Characterization of RNF43 frameshift mutations that drive Wnt ligand- and R-spondin-dependent colon cancer.

Loss-of-function mutations in RNF43 induce activation of Wnt ligand-dependent Wnt/ß-catenin signaling through stabilization of the Frizzled receptor, which is often found in microsatellite instability (MSI)-type colorectal cancer (CRC) that develops from sessile serrated adenomas. However, the mechanism underlying how RNF43 mutations promote tumorigenesis remains poorly understood. In this study, we established nine human CRC-derived organoids and found that three organoid lines carried RNF43 frameshift mutations associated with MSI-high and BRAFV600E mutations, suggesting that these CRCs developed through the serrated pathway. RNF43 frameshift mutant organoids required both Wnt ligands and R-spondin for proliferation, indicating that suppression of ZNRF3 and retained RNF43 function by R-spondin are required to achieve an indispensable level of Wnt activation for tumorigenesis. However, active ß-catenin levels in RNF43-mutant organoids were lower than those in APC two-hit mutant CRC, suggesting a lower threshold for Wnt activation in CRC that developed through the serrated pathway. Interestingly, transplantation of RNF43-mutant organoids with intestinal myofibroblasts accelerated the ß-catenin nuclear accumulation and proliferation of xenograft tumors, indicating a key role of stromal cells in the promotion of the malignant phenotype of RNF43-mutant CRC cells. Sequencing of subcloned organoid cell-expressed transcripts revealed that two organoid lines carried monoallelic RNF43 cis-mutations, with two RNF43 frameshift mutations introduced in the same allele and the wild-type RNF43 allele remaining, while the other organoid line carried two-hit biallelic RNF43 trans-mutations. These results suggest that heterozygous RNF43 frameshift mutations contribute to CRC development via the serrated pathway; however, a second-hit RNF43 mutation may be advantageous in tumorigenesis compared with a single-hit mutation through further activation of Wnt signaling. Finally, treatment with the PORCN inhibitor significantly suppressed RNF43-mutant cell-derived PDX tumor development. These results suggest a novel mechanism underlying RNF43 mutation-associated CRC development and the therapeutic potential of Wnt ligand inhibition against RNF43-mutant CRC. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Determination of individual factors associated with hallux valgus using SVM-RFE.

INTRODUCTION: This cross-sectional study aimed to determine the factors related to hallux valgus (HV) and their importance using support vector machine-recursive feature elimination (SVM-RFE). METHODS: A total of 864 participants aged ≥ 18 years were enrolled. The Manchester scale was used to determine the presence of HV (summed scores for both feet ≥ 4). The questionnaire included items such as age, sex, height, weight, and foot measurements. These internal factors were analyzed to determine if they are related to HV using SVM-RFE. RESULTS: The results of tenfold cross-validation using SVM-RFE revealed that the numbers of feature selections were 10, 10, and 9 for age, sex, and body weight, respectively, and these factors were shown to be related to HV. HV was found to be more common in women than in men (women, 24.9%; men, 7.6%), but the sex difference was not significant in older people. CONCLUSION: Age and sex were found to be important factors associated with HV identified via feature selection using SVM-RFE.

Glycogen synthase kinase-3 inhibition overcomes epithelial-mesenchymal transition-associated resistance to osimertinib in EGFR-mutant lung cancer.

A novel epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, osimertinib, has marked efficacy in patients with EGFR-mutant lung cancer. While epithelial-mesenchymal transition (EMT) plays a role in the resistance to various targeted drugs, its involvement in EGFR-inhibitor resistance remains largely unknown. Preclinical experiments with osimertinib-resistant lung cancer cells showed that EMT was associated with decreased microRNA-200c and increased ZEB1 expression. In several resistant clone cells, pretreatment with the histone deacetylase inhibitor quisinostat helped overcome the resistance by reverting EMT. Furthermore, drug screening from a library of 100 kinase inhibitors indicated that Glycogen synthase kinase-3 (GSK-3) inhibitors, such as LY2090314, markedly inhibited the growth and induced apoptosis of resistant cells, specifically those with a mesenchymal phenotype. These results suggest that GSK-3 inhibition could be useful to circumvent EMT-associated resistance to osimertinib in EGFR-mutant lung cancer.

