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Background Cell-free DNA (cfDNA) coverage patterns are emerging as potential low-invasive approaches to determine transcription factor (TF) activation status in tumors; however, the accuracy of this approach has been confirmed for only a few TFs. Here, using paired tumor/blood samples collected from two xenograft tumor models of human liver cancer (HepG2, gain-of-function mutation in CTNNB1 ; HuH7, wild type), we compared the accuracy of inferences of TF activity made using tumor-derived ATAC-seq data and plasma-derived cfDNA whole-genome sequencing (WGS) data. Results ATAC-seq and cfDNA-WGS data from the HepG2 model both showed higher activation of two downstream target TFs of CTNNB1 in the Wnt signaling pathway compared with the HuH7 model. Expanding the study to 377 TFs, each with 10,000 TF-binding sites, revealed significant concordance between the data sets for both models (Spearman’s ρ : HepG2, − 0.90; HuH7, − 0.85). To elucidate potential factors that may influence the accuracy of cfDNA-based inferences of TF activity in the clinic, we prepared simulation samples by mixing tumor-derived cfDNA with healthy-donor cfDNA; we found that a minimum of 5 × sequencing coverage and a tumor fraction of at least 3% were required to obtain accurate inferences of tumor type–specific TF activity. Conclusions cfDNA-based TF activity inference is applicable to more than 370 TFs and can be used to accurately estimate tumor-specific TF activities. We believe that this study supports a fundamental principle that will be useful for future investigations of the potential of cfDNA analysis.

Unresectable hepatocellular carcinoma (uHCC) is one of the most lethal and prevalent cancers worldwide, and current systemic therapeutic options for uHCC are limited. Lenvatinib, a multiple receptor tyrosine kinase inhibitor targeting vascular endothelial growth factor receptors (VEGFRs) and fibroblast growth factor receptors (FGFRs), recently demonstrated a treatment effect on overall survival by statistical confirmation of noninferiority to sorafenib in a phase 3 study of uHCC. Here, we investigated mechanisms underlying the antitumor activity of lenvatinib in preclinical HCC models. In vitro proliferation assay of nine human HCC cell lines showed that lenvatinib selectively inhibited proliferation of FGF signal‐activated HCC cells including FGF19‐expressing Hep3B2.1‐7. Lenvatinib suppressed phosphorylation of FRS2, a substrate of FGFR1–4, in these cells in a concentration‐dependent manner. Lenvatinib inhibited in vivo tumor growth in Hep3B2.1‐7 and SNU‐398 xenografts and decreased phosphorylation of FRS2 and Erk1/2 within the tumor tissues. Lenvatinib also exerted antitumor activity and potently reduced tumor microvessel density in PLC/PRF/5 xenograft model and two HCC patient‐derived xenograft models. These results suggest that lenvatinib has antitumor activity consistently across diverse HCC models, and that targeting of tumor FGF signaling pathways and anti‐angiogenic activity underlies its antitumor activity against HCC tumors. Here, we investigated mechanisms underlying the antitumor activity of lenvatinib, a multiple receptor tyrosine kinase inhibitor targeting vascular endothelial growth factor receptors (VEGFRs) and fibroblast growth factor receptors (FGFRs), in preclinical HCC models. Our results suggest that lenvatinib has antitumor activity consistently across diverse HCC models, and that targeting of tumor FGF signaling pathways and anti‐angiogenic activity underlies its antitumor activity against HCC tumors.

The combination of lenvatinib, a multiple receptor tyrosine kinase inhibitor, plus everolimus, a mammalian target of rapamycin (mTOR) inhibitor, significantly improved clinical outcomes versus everolimus monotherapy in a phase II clinical study of metastatic renal cell carcinoma (RCC). We investigated potential mechanisms underlying the antitumor activity of the combination treatment in preclinical RCC models. Lenvatinib plus everolimus showed greater antitumor activity than either monotherapy in three human RCC xenograft mouse models (A‐498, Caki‐1, and Caki‐2). In particular, the combination led to tumor regression in the A‐498 and Caki‐1 models. In the A‐498 model, everolimus showed antiproliferative activity, whereas lenvatinib showed anti‐angiogenic effects. The anti‐angiogenic activity was potentiated by the lenvatinib plus everolimus combination in Caki‐1 xenografts, in which fibroblast growth factor (FGF)‐driven angiogenesis may contribute to tumor growth. The combination showed mostly additive activity in vascular endothelial growth factor (VEGF)‐activated, and synergistic activity against FGF‐activated endothelial cells, in cell proliferation and tube formation assays, as well as strongly suppressed mTOR‐S6K‐S6 signaling. Enhanced antitumor activities of the combination versus each monotherapy were also observed in mice bearing human pancreatic KP‐1 xenografts overexpressing VEGF or FGF. Our results indicated that simultaneous targeting of tumor cell growth and angiogenesis by lenvatinib plus everolimus resulted in enhanced antitumor activity. The enhanced inhibition of both VEGF and FGF signaling pathways by the combination underlies its superior anti‐angiogenic activity in human RCC xenograft models. Lenvatinib is a multiple receptor tyrosine kinase (RTK) inhibitor that selectively inhibits VEGFR, FGFR, and other proangiogenic and oncogenic pathway‐related RTKs. Here we show that simultaneous targeting of tumor cell growth and angiogenesis by lenvatinib plus everolimus, and enhanced inhibition of VEGF‐ and FGF‐driven angiogenesis through greater inhibition of the mTOR–S6K–S6 signaling pathway underlie the antitumor activity conferred by the combination of lenvatinib plus everolimus in preclinical RCC models.

