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Zolbetuximab is a chimeric monoclonal antibody that targets claudin-18.2, a candidate biomarker in patients with advanced gastric/gastroesophageal cancer. This nonrandomized phase 1 study (NCT03528629) enrolled previously treated Japanese patients with claudin-18.2-positive locally advanced/metastatic gastric/gastroesophageal cancer in 2 parts: Safety (Arms A and B, n=3 each) and Expansion (n=12). Patients received zolbetuximab i.v. 800 mg/m

PDF file - 6669K, Antitumor activities of ASP3026 in hEML4-ALK transgenic mice.

Activation of anaplastic lymphoma receptor tyrosine kinase (ALK) is involved in the pathogenesis of several carcinomas, including non–small cell lung cancer (NSCLC). Echinoderm microtubule–associated protein like 4 (EML4)-ALK, which is derived from the rearrangement of ALK and EML4 genes, has been validated as a therapeutic target in a subset of patients with NSCLC. Here, we investigated the effects of ASP3026, a novel small-molecule ALK inhibitor, against ALK-driven NSCLC. ASP3026 inhibited ALK activity in an ATP-competitive manner and had an inhibitory spectrum that differed from that of crizotinib, a dual ALK/MET inhibitor. In mice xenografted with NCI-H2228 cells expressing EML4-ALK, orally administered ASP3026 was well absorbed in tumor tissues, reaching concentrations >10-fold higher than those in plasma, and induced tumor regression with a wide therapeutic margin between efficacious and toxic doses. In the same mouse model, ASP3026 enhanced the antitumor activities of paclitaxel and pemetrexed without affecting body weight. ASP3026 also showed potent antitumor activities, including tumor shrinkage to a nondetectable level, in hEML4-ALK transgenic mice and prolonged survival in mice with intrapleural NCI-H2228 xenografts. In an intrahepatic xenograft model using NCI-H2228 cells, ASP3026 induced continuous tumor regression, whereas mice treated with crizotinib showed tumor relapse after an initial response. Finally, ASP3026 exhibited potent antitumor activity against cells expressing EML4-ALK with a mutation in the gatekeeper position (L1196M) that confers crizotinib resistance. Taken together, these findings indicate that ASP3026 has potential efficacy for NSCLC and is expected to improve the therapeutic outcomes of patients with cancer with ALK abnormality. Mol Cancer Ther; 13(2); 329–40. ©2014 AACR.

PDF file - 64K, Inhibition of ALK phosphorylation and signal transduction in NCI-H2228 cells by ASP3026.

PDF file - 82K, Inhibitory activity of ASP3026 and crizotinib against various TKs.

PDF file - 445K, Growth of H2228-luc cells directly inoculated into the pleural cavity of NOD/SCID mice.

PDF file - 56K, Pharmacokinetic parameters of ASP3026 after 5-day repeated oral dosing of ASP3026 in NOD/SCID mice xenografted with NCI-H2228 cells.

PDF file - 68K

Supplementary Data from MK-2206, an Allosteric Akt Inhibitor, Enhances Antitumor Efficacy by Standard Chemotherapeutic Agents or Molecular Targeted Drugs In vitro and In vivo

Midwives significantly support women with unplanned pregnancies-promoting a shared perspective on the decision-making process. This study aimed to develop a scale to support midwives self-assess their practice of this vital role. Following the derivation of scale items and pilot testing, the final version of the scale was administered to 531 midwives to establish internal consistency and construct criterion-related validity. Through exploratory factor analysis, 35 items with a five-factor structure were retained to form the midwifery practice self-assessment scale to promote shared decision-making in women with unplanned pregnancies. These factors illustrate midwives' general aptitude and competencies in understanding environmental factors, collaborating with significant others and the interprofessional group, forming rapport and problem sharing, focusing on consultation content, and promoting autonomous decision-making. There were high and low scores on the scales after attending the workshops to support the decision-making of women with unplanned pregnancies. The reliability analysis showed acceptable Cronbach's alpha values for the five factors, from .85-.87. The scale was demonstrated to be a reliable and valid measure that would help improve the quality of midwives' practice. This article is protected by copyright. All rights reserved.

466 Background: CLDN18.2, a targetable biomarker, is a tight junction protein normally confined to gastric mucosa of healthy tissue and often retained in G/GEJa. Zolbetuximab, a chimeric IgG1 monoclonal antibody, binds to CLDN18.2 and mediates cancer cell death via antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Phase 2 FAST study results showed prolonged survival with zolbetuximab+EOX (epirubicin, oxaliplatin, capecitabine) vs EOX in CLDN18.2-positive G/GEJa. In ILUSTRO Cohort 2, confirmed partial responses (PRs) and stable disease (SD) were previously reported in 63.2% and 10.5%, respectively, of patients (pts) treated with zolbetuximab+mFOLFOX6 (modified 5-FU, folinic acid, oxaliplatin). Here is an analysis of CLDN18.2 expression and exploratory biomarkers from 2 studies of zolbetuximab alone or with mFOLFOX6 in G/GEJa. Methods: Data are from phase 1 (NCT03528629) and phase 2 (NCT03505320, ILUSTRO Cohorts 1 and 2) studies. Pts with CLDN18.2-positive locally advanced/metastatic G/GEJa received zolbetuximab alone (phase 1 [ineligible for SOC], n = 18; phase 2 Cohort 1 [≥3rd-line], n = 30) or with mFOLFOX6 (phase 2 Cohort 2 [1st-line], n = 21). Archival (any time before treatment), baseline (during screening or ≤3 months before first study treatment), and on-treatment (Cycle 3) tumor samples were collected when possible. CLDN18.2 expression and immune cell populations were assessed by IHC. Blood samples collected longitudinally while on treatment (phase 2 study) were analyzed for ADCC activity (ex vivo cell-based assay), circulating tumor antigens, and circulating tumor DNA (ctDNA). Results: Of 416 screened pts in both studies, 26.2% (n = 109), 9.6% (n = 40), and 36.1% (n = 150) had tumors that were CLDN18.2-positive in 1%–49%, 50%–74%, and ≥75% of tumor cells ( = strong to moderate staining intensity), respectively; 28.1% (n = 117) were CLDN18.2-negative (0/1+). Analysis of pre- and on-treatment tissue samples (n = 8 matched pairs) with zolbetuximab alone showed an increased trend in immune cell infiltration, with CD8+ T cells and CD163+ cells showing the most consistent trend. A trend of increased on-treatment vs baseline ADCC activity was observed in PBMCs collected from pts treated with zolbetuximab alone and with mFOLFOX6. Rapid and deep decreases in circulating tumor antigens (in pts with elevated antigen levels) and ctDNA were observed with best overall responses of both PR and SD. Conclusions: CLDN18.2 is a high-prevalence biomarker in G/GEJa. Treatment with zolbetuximab is associated with tumor and peripheral biomarker changes related to its proposed mechanism of action. Clinical responses observed in pts treated with zolbetuximab and mFOLFOX6 are associated with correlative biomarkers of activity including rapid deep molecular responses (ctDNA). Clinical trial information: NCT03528629 .

