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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.

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.

PDF - 86K, Cleavaged -PARP induction in A375 and G361 parental cells.

XLSX file - 132K, Table S2: Effects of EPZ-6438 on Gene Expression in EZH2 Mutant Lymphoma Cells.

PDF - 83K, Effect of vemurafenib, selumetinib, or E6201 on Cyclin D1 expression in exogenous MEK1-WT-positive or -negative A375 cells.

PDF - 68K, Effect of vemurafenib, selumetinib, or E6201 on Cyclin D1 expression in (A) exogenous MEK1-C121S-positive or -negative G361 cells and in (B) exogenous MEK1-WT-positive or -negative G361 cells.

PDF - 50K, Legends for Supplementary Figures 1 through 4.

IC50 values for the inhibition of 93 human kinases by E7090.

Western blot analysis of FGFR1 in human lung cancer cell lines harboring FGFR1 amplifications.

PDF file - 811K, Fig. S1. Effects of EPZ-6438 on Gene Promoter H3K27 Methylation, Recruitment of PRC2 Components to Gene Promoters, and Cell Cycle, in WSU-DLCL2 Cells. Fig. S2: Effects of EPZ-6438 on Gene Expression in EZH2 Mutant Lymphoma Cell Lines. Fig. S3: Pharmacokinetic Profiles of EPZ-6438 in Rats and Mice. Fig. S4: EPZ-6438 Compound Levels in Plasma and WSU-DLCL2 Xenograft Tumor Homogenates from Mice Dosed for 7 or 28 Days. Fig. S5: In vivo Effects of EPZ-6438 in Lymphoma Xenograft Models. Fig. S6: Body Weights of Mice during Xenograft Efficacy Studies.

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.

PDF file - 25K, Table S1: LCC Values for EPZ-6438 for WSU-DLCL2 Human Lymphoma Cells Dosed Either Continuously or After Compound Washout.

Many clinical cases of acquired resistance to the BRAF inhibitor vemurafenib have recently been reported. One of the causes of this acquired resistance is the BRAF downstream kinase point mutation MEK1-C121S. This mutation confers resistance to not only vemurafenib, but also to the allosteric MEK inhibitor selumetinib (AZD6244). Here, we investigated the pharmacologic activities and effectiveness of the novel MEK inhibitor E6201 against BRAF (v-raf murine sarcoma viral oncogene homolog B1)-V600E mutant melanoma harboring the MEK1-C121S mutation. A cell-free assay confirmed that E6201 is an ATP-competitive MEK inhibitor, meaning it has a different binding mode with MEK compared with allosteric MEK inhibitors. E6201 is more effective against BRAF-V600E mutant melanoma compared with BRAF wild-type melanoma based on MEK inhibition. We found that the acquired MEK1-C121S mutation in BRAF-V600E mutant melanoma conferred resistance to both vemurafenib and selumetinib but not E6201. The effectiveness of E6201 in this preclinical study is a result of its binding with MEK1 far from the C121S point mutation so the mutation is unable to influence the MAPK pathway inhibitory activity. These results support further clinical investigation of E6201. Mol Cancer Ther; 13(4); 823–32. ©2014 AACR.

The FGFR signaling pathway has a crucial role in proliferation, survival, and migration of cancer cells, tumor angiogenesis, and drug resistance. FGFR genetic abnormalities, such as gene fusion, mutation, and amplification, have been implicated in several types of cancer. Therefore, FGFRs are considered potential targets for cancer therapy. E7090 is an orally available and selective inhibitor of the tyrosine kinase activities of FGFR1, -2, and -3. In kinetic analyses of the interaction between E7090 and FGFR1 tyrosine kinase, E7090 associated more rapidly with FGFR1 than did the type II FGFR1 inhibitor ponatinib, and E7090 dissociated more slowly from FGFR1, with a relatively longer residence time, than did the type I FGFR1 inhibitor AZD4547, suggesting that its kinetics are more similar to the type V inhibitors, such as lenvatinib. E7090 showed selective antiproliferative activity against cancer cell lines harboring FGFR genetic abnormalities and decreased tumor size in a mouse xenograft model using cell lines with dysregulated FGFR. Furthermore, E7090 administration significantly prolonged the survival of mice with metastasized tumors in the lung. Our results suggest that E7090 is a promising candidate as a therapeutic agent for the treatment of tumors harboring FGFR genetic abnormalities. It is currently being investigated in a phase I clinical trial. Mol Cancer Ther; 15(11); 2630–9. ©2016 AACR.

