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Eribulin is a microtubule dynamics inhibitor with tumor microenvironment modulation activity such as vascular remodeling activity. Here, we investigated antitumor and immunomodulatory activities of eribulin and its liposomal formulation (eribulin-LF) as monotherapies or in combination with anti–programmed death 1 (PD-1) Ab. The antitumor activity of eribulin or eribulin-LF as monotherapy or in combination with anti–PD-1 Ab was examined in a P-glycoprotein–knockout 4T1 model. Eribulin and eribulin-LF showed stronger antitumor activity in immunocompetent mice compared with immunodeficient mice, indicating that they have immunomodulatory activity that underlies its antitumor activity. Combination therapy of eribulin and eribulin-LF with anti–PD-1 Ab showed antitumor activity, and the combination activity of eribulin-LF with anti–PD-1 Ab was observed at a lower dose and longer interval of administration compared with that using eribulin. To examine the immunomodulatory activity of eribulin and eribulin-LF and its underlying mechanisms, we performed flow cytometry, IHC, and gene expression profiling. IHC and flow cytometry revealed that eribulin-LF increased microvessel density and intratumoral populations of cytotoxic T cells and natural killer cells rather than eribulin. Gene expression profiling demonstrated that eribulin-LF induces IFNγ signaling. Furthermore, IHC also showed that eribulin-LF increased infiltration of CD8-positive cells together with increased CD31-positive cells. Eribulin-LF also increased ICAM-1 expression, which is essential for lymphocyte adhesion to vascular endothelial cells. In conclusion, eribulin showed combination antitumor activity with anti–PD-1 Ab via immunomodulation due to its vascular remodeling activity, and the liposomal formulation showed improved antitumor activity over the standard formulation.

Geometric isomers of mononuclear ruthenium(II) complexes

Supplementary Figure S1-S7

Supplementary materials and methods

Combination therapies consisting of immune checkpoint inhibitors plus anti-VEGF therapy show enhanced antitumor activity and are approved treatments for patients with renal cell carcinoma (RCC). The immunosuppressive roles of VEGF in the tumor microenvironment are well studied, but those of FGF/FGFR signaling remain largely unknown. Lenvatinib is a receptor tyrosine kinase inhibitor that targets both VEGFR and FGFR. Here, we examine the antitumor activity of anti-PD-1 mAb combined with either lenvatinib or axitinib, a VEGFR-selective inhibitor, in RCC. Both combination treatments showed greater antitumor activity and longer survival in mouse models versus either single agent treatment, whereas anti-PD-1 mAb plus lenvatinib had enhanced antitumor activity compared with anti-PD-1 mAb plus axitinib. Flow cytometry analysis showed that lenvatinib decreased the population of tumor-associated macrophages and increased that of IFNγ-positive CD8+ T cells. Activation of FGFR signaling inhibited the IFNγ-stimulated JAK/STAT signaling pathway and decreased expression of its target genes, including B2M, CXCL10, and PD-L1. Furthermore, inhibition of FGFR signaling by lenvatinib restored the tumor response to IFNγ stimulation in mouse and human RCC cell lines. These preclinical results reveal novel roles of tumor FGFR signaling in the regulation of cancer immunity through inhibition of the IFNγ pathway, and the inhibitory activity of lenvatinib against FGFRs likely contributes to the enhanced antitumor activity of combination treatment comprising lenvatinib plus anti-PD-1 mAb.Significance:FGFR pathway activation inhibits IFNγ signaling in tumor cells, and FGFR inhibition with lenvatinib enhances antitumor immunity and the activity of anti-PD-1 antibodies.

