A Wnt/ß-catenin signaling inhibitor, IMU1003, suppresses the emergence of osimertinib-resistant colonies from gefitinib-resistant non-small cell lung cancer cells.

Drug resistance has become a challenge in effective longterm molecular targeted therapy. Longterm non-small cell lung cancer (NSCLC) treatments with the first-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) shorten the effective duration of the third-generation EGFR-TKI, osimertinib, via genetic or epigenetic mechanisms in addition to the gatekeeper mutation T790M. This study reproduced this persistence in vitro using gefitinib-resistant NSCLC PC-9 cells (GR cells) and revealed that pharmacological nuclear localization inhibition of ß-catenin suppressed the osimertinib resistance. Osimertinib effectively reduced GR cell survival but left significantly more resistant colonies than parental PC-9 cells. The nuclear fraction of ß-catenin was enriched in GR cells during acquisition of osimertinib resistance. A chemical nuclear localization inhibitor of ß-catenin, IMU1003, dramatically decreased the emergence of osimertinib-resistant colonies. Forced nuclear localization of ß-catenin reduced IMU1003 efficacy. Thus, suppression of the nuclear ß-catenin function may overcome the transgenerational EGFR-TKI-resistance.

Effect of gait distance during robot training on walking independence after acute brain injury.

This study aimed to determine whether the distance of gait training using a hybrid assistive limb (HAL) is related to the improvement of walking independence in patients with acute brain injury. This was an exploratory, observational study. Thirty patients having hemiplegia (functional ambulation category, FAC score ≤2) with acute stroke or after brain tumor surgery were included. Patients performed 4 sessions of gait training using HAL (60 min/session), 1-3 sessions/week, combined with conventional physical therapy. The gait distance achieved in the four training sessions using HAL was measured. FAC score was measured before and after intervention. Patients were divided into groups A, B, and C, for FAC score improvements of 0, 1, and ≥2, respectively. Gait distance was compared among groups using one-way analysis of variance. Gait distance in group C was significantly longer than that ingroup A [mean (standard deviation): 2527 (1725) m vs. 608 (542) m]. This study suggested that the gait distance achieved during training using the HAL may be a clinical indicator of the effectiveness of the HAL on gait training in patients with acute brain injury.Clinical trial registration number: UMIN000012764 R000014756.

Ivermectin represses Wnt/ß-catenin signaling by binding to TELO2, a regulator of phosphatidylinositol 3-kinase-related kinases.

Ivermectin (IVM), an avermectin-derivative anthelmintic, specifically binds to glutamate-gated chloride ion channels (GluCls), causing paralysis in invertebrates. IVM also exhibits other biological activities such as Wnt/ß-catenin pathway inhibition in vertebrates that do not possess GluCls. This study showed that affinity purification using immobilized IVM B1a isolated TELO2, a cofactor of phosphatidylinositol 3-kinase-related kinases (PIKKs), as a specific IVM B1a-binding protein. TELO2 knockdown reduced cytoplasmic ß-catenin and the transcriptional activation of ß-catenin/TCF. IVM B1a bound to TELO2 through the C-terminal α-helix, in which mutations conferred IVM resistance. IVM reduced the TELO2 and PIKK protein levels and the AKT and S6 kinase phosphorylation levels. The inhibition of mTOR kinase reduced the cytoplasmic ß-catenin level. Therefore, IVM binds to TELO2, inhibiting PIKKs and reducing the cytoplasmic ß-catenin level. In conclusion, our data indicate TELO2 as a druggable target for human diseases involving abnormalities of the Wnt/ß-catenin pathway and PIKKs, including mTOR.

Evaluation of indices for predicting recovery of exercise tolerance in patients surviving allogenic hematopoietic stem cell transplantation.

PURPOSE: Decline in physical function in the early stage after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a major challenge. Exercise tolerance tests, such as the 6-min walk test, are useful markers for predicting exercise tolerance and various other traits, including cardiometabolic risk and non-relapse mortality. This retrospective cohort study aimed to investigate and identify predictors of recovery of exercise tolerance in the early stage after allo-HSCT. METHODS: Ninety-eight patients were classified into recovery and non-recovery groups according to the median 6-min walk distance (6MWD) at discharge. RESULTS: Logistic regression analysis revealed that pre-post change in knee extensor strength (ΔKES) and hematopoietic cell transplantation comorbidity index were useful predictors of recovery of exercise tolerance at discharge and moderate predictors of 6MWD recovery in the early post-transplant period. Receiver operating characteristic analysis showed that pre-transplant ΔKES was an accurate predictor of 6MWD recovery in the early post-transplant period. The cutoff point for ΔKES calculated using the Youden index was - 1.17 Nm/kg. CONCLUSIONS: The results of this study emphasize the importance of the need for programs designed to prevent muscle weakness in the early period after allo-HSCT. The results from markers of recovery of exercise tolerance are promising and can be used for patient education in rehabilitation programs after allo-HSCT.