Purpose To evaluate masticatory performance (MP), maximum occlusal force (MOF), maximum tongue pressure (MTP) and oral diadochokinesis (ODK) among community-dwelling elderly patients without posterior occlusal support. Methods This study enrolled community-dwelling elderly patients (≥65 years old) who belonged to Eichner A, B4, C1, C2, and C3. Each oral function was statistically compared among groups. Correlations between MP and other variables were examined in Eichner B4 and C patients. Results MP and MOF values in Eichner B4 and C patients were significantly lower than in Eichner A patients. There were significant differences in MTP values between Eichner "A and C2, C3", and "B4 and C2, C3". ODK in Eichner C patients showed significantly lower values compared to Eichner A patients in general. Although there were statistically significant correlations of MP with age, the number of remaining teeth and all oral functions were identified, and multiple regression analysis indicated that MOF and MTP were independently related to MP. Conclusion Oral functions in Eichner B4 and C patients were lower compared to Eichner A patients. MP was significantly correlated with MOF and MTP in elderly patients without posterior occlusal support, suggesting the importance of rehabilitation of MOF and MTP in MP.

// Yoko Ueno 1 , Masamichi Mori 1 , Yoshiteru Kamiyama 1 , Rika Saito 1 , Naoki Kaneko 1 , Eriko Isshiki 2 , Sadao Kuromitsu 1 and Masahiro Takeuchi 1 1 Drug Discovery Research, Astellas Pharma, Inc., Ibaraki, Japan 2 Biological Research Division, Astellas Research Technologies Co., Ltd., Ibaraki, Japan Correspondence to: Yoko Ueno, email: yoko.ueno@astellas.com Keywords: acute myeloid leukemia; FLT3 inhibition; internal tandem duplication; combination therapy; apoptosis Received: January 02, 2019 Accepted: March 04, 2019 Published: April 02, 2019 ABSTRACT Activating internal tandem duplication (ITD) and tyrosine kinase domain (TKD) point mutations in Fms-like tyrosine kinase 3 ( FLT3 ) occur in approximately 30% of patients with acute myeloid leukemia (AML), and confer a poor prognosis with standard cytarabine/anthracycline or azacitidine-based chemotherapy regimens. Gilteritinib is a highly-specific, potent FLT3/AXL inhibitor with demonstrated activity against FLT3-ITD and FLT3-TKD mutations. Compared with salvage chemotherapy, treatment with once-daily oral gilteritinib demonstrated a clinical benefit in patients with FLT3 -mutated relapsed/refractory AML, which led to its recent approval in Japan and the United States. We investigated the effects of gilteritinib combined with cytarabine plus daunorubicin/idarubicin, or combined with azacitidine in human FLT3-ITD –positive ( FLT3-ITD + ) AML cell lines and xenografted mouse models. Gilteritinib induced G 1 arrest and apoptosis in a dose-dependent manner. The addition of cytarabine, daunorubicin, idarubicin, or azacitidine potentiated apoptosis. Gilteritinib alone or combined with cytarabine, daunorubicin, idarubicin, or azacitidine, inhibited anti-apoptotic protein expression in MV4-11 cells. In xenografted mice, administration of cytarabine, idarubicin, or azacitidine in combination with gilteritinib had little impact on plasma or intratumor PK profiles of gilteritinib, cytarabine, idarubicin, or azacitidine. Gilteritinib combined with chemotherapy reduced tumor volume to a greater extent than either gilteritinib or chemotherapy alone. Of note, the addition of cytarabine plus daunorubicin/idarubicin led to tumor regression in mice, with complete regression observed in six out of eight mice in both triple combination groups. These findings support the investigation of gilteritinib combined with chemotherapy in patients with FLT3-ITD + AML, including those who are ineligible for intensive chemotherapy.

This study aimed to explore the competencies of health care practitioners who promote shared decision-making (SDM)-based care for women facing an unplanned pregnancy in Japan. We conducted semistructured interviews with 12 care providers who are pioneers in care for women facing an unplanned pregnancy and adopted a modified grounded theory approach for data analysis. A three-step model for shared decision making in practice (team talk, option talk, and decision talk) was used as a reference. The answers given by the care providers were analyzed to identify the competencies involved in shared decision making. We identified three stages: (i) building trust and promoting women's expression; (ii) discussing women's real needs; and (iii) exploring the intentions and goals expressed by women and finding the best choices while promoting women's autonomy in decision-making. The coordination between the support of partners, families, and professionals, and the care provided to strengthen women's identities facilitated shared decision making. Women facing an unplanned pregnancy can benefit from interventions designed to improve shared decision making. A key component of shared decision making-based care relates to practitioners' raised awareness toward their roles and responsibilities: developing good communication skills and fostering collaboration between all stakeholders.

Anaplastic lymphoma kinase (ALK) is a validated therapeutic target for treating echinoderm microtubule-associated protein-like 4 (EML4)-ALK positive non-small cell lung cancer (NSCLC). We synthesized a series of 1,3,5-triazine derivatives and identified ASP3026 (14a) as a potent and selective ALK inhibitor. In mice xenografted with NCI-H2228 cells expressing EML4-ALK, once-daily oral administration of 14a demonstrated dose-dependent antitumor activity. Here, syntheses and structure-activity relationship (SAR) studies of 1,3,5-triazine derivatives are described.