Legends for supplementary figures S1-S9

Supplementary Materials and Methods not included in the main manuscript

Immune checkpoint blockade (ICB) therapy has substantially improved the outcomes of patients with many types of cancers, including renal cell carcinoma (RCC). Initially studied as monotherapy, immunotherapy-based combination regimens have improved the clinical benefit achieved by ICB monotherapy and have revolutionized RCC treatment. While biomarkers like PD-L1 and tumor mutational burden (TMB) are FDA approved as biomarkers for ICB monotherapy, there are no known biomarkers for combination immunotherapies. Here, we describe the clinical outcomes and genomic determinants of response from a phase Ib/II clinical trial on patients with advanced RCC evaluating the efficacy of lenvatinib, a multi-kinase inhibitor mainly targeting VEGFR and FGFR plus pembrolizumab, an anti-PD1 immunotherapy. Concurrent treatment with lenvatinib and pembrolizumab resulted in an objective response rate of 79% (19/24) and tumor shrinkage in 96% (23/24) of patients. While tumor mutational burden (TMB) did not predict for clinical benefit, germline HLA-I diversity strongly impacted treatment efficacy. Specifically, HLA-I evolutionary divergence (HED), which measures the breadth of a patient's immunopeptidome, was associated with both improved clinical benefit and durability of response. Our results identify lenvatinib plus pembrolizumab as a highly active treatment strategy in RCC and reveal HLA-I diversity as a critical determinant of efficacy for this combination. HED also predicted better survival in a separate cohort of patients with RCC following therapy with anti-PD-1–based combination therapy.Implications:These findings have substantial implications for RCC therapy and for understanding immunogenetic mechanisms of efficacy and warrants further investigation.

HLA class-I allele data along with mean HED values for the lenvatinib/pembrolizumab and MSK-IMPACT cohorts.

Eribulin is a microtubule dynamics inhibitor with tumor microenvironment modulation activity such as vascular remodeling activity. Here, we investigated antitumor and immunomodulatory activities of eribulin and its liposomal formulation (eribulin-LF) as monotherapies or in combination with anti–programmed death 1 (PD-1) Ab. The antitumor activity of eribulin or eribulin-LF as monotherapy or in combination with anti–PD-1 Ab was examined in a P-glycoprotein–knockout 4T1 model. Eribulin and eribulin-LF showed stronger antitumor activity in immunocompetent mice compared with immunodeficient mice, indicating that they have immunomodulatory activity that underlies its antitumor activity. Combination therapy of eribulin and eribulin-LF with anti–PD-1 Ab showed antitumor activity, and the combination activity of eribulin-LF with anti–PD-1 Ab was observed at a lower dose and longer interval of administration compared with that using eribulin. To examine the immunomodulatory activity of eribulin and eribulin-LF and its underlying mechanisms, we performed flow cytometry, IHC, and gene expression profiling. IHC and flow cytometry revealed that eribulin-LF increased microvessel density and intratumoral populations of cytotoxic T cells and natural killer cells rather than eribulin. Gene expression profiling demonstrated that eribulin-LF induces IFNγ signaling. Furthermore, IHC also showed that eribulin-LF increased infiltration of CD8-positive cells together with increased CD31-positive cells. Eribulin-LF also increased ICAM-1 expression, which is essential for lymphocyte adhesion to vascular endothelial cells. In conclusion, eribulin showed combination antitumor activity with anti–PD-1 Ab via immunomodulation due to its vascular remodeling activity, and the liposomal formulation showed improved antitumor activity over the standard formulation.

Antibody lists used in the flow cytometric analysis

Mutation annotation file (MAF) showing all nonsynonymous exonic mutations identified in the lenvatinib/pembrolizumab cohort.

Fig S1 shows isotype control has no antitumor activity. Fig S2 shows gating strategy for flow cytometric analysis of TILs. Fig S3 shows induction of ICAM-1 by eribulin. Fig S4 shows establishment of P-gp KO cell lines and ICD activity of eribulin. Fig S5 shows dose-dependent antitumor activity of eribulin-LF + anti-PD-1 Ab. Fig S6 shows PK analysis of eribulin and eribulin-LF in plasma and tumor. Fig S7 shows immunomodulatory activity of eribulin in different 3 regimens. Fig S8 shows TIL population changes not included in the main figures. Fig S9 shows activation of IFN signature by eribulin-LF.

