Prediction of Acute Hepatotoxicity With Human Pluripotent Stem Cell-Derived Hepatic Organoids.

Recent development of hepatic organoids (HOs) derived from human pluripotent stem cells (hPSCs) provides an alternative in vitro model that can mimic the human liver detoxification pathway for drug safety assessment. By recapitulating the high level of maturity and drug-metabolizing capacity of the liver in a three-dimensional organoid culture, HOs may allow researchers to assess drug toxicity and metabolism more accurately than animal models or hepatocellular carcinoma cells. Although this promising potential has contributed to the development of various protocols, only a few protocols are available to generate functional HOs with guaranteed CYP450 enzymatic activity, the key feature driving toxic responses during drug metabolism. Based on previously published protocols, we describe an optimized culture method that can substantially increase the expression and activity of CYP450s, in particular CYP3A4, CYP2C9, and CYP2C19, in HOs. To generate mass-produced and highly reproducible HOs required as models for toxicity evaluation, we first generated hepatic endodermal organoids (HEOs) from hPSCs capable of in vitro proliferation and cryopreservation. The stepwise protocol includes generating HEOs as well as efficient methods to enhance CYP450 expression and activity in terminally differentiated HOs. Furthermore, we present a simple protocol for the assessment of HO cytotoxicity, one of the hallmarks of drug-induced acute hepatotoxicity. The protocols are relatively straightforward and can be successfully used by laboratories with basic experience in culturing hPSCs. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of hepatic endodermal organoids from human pluripotent stem cells Basic Protocol 2: Expansion and cryopreservation of hepatic endodermal organoids Basic Protocol 3: Differentiation of hepatic organoids from hepatic endodermal organoids Basic Protocol 4: Evaluation of hepatotoxicity using hepatic organoids Support Protocol: Human pluripotent stem cell culture.

Overcoming BRAF and CDK4/6 inhibitor resistance by inhibiting MAP3K3-dependent protection against YAP lysosomal degradation.

Transcriptional programs governed by YAP play key roles in conferring resistance to various molecular-targeted anticancer agents. Strategies aimed at inhibiting YAP activity have garnered substantial interest as a means to overcome drug resistance. However, despite extensive research into the canonical Hippo-YAP pathway, few clinical agents are currently available to counteract YAP-associated drug resistance. Here, we present a novel mechanism of YAP stability regulation by MAP3K3 that is independent of Hippo kinases. Furthermore, we identified MAP3K3 as a target for overcoming anticancer drug resistance. Depletion of MAP3K3 led to a substantial reduction in the YAP protein level in melanoma and breast cancer cells. Mass spectrometry analysis revealed that MAP3K3 phosphorylates YAP at serine 405. This MAP3K3-mediated phosphorylation event hindered the binding of the E3 ubiquitin ligase FBXW7 to YAP, thereby preventing its p62-mediated lysosomal degradation. Robust YAP activation was observed in CDK4/6 inhibitor-resistant luminal breast cancer cells. Knockdown or pharmacological inhibition of MAP3K3 effectively suppressed YAP activity and restored CDK4/6 inhibitor sensitivity. Similarly, elevated MAP3K3 expression supported the prosurvival activity of YAP in BRAF inhibitor-resistant melanoma cells. Inhibition of MAP3K3 decreased YAP-dependent cell proliferation and successfully restored BRAF inhibitor sensitivity. In conclusion, our study reveals a previously unrecognized mechanism for the regulation of YAP stability, suggesting MAP3K3 inhibition as a promising strategy for overcoming resistance to CDK4/6 and BRAF inhibitors in cancer treatment.

Nuclear Localization of Yes-Associated Protein Is Associated With Tumor Progression in Cutaneous Melanoma.