Patient-derived xenograft models of non-small cell lung cancer for evaluating targeted drug sensitivity and resistance.

Patient-derived xenograft (PDX) models are a useful tool in cancer biology research. However, the number of lung cancer PDX is limited. In the present study, we successfully established 10 PDX, including three adenocarcinoma (AD), six squamous cell carcinoma (SQ) and one large cell carcinoma (LA), from 30 patients with non-small cell lung cancer (NSCLC) (18 AD, 10 SQ, and 2 LA), mainly in SCID hairless outbred (SHO) mice (Crlj:SHO-Prkdcscid Hrhr ). Histology of SQ, advanced clinical stage (III-IV), status of lymph node metastasis (N2-3), and maximum standardized uptake value ≥10 when evaluated using a delayed 18 F-fluoro-2-deoxy-d-glucose positron emission tomography (FDG-PET) scan was associated with successful PDX establishment. Histological analyses showed that PDX had histology similar to that of patients' surgically resected tumors (SRT), whereas components of the microenvironment were replaced with murine cells after several passages. Next-generation sequencing analyses showed that after two to six passages, PDX preserved the majority of the somatic mutations and mRNA expressions of the corresponding SRT. Two out of three PDX with AD histology had epidermal growth factor receptor (EGFR) mutations (L858R or exon 19 deletion) and were sensitive to EGFR tyrosine kinase inhibitors (EGFR-TKI), such as gefitinib and osimertinib. Furthermore, in one of the two PDX with an EGFR mutation, osimertinib resistance was induced that was associated with epithelial-to-mesenchymal transition. This study presented 10 serially transplantable PDX of NSCLC in SHO mice and showed the use of PDX with an EGFR mutation for analyses of EGFR-TKI resistance.

Resistance mediated by alternative receptor tyrosine kinases in FGFR1-amplified lung cancer.

Fibroblast growth factor receptor 1 (FGFR1) amplification has been identified in 10-20% of patients with squamous non-small-cell lung cancer. Preclinical models showed promising activity of specific FGFR inhibitors, but early clinical trials showed that only a small fraction of patients with FGFR1-amplified lung cancer responded to FGFR inhibitors. These unsatisfactory results were partly explained by heterogeneous amplicons around the 8p11 genomic region, leading to false-positive amplification results. Furthermore, discrepancies in the gene amplification and protein expression of FGFR1 were also reported. In this study, we identified the roles of alternative receptor tyrosine kinases (RTKs) in FGFR1-amplified lung cancer. These alternative RTKs dominantly activate phosphoinositide 3-kinase-AKT signaling and also mitigate sustained inhibition of mitogen-activated protein kinase signaling by FGFR inhibitors. The rebound activation of extracellular signal-regulated kinase phosphorylation was associated with sensitivity to the drugs. Combinatorial inhibition of alternative RTKs and FGFR1 was required to suppress both AKT and extracellular signal-regulated kinase phosphorylation and to induce key pro-apoptotic proteins BIM and p53 upregulated modulator of apoptosis (PUMA). Furthermore, even in FGFR inhibitor-sensitive NCI-H1581 lung cancer cells, MET-expressing clones were already detectable at a very low frequency before resistance induction. Selection of these pre-existing subclones resulted in FGFR inhibitor resistance because of the activation of AKT and extracellular signal-regulated kinase by MET signaling that was mediated by GRB2 associated binding protein 1 (GAB1). These results suggest that incomplete suppression of key survival signals led to intrinsic and acquired resistance to FGFR inhibitors. Our results may help explain the low clinical response rates to FGFR inhibitors in FGFR1-amplified lung cancer.

Organ-specific efficacy of HSP90 inhibitor in multiple-organ metastasis model of chemorefractory small cell lung cancer.

