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FMS-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes in acute myeloid leukemia (AML), and its activating mutations including internal tandem duplications (ITDs) and missense point mutations in the tyrosine kinase domain (TKD) are found in approximately 30% of AML patients. Although the patients initially well responded to FLT3 inhibitors such as gilteritinib, the most cases relapsed within a few months after the initiation of treatment. For these patients who relapsed after FLT3 inhibitor-based therapy, no effective treatment is available. Mechanisms of resistance are not fully elucidated, but FLT3-ITD-TKD double mutant is considered as one of possible mechanisms. We have discovered a novel FLT3 inhibitor OTS447 that shows the potent and selective inhibition in both FLT3-ITD and FLT3-ITD-TKD double mutants. OTS447 was identified through the screening of our in-house proprietary compound library. It showed potent inhibitory activity against FLT3 with an IC50 value of 0.19 nM. The selectivity of OTS447 was investigated by human kinase profile assay. Among 371 human kinases tested, there were only seven (including FLT3) whose activity were inhibited by 80% or more at 5 nM of OTS447. We then examined the anti-proliferative effects in FLT3-ITD and -wild type (WT) AML cell lines. The proliferation of FLT3-ITD cell lines, MV4-11 and MOLM13, was more strongly suppressed than that of FLT3-WT cell lines. We also investigated the inhibitory effects to FLT3-ITD-TKD double mutants using Ba/F3 cells. FLT3-ITD-D835Y mutant was inhibited as strongly as FLT3-ITD mutant, and FLT3-ITD-F691I mutant was more strongly inhibited than Ba/F3 parental cells. These data suggest that OTS447 has selective inhibitory activity against FLT3 mutants. To confirm that FLT3 inhibition by OTS447 leads to the anti-proliferative effect, FLT3 autophosphorylation and phosphorylation of downstream molecules such as STAT5, ERK and AKT were examined. Both autophosphorylation of FLT3 and phosphorylation of downstream molecules were decreased by OTS447 in dose-dependent manner. Moreover, treatment of OTS447 induced the apoptosis and increased sub-G1 population in MV4-11 cell. Finally, we tested OTS447 anti-tumor effect using MV4-11 mouse xenograft model. OTS447 showed potent growth inhibition against MV4-11 tumor in dose-dependent manner. In summary, we discovered OTS447, a potent and selective FLT3 inhibitor, that can inhibit not only ITD mutation but also ITD-TKD mutations of the FLT3 gene. OTS447 possesses cytotoxic activity induced by inhibition of FLT3 signaling pathway and has anti-tumor activity in mouse xenograft model. We are pursuing further optimization of the inhibitor aiming for the effective treatment of AML patients with several types of FLT3-activating mutations. Citation Format: Yasuhide Okamoto, Naofumi Takamatsu, Takashi Miyamoto, Akiko Taira, Kozue Toyota, Yukiho Imai, Shinobu Fujii, Rumiko Ono, Kyoko Adachi, Yo Matsuo, Suyoun Chung, Jae-Hyun Park. Discovery of OTS447, a highly potent and selective inhibitor of FLT3 for the treatment of AML patients with FLT3-ITD/TKD mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5456.

Transient ischemic attack (TIA) is a predictor for cerebral infarction (CI), and early diagnosis of TIA is extremely important for the prevention of CI. We set out to identify novel antibody biomarkers for TIA and CI, and detected matrix metalloproteinase 1 (MMP1), chromobox homolog 1 (CBX1), and chromobox homolog 5 (CBX5) as candidate antigens using serological identification of antigens by recombinant cDNA expression cloning (SEREX) and Western blotting to confirm the presence of serum antibodies against the antigens. Amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) revealed that serum antibody levels were significantly higher in patients with TIA or acute-phase CI (aCI) compared with healthy donors (P < 0.01). Spearman’s correlation analysis and multivariate logistic regression analysis demonstrated that levels of anti-MMP1, anti-CBX1, and anti-CBX5 antibodies were associated with age, cigarette-smoking habits, and blood pressure. Thus, serum levels of antibodies against MMP1, CBX1, and CBX5 could potentially serve as useful tools for diagnosing TIA and predicting the onset of aCI.