Yes-associated protein (YAP), an effector molecule of the Hippo signaling pathway, is expressed at high levels in cutaneous melanoma. However, the role of YAP in melanoma progression according to cellular localization is poorly understood. Tissues from 140 patients with invasive melanoma were evaluated by immunohistochemistry. Flow cytometry, western blotting, viability assays, wound healing assays, verteporfin treatment, and xenograft assays were conducted using melanoma cell lines B16F1 and B16F10 subjected to YapS127A transfection and siYap knockdown. Nuclear YAP localization was identified in 63 tumors (45.0%) and was more frequent than cytoplasmic YAP in acral lentiginous and nodular subtypes (P = .007). Compared with cytoplasmic YAP melanomas, melanomas with nuclear YAP had higher mitotic activity (P = .016), deeper invasion (P < .001), and more frequently metastasized to lymph nodes (P < .001) and distant organs (P < .001). Patients with nuclear YAP melanomas had poorer disease-free survival (P < .001) and overall survival (P < .001). Nuclear YAP was an independent risk factor for distant metastasis (hazard ratio: 3.206; 95% CI, 1.032-9.961; P = .044). Proliferative ability was decreased in siYapB16F1 (P < .001) and siYapB16F10 (P = .001) cells and increased in YapS127AB16F1 (P = .003) and YapS127AB16F10 (P = .002) cells. Cell cycle analysis demonstrated relative G1 retention in siYapB16F1 (P < .001) and siYapB16F10 (P < .001) cells and S retention in YapS127AB16F1 cells (P = .008). Wound healing assays showed that Yap knockdown inhibited cell invasion (siYapB16F1, P = .001; siYapB16F10, P < .001), whereas nuclear YAP promoted it (YapS127AB16F, P < .001; YapS127AB16F1, P = .017). Verteporfin, a direct YAP inhibitor, reduced cellular proliferation in B16F1 (P = .003) and B16F10 (P < .001) cells. Proliferative effects of nuclear YAP were confirmed in xenograft mice (P < .001). In conclusion, nuclear YAP in human melanomas showed subtype specificity and correlated with proliferative activity and proinvasiveness. It is expected that YAP becomes a useful prognostic marker, and its inhibition may be a potential therapy for melanoma patients.

Short-term inhalation exposure to cigarette smoke induces oxidative stress and inflammation in lungs without systemic oxidative stress in mice.

Smoking is a well-established risk factor for various pathologies, including pulmonary diseases, cardiovascular disorders, and cancers. The toxic effects of cigarette smoke (CS) are mediated through multiple pathways and diverse mechanisms. A key pathogenic factor is oxidative stress, primarily induced by excessive formation of reactive oxygen species. However, it remains unclear whether smoking directly induces systemic oxidative stress or if such stress is a secondary consequence. This study aimed to determine whether short-term inhalation exposure to CS induces oxidative stress in extrapulmonary organs in addition to the lung in a murine model. In the experiment, 3R4F reference cigarettes were used to generate CS, and 8-week-old male BALB/c mice were exposed to CS at a total particulate matter concentration of either 0 or 800 µg/L for four consecutive days. CS exposure led to an increase in neutrophils, eosinophils, and total cell counts in bronchoalveolar lavage fluid. It also elevated levels of lactate dehydrogenase and malondialdehyde (MDA), markers indicative of tissue damage and oxidative stress, respectively. Conversely, no significant changes were observed in systemic oxidative stress markers such as total oxidant scavenging capacity, MDA, glutathione (GSH), and the GSH/GSSG ratio in blood samples. In line with these findings, CS exposure elevated NADPH oxidase (NOX)-dependent superoxide generation in the lung but not in other organs like the liver, kidney, heart, aorta, and brain. Collectively, our results indicate that short-term exposure to CS induces inflammation and oxidative stress in the lung without significantly affecting oxidative stress in extrapulmonary organs under the current experimental conditions. NOX may play a role in these pulmonary-specific events.

Clinical Activity of TGF-ß Inhibitor Vactosertib in Combination with Imatinib in Desmoid Tumors: A Multicenter Phase Ib/II Study.

