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Phosphorylated and total CHK1 or CHK2 protein levels in TE-11R cells treated with prexasertib.

While Talaporfin sodium photodynamic therapy (PDT) is an effective and safety salvage treatment for local failure after chemoradiotherapy for esophageal cancer, some case developed local failure. For such case, repeated PDT could be indicated. However, safety and efficacy of repeated PDT as a salvage treatment for esophageal cancer has not been elucidated. We retrospectively reviewed 60 patients with esophageal cancer who were treated with salvage PDT at Kyoto University Hospital between October 2015 and August 2021. Among 60 patients, repeated PDT after the first PDT was indicated for 21 lesions in 16 patients (26.7%), of which eight lesions were residual tumor, four were local recurrence after complete response (CR) after the first PDT at the primary site, and nine were metachronous lesion. The total session of repeated PDT was 33; 20 were for primary sites and 13 were for metachronous sites. Among them, seven patients (43.8%) achieved local (L) -CR and 13 lesions (61.9%) achieved lesion L-CR. By session, 14 sessions (42.4%) achieved L-CR. There were no severe adverse events except for one patient of perforation. Repeated PDT could be an effective and safe treatment option for local failure after salvage PDT for esophageal cancer.

Background: Recently we reported that trifluridine (FTD)/Tipiracil (TPI) is tolerable for unresectable Esophageal squamous cell carcinoma (ESCC) patients although the anti-tumor effect was modest (Mori Y, et al. Esophagus 2022). Therefore, we aimed at developing a combination therapy with FTD/TPI with another small molecule to achieve better efficacy against ESCC. CHK1 inhibitor was considered to be the candidate, because combination treatment with FTD/TPI and prexasertib showed potent antitumor effects in p53-mutant ESCC cells through the synthetic lethality (Ohashi S, et al. Mol Cancer Ther 2020); however, CHK1 inhibitors are not clinically available for further clinical development. Here we explored the concept of synthetic lethality with CHK1 perturbation to the whole ATR-CHK1-WEE1 pathway, especially to the downward WEE1 which directly targets a cell cycle regulator CDK1. The aim of this study is to elucidate the efficacy of the combination of a WEE1 inhibitor (WEE1i) MK1775 with FTD/TPI in ESCC. Methods: ESCC cells (TE-8 and TE-11) are used for in vitro assay, with compounds including FTD, MK1775 (WEE1i). Mitosis assay (flowcytometry with phospho-Histone H3 [p-hH3] antibody), cell viability assay (WST-1 and clonogenic assays), cytotoxicity assay (CytoTox-Glo assay), and western blotting (double-strand DNA break [γ-H2AX], DDR activity [phospho-CHK1], and CDK1 activity [phospho-Tyr15-CDK1]) were performed. Antitumor effects and tolerability were observed in vivo with TE-8 xenograft model with nu/nu nude mice. Results: FTD induced activation of CHK1 and inhibition of CDK1 sequentially in ESCC cells. FTD also decreased the proportion of p-hH3 positive cells in mitosis assay. Combination treatment with WEE1i and FTD had activated CDK1, increased p-hH3 positive cells, and induced γ-H2AX. The WST-1 cell viability assay showed significant sensitizing effect of WEE1i to FTD in ESCC cells. The cytotoxicity assay also revealed the significant increase of dead cells by the combination treatment (p = 0.003). Furthermore, we confirmed the combination treatment significantly suppressed xenografted tumor growth (-86%) without major adverse events in vivo (two-way ANOVA and Tukey post-hoc analyses: FTD/TPI vs. control, P < 0.05; MK1775 vs. control, P < 0.05, without significant interaction between the FTD/TPI treatment and MK1775 treatment). Conclusion: FTD/TPI and WEE1i combination showed potent cytotoxicity, and is considered as a candidate treatment strategy against ESCC. Citation Format: Hoang Trang Nguyen Vu, Osamu Kikuchi, Tomoki Saito, Yukie Nakai, Tomomi Ida, Yuki Kondo, Shigeki Kataoka, Yosuke Mitani, Shinya Ohashi, Manabu Muto. The combination of Trifluridine/Tipiracil and a WEE1 inhibitor is an effective and tolerable candidate strategy against ESCC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6187.