Liver-specific Expression of Constitutively Active Gsα Leads to Hyperglycemia With Impaired Insulin Secretion The ubiquitously-expressed G protein Gsα couples hormone receptors to the stimulation of intracellular cAMP generation. We previously showed that mice with liver-specific Gsα deficiency (LGsKO) have improved glucose tolerance and enlarged pancreatic islets1. In the present study, we have generated mice with liver-specific expression of a constitutively activated Gsα (LGsR201C) by breeding mice containing a Lox-STOP-Lox-GsαR201C transgene within the Hipp11 locus with albumin-Cre mice. Male LGsR201C mice had normal survival but reduced body weight and increased liver weight. Compared to control littermates, LGsR201C mice had significantly increased hepatic cAMP levels and enhanced hyperglycemic response to glucagon, confirming the activation of liver Gsα/cAMP signaling in LGsR201C mice. As a consequence, LGsR201C mice showed elevated blood glucose levels during both fed and fasting states, as well as enhanced hepatic gluconeogenesis evidenced by pyruvate tolerance test. Serum levels of insulin, glucagon, free fatty acids and triglycerides were comparable between control and LGsR201C mice in the fed state. Results of glucose and insulin tolerance tests showed that LGsR201C mice had severe glucose intolerance with normal insulin sensitivity. Unlike control mice, when given a high dose of glucose (3mg/g ip.), LGsR201C mice had completely blunted first- and second-phase insulin secretory responses to glucose. Since LGsR201C mice exhibited normal pancreatic islet size and insulin content examined with immunohistochemistry, the impaired glucose tolerance in LGsR201C mice probably resulted from an impaired insulin secretion. Results of RNA-seq analysis revealed that an array of genes was oppositely regulated in the liver of LGsKO mice vs. LGsR201C mice. Thus, our data indicate possible organ-to-organ communication between liver and pancreatic β-cells that is regulated by liver Gsα signaling. 1.Chen M., et al., JCI 115:3217, 2005

MELK is upregulated in various types of human cancer and is known to be associated with cancer progression, maintenance of stemness, and poor prognosis. OTS167, a MELK kinase inhibitor, shows potent growth-suppressive effect on human tumors in a xenograft model, but the detailed mode of action has not been fully elucidated. In this study, we demonstrate the molecular mechanism of action of MELK inhibitor OTS167 in a preclinical model. OTS167-treated cells caused morphological transformation, induced the differentiation markers, and reduced stem-cell marker expression. Furthermore, we identified DEPDC1, known as an oncogene, as an additional downstream molecule of the MELK signaling pathway. MELK enhanced DEPDC1 phosphorylation and its stability. The expression of MELK and downstream molecules was decreased in OTS167-treated xenograft tumor tissues, which revealed central necrosis and significant growth suppression. Our data should further shed light on the mechanism of action how OTS167 suppresses tumor growth through the inhibition of the MELK signaling pathway and suggest the possibility of biomarkers for the assessment of clinical efficacy.

Pancreatic β-cell dysfunction leads to diabetes with reduced β-cell mass and impaired glucose-stimulated insulin secretion. The G protein α subunit Gsα couples receptors to generate cAMP, which mediates the insulinotropic effects of incretin hormones, such as GLP-1. We previously reported that β-cell-specific Gsα deficiency causes insulin-deficient diabetes due to reduced islet size and β-cell mass, and that mice with whole pancreatic Gsα deficiency develop insulin deficient diabetes and reduced β-cell mass, but also showed increased proliferation of α-cells, indicating that Gsα plays divergent roles in pancreatic α-and β-cell growth. In the present study, we generated mice with α-cell-specific Gsα deficiency (αGsKO) by mating Gsα-floxed mice with glucagon-cre mice and studied the metabolic consequences. Deletion of Gsα in α-cells did not affect growth, blood glucose, or serum insulin or glucagon levels. However, αGsKO mice displayed enlarged islet size of normal architecture with significant increases in both α- and β-cell numbers, accompanied with increased β-cell proliferation and reduced apoptosis. This increased β-cell proliferation could be detected as early as at 3 days after birth. Consistent with this, αGsKO mice showed increased insulin response to acute glucose loading (3mg/g i.p.), although they had unaltered glucose tolerance when given a lower dose of glucose (2 mg/g i.p.). The results of RT-PCR on pancreatic islets isolated from aged mice (9-10 months old) showed that expression of Maf B and Pax6, transcription factors required for β-cell development, tended to be increased; whereas cell cycle factors, such as Cyclin D1, D2, and CDK4, tended to be decreased. Taken together, our results show that Gsα/cAMP signaling pathway in pancreatic α-cells is critical for both α- and β-cell growth as well as for maintenance of β-cell mass. Disclosure A. Taira: None. H. Sun: None. C. Le: None. O. Gavrilova: None. M. Chen: None. L. Weinstein: None. Funding National Institutes of Health