Antibody lists used in the flow cytometric analysis

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

PDF - 164K, MAPK pathway inhibitory activity of the test compounds in G361.

Mutations within the catalytic domain of the histone methyltransferase EZH2 have been identified in subsets of patients with non-Hodgkin lymphoma (NHL). These genetic alterations are hypothesized to confer an oncogenic dependency on EZH2 enzymatic activity in these cancers. We have previously reported the discovery of EPZ005678 and EPZ-6438, potent and selective S-adenosyl-methionine-competitive small molecule inhibitors of EZH2. Although both compounds are similar with respect to their mechanism of action and selectivity, EPZ-6438 possesses superior potency and drug-like properties, including good oral bioavailability in animals. Here, we characterize the activity of EPZ-6438 in preclinical models of NHL. EPZ-6438 selectively inhibits intracellular lysine 27 of histone H3 (H3K27) methylation in a concentration- and time-dependent manner in both EZH2 wild-type and mutant lymphoma cells. Inhibition of H3K27 trimethylation (H3K27Me3) leads to selective cell killing of human lymphoma cell lines bearing EZH2 catalytic domain point mutations. Treatment of EZH2-mutant NHL xenograft-bearing mice with EPZ-6438 causes dose-dependent tumor growth inhibition, including complete and sustained tumor regressions with correlative diminution of H3K27Me3 levels in tumors and selected normal tissues. Mice dosed orally with EPZ-6438 for 28 days remained tumor free for up to 63 days after stopping compound treatment in two EZH2-mutant xenograft models. These data confirm the dependency of EZH2-mutant NHL on EZH2 activity and portend the utility of EPZ-6438 as a potential treatment for these genetically defined cancers. Mol Cancer Ther; 13(4); 842–54. ©2014 AACR.

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.

Supplementary Table legends and Supplementary Figure legends.

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.

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.

Combination treatment using lenvatinib (a multikinase inhibitor) plus pembrolizumab (a programmed death-1 immune checkpoint inhibitor) has shown efficacy in the treatment of endometrial and renal cell cancers. This phase 1b study investigated the tolerability and safety of lenvatinib plus pembrolizumab in Japanese patients with metastatic selected solid tumors.Patients received a starting dose of 20 mg oral lenvatinib per day plus 200 mg intravenous pembrolizumab every 3 weeks in 21-day cycles. Dose-limiting toxicities were evaluated during the first cycle. Tumor assessments were performed by investigators based on modified RECIST v1.1. Pharmacokinetic parameters and serum biomarkers were assessed.Among enrolled patients (N = 6), 3 had non-small cell lung cancer, and 3 had urothelial cancer. No patients experienced a dose-limiting toxicity. All patients experienced at least 1 treatment-related treatment-emergent adverse event. The objective response rate was 33.3% (95% confidence interval 4.3-77.7); both responses (1 complete, 1 partial) were observed in patients with urothelial cancer. Pharmacokinetics were consistent with previous studies. Serum angiopoietin-2 levels tended to decrease, and serum fibroblast growth factor-23 levels tended to increase from baseline to Cycle 2 Day 1.This study supports the tolerability of 20 mg lenvatinib/day plus 200 mg pembrolizumab every 3 weeks in Japanese patients, consistent with the results from a global study of lenvatinib plus pembrolizumab combination therapy in patients with selected solid tumors. Favorable antitumor activity was observed and there were no new safety signals identified.Clinical Trials.gov number: NCT03006887.