Small-cell lung cancer (SCLC) accounts for nearly 15% of lung cancer cases and exhibits aggressive clinical behavior characterized by rapid growth and metastatic spread to multiple organs. About 70% of patients with SCLC present with extensive disease and distant metastases at diagnosis. HSP90 is a 90-kDa molecular chaperone whose association is required for the stability and function of its numerous "client proteins." Here, we assessed the therapeutic potential of the HSP90 inhibitor 17-DMAG in SCLC. Notably, 17-DMAG hindered the viability of human SCLC cell lines-regardless of their chemosensitivity-via the decreased expression of client proteins, including the proto-oncogene c-Raf (also known as RAF1). In an in vivo imaging model of SCLC multiple-organ metastasis with the human SCLC cell line SBC-5, treatment with 17-DMAG remarkably inhibited the formation of metastatic sites in the liver, but was ineffective in hindering the progression of bone lesions. The latter was likely the result of activation of osteoclasts. IGF-1, which is supposed to be rich in bone environment, preserved c-Raf expression and maintained viability of SBC-5 cells treated with 17-DMAG. Furthermore, the combined use of a bisphosphonate with 17-DMAG significantly attenuated the progression of metastases in both the liver and the bone. These findings suggest that therapeutic effects of HSP90 inhibitors may be organ-specific and should be carefully monitored in SCLC clinical trials.

In vivo imaging models of bone and brain metastases and pleural carcinomatosis with a novel human EML4-ALK lung cancer cell line.

EML4-ALK lung cancer accounts for approximately 3-7% of non-small-cell lung cancer cases. To investigate the molecular mechanism underlying tumor progression and targeted drug sensitivity/resistance in EML4-ALK lung cancer, clinically relevant animal models are indispensable. In this study, we found that the lung adenocarcinoma cell line A925L expresses an EML4-ALK gene fusion (variant 5a, E2:A20) and is sensitive to the ALK inhibitors crizotinib and alectinib. We further established highly tumorigenic A925LPE3 cells, which also have the EML4-ALK gene fusion (variant 5a) and are sensitive to ALK inhibitors. By using A925LPE3 cells with luciferase gene transfection, we established in vivo imaging models for pleural carcinomatosis, bone metastasis, and brain metastasis, all of which are significant clinical concerns of advanced EML4-ALK lung cancer. Interestingly, crizotinib caused tumors to shrink in the pleural carcinomatosis model, but not in bone and brain metastasis models, whereas alectinib showed remarkable efficacy in all three models, indicative of the clinical efficacy of these ALK inhibitors. Our in vivo imaging models of multiple organ sites may provide useful resources to analyze further the pathogenesis of EML4-ALK lung cancer and its response and resistance to ALK inhibitors in various organ microenvironments.

Therapeutic activity of glycoengineered anti-GM2 antibodies against malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a rare and highly aggressive neoplasm that arises from the pleural, pericardial, or peritoneal lining. Although surgery, chemotherapy, radiotherapy, and combinations of these therapies are used to treat MPM, the median survival of such patients is dismal. Therefore, there is a compelling need to develop novel therapeutics with different modes of action. Ganglioside GM2 is a glycolipid that has been shown to be overexpressed in various types of cancer. However, there are no published reports regarding the use of GM2 as a potential therapeutic target in cases of MPM. In this study, we evaluated the efficacy of the anti-GM2 antibody BIW-8962 as an anti-MPM therapeutic using in vitro and in vivo assays. Consequently, the GM2 expression in the MPM cell lines was confirmed using flow cytometry. In addition, eight of 11 cell lines were GM2-positive (73%), although the GM2 expression was variable. BIW-8962 showed a significant antibody-dependent cellular cytotoxicity activity against the GM2-expressing MPM cell line MSTO-211H, the effect of which depended on the antibody concentration and effector/target ratio. In an in vivo orthotropic mouse model using MSTO-211H cells, BIW-8962 significantly decreased the incidence and size of tumors. Additionally, the GM2 expression was confirmed in the MPM clinical specimens. Fifty-eight percent of the MPM tumors were positive for GM2, with individual variation in the intensity and frequency of staining. These data suggest that anti-GM2 antibodies may become a therapeutic option for MPM patients.