The list of Topologically significant genes by RNA-Seq analysis

The Wnt/β-catenin signaling pathway plays crucial roles in embryonic development and the development of multiple types of cancer, and its aberrant activation provides cancer cells with escape mechanisms from immune checkpoint inhibitors. E7386, an orally active selective inhibitor of the interaction between β-catenin and CREB binding protein, which is part of the Wnt/β-catenin signaling pathway, disrupts the Wnt/β-catenin signaling pathway in HEK293 and adenomatous polyposis coli (APC)-mutated human gastric cancer ECC10 cells. It also inhibited tumor growth in an ECC10 xenograft model and suppressed polyp formation in the intestinal tract of ApcMin/+ mice, in which mutation of Apc activates the Wnt/β-catenin signaling pathway. E7386 demonstrated antitumor activity against mouse mammary tumors developed in mouse mammary tumor virus (MMTV)-Wnt1 transgenic mice. Gene expression profiling using RNA sequencing data of MMTV-Wnt1 tumor tissue from mice treated with E7386 showed that E7386 downregulated genes in the hypoxia signaling pathway and immune responses related to the CCL2, and IHC analysis showed that E7386 induced infiltration of CD8+ cells into tumor tissues. Furthermore, E7386 showed synergistic antitumor activity against MMTV-Wnt1 tumor in combination with anti-PD-1 antibody. In conclusion, E7386 demonstrates clear antitumor activity via modulation of the Wnt/β-catenin signaling pathway and alteration of the tumor and immune microenvironments, and its antitumor activity can be enhanced in combination with anti-PD-1 antibody.Significance:These findings demonstrate that the novel anticancer agent, E7386, modulates Wnt/β-catenin signaling, altering the tumor immune microenvironment and exhibiting synergistic antitumor activity in combination with anti-PD-1 antibody.

Purpose: Positive results of phase I studies evaluating lenvatinib in solid tumors, including thyroid cancer, prompted a phase II trial in advanced medullary thyroid carcinoma (MTC).Experimental Design: Fifty-nine patients with unresectable progressive MTC per Response Evaluation Criteria In Solid Tumors (RECIST) v1.0 within the prior 12 months received lenvatinib (24-mg daily, 28-day cycles) until disease progression, unmanageable toxicity, withdrawal, or death. Prior anti-VEGFR therapy was permitted. The primary endpoint was objective response rate (ORR) by RECIST v1.0 and independent imaging review.Results: Lenvatinib ORR was 36% [95% confidence interval (CI), 24%–49%]; all partial responses. ORR was comparable between patients with (35%) or without (36%) prior anti-VEGFR therapy. Disease control rate (DCR) was 80% (95% CI, 67%–89%); 44% had stable disease. Among responders, median time to response (TTR) was 3.5 months (95% CI, 1.9–3.7). Median progression-free survival (PFS) was 9.0 months (95% CI, 7.0–not evaluable). Common toxicity criteria grade 3/4 treatment-emergent adverse events included diarrhea (14%), hypertension (7%), decreased appetite (7%), fatigue, dysphagia, and increased alanine aminotransferase levels (5% each). Ret proto-oncogene status did not correlate with outcomes. Low baseline levels of angiopoietin-2, hepatocyte growth factor, and IL8 were associated with tumor reduction and prolonged PFS. High baseline levels of VEGF, soluble VEGFR3, and platelet-derived growth factor BB, and low baseline levels of soluble Tie-2, were associated with tumor reduction.Conclusions: Lenvatinib had a high ORR, high DCR, and a short TTR in patients with documented progressive MTC. Toxicities were managed with dose modifications and medications. Clin Cancer Res; 22(1); 44–53. ©2015 AACR.

Tumor response

Supplementary Table S1, Table S4, Table S5, Table S6, Table S7. Supplementary Table S1: The list of DEGs by nCounter analysis (|Fold change| > 1.25, P < 0.1). Supplementary Table S4: Top 10 enriched pathways identified by RNA-Seq analysis in MMTV-Wnt1 model. Supplementary Table S5: The result of CIBERSORT analysis. Supplementary Table S6: The lists of antibody for flow cytometry analysis. Supplementary Table S7: Comparison of Physicochemical property between E7386 and C-82.