Summary Advances in the understanding of the molecular basis for acute myeloid leukemia (AML) have generated new potential targets for treatment. Fms-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes in AML and mutations in this gene are associated with poor overall survival. AXL plays a role in the activation of FLT3 and has been implicated in the pathogenesis of AML. The studies reported here evaluated the ability of a novel FLT3/AXL inhibitor, gilteritinib, to block mutated FLT3 in cellular and animal models of AML. Initial kinase studies showed that gilteritinib, a type I tyrosine kinase inhibitor, was highly selective for both FLT3 and AXL while having weak activity against c-KIT. Gilteritinib demonstrated potent inhibitory activity against the internal tandem duplication (FLT3-ITD) and FLT3-D835Y point mutations in cellular assays using MV4–11 and MOLM-13 cells as well as Ba/F3 cells expressing mutated FLT3. Gilteritinib also inhibited FLT3-F691 mutations, although to a lesser degree, in these assays. Furthermore, gilteritinib decreased the phosphorylation levels of FLT3 and its downstream targets in both cellular and animal models. In vivo, gilteritinib was distributed at high levels in xenografted tumors after oral administration. The decreased FLT3 activity and high intratumor distribution of gilteritinib translated to tumor regression and improved survival in xenograft and intra-bone marrow transplantation models of FLT3-driven AML. No overt toxicity was seen in mouse models treated with gilteritinib. These results indicate that gilteritinib may be an important next-generation FLT3 inhibitor for use in the treatment of FLT3 mutation-positive AML. Electronic supplementary material The online version of this article (doi:10.1007/s10637-017-0470-z) contains supplementary material, which is available to authorized users.

// Tatsuya Kawase 1 , Taisuke Nakazawa 1 , Tomohiro Eguchi 1 , Hirofumi Tsuzuki 1 , Yoko Ueno 1 , Yasushi Amano 1 , Tomoyuki Suzuki 1 , Masamichi Mori 1 and Taku Yoshida 1 1 Drug Discovery Research, Astellas Pharma, Tsukuba-shi, Ibaraki, Japan Correspondence to: Tatsuya Kawase, email: tatsuya.kawase@astellas.com Keywords: FLT3; FL; FLT3 inhibitor; gilteritinib; antitumor activity Received: June 20, 2019 Accepted: September 10, 2019 Published: October 22, 2019 ABSTRACT Therapeutic effects of FLT3 inhibitors have been reported in acute myeloid leukemia (AML) with constitutively activating FLT3 mutations, including internal tandem duplication (ITD) and point mutation, which are found in approximately one-third of AML patients. One of the critical issues of treatment with FLT3 inhibitors in FLT3 -mutated AML is drug resistance. FLT3 ligand (FL) represents a mechanism of resistance to FLT3 inhibitors, including quizartinib, midostaurin, and sorafenib, in AML cells harboring both wild-type and mutant FLT3 ( FLT3 wt / FLT3 mut ). Here, we investigated the effect of FL on the efficacy of gilteritinib, a FLT3 inhibitor, in AML-derived cells in vitro and in mice. In contrast to other FLT3 inhibitors, FL stimulation had little effect on growth inhibition or apoptosis induction by gilteritinib. The antitumor activity of gilteritinib was also comparable between xenograft mouse models injected with FL-expressing and mock MOLM-13 cells. In the FLT3 signaling analyses, gilteritinib inhibited FLT3 wt and FLT3-ITD to a similar degree in HEK293 and Ba/F3 cells, and similarly suppressed FLT3 downstream signaling molecules (including ERK1/2 and STAT5) in both the presence and absence of FL in MOLM-13 cells. Co-crystal structure analysis showed that gilteritinib bound to the ATP-binding pocket of FLT3. These results suggest that gilteritinib has therapeutic potential in FLT3-mutated AML patients with FL overexpression.

e16078 Background: Zolbetuximab is a chimeric IgG1 monoclonal antibody that targets Claudin 18.2 (CLDN18.2), a tight junction protein that is normally confined to gastric mucosa but is retained in G/GEJ. A phase 2 (NCT01630083, FAST) trial demonstrated significantly prolonged survival with zolbetuximab + EOX (epirubicin, oxaliplatin, capecitabine) vs EOX in G/GEJ. Two global phase 3 studies are ongoing, comparing zolbetuximab + chemotherapy vs chemotherapy as first-line treatment in CLDN18.2-positive G/GEJ. This study assessed the influence of ethnic differences and chemotherapy on the pharmacokinetics (PK) of zolbetuximab in patients (pts) with G/GEJ and the potential effect of zolbetuximab on chemotherapy PK. Methods: In the Japanese phase 1 and global phase 2 studies, adult pts with CLDN18.2-postitive G/GEJ received zolbetuximab 800 mg/m2 IV on Cycle 1 Day 1 (Cycle 1 Day 3 in Cohort 2 of phase 2) then 600 mg/m2 Q3W. Phase 1 and Cohort 1A of the phase 2 study assessed zolbetuximab monotherapy in 21-day cycles. Cohort 2 of the phase 2 study assessed zolbetuximab + mFOLFOX (5-FU, folinic acid, oxaliplatin; 4 cycles) in 42-day cycles (Q2W from Cycle 1 Day 1). Blood samples for zolbetuximab PK following single and multiple doses, and for chemotherapy (5-FU and oxaliplatin) PK alone or with zolbetuximab were collected. Maximum serum drug concentration (Cmax) and area under the concentration-time curve from 0-21 days (AUC0-21) were assessed. Potential ethnic differences in zolbetuximab exposures across Japanese, other Asian (Korean and Taiwanese), and Western pts, and the potential drug-drug interaction between zolbetuximab and chemotherapy were evaluated. Results: Zolbetuximab concentration profiles and PK parameters were generally comparable across Japanese, other Asian, and Western populations (Table). Zolbetuximab PK properties were similar between monotherapy and combination therapy cohorts (Table). Chemotherapy PK was comparable with and without zolbetuximab. Conclusions: The analyses suggest no apparent ethnic differences in zolbetuximab PK across Japanese/Asian and Western pts and no PK interactions between zolbetuximab and mFOLFOX6. These results support the use of zolbetuximab and mFOLFOX without the need for dose adjustment in global phase 3 trials. Clinical trial information: NCT03505320, NCT03528629. [Table: see text]

Anaplastic lymphoma kinase (ALK) is a validated therapeutic target for treating echinoderm microtubule-associated protein-like 4 (EML4)-ALK positive non-small cell lung cancer (NSCLC). We synthesized a series of 1,3,5-triazine derivatives and identified ASP3026 (14a) as a potent and selective ALK inhibitor. In mice xenografted with NCI-H2228 cells expressing EML4-ALK, once-daily oral administration of 14a demonstrated dose-dependent antitumor activity. Here, syntheses and structure-activity relationship (SAR) studies of 1,3,5-triazine derivatives are described.