PURPOSE: The study was to determine the activity and safety of the TGF-ß inhibitor vactosertib in combination with imatinib in patients with desmoid tumors. PATIENTS AND METHODS: In this investigator-initiated, open-label, multicenter, phase Ib/II trial, patients with desmoid tumors not amenable to locoregional therapies (surgery and/or radiotherapy) or with disease progression following at least one treatment were enrolled. Participants were administered 400 mg imatinib daily in combination with vactosertib (5 days on and 2 days off, twice a day) every 28 days. In phase Ib, the vactosertib dose was set at 100 mg (level -1) and 200 mg (level 1) to determine the recommended phase II dose (RP2D). Phase II assessed the efficacy, with the primary endpoint being progression-free rate (PFR) at 16 weeks. RESULTS: No dose-limiting toxicities were observed during phase Ib; therefore RP2D was defined at doses of 400 mg imatinib daily in combination with 200 mg vactosertib. Of the 27 patients evaluated, 7 (25.9%) achieved a confirmed partial response and 19 (70.4%) were stable. The PFR at 16 weeks and 1 year were 96.3% and 81.0%, respectively. Most toxicities were mild to moderate myalgia (n = 10, 37%), anemia (n = 10, 37%), and nausea (n = 9, 33.3%). Common grade 3 to 4 toxicities included neutropenia (n = 6, 22.2%) and anemia (n = 5, 18.5%). CONCLUSIONS: The vactosertib and imatinib combination was well tolerated, with promising clinical activity in patients with progressive, locally advanced desmoid tumors. This is the first study investigating a novel target agent, a TGF-ß inhibitor, in this rare and difficult-to-treat desmoid tumor.

Hepa-ToxMOA: a pathway-screening method for evaluating cellular stress and hepatic metabolic-dependent toxicity of natural products.

In the field of drug discovery, natural products have emerged as therapeutic agents for diseases such as cancer. However, their potential toxicity poses significant obstacles in the developing effective drug candidates. To overcome this limitation, we propose a pathway-screening method based on imaging analysis to evaluate cellular stress caused by natural products. We have established a cellular stress sensing system, named Hepa-ToxMOA, which utilizes HepG2 cells expressing green fluorescent protein (GFP) fluorescence under the control of transcription factor response elements (TREs) for transcription factors (AP1, P53, Nrf2, and NF-κB). Additionally, to augment the drug metabolic activity of the HepG2 cell line, we evaluated the cytotoxicity of 40 natural products with and without S9 fraction-based metabolic activity. Our finding revealed different activities of Hepa-ToxMOA depending on metabolic or non-metabolic activity, highlighting the involvement of specific cellular stress pathways. Our results suggest that developing a Hepa-ToxMOA system based on activity of drug metabolizing enzyme provides crucial insights into the molecular mechanisms initiating cellular stress during liver toxicity screening for natural products. The pathway-screening method addresses challenges related to the potential toxicity of natural products, advancing their translation into viable therapeutic agents.

Durvalumab plus pazopanib combination in patients with advanced soft tissue sarcomas: a phase II trial.

We aimed to determine the activity of the anti-VEGF receptor tyrosine-kinase inhibitor, pazopanib, combined with the anti-PD-L1 inhibitor, durvalumab, in metastatic and/or recurrent soft tissue sarcoma (STS). In this single-arm phase 2 trial (NCT03798106), treatment consisted of pazopanib 800 mg orally once a day and durvalumab 1500 mg once every 3 weeks. Primary outcome was overall response rate (ORR) and secondary outcomes included progression-free survival (PFS), overall survival, disease control rate, immune-related response criteria, and safety. The ORR was 30.4% and the trial met the pre-specified endpoint. The median PFS was 7.7 months (95% confidence interval: 5.7-10.4). The common treatment-related adverse events of grades 3-4 included neutropenia (9 [19.1%]), elevated aspartate aminotransferase (7 [14.9%]), alanine aminotransferase (5 [10.6%]), and thrombocytopenia (4 [8.5%]). In a prespecified transcriptomic analysis, the B lineage signature was a significant key determinant of overall response (P = 0.014). In situ analysis also showed that tumours with high CD20+ B cell infiltration and vessel density had a longer PFS (P = 6.5 × 10-4) than those with low B cell infiltration and vessel density, as well as better response (50% vs 12%, P = 0.019). CD20+ B cell infiltration was identified as the only independent predictor of PFS via multivariate analysis. Durvalumab combined with pazopanib demonstrated promising efficacy in an unselected STS cohort, with a manageable toxicity profile.

Reaching New Heights: A Comprehensive Study of Hand Transplantations in Korea after Institutionalization of Hand Transplantation Law.