Gluconeogenesis is de novo glucose synthesis from substrates such as amino acids and is vital when glucose is lacking in the diurnal nutritional fluctuation. Accordingly, genes for hepatic gluconeogenic enzymes exhibit daily expression rhythms, whose detailed regulations under nutritional variations remain elusive. As a first step, we performed general systematic characterization of daily expression profiles of gluconeogenic enzyme genes for phosphoenolpyruvate carboxykinase (PEPCK), cytosolic form (Pck1), glucose-6-phosphatase (G6Pase), catalytic subunit (G6pc), and tyrosine aminotransferase (TAT) (Tat) in the mouse liver. On a standard diet fed ad libitum, mRNA levels of these genes showed robust daily rhythms with a peak or an elevation phase during the late sleep-fasting period in the diurnal feeding/fasting (wake/sleep) cycle. The rhythmicity was preserved in constant darkness, modulated with prolonged fasting, attenuated by Clock mutation, and entrained to varied photoperiods and time-restricted feedings. These results are concordant with the notion that gluconeogenic enzyme genes are under the control of the intrinsic circadian oscillator, which is entrained by the light/dark cycle, and which in turn entrains the feeding/fasting cycle and also drives systemic signaling pathways such as the hypothalamic-pituitary-adrenal axis. On the other hand, time-restricted feedings also showed that the ingestion schedule, when separated from the light/dark cycle, can serve as an independent entrainer to daily expression rhythms of gluconeogenic enzyme genes. Moreover, nutritional changes dramatically modified expression profiles of the genes. In addition to prolonged fasting, a high-fat diet and a high-carbohydrate (no-protein) diet caused modification of daily expression rhythms of the genes, with characteristic changes in profiles of glucoregulatory hormones such as corticosterone, glucagon, and insulin, as well as their modulators including ghrelin, leptin, resistin, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide-1 (GLP-1). Remarkably, high-protein (60% casein or soy-protein) diets activated the gluconeogenic enzyme genes atypically during the wake-feeding period, with paradoxical up-regulation of glucagon, which frequently formed correlation networks with other humoral factors. Based on these results, we propose that daily expression rhythms of gluconeogenic enzyme genes are under the control of systemic oscillator-driven and nutrient-responsive hormones.

// Suyoun Chung 1 , Kyoko Kijima 1 , Aiko Kudo 1 , Yoshiko Fujisawa 1 , Yosuke Harada 1 , Akiko Taira 1 , Naofumi Takamatsu 1 , Takashi Miyamoto 1 , Yo Matsuo 1 and Yusuke Nakamura 2 1 OncoTherapy Science, Inc., Kawasaki, Kanagawa, Japan 2 Department of Medicine and Surgery, The University of Chicago, Chicago, IL, USA Correspondence to: Yusuke Nakamura, email: // Keywords : MELK, xenograft model, kinase inhibitor, molecular pharmacology, biomarker Received : December 22, 2015 Accepted : February 08, 2016 Published : February 24, 2016 Abstract MELK is upregulated in various types of human cancer and is known to be associated with cancer progression, maintenance of stemness, and poor prognosis. OTS167, a MELK kinase inhibitor, shows potent growth-suppressive effect on human tumors in a xenograft model, but the detailed mode of action has not been fully elucidated. In this study, we demonstrate the molecular mechanism of action of MELK inhibitor OTS167 in a preclinical model. OTS167-treated cells caused morphological transformation, induced the differentiation markers, and reduced stem-cell marker expression. Furthermore, we identified DEPDC1, known as an oncogene, as an additional downstream molecule of the MELK signaling pathway. MELK enhanced DEPDC1 phosphorylation and its stability. The expression of MELK and downstream molecules was decreased in OTS167-treated xenograft tumor tissues, which revealed central necrosis and significant growth suppression. Our data should further shed light on the mechanism of action how OTS167 suppresses tumor growth through the inhibition of the MELK signaling pathway and suggest the possibility of biomarkers for the assessment of clinical efficacy.