Significance The neuromuscular junction (NMJ) is a synapse between the motor nerve and myotube essential for controlling skeletal muscle contraction. Motor nerve-derived glycoprotein agrin is indispensable for the formation and maintenance of NMJs, and genetic defects in agrin underlie a congenital myasthenic syndrome (CMS). Agrin’s role has been thought to be activation of the muscle-specific receptor kinase MuSK. Here, we demonstrate that forced activation of MuSK in agrin-deficient mice restored embryonic formation, but not postnatal maintenance, of NMJs, demonstrating that agrin plays an essential role distinct from MuSK activation in the postnatal maintenance of NMJs. Given that CMSs frequently show postnatal onset, this finding provides key insights not only into NMJ homeostasis but also into CMS pathology with unknown etiology. The motoneural control of skeletal muscle contraction requires the neuromuscular junction (NMJ), a midmuscle synapse between the motor nerve and myotube. The formation and maintenance of NMJs are orchestrated by the muscle-specific receptor tyrosine kinase (MuSK). Motor neuron-derived agrin activates MuSK via binding to MuSK’s coreceptor Lrp4, and genetic defects in agrin underlie a congenital myasthenic syndrome (an NMJ disorder). However, MuSK-dependent postsynaptic differentiation of NMJs occurs in the absence of a motor neuron, indicating a need for nerve/agrin-independent MuSK activation. We previously identified the muscle protein Dok-7 as an essential activator of MuSK. Although NMJ formation requires agrin under physiological conditions, it is dispensable for NMJ formation experimentally in the absence of the neurotransmitter acetylcholine, which inhibits postsynaptic specialization. Thus, it was hypothesized that MuSK needs agrin together with Lrp4 and Dok-7 to achieve sufficient activation to surmount inhibition by acetylcholine. Here, we show that forced expression of Dok-7 in muscle enhanced MuSK activation in mice lacking agrin or Lrp4 and restored midmuscle NMJ formation in agrin-deficient mice, but not in Lrp4-deficient mice, probably due to the loss of Lrp4-dependent presynaptic differentiation. However, these NMJs in agrin-deficient mice rapidly disappeared after birth, and postsynaptic specializations emerged ectopically throughout myotubes whereas exogenous Dok-7–mediated MuSK activation was maintained. These findings demonstrate that the MuSK activator agrin plays another role essential for the postnatal maintenance, but not for embryonic formation, of NMJs and also for the postnatal, but not prenatal, midmuscle localization of postsynaptic specializations, providing physiological and pathophysiological insight into NMJ homeostasis.

Lenvatinib is a receptor tyrosine kinase (RTK) inhibitor targeting vascular endothelial growth factor (VEGF) receptors 1–3, fibroblast growth factor (FGF) receptors 1–4, platelet‐derived growth factor receptor‐α (PDGFRα), KIT, and RET that have been implicated in pathogenic angiogenesis, tumor growth, and cancer. The primary objective of this work was to evaluate, by establishing quantitative relationships, whether lenvatinib exposure and longitudinal serum biomarker data (VEGF, Ang‐2, Tie‐2, and FGF‐23) are predictors for change in longitudinal tumor size which was assessed based on data from 558 patients with radioiodine‐refractory differentiated thyroid cancer (RR‐DTC) receiving either lenvatinib or placebo treatment. Lenvatinib PK was best described by a 3‐compartment model with simultaneous first‐ and zero‐order absorption and linear elimination from the central compartment with significant covariates (body weight, albumin <30 g/dL, ALP>ULN, RR‐DTC, RCC, HCC subjects, and concomitant CYP3A inhibitors). Except for body weight, none of the covariates have any clinically meaningful effect on exposure to lenvatinib. Longitudinal biomarker measurements over time were reasonably well defined by a PK/PD model with common EC50, Emax, and a slope for disease progression for all biomarkers. Longitudinal tumor measurements over time were reasonably well defined by a tumor growth inhibition Emax model, which in addition to lenvatinib exposure, included model‐predicted relative changes from baseline over time for Tie‐2 and Ang‐2 as having significant association with tumor response. The developed PK/PD models pave the way for dose optimization and potential prediction of clinical response.