PURPOSE: With the revision of the Organ and Transplantation Act in 2018, the hand has become legal as an area of transplantable organs in Korea. In January 2021, the first hand allotransplantation since legalization was successfully performed, and we have performed a total of three successful hand transplantation since then. By comparing and incorporating our experiences, this study aimed to provide a comprehensive reconstructive solution for hand amputation in Korea. MATERIALS AND METHODS: Recipients were selected through a structured preoperative evaluation, and hand transplantations were performed at the distal forearm level. Postoperatively, patients were treated with three-drug immunosuppressive regimen, and functional outcomes were monitored. RESULTS: The hand transplantations were performed without intraoperative complications. All patients had partial skin necrosis and underwent additional surgical procedures in 2 months after transplantation. After additional operations, no further severe complications were observed. Also, patients developed acute rejection within 3 months of surgery, but all resolved within 2 weeks after steroid pulse therapy. Motor and sensory function improved dramatically, and patients were very satisfied with the appearance and function of their transplanted hands. CONCLUSION: Hand transplantation is a viable reconstructive option, and patients have shown positive functional and psychological outcomes. Although this study has limitations, such as the small number of patients and short follow-up period, we should focus on continued recovery of hand function, and be careful not to develop side effects from immunosuppressive drugs. Through the present study, we will continue to strive for a bright future regarding hand transplantation in Korea.

Role of monocarboxylate transporter I/lactate dehydrogenase B-mediated lactate recycling in tamoxifen-resistant breast cancer cells.

Although tamoxifen (TAM) is widely used in patients with estrogen receptor-positive breast cancer, the development of tamoxifen resistance is common. The previous finding suggests that the development of tamoxifen resistance is driven by epiregulin or hypoxia-inducible factor-1α-dependent glycolysis activation. Nonetheless, the mechanisms responsible for cancer cell survival and growth in a lactic acid-rich environment remain elusive. We found that the growth and survival of tamoxifen-resistant MCF-7 cells (TAMR-MCF-7) depend on glycolysis rather than oxidative phosphorylation. The levels of the glycolytic enzymes were higher in TAMR-MCF-7 cells than in parental MCF-7 cells, whereas the mitochondrial number and complex I level were decreased. Importantly, TAMR-MCF-7 cells were more resistant to low glucose and high lactate growth conditions. Isotope tracing analysis using 13C-lactate confirmed that lactate conversion to pyruvate was enhanced in TAMR-MCF-7 cells. We identified monocarboxylate transporter1 (MCT1) and lactate dehydrogenase B (LDHB) as important mediators of lactate influx and its conversion to pyruvate, respectively. Consistently, AR-C155858 (MCT1 inhibitor) inhibited the proliferation, migration, spheroid formation, and in vivo tumor growth of TAMR-MCF-7 cells. Our findings suggest that TAMR-MCF-7 cells depend on glycolysis and glutaminolysis for energy and support that targeting MCT1- and LDHB-dependent lactate recycling may be a promising strategy to treat patients with TAM-resistant breast cancer.

Enhancement of the therapeutic efficacy of the MAP regimen using thiamine pyrophosphate-decorated albumin nanoclusters in osteosarcoma treatment.

Recent studies on osteosarcoma regimens have mainly focused on modifying the combination of antineoplastic agents rather than enhancing the therapeutic efficacy of each component. Here, an albumin nanocluster (NC)-assisted methotrexate (MTX), doxorubicin (DOX), and cisplatin (MAP) regimen with improved antitumor efficacy is presented. Human serum albumin (HSA) is decorated with thiamine pyrophosphate (TPP) to increase the affinity to the bone tumor microenvironment (TME). MTX or DOX (hydrophobic MAP components) is adsorbed to HSA-TPP via hydrophobic interactions. MTX- or DOX-adsorbed HSA-TPP NCs exhibit 20.8- and 1.64-fold higher binding affinity to hydroxyapatite, respectively, than corresponding HSA NCs, suggesting improved targeting ability to the bone TME via TPP decoration. A modified MAP regimen consisting of MTX- or DOX-adsorbed HSA-TPP NCs and free cisplatin displays a higher synergistic anticancer effect in HOS/MNNG human osteosarcoma cells than conventional MAP. TPP-decorated NCs show 1.53-fold higher tumor accumulation than unmodified NCs in an orthotopic osteosarcoma mouse model, indicating increased bone tumor distribution. As a result, the modified regimen more significantly suppresses tumor growth in vivo than solution-based conventional MAP, suggesting that HSA-TPP NC-assisted MAP may be a promising strategy for osteosarcoma treatment.