Activation of anaplastic lymphoma receptor tyrosine kinase (ALK) is involved in the pathogenesis of several carcinomas, including non–small cell lung cancer (NSCLC). Echinoderm microtubule–associated protein like 4 (EML4)-ALK, which is derived from the rearrangement of ALK and EML4 genes, has been validated as a therapeutic target in a subset of patients with NSCLC. Here, we investigated the effects of ASP3026, a novel small-molecule ALK inhibitor, against ALK-driven NSCLC. ASP3026 inhibited ALK activity in an ATP-competitive manner and had an inhibitory spectrum that differed from that of crizotinib, a dual ALK/MET inhibitor. In mice xenografted with NCI-H2228 cells expressing EML4-ALK, orally administered ASP3026 was well absorbed in tumor tissues, reaching concentrations >10-fold higher than those in plasma, and induced tumor regression with a wide therapeutic margin between efficacious and toxic doses. In the same mouse model, ASP3026 enhanced the antitumor activities of paclitaxel and pemetrexed without affecting body weight. ASP3026 also showed potent antitumor activities, including tumor shrinkage to a nondetectable level, in hEML4-ALK transgenic mice and prolonged survival in mice with intrapleural NCI-H2228 xenografts. In an intrahepatic xenograft model using NCI-H2228 cells, ASP3026 induced continuous tumor regression, whereas mice treated with crizotinib showed tumor relapse after an initial response. Finally, ASP3026 exhibited potent antitumor activity against cells expressing EML4-ALK with a mutation in the gatekeeper position (L1196M) that confers crizotinib resistance. Taken together, these findings indicate that ASP3026 has potential efficacy for NSCLC and is expected to improve the therapeutic outcomes of patients with cancer with ALK abnormality. Mol Cancer Ther; 13(2); 329–40. ©2014 AACR.

Novel roles for the serine protease plasmin have been implicated recently in physiological and pathological processes. However, whether plasmin is involved in erythropoiesis is not known. In the present study, we studied the consequences of plasminogen deficiency on erythropoiesis in plasminogen-deficient (Plg knockout [KO]) mice. Erythroid differentiation was attenuated in male Plg KO mice and resulted in erythroblastic accumulation within the spleen and bone marrow, with increased apoptosis in the former, erythrocytosis, and splenomegaly, whereas similar erythropoietic defect was less prominent in female Plg KO mice. In addition, erythrocyte lifespan was shorter in both male and female Plg KO mice. Erythropoietin levels were compensatory increased in both male and female Plg KO mice, and resulted in a higher frequency of burst-forming units-erythroid within the spleen and bone marrow. Surprisingly, we found that male Plg KO mice, but not their female counterparts, exhibited normochromic normocytic anemia. The observed sex-linked erythropoietic defect was attributed to decreased serum testosterone levels in Plg KO mice as a consequence of impaired secretion of the pituitary luteinizing hormone (LH) under steady-state condition. Surgical castration causing testosterone deficiency and stimulating LH release attenuated erythroid differentiation and induced anemia in wild-type animals, but did not further decrease the hematocrit levels in Plg KO mice. In addition, complementation of LH using human choriogonadotropin, which increases testosterone production, improved the erythropoietic defect and anemia in Plg KO mice. The present results identify a novel role for plasmin in the hormonal regulation of postnatal erythropoiesis by the LH-testosterone axis.

In small cell lung carcinoma (SCLC), acetylcholine (ACh) is synthesized and secreted, and it acts as an autocrine growth factor through activation of its receptors, muscarinic receptor (mAChR) and nicotinic receptor (nAChR). Alteration of tumor growth by blockade of M3 mAChR in a human SCLC cell line, NCI-H82, was investigated in the present study. We used a highly selective M3 muscarinic antagonist, N-(2-[3-([3R]-1-(cyclohexylmethyl)-3-piperidinyl]methylamino)-3-oxopropyl]amino-2-oxoethyl)-3,3,3-triphenyl-propioamide (J-115311). Our results show that J-115311 inhibited the increased intracellular calcium elicited by carbachol, a muscarinic agonist, in SCLC cells. J-115311 also inhibited SCLC cell growth in vitro. In a mouse orthotopic xenograft model, J-115311 dose-dependently reduced tumor growth when NCI-H82 cells were inoculated into the upper left lobe of the lung. These findings indicate that blockade of M3 mAChR can suppress tumor growth in SCLC, suggesting the potential therapeutic utility of M3 muscarinic antagonists as anti-cancer agents. Keywords:: selective M3 muscarinic antagonist, M3 muscarinic receptor, small cell lung carcinoma, orthotopic xenograft model, non-neuronal cholinergic system

ErbB3 receptor tyrosine kinase has been shown to induce tumor progression in several types of cancer through heterodimerization with ErbB2. However, the role of ErbB3 and its ligand heregulin (HRG) in tumor metastasis remains poorly understood. In the present study, we tried to clarify their contributions to the metastasis of ErbB3-overexpressing B16-BL6 melanoma cells. Stimulation with HRG induced phosphorylation of ErbB3 and metastatic properties including MMP-9 expression, invasion, adhesion and experimental lung metastasis in vivo. These cellular responses were blocked by inhibiting the tyrosine kinase activity of EGFR with PD153035. In addition, phosphorylation of EGFR was rapidly induced by HRG, suggesting that EGFR is a possible heterodimeric counterpart of ErbB3. RNA interference demonstrated that subcutaneous tumor growth and angiogenesis was attenuated by inactivation of ErbB3 in cancer cells. Although experimental pulmonary metastasis was not affected by the knockdown of ErbB3, spontaneous metastasis was, even when primary tumors in the foot pad were amputated at a similar size. These results indicate that HRG-induced activation of ErbB3 via EGFR promotes tumor growth and metastasis of melanoma cells.