Introduction: Despite recent advances in treatment for unresectable hepatocellular carcinoma (uHCC), median overall survival (OS) in the first-line setting across immune-based combination therapies has plateaued at 16–24 months. Evaluation of potentially more potent therapies is warranted. We report results of the first prospective phase 1b study of lenvatinib (multi-kinase inhibitor) + nivolumab (anti-PD-1 antibody) for treating advanced uHCC. Methods: This open-label study was conducted in Japan among adults (≥20 years) with histologically/cytologically confirmed HCC. Patients received monotherapy-approved doses of either 8 mg (body weight <60 kg) or 12 mg (body weight ≥60 kg) oral lenvatinib once daily + 240 mg intravenous nivolumab every 2 weeks (days 1 and 15) in 4-week cycles. Part 1 planned to enroll 6 patients to evaluate the tolerability of lenvatinib+nivolumab. Part 2 evaluated safety and preliminary anti-tumor activity. Primary endpoints were dose-limiting toxicities (DLTs; part 1 only) and safety. Secondary endpoints were objective response rate (ORR) and pharmacokinetics of lenvatinib and nivolumab. Additional exploratory endpoints (including OS and progression-free survival; part 2 only) were assessed. Results: No DLT was observed among patients (n = 6) in part 1. Treatment-related adverse events (TRAEs) were observed in all patients (n = 30) in part 1 and 2. The most common TRAEs were palmar-plantar erythrodysesthesia syndrome (60%), dysphonia (53.3%), and decreased appetite (50.0%). Distributions of lenvatinib AUC(0-t) were similar to those observed for lenvatinib in HCC previously and were within the distributions of AUC(0-τ) observed with lenvatinib monotherapy in the REFLECT trial. ORR by mRECIST per investigator review was 66.7% in part 1 and 79.2% in part 2. In part 2, median progression-free survival was 9.07 months by mRECIST per investigator review, and median OS was 26.94 months. Conclusion: Lenvatinib+nivolumab was well-tolerated and had encouraging anti-tumor activity in patients with advanced uHCC in this phase 1b study.

Background: Stem anteversion (SA) in total hip arthroplasty (THA) is crucial for postoperative outcomes, affecting dislocation risk and hip function. Accurate SA placement is challenged by intraoperative estimation methods, with discrepancies reported between predicted and true SA. This study investigates the effect of conventional methods and intraoperative fluoroscopic confirmation on SA accuracy in THA performed with a direct anterior approach using a traction table. Methods: This involves 200 patients undergoing primary THA from August 2019 to January 2023, divided into a conventional group (n = 100) and a fluoroscopic group (n = 100). Postoperative SA measurements were conducted using computed tomography scans. Statistical analysis focused on comparing the SA angles and the prevalence of excessive SA (≥>35° and ≥>40°) between the groups. Results: The fluoroscopic group showed a lower average SA angle (24.3° ± 8.3°) compared to the conventional group (30.0° ± 11.3°), with a statistically significant difference (p < 0.01). Excessive SA (≥>40°) was found in 17% of the conventional group, significantly reduced to 5% in the fluoroscopic group (p < 0.01). Similarly, SA exceeding 35° was present in 39% of the conventional group, compared to only 11% in the fluoroscopic group (p < 0.01), indicating a substantial reduction in excessive SA placements with fluoroscopic guidance. Discussion: The study demonstrates that intraoperative fluoroscopic guidance significantly enhances the accuracy of SA placement in THA, reducing the variability and proportion of excessive SA. This suggests a critical reevaluation of conventional estimation methods in favor of fluoroscopic confirmation to improve surgical outcomes. Conclusion: Intraoperative fluoroscopic confirmation of knee external rotation angle markedly decreases the proportion of excessive SA and enhances the precision of stem placement in THA with a direct anterior approach. This technique represents a significant advancement in surgical practice, offering a simple and effective method to achieve optimal postoperative results.