Background: The discovery and development of novel biomarkers that could facilitate early diagnosis and thus prevent the progression of atherosclerosis-related diabetes mellitus (DM), cerebral infarction (CI), and cardiovascular disease (CVD) has garnered much research interest. Notably, recent reports have described a number of highly sensitive antibody markers. In this study, we aimed to identify additional antibody markers that would facilitate screening. Methods: The amplified luminescent proximity homogeneous assay (AlphaLISA) method, which incorporates glutathione- or streptavidin-donor beads and anti-human-IgG-acceptor beads, was used to evaluate serum antibody levels in serum samples. The protein array method was used for the initial screening, and peptide arrays were used to identify epitope sites. Results: The protein array identified SH3 domain-binding protein 5 (SH3BP5) as a target antigen of serum IgG antibodies in the sera of patients with atherosclerosis. We prepared recombinant glutathione S-transferase (GST)- fused SH3BP5 protein. Peptide arrays revealed that the epitope site recognized by serum antibodies is located within amino acids 161–174 of SH3BP5. AlphaLISA revealed significantly higher serum antibody levels against both the SH3BP5 protein and peptide in patients with DM, acute-phase CI, transient ischemic attack, CVD or chronic kidney disease (CKD), than in healthy donors. Furthermore, areas under the receiver operating characteristic curves of these antibodies were higher in patients with CKD and DM than in other patients. Spearman correlation analysis revealed associations between the serum antibody levels against SH3BP5 peptide and artery stenosis, hypertension, and smoking. Conclusions: The serum anti-SH3BP5 antibody marker appears to be useful for estimating the progress of atherosclerosis and may discriminate atherosclerosis associated with hypertension and/or habitual smoking.

We propose new vehicle dynamics management of in-wheel electric motor vehicle based on tire force usage rate. In the vehicle dynamics management, we adopted non-linear optimizing controller using direct yaw-moment control, tire force usage rate and each tire force. In cornering performance test simulation of 3 degree freedom model, we confirmed that by using this vehicle dynamics management, saturating tire force could be avoided and higher critical speed also could be achieved. We also conducted pylon course slalom test simulation. By this simulation, it was confirmed that the 4 wheel drive in-wheel motor electric vehicle with the proposed vehicle dynamics management system could have higher stability performance comparing to the same vehicle without control.

症例は77歳男性．平成17年9月下旬に発熱および食思不振が出現し紹介受診した．来院時に炎症反応と肝胆道系酵素の上昇を認め入院した．胆道感染症を考え第三世代セフェム系抗生剤投与を開始したが白血球上昇と発熱が続いた．入院2日目のMRI検査にて肝両葉に多発する小嚢胞性病変がみられ，一部の嚢胞周囲に造影効果を認めた．画像所見からは多発肝膿瘍も否定できずカルバペネム系抗生剤への変更と抗真菌薬併用を行ったところ炎症反応は改善した．入院1カ月後および半年後の造影MRI検査では入院時に認められた嚢胞周囲の造影効果は消失したが，依然として肝両葉に多発する小嚢胞性病変がみられた．臨床経過及び画像所見から胆管過誤腫を基礎疾患とした胆管過誤腫の感染と考えられた．胆管過誤腫は通常無症状であるが感染を合併すると多発肝膿瘍との鑑別に苦慮する場合がある．本症例は臨床経過および画像所見の経過から診断に至った貴重な症例と考えられた．

Cerebral infarction (CI), cardiovascular disease (CVD), diabetes mellitus (DM) and chronic kidney disease (CKD) are atherosclerosis-related diseases, which are major causes of health damage. For early and sensitive diagnosis, development of novel biomarkers is expected and of significant practical importance. First screening was carried out by phage expression cloning to identify antigen proteins recognized by serum IgG antibodies in patients with atherosclerosis. RPA2, LRPAP1, EEF1A1, SPOCK1, LOC729260, tubulin beta 2C (TUBB2C) and KIAA0020 markers were identified. We then compared the serum antibody levels against the candidate proteins between healthy donors (HD) and patients with CI, CVD, DM, or CKD by Alpha (amplified luminescent proximity homogeneous assay)-LISA method. The results showed that the serum TUBB2C antibody levels were significantly higher in patients with CI, DM, or CKD than those in HD. Using the average + 2SD of HD as the cut-off value, the positive thresholds of TUBB2C antibody markers were 14.8% in CI, 25.8% in DM, and 18.3% in CKD. TUBB2C antibody levels were well correlated with artery stenosis degrees such as plaque score, maximum intima-media thickness and cardio ankle vascular index. Consequently, TUBB2C antibody markers are useful to diagnose atherosclerosis, DM, and CKD, and can be applied to the prediction of the onset of CI. The serum anti-TUBB2C antibody markers are useful for the diagnosis of DM and CKD. Keywords: Atherosclerosis; Diabetes mellitus; Chronic kidney disease; Cerebral infarction; Cardiovascular disease; Antibody biomarker