A 28-year-old woman presented with right flank pain. A large, firm, fixed mass was palpable in the right side of the abdomen. Computed tomography revealed a solid mass of the right kidney with extension into the renal vein and inferior vena cava. The patient underwent right radical nephrectomy with en bloc resection of the inferior vena cava containing tumor thrombus and right adrenalectomy. Histologically the tumor consisted of small tumor cells with rosette formation. Immunohistochemical staining was positive for CD99 and NSE. Analysis with polymerase chain reaction (PCR) demonstrated the EWS/FLI1 fusion products resulting from a chromosomal translocation. These findings were consistent with primary renal primitive neuroectodermal tumor (PNET). Two months after surgery, multiple lung, liver and lymph node metastases were found. The patient received 2 cycles of chemotherapy with cisplatin, ifosfamide, etoposide, resulting in a partial remission. She subsequently received 1 cycle chemotherapy with paclitaxel and carboplatin, resulting in no response. The metastatic lung and liver diseases progressed and she died 5 months after diagnosis.

Epidermal growth factor receptor (EGFR) has been shown to be activated by specific ligands as well as other cellular stimuli including tumor necrosis factor-alpha (TNF-alpha). In the present study, we found that cellular stress suppressed ligand-mediated EGFR activity. Both TNF-alpha and osmotic stress rapidly induced phosphorylation of EGFR. This phosphorylation of EGFR and the activation of mitogen-activated protein kinases and NF-kappaB occurred independently of the shedding of extracellular membrane-bound EGFR ligands and intracellular EGFR tyrosine kinase activity. Transforming growth factor-beta-activated kinase 1 (TAK1) was involved in the TNF-alpha-induced signaling pathway to EGFR. In addition, experiments using chemical inhibitors and small interfering RNA demonstrated that p38 alpha is a common mediator for the cellular stress-induced phosphorylation of EGFR. Surprisingly, the modified EGFR was not able to respond to its extracellular ligand due to transient internalization through the clathrin-mediated mechanism. Furthermore, turnover of p38 activation led to dephosphorylation and recycling back to the cell surface of EGFR. These results demonstrated that TNF-alpha has opposite bifunctional activities in modulating the function of the EGFR.

The cell cycle G 2 /M specific inhibitor cytolethal distending toxin (CDT) from Actinobacillus actinomycetemcomitans is composed of CdtA, CdtB, and CdtC coded on the cdtA , cdtB , and cdtC genes that are tandem on the chromosomal cdt locus. A. actinomycetemcomitans CdtA has the lipid binding consensus domain, the so-called “lipobox”, at the N-terminal signal sequence. Using Escherichia coli carrying plasmid pTK3022, we show that the 16th residue, cysteine, of CdtA bound [ 3 H]palmitate or [ 3 H]glycerol. Further, posttranslational processing of the signal peptide, CdtA, was inhibited using globomycin, an inhibitor of lipoprotein-specific signal peptidase II. Fractionation and immunoblotting show the lipid-modified CdtA is present in the outer membrane. Immunoprecipitation and the pull-down assay of the CDT complex from E. coli carrying a plasmid containing cdtABC demonstrated that the CDT complex in the periplasm is composed of CdtA, CdtB, and CdtC and that the CDT complex in culture supernatant is an N-terminally truncated (36 to 43 amino acids) form of CdtA (CdtA′), CdtB, and CdtC. This suggests that CDT is present as a complex both in the periplasm and the supernatant where CdtA undergoes posttranslation processing to CdtA′ in the process of biogenesis and secretion of CDT holotoxin into the culture supernatant. Site-directed mutagenesis of the 16th cysteine residue to glycine in CdtA altered localization of CdtA in the cell and reduced the amount of CDT activity in the culture supernatant. This suggests that CDT forms a complex inside the periplasm for lipid modification where posttranslational processing of CdtA plays an important role for the efficient production of CDT holotoxin into the culture supernatant.

Time-resolved measurements of FT-IR and Raman spectra and X-ray diffraction have been performed to trace the structural changes in the solvent-induced crystallization of syndiotactic polystyrene glass sample. After the exposure of the glassy sample into the atmosphere of such organic solvent as toluene, benzene, or chloroform, the IR and Raman bands characteristic of the regular helical conformation of (T2G2)2 type were found to increase the intensities with time. Some time delay was found for the appearance of the bands with different critical sequence lengths, leading to the concrete description of the growth of regular helical chain from the random coil. The X-ray scattering intensity began to increase largely at the timing of the appearance of the IR and Raman bands of long helical sequences, indicating that the crystal lattice starts to be formed at the timing when the long segments of regular helical form are gathered together. The rate of this crystallization process depends on the kind of the used...

Solvent-induced crystallization behaviour has been investigated for the glass sample of syndiotactic polystyrene. The time-resolved measurements of the Fourier-transform infrared and Raman spectra and the X-ray diffraction profile revealed the molecular and crystal structural change during the ordering process of polymer chains. On the basis of the idea of critical sequence length, the structural change in the conformational ordering process could be deduced. The crystallization rate was found to be affected remarkably by the kind of solvent: chlorofor gave higher crystallization rate than benzene and toluene. This tendency was in contrast to that observed for the gelation process from the dilute solution: the gelation rate was chloroform << benzene. This high contrast between the crystallization and gelation rates could be interpreted qualitatively in terms of the difference in the solubility of polymer in these solvents.