Effects of periodic robot rehabilitation using the Hybrid Assistive Limb for a year on gait function in chronic stroke patients.

Using a robot for gait training in stroke patients has attracted attention for the last several decades. Previous studies reported positive effects of robot rehabilitation on gait function in the short term. However, the long-term effects of robot rehabilitation for stroke patients are still unclear. The purpose of the present study was to investigate the long-term effects of periodic gait training using the Hybrid Assistive Limb (HAL) on gait function in chronic stroke patients. Seven chronic stroke patients performed 8 gait training sessions using the HAL 3 times every few months. The maximal 10-m walk test and the 2-minute walking distance (2MWD) were measured before the first intervention and after the first, second, and third interventions. Gait speed, stride length, and cadence were calculated from the 10-m walk test. Repeated one-way analysis of variance showed a significant main effect on evaluation time of gait speed (F = 7.69, p < 0.01), 2MWD (F = 7.52, p < 0.01), stride length (F = 5.24, p < 0.01), and cadence (F = 8.43, p < 0.01). The effect sizes after the first, second, and third interventions compared to pre-intervention in gait speed (d = 0.39, 0.52, and 0.59) and 2MWD (d = 0.35, 0.46, and 0.57) showed a gradual improvement of gait function at every intervention. The results of the present study showed that gait function of chronic stroke patients improved over a year with periodic gait training using the HAL every few months.

Domestication of chemicals attacking metazoan embryogenesis: identification of safe natural products modifying developmental signaling pathways in human.

Soil microorganisms are rich sources of bioactive natural products. Interspecies interactions are the cues of their production and refine biological activities. These interactions in natural environments include the interplay between microorganisms and Metazoans (animals), such as nematodes, insects, and ticks. Chemical intercellular communication modulators could exert ideal Metazoan-selective toxicity for defending microorganisms. Developmental signaling pathways, such as the Notch, TGF-beta, and Wnt pathways, are intercellular communication networks that contribute to the reproducible formation of complex higher-order Metazoan body structures. Natural modifiers of the developmental signaling pathway are attractive therapeutic seeds for carcinoma and sarcoma treatment. However, these fundamental signaling pathways also play indispensable physiological roles and their perturbation could lead to toxicity, such as defects in stem cell physiology and tissue regeneration processes. In this review, we introduce a screening system that selects developmental signaling inhibitors with wide therapeutic windows using zebrafish embryonic phenotypes and provide examples of microorganism-derived Wnt pathway inhibitors. Moreover, we discuss safety prospects of the developmental signaling inhibitors.

Analysis of factors associated with patient-reported physical functioning scores at discharge of allogeneic hematopoietic stem cell transplantation patients: a cross-sectional study.

PURPOSE: The purpose of this study was to clarify the independent factors related to patient-reported physical functioning (PF) scores at discharge of patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: A total of 103 patients who underwent allo-HSCT were included in this cross-sectional study. As a screening method, a single regression analysis was conducted with the PF domain in the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 at discharge as the dependent variable, and body mass index, adverse events related to HSCT, and objective physical functions as independent variables. Multiple regression analysis was performed with PF as the dependent variable and variables that passed the screening by single regression analysis and confounders as independent variables. RESULTS: The mean PF score at discharge of the patients was 76.5 (standard deviation: 15.2). Based on the results of screening by the single regression analysis, length of stay, infections (+ / -), acute graft-versus-host disease grade, brief fatigue inventory score (BFI), knee extensor strength, and 6-min walk distance (6MWD) were included in the multiple regression analysis. BFI (B = - 11.94, p < 0.001) and 6MWD (per 10 m) (B = 0.56, p = 0.001) were extracted as significant independent variables governing the PF at discharge in the multiple regression model (adjusted R2 = 0.59). CONCLUSION: Higher exercise tolerance and lower fatigue in patients who underwent allo-HSCT were associated independently with patient-reported better PF scores at discharge.

Higher exercise tolerance early after allogeneic hematopoietic stem cell transplantation is the predictive marker for higher probability of later social reintegration.

