Inhibition of Notch4 using Novel Neutralizing Antibodies Reduces Tumor Growth in Murine Cancer Models by Targeting the Tumor Endothelium.

Endothelial Notch signaling is critical for tumor angiogenesis. Notch1 blockade can interfere with tumor vessel function but causes tissue hypoxia and gastrointestinal toxicity. Notch4 is primarily expressed in endothelial cells, where it may promote angiogenesis; however, effective therapeutic targeting of Notch4 has not been successful. We developed highly specific Notch4-blocking antibodies, 6-3-A6 and humanized E7011, allowing therapeutic targeting of Notch4 to be assessed in tumor models. Notch4 was expressed on tumor endothelial cells in multiple cancer models, and endothelial expression was associated with response to E7011/6-3-A6. Anti-Notch4 treatment significantly delayed tumor growth in mouse models of breast, skin, and lung cancer. Enhanced tumor inhibition occurred when anti-Notch4 treatment was used in combination with chemotherapeutics. Endothelial transcriptomic analysis of murine breast tumors treated with 6-3-A6 identified significant changes in pathways of vascular function but caused only modest change in canonical Notch signaling. Analysis of early and late treatment timepoints revealed significant differences in vessel area and perfusion in response to anti-Notch4 treatment. We conclude that targeting Notch4 improves tumor growth control through endothelial intrinsic mechanisms.

Population pharmacokinetic-pharmacodynamic modeling of serum biomarkers as predictors of tumor dynamics following lenvatinib treatment in patients with radioiodine-refractory differentiated thyroid cancer (RR-DTC).

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.

Catalytic Oxidation of Methanol to Formaldehyde Catalyzed by Iron Complex with N3S3-type Tripodal Ligand.

A Fe3+ complex with N3S3-type tripod ligand, 1, reacts with O2 in CH3OH to generate formaldehyde, which has been studied structurally, spectroscopically, and electrochemically. Complex 1 crystallizes as an octahedral structure with crystallographic C3 symmetry around the metal, with Fe-N=2.2917(17) Å and Fe-S=2.3574(6) Å. UV-vis spectrum of 1 in CH3OH under Ar shows an intense band at 572 nm (ϵ 4,100 M-1cm-1), which shifts to 590 nm (ϵ 2,860 M-1cm-1) by the addition of O2, and a new peak appeared at 781 nm (ϵ 790 M-1cm-1). Such a spectral change is not observed in CH2Cl2. Cyclic voltammogram (CV) of 1 in CH2Cl2 under Ar gives reversible redox waves assigned to Fe2+/Fe3+ and Fe3+/Fe4+ couples at -1.60 V (ΔE=69 mV) and -0.53 V (ΔE=71 mV) vs Fc/Fc+, respectively. In contrast, in CH3OH, the reversible redox waves, albeit accompanied by a positive shift of the Fe2+/Fe3+ couple, are observed at -1.20 V (ΔE=85 mV) and -0.53 V (ΔE=64 mV) vs Fc/Fc+ under Ar. Interestingly, a catalytic current was observed for the CV of 1 in CH3OH in the presence of CH3ONa under Ar, when the sweep rate was slowed down.

Amelioration of Tumor-promoting Microenvironment via Vascular Remodeling and CAF Suppression Using E7130: Biomarker Analysis by Multimodal Imaging Modalities.