冬期および夏期に漁獲されたマサバを用い, 死後変化に対する貯蔵温度の影響を検討した。魚体が完全硬直に達するまでの時間は, 冬期夏期ともに5℃で貯蔵したときが最も長かった。背肉中のATP, IMP, イノシン, およびクレアチンリン酸の含量の変化は冬期では5℃で貯蔵したときに最も遅く, 夏期では0℃で貯蔵したときに比較して5℃および10℃で貯蔵したときの方が遅かった。K値は冬期夏期ともに10℃で貯蔵したときの上昇速度が速かった。筋肉破断強度の経時変化は冬期は顕著に, 夏期はわずかではあるが5℃で貯蔵したときに高い値を維持した。以上の結果から, マサバの死後変化を遅くするための貯蔵温度は, 漁期を問わず5℃程度が適当であると考えられた。

Actinobacillus actinomycetemcomitans has been shown to produce a soluble cytotoxic factor(s) distinct from leukotoxin. We have identified in A. actinomycetemcomitans Y4 a cluster of genes encoding a cytolethal distending toxin (CDT). This new member of the CDT family is similar to the CDT produced by Haemophilus ducreyi . The CDT from A. actinomycetemcomitans was produced in Escherichia coli and was able to induce cell distension, growth arrest in G 2 /M phase, nucleus swelling, and chromatin fragmentation in HeLa cells. The three proteins, CDTA, -B and -C, encoded by the cdt locus were all required for toxin activity. Antiserum raised against recombinant CDTC completely inhibited the cytotoxic activity of culture supernatant and cell homogenate fractions of A. actinomycetemcomitans Y4. These results strongly suggest that the CDT is responsible for the cytotoxic activity present in the culture supernatant and cell homogenate fractions of A. actinomycetemcomitans Y4. This CDT is a new putative virulence factor of A. actinomycetemcomitans and may play a role in the pathogenesis of periodontal diseases.

We report a rare case of squamous cell carcinoma in the renal pelvis of a horseshoe kidney. An 80-year-old woman was referred to the National Nagano Hospital for the examination of occult blood in her urine. Microscopic hematuria was found, but pyuria was not seen. Computed tomography and magnetic resonance imaging showed a mass in the left renal pelvis of the horseshoe kidney. No renal stone or hydronephrosis was found. Cytopathological examination in the voided urine specimen was positive. Left nephroureterectomy with the splitting of the isthmus of the horseshoe kidney was performed without renal pedicle clamping using a microwave tissue coagulator. No bleeding was encountered after separating the isthmus. A final pathological diagnosis of squamous cell carcinoma with a tumor thrombus was made. Lymph node metastasis had developed and rapidly progressed and the patient died of disseminated malignancy 4 months after the operation. We reviewed 24 cases of renal pelvic tumor in horseshoe kidneys previously reported in Japan. Seven cases (30%) included components of squamous cell carcinoma. The incidence is higher than that of renal pelvic tumors in the general population.

Background FMS-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes in acute myeloid leukemia (AML). Activating mutations in FLT3 such as internal tandem duplications (ITD) at the juxtamembrane domain are present in approximately 25-30% of newly diagnosed AML cases. Patients with AML harboring the FLT3-ITD mutation have poorer prognosis following the current induction chemotherapy treatment of cytarabine (AraC) and an anthracycline (daunorubicin [DNR] or idarubicin [IDR]). Azacitidine (Aza) is a treatment option for AML patients who are not eligible for intensive chemotherapy. Gilteritinib, a novel, small molecule inhibitor of the tyrosine kinases FLT3/AXL, is in Phase 3 development for treatment of FLT3 mutation-positive AML, including patients with FLT3-ITD mutations. In previous preclinical studies, gilteritinib has demonstrated superior antitumor effects when given in combination with AraC and either DNR or IDR compared with combination chemotherapy. Here, we report preclinical antileukemic activity of gilteritinib in combination with Aza against FLT3-ITD mutation-positive AML. Methods: The antitumor effects of gilteritinib alone and in combination with Aza were investigated in MV4-11 cells harboring the FLT3-ITD mutation. Apoptosis was detected using annexin V staining via flow cytometry; PARP cleavage was evaluated via Western blot analysis. Anti-apoptotic protein expression was evaluated by Western blot. Furthermore, the combination antitumor effects were evaluated in MV4-11-xenografted nude mice administered once-daily, oral gilteritinib at 3 mg/kg for 21 days alone and concomitantly with once-daily intravenous Aza at 3 mg/kg for 5 days. Pharmacokinetic parameters were also investigated in MV4-11 xenografted nude mice. Statistical differences in antitumor effects of combination therapy versus gilteritinib alone and combination therapy versus Aza alone were assessed on Day 21 using the Student's t-test. Results: Gilteritinib treatment for 48h resulted in an induction of apoptosis in MV4-11 cells as determined by an increase in annexin V-positive cells. When used in combination, gilteritinib augmented the Aza-induced increase in annexin V-positive cells. Gilteritinib also decreased the expression of anti-apoptotic proteins such as MCL-1, BCL2L10, and survivin, which are reported to be important in chemotherapy sensitivity, following 24h treatment. In combination with annexin results, an increase in PARP cleavage was also observed in MV4-11 cells following gilteritinib treatment; cells co-treated with gilteritinib and Aza showed a further increase in PARP cleavage. In mice xenografted with MV4-11 cells, oral gilteritinib (3 mg/kg/day) inhibited the tumor growth by 71%; Aza alone did not suppress tumor growth. The tumor volume in the doublet combination group (94% growth inhibition) was significantly smaller than in each single treatment group on Day 21 (Figure). No obvious influences on body weight, behavior, or diarrhea were noted in the combination group. Plasma concentrations for either gilteritinib or Aza were not considerably increased by their use in combination therapy when compared with the concentrations observed with single treatment. Conclusions: Gilteritinib treatment augmented Aza-induced apoptosis in part by reductions of anti-apoptotic protein expressions in vitro. In the non-clinical models used, gilteritinib, in combination with Aza, showed superior antitumor efficacy compared with each single agent alone. These findings support the development of gilteritinib in combination with Aza as a potential treatment of FLT3-ITD mutation-positive AML. Figure Antitumor Effect of Gilteritinib in Combination with Azacitidine in Mice Xenografted with MV4-11 Cells ***P Disclosures Ueno: Astellas Pharma Inc: Employment. Mori:Astellas Pharma Inc: Employment. Kamiyama:Astellas Pharma Inc: Employment. Kaneko:Astellas Pharma Inc: Employment. Isshiki:Astellas Pharma Inc: Employment. Takeuchi:Astellas Pharma Inc: Employment.