As the proportion of long-term survivors after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is on the rise, it is essential to consider the significance of quality of life (QOL), including reintegration with society (returning to school or work). This retrospective cohort study aims to illustrate the precise epidemiology of social reintegration later after allo-HSCT and determine its predictive indicators. We enrolled 56 patients, and 40 patients (71%) attained social reintegration at 2 years post-HSCT. Reintegration failure markedly correlated with an inferior performance status and concurrent chronic graft-versus-host disease. In non-reintegrated patients, the physical function at discharge measured by the 6-min walking distance (6MWD) was markedly decreased. On the multivariate risk analyses, sex (female; odds ratio (OR) 0.07; 95% confidence interval (CI) 0.01-0.54; p = 0.01), HCT-CI (≥ 2; OR 0.10; 95% CI 0.01-0.84; p = 0.03), and change in 6MWD (per 5% increase; OR 1.47; 95% CI 1.01-2.13; p = 0.04) were significant predictors of later social reintegration. This study suggests that a multidisciplinary strategy including rehabilitation is essential, especially in patients with poor predictive markers at an early phase, and we should consider suitable rehabilitation programs to prevent a decline in exercise tolerance and improve social reintegration and overall QOL in patients after allo-HSCT.

Lamellarin 14, a derivative of marine alkaloids, inhibits the T790M/C797S mutant epidermal growth factor receptor.

The emergence of acquired resistance is a major concern associated with molecularly targeted kinase inhibitors. The C797S mutation in the epidermal growth factor receptor (EGFR) confers resistance to osimertinib, a third-generation EGFR-tyrosine kinase inhibitor (EGFR-TKI). We report that the derivatization of the marine alkaloid topoisomerase inhibitor lamellarin N provides a structurally new class of EGFR-TKIs. One of these, lamellarin 14, is effective against the C797S mutant EGFR. Bioinformatic analyses revealed that the derivatization transformed the topoisomerase inhibitor-like biological activity of lamellarin N into kinase inhibitor-like activity. Ba/F3 and PC-9 cells expressing the EGFR in-frame deletion within exon 19 (del ex19)/T790M/C797S triple-mutant were sensitive to lamellarin 14 in a dose range similar to the effective dose for cells expressing EGFR del ex19 or del ex19/T790M. Lamellarin 14 decreased the autophosphorylation of EGFR and the downstream signaling in the triple-mutant EGFR PC-9 cells. Furthermore, intraperitoneal administration of 10 mg/kg lamellarin 14 for 17 days suppressed tumor growth of the triple-mutant EGFR PC-9 cells in a mouse xenograft model using BALB/c nu/nu mice. Thus, lamellarin 14 serves as a novel structural backbone for an EGFR-TKI that prevents the development of cross-resistance against known drugs in this class.

IMU1003, an atrarate derivative, inhibits Wnt/ß-catenin signaling.

Aberrant activation of the canonical Wnt/ß-catenin signaling pathway triggers tumorigenesis in various tissues. This study identified an atrarate compound, IMU14, derived from filamentous fungi as an inhibitor of Wnt/ß-catenin signaling in phenotypic chemical inhibitor screening of the zebrafish eyeless phenotype. Its derivatization resulted in synthesis of IMU1003 with enhanced Wnt inhibitory activity. IMU1003 inhibited ß-catenin/TCF-dependent transcriptional activation and decreased nuclear ß-catenin level. In addition, IMU1003 selectively decreased viability and target gene products of the Wnt/ß-catenin signaling pathway in human non-colorectal cancer cell lines harboring intact APC and ß-catenin. Therefore, atrarate derivatives inhibit Wnt/ß-catenin signaling and show anticancer potential, and we developed a new class of chemical backbones for Wnt/ß-catenin signaling inhibitors.

Bucillamine Prevents Afatinib-Mediated Inhibition of Epidermal Growth Factor Receptor Signaling.

Molecular targeting therapies often cause characteristic adverse effects, such as skin rash during anti-epidermal growth factor receptor (EGFR) therapies, making treatment continuation difficult. In contrast, skin symptoms induced by EGFR inhibition are strongly correlated with the overall survival of the therapies. Therefore, controlling adverse effects not only facilitates treatment continuation but also increases clinical benefits. In this study, we proposed a novel strategy for reducing EGFR-tyrosine kinase inhibitor (TKI)-induced adverse effects in nontumorous organs by repositioning approved medicines using a zebrafish model. We developed a model system for evaluating chemical quenchers of afatinib, a clinically available irreversible EGFR-TKI, by scoring the inhibition of afatinib-induced hyperformation of lateral line neuromasts in zebrafish larvae. Bucillamine, an antirheumatic drug, was identified as an afatinib quencher in the zebrafish system and inhibited TKI activity in vitro. In addition, bucillamine restored EGFR autophosphorylation and downstream signaling in afatinib-treated A431 cells. Thus, topical bucillamine is a potential reliever of irreversible EGFR-TKI-induced skin rash. The zebrafish model can be applied to a screening for quenchers of other anti-EGFR-targeting therapies, including reversible TKIs and biologics.