E7130 is a novel anticancer agent created from total synthetic study of the natural compound norhalichondrin B. In addition to inhibiting microtubule dynamics, E7130 also ameliorates tumor-promoting aspects of the tumor microenvironment (TME) by suppressing cancer-associated fibroblasts (CAF) and promoting remodeling of tumor vasculature. Here, we demonstrate TME amelioration by E7130 using multi-imaging modalities, including multiplexed mass cytometry [cytometry by time-of-flight (CyTOF)] analysis, multiplex IHC analysis, and MRI. Experimental solid tumors characterized by large numbers of CAFs in TME were treated with E7130. E7130 suppressed LAP-TGFß1 production, a precursor of TGFß1, in CAFs but not in cancer cells; an effect that was accompanied by a reduction of circulating TGFß1 in plasma. To our best knowledge, this is the first report to show a reduction of TGFß1 production in TME. Furthermore, multiplex IHC analysis revealed reduced cellularity and increased TUNEL-positive apoptotic cells in E7130-treated xenografts. Increased microvessel density (MVD) and collagen IV (Col IV), an extracellular matrix (ECM) component associated with endothelial cells, were also observed in the TME, and plasma Col IV levels were also increased by E7130 treatment. MRI revealed increased accumulation of a contrast agent in xenografts. Moreover, diffusion-weighted MRI after E7130 treatment indicated reduction of tumor cellularity and interstitial fluid pressure. Overall, our findings strongly support the mechanism of action that E7130 alters the TME in therapeutically beneficial ways. Importantly, from a translational perspective, our data demonstrated MRI as a noninvasive biomarker to detect TME amelioration by E7130, supported by consistent changes in plasma biomarkers.

Liposome-Encapsulated Eribulin Shows Enhanced Antitumor Activity over Eribulin for Combination Therapy with Anti-PD-1 Antibody.

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.

New Cluster Analysis Method for Quantitative Dynamic Contrast-Enhanced MRI Assessing Tumor Heterogeneity Induced by a Tumor-Microenvironmental Ameliorator (E7130) Treatment to a Breast Cancer Mouse Model.

BACKGROUND: 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 Ktrans and ve values remains insufficiently explored. HYPOTHESIS: Two-dimensional cluster analysis may quantify the distribution of a tumor voxel subregion with a distinct range of Ktrans and ve values in human breast cancer xenografts. STUDY TYPE: Prospective longitudinal study. ANIMAL MODEL: Twenty-two female athymic nude mice with MCF-7 xenograft, treated with E7130, a tumor-microenvironmental ameliorator, or saline. FIELD STRENGTH/SEQUENCE: 9.4 Tesla, turbo rapid acquisition with relaxation enhancement, and spoiled gradient-echo sequences. ASSESSMENT: We performed two-dimensional k-means clustering to identify tumor voxel clusters with a distinct range of Ktrans and ve values on Days 0, 2, and 5 after treatment, calculated the ratio of the number of tumor voxels in each cluster to the total number of tumor voxels, and measured the normalized distances defined as the ratio of the distance between each tumor voxel and the nearest tumor margin to a tumor radius. STATISTICAL TESTS: Unpaired t-tests, Dunnett's multiple comparison tests, and Chi-squared test were used. RESULTS: The largest and second largest clusters constituted 44.4% and 27.5% of all tumor voxels with cluster centroid values of Ktrans at 0.040 min-1 and 0.116 min-1 , and ve at 0.131 and 0.201, respectively. At baseline (Day 0), the average normalized distances for the largest and second largest clusters were 0.33 and 0.24, respectively. E7130-treated group showed the normalized distance of the initial largest cluster decreasing to 0.25, while that of the second largest cluster increasing to 0.31. Saline-treated group showed no change. DATA CONCLUSION: A two-dimensional cluster analysis might quantify the spatial distribution of a tumor subregion with a distinct range of Ktrans and ve values. LEVEL OF EVIDENCE: 1 TECHNICAL EFFICACY: Stage 1.

Rho-Kinase/ROCK Phosphorylates PSD-93 Downstream of NMDARs to Orchestrate Synaptic Plasticity.