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.

375 Background: In the Study 111/KEYNOTE-146 trial (NCT02501096; N=147), lenvatinib (lenva) + pembrolizumab (pembro) showed encouraging antitumor activity and a manageable safety profile in treatment-naive (n=23) or previously treated metastatic RCC (n=105, previously treated with immune checkpoint inhibitor [ICI]; n=19, previously treated ICI naive); 145 had clear cell RCC and 2 had non-clear cell RCC. In this exploratory analysis, we evaluated the association between clinical outcomes and gene expression signatures and DNA variants for individual RCC-specific driver genes of interest based on published reports. Methods: Patients (pts) with metastatic RCC were treated with lenva 20 mg orally once daily + pembro 200 mg intravenously once every 3 weeks. The analysis population included pts with treatment-naive (n=10) and ICI pretreated (n=70) disease with evaluable RNA-sequencing data for the 18-gene T-cell–inflamed gene expression profile (TcellinfGEP) and for 11 other signatures (angiogenesis; glycolysis; gMDSC; hypoxia; mMDSC; MVD; MYC; proliferation; RAS; stroma/EMT/TGFβ; WNT) and whole exome sequencing (WES) data for DNA variants for individual genes ( VHL, PBRM1, BAP1, and SETD2). Specimens were collected prior to the start of treatment. The associations between each signature score and ORR and PFS per immune-related RECIST were evaluated using logistic regression and Cox proportional hazards, respectively. One-sided P values for TcellinfGEP (hypothesized positive association) and two-sided P values for all other signatures (no hypothesized association) were adjusted for multiplicity using the Hochberg step-up procedure; significance was prespecified at α=0.05. The association between DNA variants for individual genes and ORR was evaluated descriptively. Clinical data cutoff was August 18, 2020. Results: Of 147 treated pts, RNA sequencing and WES data were available for 80 (54%) and 60 (41%), respectively. TcellinfGEP was not associated with ORR ( P=0.827) or PFS ( P=0.741), nor were the other 11 signatures before or after adjustment for TcellinfGEP. ORR for DNA variants reported in the table. Conclusions: In this exploratory analysis of pts with metastatic RCC enrolled in Study 111/KEYNOTE-146 treated with lenva + pembro, responses were observed regardless of biomarker status. There were no statistically significant associations between gene signatures and clinical outcomes. Clinical benefit was observed regardless of VHL, PBRM1, BAP1, or SETD2 mutation status. Analyses in larger randomized datasets will provide additional information on the role of biomarkers in RCC. Clinical trial information: NCT02501096. [Table: see text]

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.

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.

