exposure. Resistance was partly explained by the presence of a unique point mutation, G322A, in the gene encoding PSMB5 in both BTZ-resistant cell lines, which substituted Thr for Ala at the codon 49 in amino acid level. This constitution has been reported to gives rise to the conformational change of BTZ-binding pocket in β5 subunit, which results in partial disruption of the contact between BTZ and chymotrypsin-like active site. Furthermore, BTZ-resistant and parental MM cells had nearly equal expression of cytoplasmic and ER chaperons, however, only BTZ-resistant cells could prevent misfolded protein accumulation and therefore avoid fatal ER stress represented as activation of CHOP and of caspase-4, -12 after BTZ treatment. [Conclusion] Two kinds of stable BTZ-resistant MM cell lines were established, which acquired the unique point mutation (G322A) in BTZ-binding pocket of PSMB5, prevented the accumulation of misfolded proteins probably via reduced affinity of 26S proteasome to BTZ and avoided the development of catastrophic ER stress unlike their parental cells. These cell lines will provide better understanding of the underlying mechanisms of the BTZ-resistance, and will lead to the development of novel treatment strategies for overcoming BTZ-resistance in the patients with MM. Disclosures: Iida: JANSSEN PHARMACEUTICAL: Honoraria; KYOWA KIRIN: Research Funding. Nakashima:KYOWA KIRIN: Employment. Miyazaki:KYOWA KIRIN: Employment. Shiotsu:KYOWA KIRIN: Employment.

Background : HSP90 plays an important role in chaperoning key proteins implicated in malignant disease and is a promising therapeutic target. We now report the in vitro and in vivo activity of a novel HSP90 inhibitor of non-ansamycin, non-purine analogue class, KW-2478, (Kyowa Hakko Kirin) in B-cell malignancies including multiple myeloma (MM), B-cell lymphoma (BCL) and mantle cell lymphoma (MCL) cells, and in primary tumour cells from MM and BCL patients. Procedures: The binding affinity of KW-2478 to HSP90 was examined using immobilised human HSP90a and a biotinylated HSP90 binding agent, radicicol (bRD). The effect of KW-2478 on cell viability, cell growth and apoptosis induction were evaluated in cell lines, with KW-2478 induced changes in major HSP90 client proteins studied by Western blotting analysis. The in vivo anti-tumour activity of KW-2478 was evaluated in a human MM xenograft mouse model,. Primary MM cells were studied using a co-culture system with the HS-5 bone marrow stromal cell line (BMSCs), while primary BCL samples were cultured on CHO cells stably transfected to produce CD40L. Results: KW-2478 inhibited the binding of bRD to HSP90α in concentration-dependent manner with an IC50 value of 3.8 nM. KW-2478 clearly inhibited cancer cell growth in all cell lines, with EC50 values from 101–252 nM in BCL, 81.4–91.4 nM in MCL and 120–622 nM in MM. The drug also exhibited potent growth inhibitory activity in primary CLL (n=3) and NHL (n=2) cells with EC50 values of 40–170 nM and 200–400 nM, respectively. In 2 of 4 human primary myeloma cells, KW-2478 at 2 μM inhibited cell growth by at least 50%. The presence of BMSCs did not affect drug activity against primary MM cells and importantly there was little or no effect on cell number or viability of normal BMSCs at up to 20 μM KW-2478. Exposure of MM and BCL cell lines to KW-2478 for 24 hours resulted in the degradation of HSP90 client proteins (IGF-1Rβ and Raf-1) and the induction of HSP70. KW-2478 also induced PARP cleavage and dephosphorylated Erk1/2 in NCI-H929 cells. Further studies in selected cell lines showed that exposure to 1 μM KW-2478 or lower resulted in the depletion of p53 and Akt proteins, a reduction in nuclear NFKB, and the cleavage of caspase-3. In the NCI-H929 xenograft model, KW-2478 (qd×5, i.v.) showed a statistically significant suppression of tumour growth at the doses of 25, 50, 100 and 200 mg/kg. Moreover, tumour regressions were observed at doses of 100 and 200 mg/kg, with a significant decrease in serum M protein concentration at doses of 50, 100 and 200 mg/kg. No severe KW-2478 toxicity was observed as assessed by treatment-related mortality and body weight change. Conclusions: The novel HSP90 inhibitor KW-2478 showed a potent anti-tumour activity both in vitro and in vivo, including activity in primary patient samples. The agent retained its activity in primary myeloma cells in the presence of BMSCs, suggesting that KW-2478 can overcome the protective effect of the bone marrow microenvironment. Additional pharmacokinetic and safety data support the further development of KW-2478 and the drug is currently undergoing clinical evaluation in a phase I trial.

The hydrolysis of cyclic trimetaphosphate in acidic aqueous solution ([H + ] = 0.1) was investigated by liquid chromatography. The phosphate in the effluent from an anion-exchange column was detected automatically with the use of Mo(V)-Mo(VI) reagent. From the rate constants at 10, 20, 30, 40 and 50°C, the Arrhenius activation energy in 0.1 M hydrochloric acid solution for scission of a P-O-P linkage was estimated as 21.3 kcal/mole.

Decarbonation experiments of composite spheres of calciumcarbonate and carbon powders were carried out to rationalizesupplement of decarbonation heat by simultaneous combustionof carbon, and the effects of chemical combustibility ofcarbon on decarbonation rate were investigated. In experiments,three kinds of powders of charcoal, petroleum cokeand graphite were used as carbon sample. The betterchemically combustible carbon was observed to be the moreeffective at the relatively low heating temperature range,while the worse chemically combustible carbon, was moreeffective at the relatively high heating temperature range.