A follow-up study of the effect of training using the Hybrid Assistive Limb on Gait ability in chronic stroke patients.

Objectives: Recently, use of the Hybrid Assistive Limb (HAL) that is effective for improvement of gait ability in chronic stroke patients has been reported. However, how long the effects are maintained remains unknown. The purpose of the present study was to investigate whether the effect of gait training using the HAL on gait ability was maintained for 3 months after the intervention. Methods: A longitudinal, observational study with an intervention for a single group that adhered to the STROBE guidelines was performed. Nine chronic stroke patients were enrolled in this study. The patients performed gait training sessions using the HAL, 2-5 sessions/week for 3 weeks. Gait speed, stride length, cadence, and 2-minute walk distance (2MWD) were measured before and after intervention and at 3-month follow-up. The clinical trial registration number of this study is UMIN000012764 R000014756. Results: Compared to the initial status, gait speed (p = .02), stride length (p = .03), cadence (p = .01), and 2MWD (p < .05) were significantly increased immediately after the intervention. Moreover, gait speed (p < .01), cadence (p = .03), and 2MWD (p = .02) remained significantly higher 3 months after the intervention. There were no significant changes in all outcome measures between after intervention and at 3-month follow-up. Conclusions: This study showed that gait training using the HAL resulted in significant improvement of gait ability after the intervention and the effect was maintained for 3 months after the training.

Spatiotemporal gait characteristic changes with gait training using the hybrid assistive limb for chronic stroke patients.

BACKGROUND: Robotic rehabilitation has been attracting attention as a means to carry out "intensive", "repetitive", "task-specific", gait training. The newly developed robotic device, the Hybrid Assistive Limb (HAL), is thought to have the possibility of having an excellent effect on gait speed improvement over the conventional automatic programed assist robot. The purpose of this study was to investigate the spatiotemporal characteristics related to gait speed improvement using the HAL in chronic stroke patients. RESEARCH QUESTION: To investigate the effects of robotic gait training on gait speed and gait parameters. METHODS: An observational study with an intervention for single group was used. Intervention was conducted in University Hospital. Eleven chronic stroke patients were enrolled in this study. The patients performed 8 gait training sessions using the HAL, 2-5 sessions/week for 3 weeks. Gait speed, stride length, cadence, time of gait cycle (double-limb stance phases and single-limb stance phases) and time asymmetry index were measured before and after intervention. RESULTS: After intervention, gait speed, stride length, and cadence were significantly improved (Effect size = 0.39, 0.29, and 0.29), the affected initial double-limb stance phase was significantly shortened (from 15.8 ± 3.46%-13.3 ± 4.20%, p = .01), and the affected single-limb stance phase was significantly lengthened (from 21.8±7.02%-24.5±7.95%, p < .01). The time asymmetry index showed a tendency to improve after intervention (from 22.9±11.8-17.6±9.62, p = .06). There was a significant correlation between gait speed and the stride length increase rate (r = .72, p = .01). SIGNIFICANCE: This study showed that increasing stride length with lengthening of the affected single-stance phase by gait training using the HAL improved gait speed in chronic stroke patients. However, the actual contributions on HAL cannot be separated from gait training because this study is an observational research without a control group.

Clotrimazole inhibits the Wnt/ß-catenin pathway by activating two eIF2α kinases: The heme-regulated translational inhibitor and the double-stranded RNA-induced protein kinase.

The Wnt/ß-catenin signaling pathway controls cell proliferation and differentiation, and therefore, when this pathway is excessively activated, it causes tumorigenesis. Our chemical suppressor screening in zebrafish embryos identified antifungal azoles including clotrimazole, miconazole, and itraconazole, as Wnt/ß-catenin signaling inhibitors. Here we show the mechanism underlying the Wnt/ß-catenin pathway inhibition by antifungal azoles. Clotrimazole reduced ß-catenin revels in a proteasome-independent fashion. By gene knockdown of two translational regulators, heme-regulated translational inhibitor and double-stranded RNA-induced protein kinase, we show that they mediate the clotrimazole-induced inhibition of the Wnt/ß-catenin pathway. Thus, clotrimazole inhibits the Wnt/ß-catenin pathway by decreasing ß-catenin protein levels through translational regulation. Antifungal azoles represent genuine candidate compounds for anticancer drugs or chemopreventive agents that reduce adenomatous polyps.