Background: Systemic lupus erythematosus (SLE) is an autoimmune disease which may be caused by development of the autoantibodies. On the other hand, SLE is a high-risk group of atherosclerosis, so it is possible that some of autoantibodies in SLE are the result of atherosclerosis-related diseases such as cerebral infarction (CI), cardiovascular disease (CVD) and diabetes mellitus (DM). Methods: The initial screening of autoantibodies was performed using the protein array method. AlphaLISA was used to analyze the serum antibody levels using synthetic polypeptides as antigens. Results: After the initial screening using protein array, we identified 67 antigens that were recognized by IgG antibodies in sera of patients with SLE. In the second screening, 170 peptides derived from amino acid sequences of 67 antigens were synthesized and used as antigens for analysis of serum antibody levels by AlphaLISA. The antibody levels for ten peptides were significantly higher in the sera of patients with SLE than in those of healthy donors. Further AlphaLISA analysis of sera of patients with CI, CVD or DM revealed that the serum antibody levels for four peptides derived from SOSTDC1, CTNND1, CLDND1 and CCNG2 were elevated in patients as compared to those of healthy donors. Conclusions: Serum antibody levels against peptide antigens of SOSTDC1, CTNND1, CLDND1 and CCNG2 are useful markers for diagnosis of the progression of CI, CVD and/or DM.

Background: Atherosclerosis-related life-style diseases such as cerebral infarction (CI), cardiovascular disease (CVD), diabetes mellitus (DM), and chronic kidney disease (CKD) are a serious problem in the recently aging society. The development of novel and sensitive diagnostic markers is necessary and expected for the early treatment. Methods and Results: Through the first screening by phage expression cloning, we identified ATPase, Ca++ transporting, plasma membrane 4 (ATP2B4) and bone morphogenetic protein 1 (BMP-1) as antigens recognized by IgG antibodies in the sera of patients with atherosclerosis. The presence of autoantibodies against these antigens in serum specimens was confirmed by Western blotting. We then compared serum antibody levels against recombinant ATP2B4 and BMP-1 proteins between healthy donors (HD) and patients with atherosclerotic diseases, such as CI, transient ischemic attack (TIA), CVD, DM, or CKD, by the Alpha (amplified luminescent proximity homogeneous assay)-LISA method. The results revealed that both antibody levels were significantly higher in patients with these diseases than in HD and exhibited most prominent differences in CKD vs. HD. Correlation analysis showed that both antibody levels were well correlated with the degree of artery stenosis, such as maximum intima-media thickness with some different patterns, i.e., anti-ATP2B4 antibody levels were related to hypertension, whereas anti-BMP-1 antibodies were related to smoking habits. Conclusions: The serum antibody levels against ATP2B4 and BMP-1 can be useful diagnostic markers for atherosclerosis and its related diseases caused by hypertension and smoking habits, respectively.

Gallstone ileus is a rare but important cause of small bowel obstruction in the geriatric population. A 65-year-old man with a twenty year history of cholecystolithiasis was admitted to our hospital with abdominal pain and vomiting. Physical exams showed abdominal defence and rebound tenderness. A plain abdominal X-ray suggested a small bowel obstruction and pneumobilia. CT scan revealed a 2.5-cm gallstone at the jejunum and air in the biliary tree. The patient underwent a emergency laparotomy based on a diagnosis of panperitonitis with a perforation associated with gallstone ileus. Operative findings revealed a jejunal perforation and a impacted stone on the anal side of perforation. Enterolithotomy and jejunal resection were performed with cholecystectomy and repairment of the cholecystoduodenal fistula.