Validation of an ultra-performance liquid chromatography-tandem mass spectrometry method for the determination of flecainide in human plasma and its clinical application.

A simple and reproducible bioanalytical method for the determination of flecainide in human plasma was developed and validated using an ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) to obtain higher sensitivity than the current available methods. After simple protein precipitation, flecainide and a stable isotope-labeled internal standard (IS) were chromatographed on an Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 µm) with isocratic elution of mobile phase consisting of 45% methanol containing 0.1% formic acid at a flow rate 0.25 mL/min. Detection was performed in positive electrospray ionization by monitoring the selected ion transitions at m/z 415.4/301.1 for flecainide and m/z 419.4/305.1 for the IS. The method was validated according to current bioanalytical method validation guidelines. The calibration standard curve was linear from 2.5 to 1000 ng/mL using 0.1 mL of plasma. No significant interferences were detected in blank human plasma. Accuracy and precision in the intra- and inter-batch reproducibility study were within acceptance criteria. Neither hemolysis effects nor matrix effects were observed. The UPLC-MS/MS method developed was successfully applied to determine plasma flecainide concentrations to support clinical studies and incurred sample reanalysis also ensured the reproducibility of the method.

Validation of a hydrophilic interaction ultra-performance liquid chromatography-tandem mass spectrometry method for the determination of gemcitabine in human plasma with tetrahydrouridine.

A simple and reproducible bioanalytical method for the determination of gemcitabine in human plasma treated with tetrahydrouridine (THU) was developed and validated using a hydrophilic interaction ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS). To prevent deamination of gemcitabine, blood was treated with THU, and the plasma samples obtained after centrifugation were used in this study. Gemcitabine and gemcitabine-(13)C, (15)N2 used as an internal standard, were extracted from human plasma treated with THU using a 96-well Hybrid SPE-Precipitation plate. Extracts were chromatographed on a hydrophilic interaction chromatography column with isocratic elution. Detection was performed using Quattro Premier with positive electrospray ionization multiple reaction monitoring mode. The standard curve ranged from 10 to 10,000 ng/mL without carryover. No significant interferences were detected in blank plasma and no interferences by 2'-2'-difluoro-2'-deoxyuridine, a metabolite of gemcitabine. Accuracy and precision in the intra-batch reproducibility study using quality control samples with three THU levels did not exceed ±5.4 and 7.3%, respectively, and the inter-batch reproducibility results also met the criteria. Stability of gemcitabine was ensured in whole blood and plasma as well as stability of THU in solutions. The UPLC-MS/MS method developed was successfully validated and can be applied for gemcitabine bioanalysis in clinical studies.

Generic UPLC-MS/MS and Gyrolab assays with blood microsampling for pharmacokinetic assessments of therapeutic antibodies in mice.

Serial blood sampling from one animal is useful to understand relationship between pharmacokinetics (PK) and pharmacological or toxicological events in individual animals. To assess its feasibility in mice, two therapeutic antibodies were used to evaluate impacts by different blood sampling methods, sampling sites, and assay platforms on PK. Denosumab and Panitumumab were intravenously administered to mice and only 0.05 mL of blood sample per point was collected from jugular vein or tail vein. Blood samples were collected serially from a mouse or collected by traditional composite sampling from each mouse. Plasma concentrations of the two drugs were assayed by a generic ligand binding assay using Gyrolab or by a generic ultra-performance liquid chromatography with tandem mass spectrometry. The two assay platforms showed acceptable accuracy and precision and gave comparable PK parameters of the drugs, suggesting that both assays were successfully applied to the PK assessments. Comparable results in the PK profiles were noted between serial and composite blood samplings and differences in the two sampling sites did not impact PK. These findings suggest that microsampling combined with generic assays is useful to assess PK profiles of therapeutic antibodies in mice.

Neutral selection and clonal expansion during the development of colon cancer metastasis.