Supplementary Materials and Methods

Supplementary Figure 3 from A Notch1 Ectodomain Construct Inhibits Endothelial Notch Signaling, Tumor Growth, and Angiogenesis

Supplementary Figure 4 from A Notch1 Ectodomain Construct Inhibits Endothelial Notch Signaling, Tumor Growth, and Angiogenesis

Supplementary Figure 1 from A Notch1 Ectodomain Construct Inhibits Endothelial Notch Signaling, Tumor Growth, and Angiogenesis

Supplementary Figure 2 from A Notch1 Ectodomain Construct Inhibits Endothelial Notch Signaling, Tumor Growth, and Angiogenesis

Capping TMβCD over AlTPyP has an excellent protective effect on seawater oxidation catalyzed by the supramolecular system AlTPyP@TMβCD against attack by Cl−.

The nucleus accumbens (NAc) plays critical roles in emotional behaviors, including aversive learning. Aversive stimuli such as an electric foot shock increase acetylcholine (ACh) in the NAc, and muscarinic signaling appears to increase neuronal excitability and aversive learning. Muscarinic signaling inhibits the voltage-dependent potassium KCNQ current which regulates neuronal excitability, but the regulatory mechanism has not been fully elucidated. Phosphorylation of KCNQ2 at threonine 217 (T217) and its inhibitory effect on channel activity were predicted. However, whether and how muscarinic signaling phosphorylates KCNQ2 in vivo remains unclear. Here, we found that PKC directly phosphorylated KCNQ2 at T217 in vitro. Carbachol and a muscarinic M1 receptor (M1R) agonist facilitated KCNQ2 phosphorylation at T217 in NAc/striatum slices in a PKC-dependent manner. Systemic administration of the cholinesterase inhibitor donepezil, which is commonly used to treat dementia, and electric foot shock to mice induced the phosphorylation of KCNQ2 at T217 in the NAc, whereas phosphorylation was suppressed by an M1R antagonist. Conditional deletion of Kcnq2 in the NAc enhanced electric foot shock induced aversive learning. Our findings indicate that muscarinic signaling induces the phosphorylation of KCNQ2 at T217 via PKC activation for aversive learning.

Protein phosphorylation plays critical roles in a variety of intracellular signaling pathways and physiological functions that are controlled by neurotransmitters and neuromodulators in the brain. Dysregulation of these signaling pathways has been implicated in neurodevelopmental disorders, including autism spectrum disorder, attention deficit hyperactivity disorder and schizophrenia. While recent advances in mass spectrometry-based proteomics have allowed us to identify approximately 280,000 phosphorylation sites, it remains largely unknown which sites are phosphorylated by which kinases. To overcome this issue, previously, we developed methods for comprehensive screening of the target substrates of given kinases, such as PKA and Rho-kinase, upon stimulation by extracellular signals and identified many candidate substrates for specific kinases and their phosphorylation sites. Here, we developed a novel online database to provide information about the phosphorylation signals identified by our methods, as well as those previously reported in the literature. The “KANPHOS” (Kinase-Associated Neural Phospho-Signaling) database and its web portal were built based on a next-generation XooNIps neuroinformatics tool. To explore the functionality of the KANPHOS database, we obtained phosphoproteomics data for adenosine-A2A-receptor signaling and its downstream MAPK-mediated signaling in the striatum/nucleus accumbens, registered them in KANPHOS, and analyzed the related pathways.

Dopamine regulates emotional behaviors, including rewarding and aversive behaviors, through the mesolimbic dopaminergic pathway, which projects dopamine neurons from the ventral tegmental area to the nucleus accumbens (NAc). Protein phosphorylation is critical for intracellular signaling pathways and physiological functions, which are regulated by neurotransmitters in the brain. Previous studies have demonstrated that dopamine stimulated the phosphorylation of intracellular substrates, such as receptors, ion channels, and transcription factors, to regulate neuronal excitability and synaptic plasticity through dopamine receptors. We also established a novel database called KANPHOS that provides information on phosphorylation signals downstream of monoamines identified by our kinase substrate screening methods, including dopamine, in addition to those reported in the literature. Recent advances in proteomics techniques have enabled us to clarify the mechanisms through which dopamine controls rewarding and aversive behaviors through signal pathways in the NAc. In this review, we discuss the intracellular phosphorylation signals regulated by dopamine in these two emotional behaviors.

