Lenvatinib is a multiple receptor tyrosine kinase inhibitor targeting mainly vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) receptors. We investigated the immunomodulatory activities of lenvatinib in the tumor microenvironment and its mechanisms of enhanced antitumor activity when combined with a programmed cell death-1 (PD-1) blockade. Antitumor activity was examined in immunodeficient and immunocompetent mouse tumor models. Single-cell analysis, flow cytometric analysis, and immunohistochemistry were used to analyze immune cell populations and their activation. Gene co-expression network analysis and pathway analysis using RNA sequencing data were used to identify lenvatinib-driven combined activity with anti-PD-1 antibody (anti-PD-1). Lenvatinib showed potent antitumor activity in the immunocompetent tumor microenvironment compared with the immunodeficient tumor microenvironment. Antitumor activity of lenvatinib plus anti-PD-1 was greater than that of either single treatment. Flow cytometric analysis revealed that lenvatinib reduced tumor-associated macrophages (TAMs) and increased the percentage of activated CD8 + T cells secreting interferon (IFN)-γ + and granzyme B (GzmB). Combination treatment further increased the percentage of T cells, especially CD8 + T cells, among CD45 + cells and increased IFN-γ + and GzmB + CD8 + T cells. Transcriptome analyses of tumors resected from treated mice showed that genes specifically regulated by the combination were significantly enriched for type-I IFN signaling. Pretreatment with lenvatinib followed by anti-PD-1 treatment induced significant antitumor activity compared with anti-PD-1 treatment alone. Our findings show that lenvatinib modulates cancer immunity in the tumor microenvironment by reducing TAMs and, when combined with PD-1 blockade, shows enhanced antitumor activity via the IFN signaling pathway. These findings provide a scientific rationale for combination therapy of lenvatinib with PD-1 blockade to improve cancer immunotherapy.

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.

Calsyntenins/alcadeins are type I transmembrane proteins with two extracellular cadherin domains highly expressed in mammalian brain. They form a tripartite complex with X11/X11L and APP (amyloid precursor protein) and are proteolytically processed in a similar fashion to APP. Although a genetic association of calsyntenin-2 with human memory performance has recently been reported, physiological roles and molecular functions of the protein in the nervous system are poorly understood. Here, we show that CASY-1, the Caenorhabditis elegans ortholog of calsyntenins/ alcadeins, is essential for multiple types of learning. Through a genetic screen, we found that casy-1 mutants show defects in salt chemotaxis learning. casy-1 mutants also show defects in temperature learning, olfactory adaptation, and integration of two sensory signals. casy-1 is widely expressed in the nervous system. Expression of casy-1 in a single sensory neuron and at the postdevelopmental stage is sufficient for its function in salt chemotaxis learning. The fluorescent protein-tagged ectodomain of CASY-1 is released from neurons. Moreover, functional domain analyses revealed that both cytoplasmic and transmembrane domains of this protein are dispensable, whereas the ectodomain, which contains the LG/LNS-like domain, is critically required for learning. These results suggest that learning is modulated by the released ectodomain of CASY-1.ectodomain shedding ͉ learning and memory

The heterotrimeric G protein Go is abundantly expressed in the mammalian nervous system and modulates neural activities in response to various ligands. However, G o's functions in living animals are less well understood. Here, we demonstrate that GOA-1 G o␣ has a fundamental role in olfactory adaptation in Caenorhabditis elegans. Impairment of GOA-1 G o␣ function and excessive activation of EGL-30 G q␣ cause a defect in adaptation to AWC-sensed odorants. These pathways antagonistically modulate olfactory adaptation in AWC chemosensory neurons. Wild-type animals treated with phorbol esters and double-mutant animals of diacylglycerol (DAG) kinases, dgk-3; dgk-1, also have a defect in adaptation, suggesting that elevated DAG signals disrupt normal adaptation. Constitutively active GOA-1 can suppress the adaptation defect of dgk-3; dgk-1 double mutants, whereas it fails to suppress the adaptation defect of animals with constitutively active EGL-30, implying that GOA-1 acts upstream of EGL-30 in olfactory adaptation. Our results suggest that down-regulation of EGL-30 -DAG signaling by GOA-1 underlies olfactory adaptation and plasticity of chemotaxis.chemotaxis ͉ G protein T he olfactory sensory system can endow animals with abilities to detect food sources and mates and, in some cases, to avoid harmful chemicals and predators. Sensitivity to an odor stimulus can be appropriately adjusted by previous experience, allowing the sensory system to adapt to changeable environments. In mammals, olfactory adaptation (habituation) is known to occur throughout the odorant sensory pathway; for example, olfactory receptor neurons (1), secondary interneurons (2), and primary and higher-order olfactory cortices (3). These adaptation mechanisms appear to allow animals to increase the range of concentrations of odor that can be sensed and to discriminate among multiple odors.The nematode Caenorhabditis elegans has only 302 neurons, and a variety of behaviors have been observed. Of these, olfactory behavior is relatively well studied. Volatile odorants are mainly sensed by five pairs of sensory neurons, AWA, AWB, AWC, ADL, and ASH (4-6), of which AWA and AWC chemosensory neurons mediate attraction behavior (4). In this response, olfactory adaptation has also been observed (7). Animals lacking OSM-9 TRPV channel (7, 8), EGL-4 cGMPdependent protein kinase (9) or animals overexpressing ODR-1 guanylyl cyclase (10) exhibited defects in olfactory adaptation to AWC-sensed odorants. By contrast, animals with mutated tax-6, which encodes calcineurin, exhibited hyperadaptation (11), suggesting that Ca 2ϩ and cGMP signaling cascades participate in olfactory adaptation. Moreover, ARR-1 arrestin (12), TBX-2 T-box transcription factor (13), and the Ras-MAPK pathway (14) are also known to act in olfactory adaptation, illustrating that olfactory adaptation in C. elegans is modulated by complicated mechanisms consisting of multiple signaling cascades and control of gene expression.In general, neural activities are modulated by many types of ligands th...