4084 Background: In a phase 1b trial (NCT03006926), L + P had promising antitumor activity as first-line (1L) therapy in uHCC. We present exploratory biomarker analyses of circulating angiogenic factors and cytokines/chemokines related to the mechanism of action of L + P (ie, pharmacodynamic [PD] biomarkers), as well as biomarker correlations with clinical outcomes in patients (pts) with uHCC, from this trial. Methods: Pts received lenvatinib 12 mg/d (bodyweight [BW] >60 kg) or 8 mg/d (BW < 60 kg) PO + pembrolizumab 200 mg IV Q3W. Tumors were assessed using mRECIST or RECIST v1.1 per independent imaging review. Peripheral blood samples were collected before administration of study drug at baseline, cycle (C) 2, day (D) 1, C3D1, C4D1, and off-treatment. 43 Biomarkers were assayed in serum from 100 1L uHCC pts (excluding 4 pts from the dose-limiting toxicity part of the trial with prior sorafenib). Of these 43, 31 biomarkers (for which ≤20% of samples had measurements above/below the quantification limit of the assay) were included in the analyses. Changes in biomarker levels from baseline were evaluated via 1-sample Wilcoxon signed-rank test. Associations were explored between changes in biomarker levels and maximum tumor shrinkage (MTS) via the Spearman’s rank correlation test, objective response (OR; complete response + partial response) via the Wilcoxon rank sum test, and PFS via Cox regression analysis and log rank test. Data cutoff date for clinical endpoints was 7 August 2020. Results: Levels of PD biomarkers related to angiogenic signaling (VEGF increase/ANG2 decrease), FGF signaling (increase in FGF23/FGF19), and IFNγ signaling (increase in IFNγ, CXCL9/10/11) were changed significantly (adjusted P< 0.05) with L + P (C2D1–C4D1; except for FGF19 at C3D1). Significant decreases of TIMP1 and increases of MCP1 were observed at C4D1 during treatment; these were associated with greater MTS. Greater decreases in TIMP1 and greater increases in MCP1 were observed in pts with OR vs others. Changes in levels of the PD biomarkers ANG2, IL10, and VEGFR2 were found to be associated with PFS by dichotomized analysis. With tertile 2 cutoff, median PFS for pts in the group with greater decreases of ANG2 was 13.9 months vs 9.6 months for pts in the group with lesser decreases of ANG2 (unadjusted P= 0.002; HR 2.65, 95% CI 1.39–5.08). Conclusions: These are the first exploratory biomarker analyses for the single-arm study of L + P in pts with uHCC. Changes in serum biomarkers associated with angiogenic-, FGF-, and IFNγ-signaling pathways indicated target engagement of L + P. Decreases in TIMP1 and increases in MCP1 were associated with MTS and OR. Associations were found between longer PFS and a greater decrease in levels of ANG2. Angiogenesis inhibition and modulation of cancer immune response were observed with L + P. Further validation from independent studies is warranted. Clinical trial information: NCT03006926.

Notch4 is selectively expressed on tumor endothelial cells and hypothesized to contribute to cancer malignancy through mechanisms including tumor angiogenesis regulation and promoting drug resistance. We developed E7011, a humanized IgG2κ antibody targeting Notch4 receptor signaling. To obtain anti-human Notch4 monoclonal antibody, human Notch4 3EGFR (epidermal growth factor repeat) domain - NRR (negative regulatory region) - SEAP (secreted alkaline phosphatase) - His protein was immunized to mice and 6698 hybridomas were obtained. By screening these clones with the Notch4 binding assay and Notch4-specific gal4-fusion luciferase reporter assay, mouse anti-human Notch4 mAb clone (6-3-A6) was selected. The CDRs (complementarity determining regions) of 6-3-A6 were grafted into the variable regions of human IgG2κ, and E7011, novel humanized anti-human Notch4 monoclonal antibody, was developed by screening in assays above. The biomolecular interaction analysis showed that E7011 is able to bind to human and mouse Notch4-NRR domain-SEAP-His, and inhibit human Notch4 receptor signaling in an antibody-dose dependent manner as well as 6-3-A6. IHC analysis showed that Notch4 was expressed on CD31 positive endothelial cells in Calu6 human non-small cell lung carcinoma xenograft tumor. Intravenous administration of E7011 at 1, 3, 10 mg/kg in twice a week significantly decreased tumor growth compared with control IgG treatment without severe body weight loss. We also investigated the functional tumor angiogenesis by hoechest dye-perfusion and staining with anti-CD31 antibody in the Calu6 xenograft model. E7011 treatment significantly decreased hoechst-positive perfusion area compared with vehicle treatment. In conclusion, E7011 is a novel antitumor agent that is a specific signal inhibitor targeting the Notch4 receptor and suppresses tumor growth through decreasing tumor vessel function in based on preclinical studies. Citation Format: Yusuke Adachi, Yoshimasa Sakamoto, Youya Nakazawa, Sho Tachino, Ken Ito, Takanori Abe, Yukinori Minoshima, Kana Hoshino-Negishi, Hideaki Ogasawara, Tomomi Kawakatsu, Miyuki Nishimura, Masashi Shimizu, Kazuhiro Tahara, Toshitaka Sato, Katsuhisa Suzuki, Kishan Agarwala, Masao Iwata, Yasuhiro Funahashi, Kenichi Nomoto, Yoichi Ozawa, Junji Matsui, Toshio Imai, Yu Kato. E7011, a novel anti-angiogenesis inhibitor targeting Notch4 receptor signal pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1485.