Immune checkpoint blockade (ICB) therapy has substantially improved the outcomes of patients with many types of cancers, including renal cell carcinoma (RCC). Initially studied as monotherapy, immunotherapy-based combination regimens have improved the clinical benefit achieved by ICB monotherapy and have revolutionized RCC treatment. While biomarkers like PD-L1 and tumor mutational burden (TMB) are FDA approved as biomarkers for ICB monotherapy, there are no known biomarkers for combination immunotherapies. Here, we describe the clinical outcomes and genomic determinants of response from a phase Ib/II clinical trial on patients with advanced RCC evaluating the efficacy of lenvatinib, a multi-kinase inhibitor mainly targeting VEGFR and FGFR plus pembrolizumab, an anti-PD1 immunotherapy. Concurrent treatment with lenvatinib and pembrolizumab resulted in an objective response rate of 79% (19/24) and tumor shrinkage in 96% (23/24) of patients. While tumor mutational burden (TMB) did not predict for clinical benefit, germline HLA-I diversity strongly impacted treatment efficacy. Specifically, HLA-I evolutionary divergence (HED), which measures the breadth of a patient's immunopeptidome, was associated with both improved clinical benefit and durability of response. Our results identify lenvatinib plus pembrolizumab as a highly active treatment strategy in RCC and reveal HLA-I diversity as a critical determinant of efficacy for this combination. HED also predicted better survival in a separate cohort of patients with RCC following therapy with anti-PD-1–based combination therapy. Implications: These findings have substantial implications for RCC therapy and for understanding immunogenetic mechanisms of efficacy and warrants further investigation.

The enantioselective reaction of 2H-azirines with oxazol-5-(4H)-ones (oxazolones) using a cinchona alkaloid sulfonamide catalyst has been developed. The reaction proceeded at the C-2 position of oxazolones to afford products with consecutive tetrasubstituted stereogenic centers in high yield with high diastereo- and enantioselectivity. The obtained aziridines were converted into various chiral compounds without loss of enantiopurity.

The Wnt/β-catenin signaling pathway plays crucial roles in embryonic development and the development of multiple types of cancer, and its aberrant activation provides cancer cells with escape mechanisms from immune checkpoint inhibitors. E7386, an orally active selective inhibitor of the interaction between β-catenin and CREB binding protein, which is part of the Wnt/β-catenin signaling pathway, disrupts the Wnt/β-catenin signaling pathway in HEK293 and adenomatous polyposis coli (APC)-mutated human gastric cancer ECC10 cells. It also inhibited tumor growth in an ECC10 xenograft model and suppressed polyp formation in the intestinal tract of ApcMin/+ mice, in which mutation of Apc activates the Wnt/β-catenin signaling pathway. E7386 demonstrated antitumor activity against mouse mammary tumors developed in mouse mammary tumor virus (MMTV)-Wnt1 transgenic mice. Gene expression profiling using RNA sequencing data of MMTV-Wnt1 tumor tissue from mice treated with E7386 showed that E7386 downregulated genes in the hypoxia signaling pathway and immune responses related to the CCL2, and IHC analysis showed that E7386 induced infiltration of CD8+ cells into tumor tissues. Furthermore, E7386 showed synergistic antitumor activity against MMTV-Wnt1 tumor in combination with anti-PD-1 antibody. In conclusion, E7386 demonstrates clear antitumor activity via modulation of the Wnt/β-catenin signaling pathway and alteration of the tumor and immune microenvironments, and its antitumor activity can be enhanced in combination with anti-PD-1 antibody. Significance: These findings demonstrate that the novel anticancer agent, E7386, modulates Wnt/β-catenin signaling, altering the tumor immune microenvironment and exhibiting synergistic antitumor activity in combination with anti-PD-1 antibody.