Intratumor heterogeneity has been shown to play a role in the malignant progression of cancer. Although clonal evolution in primary cancer has been well studied, that in metastatic tumorigenesis is not fully understood. In this study, we established human colon cancer-derived organoids and investigated clonal dynamics during liver metastasis development by tracking barcode-labelled subclones. Long-term subclone co-cultures showed clonal drift, with a single subclone becoming dominant in the cell population. Interestingly, the selected subclones were not always the same, suggesting that clonal selection was not based on cell intrinsic properties. Furthermore, liver tumors developed by co-transplantation of organoid subclones into the immunodeficient mouse spleen showed a progressive drastic reduction in clonal diversity, and only one or two subclones predominated in the majority of large metastatic tumors. Importantly, selections were not limited to particular subclones but appeared to be random. A trend towards a reduction in clonal diversity was also found in liver metastases of multiple color-labeled organoids of mouse intestinal tumors. Based on these results, we propose a novel mechanism of metastasis development, i.e. a subclone population of the disseminated tumor cells in the liver is selected by neutral selection during colonization and constitutes large metastatic tumors.

Targeting WEE1 enhances the antitumor effect of KRAS-mutated non-small cell lung cancer harboring TP53 mutations.

The clinical development of Kirsten rat sarcoma virus (KRAS)-G12C inhibitors for the treatment of KRAS-mutant lung cancer is limited by the presence of co-mutations, intrinsic resistance, and the emergence of acquired resistance. Therefore, innovative strategies for enhancing apoptosis in KRAS-mutated non-small cell lung cancer (NSCLC) are urgently needed. Through CRISPR-Cas9 knockout screening using a library of 746 crRNAs and drug screening with a custom library of 432 compounds, we discover that WEE1 kinase inhibitors are potent enhancers of apoptosis, particularly in KRAS-mutant NSCLC cells harboring TP53 mutations. Mechanistically, WEE1 inhibition promotes G2/M transition and reduces checkpoint kinase 2 (CHK2) and Rad51 expression in the DNA damage response (DDR) pathway, which is associated with apoptosis and the repair of DNA double-strand breaks, leading to mitotic catastrophe. Notably, the combined inhibition of KRAS-G12C and WEE1 consistently suppresses tumor growth. Our results suggest targeting WEE1 as a promising therapeutic strategy for KRAS-mutated NSCLC with TP53 mutations.

Dual inhibition of Met kinase and angiogenesis to overcome HGF-induced EGFR-TKI resistance in EGFR mutant lung cancer.

Acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) is a serious problem in the management of EGFR mutant lung cancer. We recently reported that hepatocyte growth factor (HGF) induces resistance to EGFR-TKIs by activating the Met/PI3K pathway. HGF is also known to induce angiogenesis in cooperation with vascular endothelial growth factor (VEGF), which is an important therapeutic target in lung cancer. Therefore, we hypothesized that dual inhibition of HGF and VEGF may be therapeutically useful for controlling HGF-induced EGFR-TKI-resistant lung cancer. We found that a dual Met/VEGF receptor 2 kinase inhibitor, E7050, circumvented HGF-induced EGFR-TKI resistance in EGFR mutant lung cancer cell lines by inhibiting the Met/Gab1/PI3K/Akt pathway in vitro. HGF stimulated VEGF production by activation of the Met/Gab1 signaling pathway in EGFR mutant lung cancer cell lines, and E7050 showed an inhibitory effect. In a xenograft model, tumors produced by HGF-transfected Ma-1 (Ma-1/HGF) cells were more angiogenic than vector control tumors and showed resistance to gefitinib. E7050 alone inhibited angiogenesis and retarded growth of Ma-1/HGF tumors. E7050 combined with gefitinib induced marked regression of tumor growth. Moreover, dual inhibition of HGF and VEGF by neutralizing antibodies combined with gefitinib also markedly regressed tumor growth. These results indicate the therapeutic rationale of dual targeting of HGF-Met and VEGF-VEGF receptor 2 for overcoming HGF-induced EGFR-TKI resistance in EGFR mutant lung cancer.

Heterogeneity among tumors with acquired resistance to EGFR tyrosine kinase inhibitors harboring EGFR-T790M mutation in non-small cell lung cancer cells.