733 Background: A phase 1b/2 clinical trial indicated that len/pembro shows promise in the treatment of renal cell carcinoma (RCC) in both PD-1/PD-L1 immune checkpoint blockade (ICB)-naïve and pretreated patients (NCT02501096). The combination is being further investigated in a phase 3 clinical trial in RCC (NCT02811861). Tumor antigen presentation depends on multiple factors, including HLA diversity, which can be measured by HLA evolutionary divergence (HED). HED quantitates the capacity of a patient’s HLA genotype to present different peptide antigens. This study is investigating the genomic components of tumor antigen presentation in ICB-naïve patients who have RCC and are treated with len/pembro. Methods: Whole exome sequencing (WES) was performed on pretreatment tumor-derived DNA. Somatic mutations, tumor mutation burden (TMB), neoantigen (NA) load, germline HLA zygosity and somatic loss of heterozygosity (LOH), and HED were correlated with objective response rate (ORR) and progression-free survival (PFS). An updated clinical cutoff was March 29, 2019. Results: Twenty four (80%) of 30 ICB-naïve patients underwent WES. A top-quartile cutoff was used. Increased mean HED was associated with improved PFS, while HLA homozygosity or LOH trended toward worse PFS. Loss-of-function mutations in PBRM1 (PBRM1 LOF) trended toward improved PFS. However, TMB and NA load were not correlated to PFS. No genomic biomarkers were correlated to ORR. Conclusions: Increased HLA diversity was associated with improved PFS, while decreased HLA diversity may be associated with worse PFS. PBRM1 mutation may be associated with improved PFS; however, TMB and NA load were not correlated to outcomes. These findings warrant further examination in larger datasets to rule out possible artifacts from multiple testing in a small cohort. Clinical trial information: NCT02811861. [Table: see text]

Inhibitors of the protein methyltransferase Enhancer of Zeste Homolog 2 (EZH2) may have significant therapeutic potential for the treatment of B cell lymphomas and other cancer indications. The ability of the scientific community to explore fully the spectrum of EZH2-associated pathobiology has been hampered by the lack of in vivo-active tool compounds for this enzyme. Here we report the discovery and characterization of EPZ011989, a potent, selective, orally bioavailable inhibitor of EZH2 with useful pharmacokinetic properties. EPZ011989 demonstrates significant tumor growth inhibition in a mouse xenograft model of human B cell lymphoma. Hence, this compound represents a powerful tool for the expanded exploration of EZH2 activity in biology.