Tumor progression and immune evasion result from multiple oncogenic and immunosuppressive signals within the tumor microenvironment. The combined blockade of VEGF and inhibitory immune checkpoint signaling has been shown to enhance immune activation and tumor destruction in preclinical models. The LEAP clinical trial program is evaluating the safety and efficacy of lenvatinib (a multikinase inhibitor) plus pembrolizumab (a PD-1 inhibitor) across several solid tumor types. Preliminary results from ongoing trials demonstrate robust antitumor activity and durable responses across diverse tumor types with a manageable safety profile. Thus, lenvatinib plus pembrolizumab is anticipated to be an important potential new regimen for several solid cancers that currently have limited therapeutic options. Clinical trial registration: NCT03884101 , NCT03713593 , NCT03820986 , NCT03776136 , NCT03797326 , NCT03829319 , NCT03829332 , NCT03976375 , NCT04428151 , NCT04199104 , NCT03898180 , NCT04246177 (ClinicalTrials.gov).

Background/aim There is no established standard chemotherapy after administration of the combination endocrine plus CDK4/6 inhibitor therapy for luminal-type breast cancer. We used patient-derived xenograft (PDX) models to determine the antitumor activity of eribulin and capecitabine after endocrine therapy plus CDK4/6 inhibitor. Materials and methods We examined the antitumor activity of fulvestrant, palbociclib, eribulin, and capecitabine in 4 luminal-type breast cancer PDX models (OD-BRE-0188, -0438, -0450, -0745). In OD-BRE-0438, we determined the antitumor activity of chemotherapy after fulvestrant-palbociclib treatment. We also performed immunohistochemical analysis to explore the effects of treatment on E-cadherin in tumor tissues. Results Fulvestrant, fulvestrant-palbociclib and chemotherapy had antitumor activity in the 4 PDX models. In OD-BRE-0438 (the most resistant to fulvestrant-palbociclib), eribulin had superior antitumor activity to capecitabine after fulvestrant plus palbociclib. Only eribulin tended to increase E-cadherin expression. Conclusion Eribulin had superior antitumor activity to capecitabine after fulvestrant-palbociclib in the OD-BRE-0438 model.

Background No biomarkers have been established to predict treatment efficacy in renal cell carcinoma (RCC). In an exploratory retrospective analysis of a Phase 2 study, we constructed composite biomarker scores (CBSs) to predict progression-free survival (PFS) and overall survival (OS) in patients with metastatic RCC randomised to receive lenvatinib-plus-everolimus. Methods Of 40 biomarkers tested, the 5 most strongly associated with PFS (HGF, MIG, IL-18BP, IL-18, ANG-2) or OS (TIMP-1, M-CSF, IL-18BP, ANG-2, VEGF) were used to make a 5-factor PFS-CBS or OS-CBS, respectively. A 2-factor CBS was generated with biomarkers common to PFS-CBS and OS-CBS. Patients were divided into groups accordingly (5-factor-CBS high: 3−5, CBS-low: 0–2; 2-factor-CBS high: 1–2, CBS-low: 0). Results PFS/OS with lenvatinib-plus-everolimus were significantly longer in the 5-factor CBS-high group versus the CBS-low group (P = 0.0022/P P P = 0.0079, respectively); PFS was also significantly longer with lenvatinib-plus-everolimus versus lenvatinib (P = 0.0046). The 5-factor-CBS had a predictive role in PFS and OS after multivariate analysis. Similar trends were observed with the 2-factor-CBS for PFS (i.e., lenvatinib-plus-everolimus versus everolimus). Conclusions The 5-factor CBS may identify patients with metastatic RCC who would benefit from lenvatinib-plus-everolimus versus everolimus; additional validation is required. Clinical trial registration The clinical trial registration number is NCT01136733.