Alcohol consumption is the key risk factor for the development of esophageal squamous cell carcinoma (ESCC), and acetaldehyde, a metabolite of alcohol, is an alcohol-derived major carcinogen that causes DNA damage. Aldehyde dehydrogenase2 (ALDH2) is an enzyme that detoxifies acetaldehyde, and its activity is reduced by ALDH2 gene polymorphism. Reduction in ALDH2 activity increases blood, salivary and breath acetaldehyde levels after alcohol intake, and it is deeply associated with the development of ESCC. Heavy alcohol consumption in individuals with ALDH2 gene polymorphism significantly elevates the risk of ESCC; however, effective prevention has not been established yet. In this study, we investigated the protective effects of Alda-1, a small molecule ALDH2 activator, on alcohol-mediated esophageal DNA damage. Here, we generated novel genetically engineered knock-in mice that express the human ALDH2*1 (wild-type allele) or ALDH2*2 gene (mutant allele). Those mice were crossed, and human ALDH2*1/*1, ALDH2*1/*2 and ALDH2*2/*2 knock-in mice were established. They were given 10% ethanol for 7 days in the presence or absence of Alda-1, and we measured the levels of esophageal DNA damage, represented by DNA adduct (N2-ethylidene-2′-deoxyguanosine). Alda-1 significantly increased hepatic ALDH2 activity both in human ALDH2*1/*2 and/or ALDH2*2/*2 knock-in mice and reduced esophageal DNA damage levels after alcohol drinking. Conversely, cyanamide, an ALDH2-inhibitor, significantly exacerbated esophageal DNA adduct level in C57BL/6N mice induced by alcohol drinking. These results indicate the protective effects of ALDH2 activation by Alda-1 on esophageal DNA damage levels in individuals with ALDH2 gene polymorphism, providing a new insight into acetaldehyde-mediated esophageal carcinogenesis and prevention., We generated human ALDH2*2 (Glu504Lys) knock-in mice and showed the protective effects of Alda-1 (ALDH2 activator) on esophageal DNA damage in those mice with alcohol drinking. Our findings can be the basis for developing a novel prevention for alcohol-mediated esophageal carcinogenesis.

Background Clinical cancer genome sequencing detects oncogenic variants that are potential targets for cancer treatment, but it also detects variants of unknown significance that may interact and affect the pathophysiology of the tumor; however, these interactions are not fully understood. In this study, we examined the interactions of a minor HER2 mutation (G776S) and APC mutations, which were detected by cancer genome sequencing of samples from a patient with colorectal cancer. Methods We transfected HER2-G776S mutant- or HER2 wild type- expressing vectors into several cell lines, HeLa, FHC, CACO-2 and COLO-320, to evaluate their effects on HER2 phosphorylation and kinase activity, HER2 downstream signaling (phosphorylation of AKT and MAPK), and anchorage-independent growth ability. APC- knockout cells and APC overexpressing cells were established to investigate the effect of APC function on the HER2 signaling pathway. We also evaluated the efficacy of a HER2 tyrosine kinase inhibitor on xenograft tumors derived from HER2-G776S transfected cells. Results HER2 G776S mutation increased the kinase activity and phosphorylation of HER2 protein, but these effects were weaker than those of the other HER2 driver mutation. HER2 G776S did not activate HER2-downstream signal pathways, such as ERK and AKT phosphorylation, in cells with wild-type APC (HeLa and FHC cells). By contrast, HER2 G776S increased the activation of HER2 downstream signaling, especially ERK phosphorylation, and anchorage-independent cell growth in cells with an APC mutation (CACO-2 and COLO-320) and APC-knockout HeLa cells. Wild-type APC overexpression in HER2 G776S-transfected COLO-320 cells neutralized ERK phosphorylation. Loss of APC function increased Wnt pathway activity but also increased RAS–GTP, which increased ERK phosphorylation triggered by HER2 G776S transfection. Afatinib, a pan-HER tyrosine kinase inhibitor, inhibited tumor growth of HER2 G776S-transfected COLO-320 xenografts Conclusions HER2 G776S mutation acts as a weak oncogenic driver, but it also increases HER2–ERK signaling activity by increasing RAS–GTP production when APC function is simultaneously impaired. These results suggest that even weakly active mutations may be therapeutic targets, and the use of this strategy may contribute to the development of HER2-targeted therapy for colorectal cancer.