EGFR-T790M mutation is a major mechanism underlying acquired resistance to first- and second-generation EGFR tyrosine kinase inhibitors (EGFR-TKIs) in lung cancer with mutated EGFR. However, differences in the biological characteristics of T790M tumors based on treatment regimens with each generation of EGFR-TKI are not fully understood. We established cell lines with acquired resistance harboring EGFR-T790M mutation derived from xenograft tumors treated with each generation of EGFR-TKI and examined their biological characteristics with respect to third-generation EGFR-TKI osimertinib sensitivity. Second-generation EGFR-TKI dacomitinib-resistant cells with T790M-exhibited higher sensitivity to osimertinib than first-generation EGFR-TKI gefitinib-resistant cells with T790M via inhibition of AKT and ERK signaling and promotion of apoptosis. Furthermore, gefitinib-resistant cells showed enhanced intratumor heterogeneity accompanied by genomic instability and activation of alternative resistance mechanisms compared with dacomitinib-resistant cells; this suggests that the maintenance of EGFR dependency after acquiring resistance might depend on the type of EGFR-TKI. Our results demonstrate that the progression of tumor heterogeneity via both genetic and non-genetic mechanisms might affect osimertinib sensitivity in tumors with acquired resistance harboring EGFR-T790M mutation.

Genetically engineered humanized anti-ganglioside GM2 antibody against multiple organ metastasis produced by GM2-expressing small-cell lung cancer cells.

Small-cell lung cancer (SCLC) grows rapidly and metastasizes to multiple organs. We examined the antimetastatic effects of the humanized anti-ganglioside GM2 (GM2) antibodies, BIW-8962 and KM8927, compared with the chimeric antibody KM966, in a SCID mouse model of multiple organ metastases induced by GM2-expressing SCLC cells. BIW-8962 and KM8927 induced higher antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity than KM966 against the GM2-expressing SCLC cell line SBC-3 in vitro. These humanized antibodies inhibited the production of multiple organ metastases, increased the number of apoptotic cells, and prolonged the survival of the SCID mice. Histological analyses using clinical specimens showed that SCLC cells expressed GM2. These findings suggest that humanized anti-GM2 antibodies could be therapeutically useful for controlling multiple organ metastases of GM2-expressing SCLC.

Quantitative and qualitative application of a novel capillary microsampling device, Microsampling Wing™ (MSW), using antiepileptic drugs in rats.

A novel microsampling device, namely, the Microsampling Wing™ (MSW), was evaluated using three anti-epileptic drugs (AEDs): carbamazepine, lamotrigine, and phenytoin. A simultaneous assay method of the three AEDs was developed and qualified via liquid chromatography with tandem mass spectrometry. Using 2.8 µL plasma, the three AEDs were quantifiable from 1 or 2 ng/mL. According to the intra-assay reproducibility assessment and additional validation parameters, the established method is reproducible. To apply the device to a pharmacokinetic (PK) study in rats, a cocktail of the three AEDs was orally administered to rats. Whole blood samples were serially collected using the MSW device and a glass capillary from the tail vein, and plasma samples (each 2.8 µL) from each device were assayed to compare PK parameters. The PK parameters of the three AEDs were similar between the two devices. A metabolite identification study was also conducted after oral administration of carbamazepine to rats. At least seven metabolites were detected in plasma, and the major metabolite was carbamazepine 10,11-epoxide, which is in accordance with the reported results. These findings suggest that the MSW device is a useful microsampling device for PK and metabolite identification studies.

FOXO3 is a latent tumor suppressor for FOXO3-positive and cytoplasmic-type gastric cancer cells.