The N-methyl-D-aspartate receptor (NMDAR)-mediated structural plasticity of dendritic spines plays an important role in synaptic transmission in the brain during learning and memory formation. The Rho family of small GTPase RhoA and its downstream effector Rho-kinase/ROCK are considered as one of the major regulators of synaptic plasticity and dendritic spine formation, including long-term potentiation (LTP). However, the mechanism by which Rho-kinase regulates synaptic plasticity is not yet fully understood. Here, we found that Rho-kinase directly phosphorylated discs large MAGUK scaffold protein 2 (DLG2/PSD-93), a major postsynaptic scaffold protein that connects postsynaptic proteins with NMDARs; an ionotropic glutamate receptor, which plays a critical role in synaptic plasticity. Stimulation of striatal slices with an NMDAR agonist induced Rho-kinase-mediated phosphorylation of PSD-93 at Thr612. We also identified PSD-93-interacting proteins, including DLG4 (PSD-95), NMDARs, synaptic Ras GTPase-activating protein 1 (SynGAP1), ADAM metallopeptidase domain 22 (ADAM22), and leucine-rich glioma-inactivated 1 (LGI1), by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Among them, Rho-kinase increased the binding of PSD-93 to PSD-95 and NMDARs. Furthermore, we found that chemical-LTP induced by glycine, which activates NMDARs, increased PSD-93 phosphorylation at Thr612, spine size, and PSD-93 colocalization with PSD-95, while these events were blocked by pretreatment with a Rho-kinase inhibitor. These results indicate that Rho-kinase phosphorylates PSD-93 downstream of NMDARs, and suggest that Rho-kinase mediated phosphorylation of PSD-93 increases the association with PSD-95 and NMDARs to regulate structural synaptic plasticity.

Inhibition of FGFR Reactivates IFNγ Signaling in Tumor Cells to Enhance the Combined Antitumor Activity of Lenvatinib with Anti-PD-1 Antibodies.

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.

Pharmacodynamic Biomarkers Predictive of Survival Benefit with Lenvatinib in Unresectable Hepatocellular Carcinoma: From the Phase III REFLECT Study.

PURPOSE: In REFLECT, lenvatinib demonstrated an effect on overall survival (OS) by confirmation of noninferiority to sorafenib in unresectable hepatocellular carcinoma. This analysis assessed correlations between serum or tissue biomarkers and efficacy outcomes from REFLECT. EXPERIMENTAL DESIGN: Serum biomarkers (VEGF, ANG2, FGF19, FGF21, and FGF23) were measured by ELISA. Gene expression in tumor tissues was measured by the nCounter PanCancer Pathways Panel. Pharmacodynamic changes in serum biomarker levels from baseline, and associations of clinical outcomes with baseline biomarker levels, were evaluated. RESULTS: Four hundred and seven patients were included in the serum analysis set (lenvatinib n = 279, sorafenib n = 128); 58 patients were included in the gene-expression analysis set (lenvatinib n = 34, sorafenib n = 24). Both treatments were associated with increases in VEGF; only lenvatinib was associated with increases in FGF19 and FGF23 at all time points. Lenvatinib-treated responders had greater increases in FGF19 and FGF23 versus nonresponders at cycle 4, day 1 (FGF19: 55.2% vs. 18.3%, P = 0.014; FGF23: 48.4% vs. 16.4%, P = 0.0022, respectively). Higher baseline VEGF, ANG2, and FGF21 correlated with shorter OS in both treatment groups. OS was longer for lenvatinib than sorafenib [median, 10.9 vs. 6.8 months, respectively; HR, 0.53; 95% confidence interval (CI), 0.33-0.85; P-interaction = 0.0397] with higher baseline FGF21. In tumor tissue biomarker analysis, VEGF/FGF-enriched groups showed improved OS with lenvatinib versus the intermediate VEGF/FGF group (HR, 0.39; 95% CI, 0.16-0.91; P = 0.0253). CONCLUSIONS: Higher baseline levels of VEGF, FGF21, and ANG2 may be prognostic for shorter OS. Higher baseline FGF21 may be predictive for longer OS with lenvatinib compared with sorafenib, but this needs confirmation.

High Response Rate and Durability Driven by HLA Genetic Diversity in Patients with Kidney Cancer Treated with Lenvatinib and Pembrolizumab.

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.

Dynamic subcellular localization and transcription activity of the SRF cofactor MKL2 in the striatum are regulated by MAPK.