Genomic and transcriptomic profiling reveal molecular characteristics of parathyroid carcinoma.

Genomic and transcriptomic profiling has enhanced the diagnostic and treatment options for many cancers. However, the molecular characteristics of parathyroid cancer remain largely unexplored, thereby limiting the development of new therapeutic interventions. Herein, we conducted genomic and transcriptomic sequencing of 50 parathyroid tissues (12 carcinomas, 28 adenomas, and 10 normal tissues) to investigate the intrinsic and comparative molecular features of parathyroid carcinoma. We confirmed multiple two-hit mutation patterns in cell division cycle 73 (CDC73) that converged to biallelic inactivation, calling into question the presence of a second hit in other genes. In addition, allele-specific repression of CDC73 in copies with germline-truncating variants suggested selective pressure prior to tumorigenesis. Transcriptomic analysis identified upregulation of the expression of E2F targets, KRAS and TNF-alpha signaling, and epithelial-mesenchymal transition pathways in carcinomas compared to adenomas and normal tissues. A molecular classification model based on carcinoma-specific genes clearly separated carcinomas from adenomas and normal tissues, the clinical utility of which was demonstrated in two patients with uncertain malignant potential. A deeper analysis of gene expression and functional prediction suggested that Wilms tumor 1 (WT1) is a potential biomarker for CDC73-mutant parathyroid carcinoma, which was further validated through immunohistochemistry. Overall, our study revealed the genomic and transcriptomic profiles of parathyroid carcinoma and may help direct future precision diagnostic and therapeutic improvements.

Genetic Characteristics of Primary Cutaneous Malignant Melanoma in Koreans Compared With Western Populations.

BACKGROUND/AIM: Cutaneous melanoma, a melanocyte malignancy, can be divided into many clinical subtypes that differ in presentation, demographics, and genetic profile. In this study, we used next-generation sequencing (NGS) analysis to review genetic alterations in 47 primary cutaneous melanomas in the Korean population and compared them to alterations from melanomas in Western populations. PATIENTS AND METHODS: We retrospectively reviewed clinicopathologic and genetic features of 47 patients diagnosed with cutaneous melanomas between 2019-2021 at Severance Hospital, Yonsei University College of Medicine. NGS analysis was performed at diagnosis to evaluate single nucleotide variations (SNVs), copy number variations (CNVs), and genetic fusions. Genetic features in Western cohorts of melanoma were then compared with previous studies performed in the USA: Cohort 1 (n=556), Cohort 2 (n=79), and Cohort 3 (n=38). RESULTS: The most common histological classification of melanoma was the acral lentiginous type (23/47, 48.9%). BRAF V600 mutation was most frequent (11/47, 23.4%), but was significantly lower compared to Cohort 1 (240/556, 43.2%) and Cohort 2 (34/79, 43.0%) (p=0.0300). CNV analysis identified amplifications in chromosomes 12q14.1-12q15 (11/47, 23.4%) including CDK4 and MDM2 genes and 11q13.3 (9/47, 19.2%) including CND1, FGF19, FGF3, and FGF4 genes more frequently in the present study population than Cohort 1 (p<0.0001). CONCLUSION: These results clearly demonstrated differences in genetic alterations between melanomas in Asian and Western populations. Therefore, BRAF V600 mutation should be considered a significant signaling pathway explaining melanoma pathogenesis occurrence in both Asian and Western populations, whereas loss of chromosome 9p21.3 is unique to melanomas in Western populations.

Generation of multilineage liver organoids with luminal vasculature and bile ducts from human pluripotent stem cells via modulation of Notch signaling.