FOXO3 is a member of the FOXO transcription factors thought to play a tumor-suppressor role in gastrointestinal cancer, while tumor-promoting function of FOXO3 has also been reported. These results suggest a context-dependent function of FOXO3 in tumor development. However, the relationship between the FOXO3 expression pattern and its role in tumorigenesis has not been elucidated. We examined the FOXO3 expression in 65 human primary gastric cancer and patient-derived xenograft tissues by immunohistochemistry and identified three subtypes according to subcellular localization: FOXO3-nuclear accumulated (FOXO3-Nuc), FOXO3-nuclear/cytoplasmic or cytoplasmic distributed (FOXO3-Cyt), and FOXO3-negative. In the FOXO3-Cyt gastric cancer cells, the expression of the constitutive active mutant FOXO3 (Act-ER FOXO3) induced the nuclear accumulation of FOXO3 and significantly suppressed colony formation and proliferation. The inhibition of the PI3K-AKT pathway by inhibitor treatment also suppressed the proliferation of FOXO3-Cyt gastric cancer cells, which was associated with the nuclear accumulation of endogenous FOXO3. Furthermore, the expression of Act-ER FOXO3 by an endogenous promoter significantly suppressed gastric tumorigenesis in Gan mice, a model of gastric cancer. Finally, treatment of FOXO3-Cyt human gastric cancer-derived organoids with an AKT inhibitor significantly suppressed the survival and proliferation. These results indicate that FOXO3 is a latent tumor suppressor for FOXO3-Cyt-type gastric cancer cells and that activation of the PI3K-AKT pathway protects this type of gastric cancer cell from FOXO3-mediated growth suppression via constitutive nuclear export. Thus, the inhibition of the PI3K-AKT pathway and nuclear translocation of endogenous FOXO3 may have therapeutic applications in the treatment of FOXO3-positive and cytoplasmic-type gastric cancer.

Transient IGF-1R inhibition combined with osimertinib eradicates AXL-low expressing EGFR mutated lung cancer.

Drug tolerance is the basis for acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) including osimertinib, through mechanisms that still remain unclear. Here, we show that while AXL-low expressing EGFR mutated lung cancer (EGFRmut-LC) cells are more sensitive to osimertinib than AXL-high expressing EGFRmut-LC cells, a small population emerge osimertinib tolerance. The tolerance is mediated by the increased expression and phosphorylation of insulin-like growth factor-1 receptor (IGF-1R), caused by the induction of its transcription factor FOXA1. IGF-1R maintains association with EGFR and adaptor proteins, including Gab1 and IRS1, in the presence of osimertinib and restores the survival signal. In AXL-low-expressing EGFRmut-LC cell-derived xenograft and patient-derived xenograft models, transient IGF-1R inhibition combined with continuous osimertinib treatment could eradicate tumors and prevent regrowth even after the cessation of osimertinib. These results indicate that optimal inhibition of tolerant signals combined with osimertinib may dramatically improve the outcome of EGFRmut-LC.

Application of a Volumetric Absorptive Microsampling Device to a Pharmacokinetic Study of Tacrolimus in Rats: Comparison with Wet Blood and Plasma.

BACKGROUND AND OBJECTIVES: Volumetric absorptive microsampling (VAMS) devices are useful for sampling a smaller volume of blood from rodents in the preclinical setting. In the present study, we evaluated the proof of concept of a VAMS device by comparing the pharmacokinetic data of tacrolimus in rats among dried blood in VAMS, wet blood, and plasma. METHODS: Tacrolimus was administered orally, to rats, at a dose of 10 mg/kg. Only 10 µL aliquots of blood were absorbed by VAMS devices at designated time points. Tacrolimus was extracted with a methanol-water mixture (1:1, v/v) via sonication. Tacrolimus levels in wet blood (10 µL) and plasma (10 µL) were quantified after protein precipitation. RESULTS: Tacrolimus in VAMS devices was quantifiable from 0.2 ng/mL using high-performance liquid chromatography with tandem mass spectrometer. Accuracy and precision were within the acceptance criteria. Bland-Altman plots showed that tacrolimus concentrations in VAMS devices were similar to those in wet blood, regardless of tacrolimus levels. On the other hand, tacrolimus levels in plasma were different from those in VAMS devices, especially at lower concentrations, likely due to partition of tacrolimus to blood cells. However, pharmacokinetic parameters were comparable among the three matrices. CONCLUSIONS: Collectively, these findings suggest that the VAMS device can be a useful device for pharmacokinetic studies in rats.