Dopamine type 1 receptor (D1R) signaling activates protein kinase A (PKA), which then activates mitogen-activated protein kinase (MAPK) through Rap1, in striatal medium spiny neurons (MSNs). MAPK plays a pivotal role in reward-related behavior through the activation of certain transcription factors. How D1R signaling regulates behavior through transcription factors remains largely unknown. CREB-binding protein (CBP) promotes transcription through hundreds of different transcription factors and is also important for reward-related behavior. To identify transcription factors regulated by dopamine signaling in MSNs, we performed a phosphoproteomic analysis using affinity beads coated with CBP. We obtained approximately 40 novel candidate proteins in the striatum of the C57BL/6 mouse brain after cocaine administration. Among them, the megakaryoblastic leukemia-2 (MKL2) protein, a transcriptional coactivator of serum response factor (SRF), was our focus. We found that the interaction between CBP and MKL2 was increased by cocaine administration. Additionally, MKL2, CBP and SRF formed a ternary complex in vivo. The C-terminal domain of MKL2 interacted with CBP-KIX and was phosphorylated by MAPK in COS7 cells. The activation of PKA-MAPK signaling induced the nuclear localization of MKL2 and increased SRF-dependent transcriptional activity in neurons. These results demonstrate that dopamine signaling regulates the interaction of MKL2 with CBP in a phosphorylation-dependent manner and thereby controls SRF-dependent gene expression. Cover Image for this issue: https://doi.org/10.1111/jnc.15067.

E7386, a Selective Inhibitor of the Interaction between ß-Catenin and CBP, Exerts Antitumor Activity in Tumor Models with Activated Canonical Wnt Signaling.

The Wnt/ß-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 ß-catenin and CREB binding protein, which is part of the Wnt/ß-catenin signaling pathway, disrupts the Wnt/ß-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/ß-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/ß-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/ß-catenin signaling, altering the tumor immune microenvironment and exhibiting synergistic antitumor activity in combination with anti-PD-1 antibody.

The LEAP program: lenvatinib plus pembrolizumab for the treatment of advanced solid tumors.

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).

KANPHOS: A Database of Kinase-Associated Neural Protein Phosphorylation in the Brain.

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.

Correlative serum biomarker analyses in the phase 2 trial of lenvatinib-plus-everolimus in patients with metastatic renal cell carcinoma.

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 < 0.0001, respectively). In the CBS-high group, PFS/OS were significantly longer with lenvatinib-plus-everolimus versus everolimus (P < 0.001/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.

Antitumor Activity of Eribulin After Fulvestrant Plus CDK4/6 Inhibitor in Breast Cancer Patient-derived Xenograft Models.

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.

Activated FGF2 signaling pathway in tumor vasculature is essential for acquired resistance to anti-VEGF therapy.

Anti-vascular endothelial growth factor (VEGF) therapy shows antitumor activity against various types of solid cancers. Several resistance mechanisms against anti-VEGF therapy have been elucidated; however, little is known about the mechanisms by which the acquired resistance arises. Here, we developed new anti-VEGF therapy-resistant models driven by chronic expression of the mouse VEGFR2 extracellular domain fused with the human IgG4 fragment crystallizable (Fc) region (VEGFR2-Fc). In the VEGFR2-Fc-expressing resistant tumors, we demonstrated that the FGFR2 signaling pathway was activated, and pericytes expressing high levels of FGF2 were co-localized with endothelial cells. Lenvatinib, a multiple tyrosine kinase inhibitor including VEGFR and FGFR inhibition, showed marked antitumor activity against VEGFR2-Fc-expressing resistant tumors accompanied with a decrease in the area of tumor vessels and suppression of phospho-FGFR2 in tumors. Our findings reveal the key role that intercellular FGF2 signaling between pericytes and endothelial cells plays in maintaining the tumor vasculature in anti-VEGF therapy-resistant tumors.

Second-line lenvatinib in patients with recurrent endometrial cancer.