BACKGROUND: The generation of liver organoids recapitulating parenchymal and non-parenchymal cell interplay is essential for the precise in vitro modeling of liver diseases. Although different types of multilineage liver organoids (mLOs) have been generated from human pluripotent stem cells (hPSCs), the assembly and concurrent differentiation of multiple cell types in individual mLOs remain a major challenge. Particularly, most studies focused on the vascularization of mLOs in host tissue after transplantation in vivo. However, relatively little information is available on the in vitro formation of luminal vasculature in mLOs themselves. METHODS: The mLOs with luminal blood vessels and bile ducts were generated by assembling hepatic endoderm, hepatic stellate cell-like cells (HscLCs), and endothelial cells derived entirely from hPSCs using 96-well ultra-low attachment plates. We analyzed the effect of HscLC incorporation and Notch signaling modulation on the formation of both bile ducts and vasculature in mLOs using immunofluorescence staining, qRT-PCR, ELISA, and live-perfusion imaging. The potential use of the mLOs in fibrosis modeling was evaluated by histological and gene expression analyses after treatment with pro-fibrotic cytokines. RESULTS: We found that hPSC-derived HscLCs are crucial for generating functional microvasculature in mLOs. HscLC incorporation and subsequent vascularization substantially reduced apoptotic cell death and promoted the survival and growth of mLOs with microvessels. In particular, precise modulation of Notch signaling during a specific time window in organoid differentiation was critical for generating both bile ducts and vasculature. Live-cell imaging, a series of confocal scans, and electron microscopy demonstrated that blood vessels were well distributed inside mLOs and had perfusable lumens in vitro. In addition, exposure of mLOs to pro-fibrotic cytokines induced early fibrosis-associated events, including upregulation of genes associated with fibrotic induction and endothelial cell activation (i.e., collagen I, α-SMA, and ICAM) together with destruction of tissue architecture and organoid shrinkage. CONCLUSION: Our results demonstrate that mLOs can reproduce parenchymal and non-parenchymal cell interactions and suggest that their application can advance the precise modeling of liver diseases in vitro.

Selective Inhibition of PI3K Isoforms in Brain Tumors Suppresses Tumor Growth by Increasing Radiosensitivity.

PURPOSE: Glioblastoma (GBM) is a malignant brain tumor with poor prognosis. Radioresistance is a major challenge in the treatment of brain tumors. The development of several types of tumors, including GBM, involves the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. Upon activation, this pathway induces radioresistance. In this study, we investigated whether additional use of selective inhibitors of PI3K isoforms would enhance radiosensitivity in GBM. MATERIALS AND METHODS: We evaluated whether radiation combined with PI3K isoform selective inhibitors can suppress radioresistance in GBM. Glioma 261 expressing luciferase (GL261-luc) and LN229 were used to confirm the effect of combination of radiation and PI3K isoform inhibitors in vitro. Cell viability was confirmed by clonogenic assay, and inhibition of PI3K/AKT signaling activation was observed by Western blot. To confirm radiosensitivity, the expression of phospho-γ-H2AX was observed by immunofluorescence. In addition, to identify the effect of a combination of radiation and PI3K-α isoform inhibitor in vivo, an intracranial mouse model was established by implanting GL261-luc. Tumor growth was observed by IVIS imaging, and survival was analyzed using Kaplan-Meier survival curves. RESULTS: Suppression of the PI3K/AKT signaling pathway increased radiosensitivity, and PI3K-α inhibition had similar effects on PI3K-pan inhibition in vitro. The combination of radiotherapy and PI3K-α isoform inhibitor suppressed tumor growth and extended survival in vivo. CONCLUSION: This study verified that PI3K-α isoform inhibition improves radiosensitivity, resulting in tumor growth suppression and extended survival in GBM mice.

Evaluation of genotoxicity and 13-week subchronic toxicity of root of Asarum heterotropoides var. seoulense (Nakai) Kitag.