Background Talaporfin sodium photodynamic therapy (tPDT) is an effective salvage treatment for local failure after chemoradiotherapy for esophageal cancer. Repeated tPDT could also be indicated for local recurrence or residue after the first salvage tPDT. However, the safety and efficacy of repeated tPDT have not been elucidated. Methods We reviewed 52 patients with esophageal cancer who were treated with the first tPDT at Kyoto University Hospital between October 2015 and April 2020. Results Among 52 patients, repeated tPDT after the first tPDT was indicated for 13 patients (25%), of which six had residual tumor, four had local recurrence after complete response (CR) after the first tPDT at the primary site, and six had metachronous lesion. The total session of repeated tPDT was 25; 16 were for primary sites and nine were for metachronous sites. Among them, six patients (46.2%) achieved local (L)-CR and nine lesions (56.3%) achieved lesion L-CR. By session, 10 sessions (40%) achieved L-CR. There were no severe adverse events except for one patient; this patient showed grade 3 esophageal stenosis and perforation after the third tPDT on the same lesion that was previously treated with porfimer sodium photodynamic therapy four times. Conclusion Repeated tPDT could be an effective and safe treatment for local failure even after salvage tPDT for esophageal cancer.

Background Cancer of unknown primary (CUP) is usually treated with nonselective and empirical chemotherapy; however, its prognosis is generally poor, with a median survival of less than a year. Thus, clinicians eagerly await the development of more effective treatment strategies. In recent years, advances in next-generation sequencing (NGS) have made it possible to analyze comprehensively the genome of individual cancers. NGS has identified many genomic alterations, some of which are potential molecular targets of specific agents. We report a case of CUP that was successfully treated with targeted therapy directed by the genomic data obtained from an NGS-based multiplex assay. Case presentation A 52-year-old Asian woman with right hip joint pain underwent fluorodeoxyglucose-positron emission tomography/computed tomography, which showed multiple metastatic tumors in her right hip joint, thyroid gland, lung, and vertebrae. Brain magnetic resonance imaging showed multiple cerebral metastases. Additional tests, including pathology examination and conventional epidermal growth factor receptor (EGFR) gene mutation analysis (single-strand conformation polymorphism assay), could not identify the primary origin of the tumors, so the patient was diagnosed with CUP. After empirical chemotherapy for CUP, an NGS-based multiplex assay performed using a resected specimen of thyroid tumor detected the EGFR mutation c.2573 T > G p.Leu858Arg (L858R). Her treatment was changed to erlotinib, an EGFR tyrosine-kinase inhibiter, which dramatically shrank the tumors and decreased her serum carcinoembryonic antigen level. She achieved long-term disease control and survived for 2 years and 9 months from the first diagnosis. Conclusion This case might support the strategy that NGS-based multiplex assays could identify actionable molecular targets for individual patients with CUP.

Esophageal squamous cell carcinoma (ESCC) is a disease characterized by a high mutation rate of the TP53 gene, which plays pivotal roles in the DNA damage response (DDR) and is regulated by checkpoint kinase (CHK) 2. CHK1 is another key DDR-related protein, and its selective inhibition is suggested to be particularly sensitive to TP53-mutated cancers, because a loss of both pathways (CHK1 and/or CHK2–p53) is lethal due to the serious impairment of DDR. Such a therapeutic strategy is termed synthetic lethality. Here, we propose a novel therapeutic strategy based on synthetic lethality combining trifluridine/tipiracil and prexasertib (CHK1 inhibitor) as a treatment for ESCC. Trifluridine is a key component of the antitumor drug combination with trifluridine/tipiracil (an inhibitor of trifluridine degradation), also known as TAS-102. In this study, we demonstrate that trifluridine increases CHK1 phosphorylation in ESCC cells combined with a reduction of the S-phase ratio as well as the induction of ssDNA damage. Because CHK1 phosphorylation is considered to be induced as DDR for trifluridine-mediated DNA damage, we examined the effects of CHK1 inhibition on trifluridine treatment. Consequently, CHK1 inhibition by short hairpin RNA or treatment with the CHK1 inhibitor, prexasertib, markedly enhanced trifluridine-mediated DNA damage, represented by an increase of γH2AX expression. Moreover, the combination of trifluridine/tipiracil and CHK1 inhibition significantly suppressed tumor growth of ESCC-derived xenograft tumors. Furthermore, the combination of trifluridine and prexasertib enhanced radiosensitivity both in vitro and in vivo. Thus, the combination of trifluridine/tipiracil and a CHK1 inhibitor exhibits effective antitumor effects, suggesting a novel therapeutic strategy for ESCC.