Neurons are highly polarized cells that have structurally and functionally distinct processes called axons and dendrites. How neurons establish polarity is one of the fundamental questions of neuroscience. In the last decade, significant progress has been made in identifying and understanding the molecular mechanisms responsible for neuronal polarization, primarily through researches conducted on cultured neurons. Advances in phosphoproteomics technologies and molecular tools have enabled comprehensive signal analysis and visualization and manipulation of signaling molecules for analyzing neuronal polarity. Furthermore, advances in gene transfer techniques have revealed the role of extracellular and intracellular signaling molecules in neuronal polarization in vivo. This review discusses the latest insights and techniques for the elucidation of the molecular mechanisms that control neuronal polarity.

Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) can provide insight into tumor perfusion. However, a method that can quantitatively measure the intra-tumor distribution of tumor voxel clusters with a distinct range of KTwo-dimensional cluster analysis may quantify the distribution of a tumor voxel subregion with a distinct range of KProspective longitudinal study.Twenty-two female athymic nude mice with MCF-7 xenograft, treated with E7130, a tumor-microenvironmental ameliorator, or saline.9.4 Tesla, turbo rapid acquisition with relaxation enhancement, and spoiled gradient-echo sequences.We performed two-dimensional k-means clustering to identify tumor voxel clusters with a distinct range of KUnpaired t-tests, Dunnett's multiple comparison tests, and Chi-squared test were used.The largest and second largest clusters constituted 44.4% and 27.5% of all tumor voxels with cluster centroid values of KA two-dimensional cluster analysis might quantify the spatial distribution of a tumor subregion with a distinct range of K1 TECHNICAL EFFICACY: Stage 1.

Introduction: Lenvatinib (LEN) is an oral, multiple-receptor, tyrosine kinase inhibitor that mainly targets vascular endothelial growth factor and fibroblast growth factor receptors. Combination therapy with LEN and everolimus is approved for people with advanced renal cell carcinoma (RCC). Combination therapy with LEN and pembrolizumab, an anti human-programmed-cell-death-1 (PD-1) antibody, is also approved for the treatment of people with advanced endometrial cancer and advanced RCC. In this study, we investigated the antitumor activity of LEN in combination with chemotherapy and immune checkpoint inhibitors (ICIs), and explored the mechanism of action underlying these effects, in the mouse lung cancer syngeneic model. Methods: In the LL/2 mouse Lewis lung carcinoma model, LEN was dosed at either 3 or 10 mg/kg (orally; once daily for 2 weeks) with or without cisplatin 4 mg/kg (intravenously; 3 or 4 times, twice weekly) and with or without anti-PD-1 200 ug/head + anti cytotoxic T-lymphocyte antigen 4 (CTLA4) antibodies 10mg/kg (intraperitoneally; twice weekly). Functional blood vessels were evaluated in tumor microenvironments by injections of Hoechst 33342 (Hoechst) into the tail veins of mice. The Hoechst-stained area and expressions of angiogenesis/immune-cell-related molecules were analyzed by immunohistochemistry (IHC) analysis. Results: In this model, the combination treatment of LEN 3 mg/kg + cisplatin showed significant tumor growth inhibitory activity compared with each single agent without any marked body-weight loss. LEN 10 mg/kg + cisplatin did not show enhanced antitumor activity vs LEN 10 mg/kg as a single agent. IHC analysis using anti-CD31 antibody showed that LEN 3 mg/kg daily decreased microvessel density in tumors after 4 days. In addition, LEN 3 mg/kg daily for 4 days showed a trend toward an increase of the number of functional blood vessels stained with Hoechst in the tumor microenvironment compared with untreated controls. The combination of LEN 3 mg/kg + cisplatin + ICIs (anti-PD-1+anti-CTLA4) significantly inhibited tumor growth compared with combinations of cisplatin + ICIs. Conclusion: Our findings show that LEN 3 mg/kg enhances the antitumor activity of cisplatin in the LL/2 mouse Lewis lung carcinoma tumor model; and LEN 3 mg/kg + cisplatin + ICIs shows greater antitumor activity compared with cisplatin + ICIs. The results of the IHC analysis suggest that LEN 3 mg/kg inhibits angiogenesis. Conversely, LEN 3 mg/kg increases functional vessels in tumor microenvironments. This situation could enhance combination antitumor activity of chemotherapy, and also chemotherapy + ICIs combination treatment. Further analysis of the mechanism of action of LEN 3 mg/kg in combination with chemotherapy + ICIs is warranted. Citation Format: Yu Kato, Yoichi Ozawa, Keito Adachi, Masahiko Kume, Yusuke Adachi, Yudai Narita, Megumi Kuronishi, Junji Matsui, Akira Yokoi, Yasuhiro Funahashi. The effect of lenvatinib in combination with chemotherapy plus immune checkpoint inhibitors on tumor microenvironments in the mouse syngeneic tumor model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5124.