ETHNOPHARMACOLOGICAL RELEVANCE: Asarum heterotropoides var. seoulense (Nakai) Kitag is a traditional herbal medicine used in Korea and China. It is effective in aphthous stomatitis, local anesthesia, headache, toothache, gingivitis, and inflammatory diseases. However, information on the toxicity of the root of Asarum heterotropoides var. seoulense (Nakai) Kitag (AR) is limited. Therefore, preclinical toxicity studies on AR are needed to reduce the risk of excessive intake. AIM OF THE STUDY: We aimed to evaluate genotoxicity and the potential toxicity due to repeated administration of AR powder. MATERIALS AND METHODS: In vitro bacterial reverse mutation assay (Ames), in vitro chromosomal aberration assay (CA), and in vivo micronucleus (MN) assay in ICR mice were conducted. As positive results were obtained in Ames and CA assays, alkaline comet assay and pig-a gene mutation test were conducted for confirmation. For evaluating the general toxicity of AR powder, a 13-week subchronic toxicity test was conducted, after determining the dose by performing a single and a 4-week dose range finding (DRF) test. A total of 152 Sprague-Dawley (SD) rats were orally administered AR powder at doses of 0, 150, 350, 500, 1000, and 2000 mg/kg/day in the 13-week subchronic toxicity test. Hematology, clinical chemistry, urinalysis, organ weight, macro-, and microscopic examination were conducted after rat necropsy. RESULTS: AR powder induced genotoxicity evidenced in the Ames test at 187.5, 750, 375, and 1500 µg/plate of TA100, TA98, TA1537, and E. coli WP2uvrA in the presence and absence of S9, respectively; CA test at 790 µg/mL for 6 h in the presence of S-9; 75 µg/mL for 6 h in the absence of S-9, and 70 µg/mL for 22 h in the absence of S-9 in the stomach in the comet assay but not in MN and pig-a assays. In the 13-week subchronic toxicity study, clinical signs including irregular respiration, noisy respiration, salivation, and decreased body weight or food consumption were observed in males and females in the 2000 mg/kg/day group. In hematology tests, clinical chemistry, urinalysis, organ weight, and macroscopic examination, changes were observed in the dose groups of 500 mg/kg/day and above. Microscopic examination revealed hyperplasia of the stomach as a test-related change. Hepatocellular adenoma and changes in liver-related clinical chemistry parameters were observed. The rat No Observed Adverse Effect Level (NOAEL) was 150 mg/kg/day in males and <150 mg/kg/day in females. CONCLUSIONS: AR powder is potentially toxic to the liver and stomach and should be used with caution in humans. A long-term study on carcinogenicity is necessitated because DNA damage or changes in tissue lesions were observed in SD rats.

Comprehensive Molecular Characterization of Soft Tissue Sarcoma for Prediction of Pazopanib-Based Treatment Response.

PURPOSE: Even though pazopanib, a multitargeted tyrosine kinase inhibitor, has been approved for refractory soft tissue sarcoma (STS), little is known about the molecular determinants of the response to pazopanib. We performed integrative molecular characterization to identify potential predictors of pazopanib efficacy. Materials and Methods: We obtained fresh pre-treatment tumor tissue from 35 patients with advanced STS receiving pazopanib-based treatment. Among those, 18 (51.4%) received pazopanib monotherapy, and the remaining 17 (48.6%) received pazopanib in combination with durvalumab, programmed death-ligand 1 blockade. Whole-exome and transcriptome sequencing were performed for each tumor and patient germline DNA. RESULTS: Of the 35 patients receiving pazopanib-based treatment, nine achieved a partial response (PR), resulting in an objective response rate (ORR) of 27.3%, and the median progression-free survival (PFS) was 6.0 months. Patients with CDK4 amplification (copy ratio tumor to normal > 2) exhibited shorter PFS (3.7 vs. 7.9 months, p=2.09×10-4) and a poorer response (ORR; 0% vs. 33.3%) compared to those without a gene amplification (copy ratio ≤ 2). Moreover, non-responders demonstrated transcriptional activation of CDK4 via DNA amplification, resulting in cell cycle activation. In the durvalumab combination cohort, seven of the 17 patients (41.2%) achieved a PR, and gene expression analysis revealed that durvalumab responders exhibited high immune/stromal cell infiltration, mainly comprising natural killer cells, compared to non-responders as well as increased expression of CD19, a B-cell marker. CONCLUSION: Despite the limitation of heterogeneity in the study population and treatment, we identified possible molecular predictors of pazopanib efficacy that can be employed in future clinical trials aimed at evaluating therapeutic strategies.

Therapeutic Implications of TGF-ß Pathway in Desmoid Tumor Based on Comprehensive Molecular Profiling and Clinicopathological Properties.