Adipogenesis is directed by both transcriptional network and posttranslational modification of chromatin structure. Although adipogenesis in vivo proceeds in collagen-rich extracellular matrix (ECM) environments, the impact of ECM proteins and their modifying enzymes on the epigenetic regulation of adipogenesis has been largely unknown. We aimed to define the role of fibrillar type I collagen and its modifying enzymes in regulating adipogenic chromatin signatures and gene regulation in the in vivo-like settings. Adipogenic cocktail induces a robust increase in the level of protranscriptional acetylated histone H3 at lysine 9 (H3K9ac) within 24 h. When cultured atop fibrillar type I collagen gel, however, H3K9ac levels in differentiating 3T3-L1 cells are substantially reduced. The suppression of adipogenic histone mark in differentiating 3T3-L1 cells is type I collagen density dependent and released by heat denaturing of the subjacent collagen substratum, pointing to the critical role played by the triple-helical structure of type I collagen. By probing adipogenic collagenolysis with a series of proteinase inhibitors, matrix metalloproteinase (MMP) family members are found to be responsible for adipogenic collagenolysis. At the same time, MMP inhibitor specifically blocked the adipogenic induction of H3K9ac. By targeting individual MMP using small interfering RNA oligos, MMP14 was identified as the major adipogenic MMP critical for H3K9 acetylation. Consistently, MMP14-null adipose tissues display diminished protranscriptional histone mark H3K9ac while maintaining repressive histone mark tri-methylated histone H3 at lysine 9 (H3K9me3). Taken together, MMP14-dependent collagenolysis plays the major role in regulating adipogenic histone marks by releasing the epigenetic constraints imposed by fibrillar type I collagen.

A new medium-sized heterocycle, 5H,7H-dibenzo[b,g][1,5]diselenocine 1, has been synthesized; the reaction of the selenoxide of 1 with two equivalents of CF3SO3SiMe3 gave the stable diselenide dication salt, this is a new method for the preparation of the diselenide dication.

The transannular reaction of 5H,7H-dibenzo [b,g][1,5] diselenocine Se-oxide 1 or N-methyl-5H,7H-dibenzo[b,g][1,5]selenazocine Se-oxide 2 with 1 equiv. of silyl triflates, CF3SO3SiMe3 and CF3SO3SiMe2But, produced a new hypervalent selenurane which consists of two Unsymmetrical apical ligands such as the hydroxy group and the selenonio or ammonio group.

The serine/threonine kinase Akt lies at a critical signaling node downstream of phosphatidylinositol-3-kinase and is important in promoting cell survival and inhibiting apoptosis. An Akt inhibitor may be particularly useful for cancers in which increased Akt signaling is associated with reduced sensitivity to cytotoxic agents or receptor tyrosine kinase inhibitors. We evaluated the effect of a novel allosteric Akt inhibitor, MK-2206, in combination with several anticancer agents. In vitro, MK-2206 synergistically inhibited cell proliferation of human cancer cell lines in combination with molecular targeted agents such as erlotinib (an epidermal growth factor receptor inhibitor) or lapatinib (a dual epidermal growth factor receptor/human epidermal growth factor receptor 2 inhibitor). Complementary inhibition of erlotinib-insensitive Akt phosphorylation by MK-2206 was one mechanism of synergism, and a synergistic effect was found even in erlotinib-insensitive cell lines. MK-2206 also showed synergistic responses in combination with cytotoxic agents such as topoisomerase inhibitors (doxorubicin, camptothecin), antimetabolites (gemcitabine, 5-fluorouracil), anti-microtubule agents (docetaxel), and DNA cross-linkers (carboplatin) in lung NCI-H460 or ovarian A2780 tumor cells. The synergy with docetaxel depended on the treatment sequence; a schedule of MK-2206 dosed before docetaxel was not effective. MK-2206 suppressed the Akt phosphorylation that is induced by carboplatin and gemcitabine. In vivo, MK-2206 in combination with these agents exerted significantly more potent tumor inhibitory activities than each agent in the monotherapy setting. These findings suggest that Akt inhibition may augment the efficacy of existing cancer therapeutics; thus, MK-2206 is a promising agent to treat cancer patients who receive these cytotoxic and/or molecular targeted agents. Mol Cancer Ther; 9(7); 1956–67. ©2010 AACR.

Inflammation in the tumor stroma greatly influences tumor development. In the present study, we investigated the roles of fibroblast growth factor (FGF)-2-induced chronic inflammation in the development of 4T1 murine mammary tumors. Administration of FGF-2 into the tumor inoculation site during the initial phase of tumor growth enhanced tumor growth and pulmonary metastasis as well as microvessel density in tumor tissues in normal but not in nude mice. Infiltration of T lymphocytes and macrophages, recruitment of pericytes/vascular mural cells in neovascular walls, and the expression levels of cyclooxygenase (COX)-2 and vascular endothelial growth factor A (VEGFA) were also enhanced in the FGF-2-activated host stroma of normal mice. In addition, FGF-2-induced tumor growth and metastasis was abrogated by administration of either an immunosuppressant, FK506, or a COX-2 inhibitor. FGF-2 enhanced prostaglandin E(2) secretion in cultured T lymphocytes. In addition, VEGFA secretion was increased in a co-culture of T lymphocytes and fibroblasts in vitro. These results indicate that the massive infiltration of T lymphocytes into FGF-2-activated host stroma during the initial phase of tumor growth enhances neovascular stability by regulating endogenous COX-2 and VEGFA levels because both compounds are known to play important roles in marked 4T1 mammary tumor development via FGF-2-induced inflammatory reactions.