OBJECTIVE: This study assessed the efficacy of lenvatinib, a multitargeted tyrosine kinase inhibitor, as second-line therapy in patients with unresectable endometrial cancer. The primary end point was the objective response rate (ORR) as assessed by independent radiologic review (IRR). Secondary end points included median progression-free survival (PFS), overall survival (OS), and clinical benefit rate. Exploratory end points examined the association of baseline levels of plasma biomarkers (50 circulating cytokine and/or angiogenic factors measured by immunoassays) with efficacy outcomes. METHODS: An international, open-label, single-arm, multicenter, phase 2 trial was conducted. Eligible patients had histologically confirmed unresectable endometrial cancer that relapsed after 1 prior systemic platinum-based chemotherapy. Patients received once-daily oral lenvatinib 24 mg in a 28-day dosing cycle. RESULTS: There were 133 patients in the study. By IRR, 19 patients had a confirmed objective response for an ORR of 14.3% (95% CI: 8.8-21.4). Durable stable disease (≥23 weeks) was observed in 31 patients (23.3%) and the clinical benefit rate was 37.6% (95% CI: 29.3-46.4). Median PFS was 5.6 months (95% CI: 3.7-6.3), and median OS was 10.6 months (95% CI: 8.9-14.9). The most common (any grade) treatment-related adverse events were fatigue/asthenia (48%), hypertension (49%), nausea/vomiting (32%), decreased appetite (32%), and diarrhea (31%). Lower baseline levels of angiopoietin-2 were associated with longer PFS, OS, and a higher ORR. CONCLUSIONS: Patients with recurrent endometrial cancer treated with second-line lenvatinib experienced modest antitumor activity and treatment was generally well tolerated, with a safety profile consistent with previous studies.

Lenvatinib induces death of human hepatocellular carcinoma cells harboring an activated FGF signaling pathway through inhibition of FGFR-MAPK cascades.

Lenvatinib inhibits VEGF- and FGF-driven angiogenesis, and proliferation of tumor cells with activated FGF signaling pathways in preclinical models, and we previously demonstrated antitumor activity in human HCC xenograft tumor models. Here, we examined the inhibitory activity of lenvatinib against FGF-driven survival of human HCC cell lines. First, we conducted a histological analysis of FGF19-overexpressing Hep3B2.1-7 xenograft tumors collected from mice treated with lenvatinib. Second, we examined the effects of pharmacological inhibition on survival of cultured HCC cells with an activated FGF signaling pathway under nutrient-starved culture condition to mimic tumor microenvironments induced by angiogenesis inhibition. In the first analysis, area of histological focal necrosis was greater in Hep3B2.1-7 xenograft tumors with the lenvatinib treatment than that after the treatment with sorafenib, which does not inhibit FGFRs. Lenvatinib and E7090 (a selective FGFR1-3 inhibitor), but not sorafenib, induced death of Hep3B2.1-7, and another FGF19 overexpressing HuH-7 cells. Lenvatinib and E7090 decreased phosphorylation of downstream molecules of the FGF signaling pathway (such as FRS2, Erk, and p38 MAPK), and induced PARP cleavage, even under limited nutrients. PD0325901, MEK inhibitor, caused the same changes in HCC cells as those described above for lenvatinib and E7090. These results reveal that the FGF signaling pathway through MAPK cascades plays an important role in survival of HCC cell lines with an activated FGF signaling pathway under limited nutrients, and FGFR-MAPK cascades likely contribute to survival of HCC cells with an activated FGF signaling pathway under tumor microenvironments with limited nutrients, where tumor angiogenesis is inhibited.

Antitumor and Antiangiogenic Activities of Lenvatinib in Mouse Xenograft Models of Vascular Endothelial Growth Factor-Induced Hypervascular Human Hepatocellular Carcinoma.

High expression of vascular endothelial growth factor (VEGF) in patients with hepatocellular carcinoma (HCC) is associated with poor prognosis. Here, we investigated the antitumor activity of lenvatinib, a multiple receptor tyrosine kinase inhibitor, in VEGF-overexpressing HCC models. In human umbilical vein endothelial cells, lenvatinib showed potent inhibitory activities against VEGF-induced proliferation and VEGF/basic fibroblast growth factor-induced tube formation. In VEGF-overexpressing HCC xenograft models, characterized by aggressive tumor growth and hypervascularity, lenvatinib had significant antitumor and antiangiogenic activities. These results suggest that potent activity of lenvatinib against VEGF signaling underlies its antitumor and antiangiogenic activities in the hypervascular HCC models.