(1) Background: Desmoid tumors have a relatively high local failure rate after primary treatment using surgery and/or radiotherapy. Moreover, desmoid tumors recur at the primary site for many patients. An effective therapeutic strategy for the desmoid tumor is needed to maintain quality of life and prolong survival. (2) Method: First of all, we collected desmoid tumor tissues and investigated the status of protein expression for beta-catenin and alpha-SMA through immunohistochemistry. Then, we performed targeted sequencing and whole RNA sequencing. To compare the data with other cancer types, we used NGS data from sarcoma patients at Yonsei Cancer Center (YCC-sarcoma cohort, n = 48) and The Cancer Genome Atlas (TCGA, n = 9235). Secondly, we established the novel patient-derived preclinical models (n = 2) for the validation of treatment strategy. The same gene alteration of primary tissue was demonstrated. (3) Results: We discovered specific gene sets related to the TGF-ß signaling pathway. Moreover, we selected the combination treatment comprising TGF-ß inhibitor, vactosertib, and imatinib. In screening for the anti-proliferation effect, the combination treatment of TGF-ß inhibitor was more effective for tumor suppression than monotherapy. (4) Conclusion: We found preclinical indications that TGF-ß inhibitors could prove useful as a potential treatment for patients with desmoid tumors. Moreover, we could find some examples in clinical trials.

First-line pembrolizumab versus dabrafenib/trametinib treatment for BRAF V600-mutant advanced melanoma.

BACKGROUND: Limited data are available to assist the selection between immune checkpoint inhibitors and BRAF/mitogen-activated protein kinase kinase inhibitors as first-line treatment for patients with BRAF-mutant advanced malignant melanoma. OBJECTIVE: To investigate the outcomes associated with first-line pembrolizumab or dabrafenib/trametinib treatment for advanced melanoma with activating BRAF V600 mutation. METHODS: Data of patients with BRAF V600-mutant melanoma who were treated with first-line pembrolizumab (n = 40) or dabrafenib/trametinib (n = 32) were analyzed. Tumor response, progression-free survival, and overall survival were evaluated. Immune evasion accompanied with emerging resistance to BRAF/mitogen-activated protein kinase kinase inhibitors was assessed. RESULTS: A longer overall survival was observed after first-line pembrolizumab treatment than after first-line dabrafenib/trametinib treatment (hazard ratio = 2.910, 95% CI: 1.552-5.459), although there were no significant differences in progression-free survival (P = .375) and response rate (P = .123). Emergence of resistance to dabrafenib/trametinib co-occurred with immune evasion, enabling melanoma cells to escape recognition and killing by Melan-A-specific CD8+ T cells. LIMITATIONS: Analysis was conducted in a retrospective manner. CONCLUSION: Pembrolizumab may be recommended over BRAF/mitogen-activated protein kinase kinase inhibitors as the first-line treatment in patients with advanced BRAF V600-mutant melanoma.

Epigenetic Silencing of CHOP Expression by the Histone Methyltransferase EHMT1 Regulates Apoptosis in Colorectal Cancer Cells.

Colorectal cancer (CRC) has a high mortality rate among cancers worldwide. To reduce this mortality rate, chemotherapy (5-fluorouracil, oxaliplatin, and irinotecan) or targeted therapy (bevacizumab, cetuximab, and panitumumab) has been used to treat CRC. However, due to various side effects and poor responses to CRC treatment, novel therapeutic targets for drug development are needed. In this study, we identified the overexpression of EHMT1 in CRC using RNA sequencing (RNA-seq) data derived from TCGA, and we observed that knocking down EHMT1 expression suppressed cell growth by inducing cell apoptosis in CRC cell lines. In Gene Ontology (GO) term analysis using RNA-seq data, apoptosis-related terms were enriched after EHMT1 knockdown. Moreover, we identified the CHOP gene as a direct target of EHMT1 using a ChIP (chromatin immunoprecipitation) assay with an anti-histone 3 lysine 9 dimethylation (H3K9me2) antibody. Finally, after cotransfection with siEHMT1 and siCHOP, we again confirmed that CHOP-mediated cell apoptosis was induced by EHMT1 knockdown. Our findings reveal that EHMT1 plays a key role in regulating CRC cell apoptosis, suggesting that EHMT1 may be a therapeutic target for the development of cancer inhibitors.