Background Esophageal squamous cell carcinoma (ESCC) is one of the most intractable cancers, so the development of novel therapeutics has been required to improve patient outcomes. Curcumin, a polyphenol from Curcuma longa, exhibits various health benefits including antitumor effects, but its clinical utility is limited because of low bioavailability. Theracurmin® (THC) is a highly bioavailable curcumin dispersed with colloidal submicron particles. Methods We examined antitumor effects of THC on ESCC cells by cell viability assay, colony and spheroid formation assay, and xenograft models. To reveal its mechanisms, we investigated the levels of reactive oxygen species (ROS) and performed microarray gene expression analysis. According to those analyses, we focused on NQO1, which involved in the removal of ROS, and examined the effects of NQO1-knockdown or overexpression on THC treatment. Moreover, the therapeutic effect of THC and NQO1 inhibitor on ESCC patient-derived xenografts (PDX) was investigated. Results THC caused cytotoxicity in ESCC cells, and suppressed the growth of xenografted tumors more efficiently than curcumin. THC increased ROS levels and activated the NRF2–NMRAL2P–NQO1 expressions. Inhibition of NQO1 in ESCC cells by shRNA or NQO1 inhibitor resulted in an increased sensitivity of cells to THC, whereas overexpression of NQO1 antagonized it. Notably, NQO1 inhibitor significantly enhanced the antitumor effects of THC in ESCC PDX tumors. Conclusions These findings suggest the potential usefulness of THC and its combination with NQO1 inhibitor as a therapeutic option for ESCC. Electronic supplementary material The online version of this article (10.1007/s00535-019-01549-x) contains supplementary material, which is available to authorized users.

This paper presents a low distortion 3rd-order continuous-time delta-sigma modulator for a worldwide digital TV-receiver whose peak SNDR is 69.8dB and SNR is 70.2dB under 1V power supply. To enhance SNDR performance, the mechanisms to occur harmonic distortions at feedback current-steering DAC and flash ADC have been analyzed. A low power tuning system using RC-relaxation oscillator has been developed in order to achieve high yield against PVT variations. A 3rd-order modulator with modified single opamp resonator contributes to cost reduction by realizing a very compact circuit. Reduction schemes of the distortions enabled the modulator to achieve FOM of 0.18pJ/conv-step.

Conventional continuous-time (CT) delta-sigma (??) analog-to-digital converters (ADCs) consume large amount of power in operational amplifiers of a loop-filter. We propose a new loop-filter with single-opamp resonator, ringing-relaxation filter and passive resistor adder to lower power consumption. These three techniques are essential for designing high-order delta sigma modulators with low oversampling ratio. Because the new resonator reduces the number of opamps, the resistor adder displaces a conventional active adder and the ringing-relaxation filter alleviates the burden on the first opamp by reducing its gain bandwidth, FOM is greatly improved. To demonstrate the concept, 300 MHz, fifth-order low-pass, 3-bit CT?? ADC of single feedback with feedforward architecture was implemented in a 1.1 V, 110 nm 1P6M CMOS process. An SNR of 68.2 dB and an SNDR of 62.5 dB were measured in a 10 MHz bandwidth and FOM was 0.24 pJ/conv.

This paper presents a 3rd-order continuous time delta-sigma modulator for a worldwide digital TV-receiver whose SNDR is 69.8 dB. An ultimate low power tuning system using RC-relaxation oscillator is developed in order to achieve high yield against PVT variations. A 3rd-order modulator with modified single opamp resonator contributes to cost reduction by realizing very compact circuit. The mechanism to occur 2nd-order harmonic distortion at current feedback DAC was analyzed and a reduction scheme of the distortion enabled the modulator to achieved FOM of 0.18 pJ/conv-step.

Seventeen simple isoquinolines, 15 1-benzylisoquinolines, and 19 protoberberines were tested for their inhibitory activities against Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Among the tested alkaloids, the inhibitory activity of all 1-benzylisoquinolines and 11 protoberberines was higher than that of beta-carotene. The 1-benzylisoquinolines 19, 21, 22, 29, and 34 and protoberberines 41, 47-49, 51, 52, and 55 showed potent inhibitory effects on EBV-EA induction (96-100% inhibition at 1 x 10(3) mol ratio/TPA). These alkaloids were more active than the naturally occurring alkaloids, 23, 25, 33, 53, and 54. In addition, fifteen simple isoquinolines, eighteen 1-benzylisoquinolines and eight protoberberines were evaluated with respect to their ability to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals. Nine simple isoquinolines, ten 1-benzylisoquinolines, and four protoberberines were more potent than alpha-tocopherol, and four 1-benzylisoquinolines, 20 and 28-30, exhibited potent activities (SC50 4.5-5.8 microM). Their activities were higher than the naturally occurring alkaloids, 23, 25, and 33. Therefore, some of the isoquinoline alkaloids indicating the high activity on both assays may be potentially valuable cancer chemopreventive agents. Structure-activity relationships are discussed for both tests.