ErbB3 receptor tyrosine kinase has been shown to induce tumor progression in several types of cancer through heterodimerization with ErbB2. However, the role of ErbB3 and its ligand heregulin (HRG) in tumor metastasis remains poorly understood. In the present study, we tried to clarify their contributions to the metastasis of ErbB3-overexpressing B16-BL6 melanoma cells. Stimulation with HRG induced phosphorylation of ErbB3 and metastatic properties including MMP-9 expression, invasion, adhesion and experimental lung metastasis in vivo. These cellular responses were blocked by inhibiting the tyrosine kinase activity of EGFR with PD153035. In addition, phosphorylation of EGFR was rapidly induced by HRG, suggesting that EGFR is a possible heterodimeric counterpart of ErbB3. RNA interference demonstrated that subcutaneous tumor growth and angiogenesis was attenuated by inactivation of ErbB3 in cancer cells. Although experimental pulmonary metastasis was not affected by the knockdown of ErbB3, spontaneous metastasis was, even when primary tumors in the foot pad were amputated at a similar size. These results indicate that HRG-induced activation of ErbB3 via EGFR promotes tumor growth and metastasis of melanoma cells.

We have shown that the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib ('Iressa', ZD1839) inhibits the development of intrahepatic metastases of hepatocellular carcinoma CBO140C12, and EGFR transactivation by tumor necrosis factor-alpha is a possible target of gefitinib. In the present study, we focused on the fibronectin (FN)-dependent signaling pathway to further elucidate the antimetastatic activity of gefitinib in CBO140C12 cells. We initially observed that FN induced activation of extracellular signal-regulated kinase (ERK), p38 and Akt, as well as cell proliferation and CBO140C12 cell invasion. These responses were mediated by EGFR tyrosine kinase, because gefitinib inhibited these effects of FN. FN-induced ERK, p38 and Akt activation was partly blocked by the Arg-Gly-Asp (RGD)-pseudo-peptide FC-336, anti-alphav integrin antibody RMV-7, the broad-spectrum matrix metalloprotease inhibitor GM6001 and the broad spectrum a disintegrin and metalloprotease (ADAM) inhibitor TAPI-1. But these inhibitors had no effect on EGF-induced signaling pathways, suggesting that integrins and ADAM may be upstream components of EGFR in these responses. These results suggest that FN-induced activation of ERK, p38, Akt, cell proliferation and invasion was mediated, at least in part, via integrins, ADAM and EGFR, and that this FN-induced signaling pathway might be involved in the antimetastatic activity of gefitinib.

We experienced a 78-year-old man in whom transient pulmonary hypertension was documented during epileptic seizure. A Swan-Ganz catheter demonstrated that pulmonary arterial pressure rose immediately after the initiation of the seizure, which was followed by systemic hypertension. The plasma level of angiotensin II increased in pulmonary circulation during the seizures; however, catecholamines did not change from the baseline. The patient responded well to treatment with an antiepileptic drug and losartan potassium.

Cytolethal distending toxin (CDT), produced by Actinobacillus actinomycetemcomitans, is a putative virulence factor in the pathogenesis of periodontal diseases. It is a cell cycle specific inhibitor at the G2/M transition. CDTB, one of the subunits of the CDT holotoxin, is implicated in a genotoxic role after entering the target cells, whereby chromosomal damage induces checkpoint phosphorylation cascades. CDTB microinjected into the cytoplasm was shown to localize in the nucleus and induce chromatin collapse. To investigate the molecular mechanism involved in nuclear transport of CDTB, we used transient expression and microinjection of a CDTB-green fluorescent protein (GFP) fusion protein. After microinjection, His-tagged CDTB-GFP entered the nucleus in 3-4 h. Leptomycin B did not increase the speed of entry of the fusion protein, suggesting that the relatively slow entry of the fusion protein is not due to the CRM1-dependent nuclear export of the protein. Nuclear localization of the CDTBGFP was temperature-dependent. An in vitro transport assay demonstrated that the nuclear localization of CDTB is mediated by active transport. An assay using transient expression of a series of truncated CDTB-GFP fusion proteins revealed that residues 48-124 constitute the minimum region involved in nuclear transport of CDTB. A domain swapping experiment of the region involved in nuclear transport of CDTB with an SV40 T nuclear localization signal indicated that CDTB is composed of two domains, an N-terminal domain for nuclear transport and a C-terminal active domain. Our results strongly suggest that nuclear localization of CDTB is required for the holotoxin to induce cytodistension and cell cycle block. This is the first demonstration that a bacterial toxin possessing a unique domain for nuclear transport is transferred to the animal cell nucleus by active transport.

EML4-ALK translocation has been validated as a therapeutic target in a subset of non-small-cell lung cancer (NSCLC) patients. In clinical settings, crizotinib has shown promising response rates in patients with EML4-ALK-positive NSCLC. However, disease relapse has been observed after an initial favorable response and several resistance mechanisms, including the gatekeeper L1196M mutation, have been reported to date. We have discovered ASP3026, a selective inhibitor for the ALK kinase. ASP3026 potently inhibited ALK kinase activity with an IC50 value of 3.5 nmol/L and showed more selective inhibition in a Tyr-kinase panel than crizotinib. Using NCI-H2228, a human NSCLC cell line endogenously expressing EML4-ALK, we determined the anti-proliferative effect of ASP3026. ASP3026 inhibited the in vitro proliferation with an IC50 value of 64.8 nmol/L and induced dose-dependent anti-tumor effects starting at 1 mg/kg with marked regression at 10, 30 and 100 mg/kg in a subcutaneous tumor model. Body weights were unaffected. ASP3026 at 100 mg/kg also showed tumor regression against EML4-ALK-driven tumor model with gatekeeper mutation while crizotinib at 100 mg/kg was ineffective. In an NCI-H2228 orthotopic lung model, ASP3026 at 30 mg/kg induced sustained tumor regression during the experimental period. In contrast, crizotinib at 30 mg/kg induced initial regression during the first 7 days but regrowth of the tumor was observed despite continuous crizotinib administration. After tumor regrowth had been established in crizotinib-treated mice, substantial tumor regression was achieved by subsequent administration of ASP3026 at 30 mg/kg. Taken together, these results suggest that ASP3026 in the non-clinical setting shows superior efficacy to crizotinib in crizotinib-resistant models.

7070 Background: Activating mutations in FLT3 receptor tyrosine kinase, characterized by internal tandem duplication (ITD) and tyrosine kinase domain point mutations near position D835, have been i...