Diffuse large B-cell lymphoma (DLBCL) is a clinically heterogeneous lymphoid malignancy that is the most common type of lymphoma in Japan. Previous studies have demonstrated that patients with DLBCL have a poor prognosis due to increased levels of indoleamine 2,3-dioxygnase and kynurenine (KYN). However, the roles of metabolites acting downstream of KYN and associated enzymes are not fully understood. The present study investigated the role of kynurenine 3-monooxygenase (KMO), which catalyzes the conversion of KYN to 3-hydroxykynurenine (3-HK), using serum samples from patients with DLBCL and human DLBCL cell lines with different KMO expression [STR-428 cells with high levels of KMO expression (KMOhigh) and KML-1 cells with low levels of KMO expression (KMOlow)]. Serum samples from 28 patients with DLBCL and 34 healthy volunteers were used to investigate the association between prognosis and KMO activity or 3-HK levels. Furthermore, to investigate the roles of KMO and its related metabolites, STR-428 and KML-1 cell lines, and the lymph nodes of patients with DLBCL were analyzed by reverse transcription-quantitative PCR for KMO, KYNU, 3-hydroxyanthranilate-3,4-dioxygenase and quinolinate phosphoribosyltransferase, by western blotting, and immunohistochemical or immunofluorescence staining for KMO, and by cell viability and NAD+/NADH assays. KYN pathway metabolites in serum samples were measured by HPLC. Serum 3-HK levels were regulated independently of serum KYN levels, and increased serum 3-HK levels and KMO activity were found to be associated with worse disease progression. Notably, the addition of KMO inhibitors and 3-HK negatively and positively regulated the viability of DLBCL cells, respectively. Furthermore, NAD+ levels in KMOhigh STR-428 cells were significantly higher than those in KMOlow KML-1 cells. These results suggested that 3-HK generated by KMO activity may be involved in the regulation of DLBCL cell viability via NAD+ synthesis.

The objectives of this study are (1) to examine age-dependent longitudinal differences in histological responses after creation of partial-thickness articular cartilage defects (PTCDs) in rats and to use this model (2) to objectively evaluate the effectiveness of interventions for cartilage repair. Linear PTCDs were created at a depth of 100 μm in the weight-bearing region of the medial femoral condyle in rats of different ages (3 weeks, 6 weeks, 10 weeks and 14 weeks). One day, one week, two weeks, four weeks and twelve weeks after PTCD generation, spontaneous healing was evaluated histologically and immunohistochemically. Effects of interventions comprising mesenchymal stem cells (MSCs) or platelet-rich plasma (PRP) or both on 14-week-old PTCD rats were evaluated and compared with natural courses in rats of other ages. Younger rats exhibited better cartilage repair. Cartilage in 3-week-old and 6-week-old rats exhibited nearly normal restoration after 4–12 weeks. Cartilage in 14-week-old rats deteriorated over time and early signs of cartilage degeneration were observed. With injection of MCSs alone or MSCs + PRP, 14-week-old PTCD rats showed almost the same reparative cartilage as 6-week-old rats. With injection of PRP, 14-week-old PTCD rats showed almost the same reparative cartilage as 10-week-old rats. This model will be of great use to objectively compare the effects of interventions for small cartilage lesions and may help to advance the development of disease-modifying osteoarthritis drugs.

Partial thickness articular cartilage injuries (PTCIs) were not previously thought to heal spontaneously. Immature rats have the capacity for spontaneous repair of PTCIs, although it is a long-term process. Our aim has been to examine the spontaneous repair response mechanism in immature rats. Single linear PTCIs were created in 3-week-old and 12-week-old rats in the direction of joint motion. On day 1 and at 1, 2, and 4 weeks after PTCI, evaluations of histological changes and immunohistology at the injury site and in the surrounding cartilage were performed. Anti-CD105 and anti-CD166 antibodies (as stem cell markers to identify mesenchymal stem cells in reparative cartilage tissue) were used for immunohistological evaluations. To determine whether endogenous repair ability existed in articular cartilage, an ex vivo experiment was also carried out. Femoral condyles with PTCIs were incubated in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum for 1 day and for 1 and 2 weeks. Histological changes were subsequently examined. Immature cartilage showed a higher repair response than did mature cartilage, and the response occurred immediately after PTCI. In immature rats, CD105- and CD166-positive cells were found in the superficial and transitional zones of the articular cartilage. Few CD166-positive cells were identified in mature articular cartilage. No significant in vivo differences in the spontaneous repair responses to PTCIs were observed between mature and immature groups. Thus, the repair response to PTCIs seems to be associated not only with CD105- and CD166-positive cells, but also with other perichondral factors.