Introduction: Combination anti-CTLA-4 antibody (Ab) + anti-PD-1 Ab is approved in multiple cancer types and has improved clinical outcomes. In addition, atezolizumab + bevacizumab combination therapy is approved for unresectable hepatocellular carcinoma. Given the rapid changes in the therapeutic landscape, it is important to investigate effective treatments for people with prior ICI-based combination therapy. E7386 modulates Wnt/β-catenin signaling, and a phase 1 study in combination with LEN (a receptor tyrosine kinase inhibitor that mainly targets both VEGFR and FGFR), in people with solid tumors (NCT04008797) is ongoing. In this study, we examined the antitumor activity of the combination of E7386 + LEN, with either prior anti-CTLA-4 Ab + anti-PD-1 Ab or anti-PD-1 Ab + aflibercept (a soluble decoy receptor against VEGF), in the mouse lung tumor KLN 205 model. Methods: Antitumor activity of E7386 (25 or 50 mg/kg), LEN (3 mg/kg), and the combination, was evaluated in the KLN 205 model (orally, once daily x 14 days), with or without prior treatments of either anti-CTLA-4 Ab (0.2 mg/head, weekly x 3, intraperitoneally [ip]) + anti-PD-1 Ab (0.2 mg/head, weekly x 3, ip)─ie, CTLA-4 + PD-1; or aflibercept (5 mg/kg) + anti-PD-1 Ab (0.2 mg/head, twice a week x 2, ip)─ie, AFL + PD-1. Mice with relatively rapid-growing tumors and prior treatments were given E7386, LEN, and their combination. RNA-seq analyses were conducted by using tumor tissues collected from mice one day after the prior treatment period or without treatment to analyze gene-expression alterations with prior treatments. Results: In the mouse model without prior treatments, LEN had clear antitumor activity. E7386 had limited antitumor activity as a monotreatment. E7386 + LEN had superior antitumor activity vs LEN in a dose-dependent manner. In addition, E7386 + LEN had superior antitumor activity to each single treatment despite either prior CTLA-4 + PD-1 or AFL + PD-1. E7386 + LEN with prior CTLA-4 + PD-1 had equivalent antitumor activity to LEN without prior treatments. Conversely, E7386 + LEN with prior AFL + PD-1 had strong antitumor activity, including marked tumor regression, compared to both LEN alone and E7386 + LEN without prior treatments. RNA-seq analyses showed that intratumoral Axin2, a target gene in the canonical Wnt-signaling pathway, was upregulated with prior AFL + PD-1—suggesting a potential activation of the Wnt-signaling pathway. Conclusion: E7386 + LEN combination shows antitumor activity in the mouse KLN 205 lung tumor model when there is prior ICI-based (CTLA-4 + PD-1 or AFL + PD-1) combination treatment. Further, E7386 + LEN shows enhanced antitumor activity with prior AFL + PD-1; possibly due to activation of Wnt-signaling pathway during prior treatments. Citation Format: Satoshi Kawano, Yusuke Adachi, Megumi Kuronishi, Kohta Toshimitsu, Kotaro Kodama, Takayuki Kimura, Masahiko Kume, Jialing Shen, Saori Watanabe Miyano, Akira Yokoi, Junji Matsui, Yu Kato, Yasuhiro Funahashi. E7386, a selective inhibitor of the interaction between β-catenin and CREB-binding protein (CBP), in combination with lenvatinib (LEN), exerts antitumor activity in preclinical tumor models with prior immune checkpoint inhibitor (ICI)-based combination treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1830.