Anti-vascular endothelial growth factor (VEGF) therapies have been in clinical use to treat patients with multiple types of cancers, but benefits of anti-VEGF therapy are still limited because of the acquired resistance to the treatment. In this study, we developed a novel resistance model to anti-VEGF therapy by expressing VEGFR2-Fc in mouse Renca and B16F10 tumor models. We investigated activated signaling pathways in established resistant tumors by differentially expressed gene analysis and using pathway analysis using Reactome based on RNA-Seq data. Differentially expressed gene analysis identified 293 genes, which are altered commonly in both resistant tumors, and pathway analysis showed that those genes were enriched in FGF2-FGFR2 signaling pathway. IHC analysis by staining with anti-CD31 and αSMA antibodies demonstrated that tumor vessels in VEGFR2-Fc expressing resistant tumors were covered with pericytes, and further IHC analysis with anti-FGF2 antibody indicated that FGF2 was stained in pericytes, which covered endothelial cells in VEGFR2-Fc expressing tumor. Moreover, we isolated CD31 positive cells as endothelial cells, CD90.2 positive cells as pericytes, and CD45 positive cells as tumor infiltrated lymphocytes, respectively. Expression level of Fgf2 and Fgfr2 mRNA in these subsets from Mock tumors and VEGFR2-Fc expressing tumors were analyzed. As a result, Fgf2 and Fgfr2 mRNA expressions in VEGFR2-Fc expressing tumors were upregulated in pericytes and endothelial cells, respectively compared with Mock tumors. These results suggested that there is a spatially effective activation of FGF signaling pathway between endothelial cells and pericytes in tumor vessels that are resistant to anti-VEGF therapy. Indeed, by expression of FGFR2-Fc, in vivo tumor growth of anti-VEGF therapy-resistant tumors was suppressed as well as angiogenesis. Lenvatinib mesilate (lenvatinib) is an oral multiple receptor tyrosine kinase (RTK) inhibitor that suppresses the kinase activities of VEGF receptors (VEGFR1-3), in addition to other proangiogenic and oncogenic pathway-related RTKs including fibroblast growth factor receptors (FGFR1-4), the platelet-derived growth factor receptor (PDGFR) α, KIT, and RET. Lenvatinib suppressed phosphorylation of FGFR2 in VEGFR2-Fc expressing tumors, and showed antitumor activity accompanied with further decrease of tumor vessels. But, sorafenib and aflibercept, which are selective inhibitors against VEGF signaling pathway, didn’t show antitumor activity against anti-VEGF therapy resistant tumors. In conclusion, our findings revealed key roles of pericytes expressing FGF2 in anti-VEGF therapy acquired resistant tumor models, where lenvatinib showed antitumor activity, indicating an importance of inhibition of FGF signaling pathway to suppress escape from anti-VEGF therapy. Citation Format: Kenji Ichikawa, Saori Watanabe Miyano, Yukinori Minoshima, Junji Matsui, Yasuhiro Funahashi. Antitumor activity of lenvatinib against acquired resistance tumor to anti-VEGF therapy in mouse syngeneic tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 196.

Target-protein degradation is an emerging field in drug discovery and development. In particular, the substrate-receptor proteins of the cullin-ubiquitin ligase system play a key role in selective protein degradation, which is an essential component of the anti-myeloma activity of immunomodulatory drugs (IMiDs), such as lenalidomide. Here, we demonstrate that a series of anticancer sulfonamides NSC 719239 (E7820), indisulam, and NSC 339004 (chloroquinoxaline sulfonamide, CQS) induce proteasomal degradation of the U2AF-related splicing factor coactivator of activating protein-1 and estrogen receptors (CAPERα) via CRL4DCAF15 mediated ubiquitination in human cancer cell lines. Both CRISPR-Cas9-based knockout of DCAF15 and a single amino acid substitution of CAPERα conferred resistance against sulfonamide-induced CAPERα degradation and cell-growth inhibition. Thus, these sulfonamides represent selective chemical probes for disrupting CAPERα function and designate DCAFs as promising drug targets for promoting selective protein degradation in cancer therapy.

The FGFR signaling pathway has a crucial role in proliferation, survival, and migration of cancer cells, tumor angiogenesis, and drug resistance. FGFR genetic abnormalities, such as gene fusion, mutation, and amplification, have been implicated in several types of cancer. Therefore, FGFRs are considered potential targets for cancer therapy. E7090 is an orally available and selective inhibitor of the tyrosine kinase activities of FGFR1, -2, and -3. In kinetic analyses of the interaction between E7090 and FGFR1 tyrosine kinase, E7090 associated more rapidly with FGFR1 than did the type II FGFR1 inhibitor ponatinib, and E7090 dissociated more slowly from FGFR1, with a relatively longer residence time, than did the type I FGFR1 inhibitor AZD4547, suggesting that its kinetics are more similar to the type V inhibitors, such as lenvatinib. E7090 showed selective antiproliferative activity against cancer cell lines harboring FGFR genetic abnormalities and decreased tumor size in a mouse xenograft model using cell lines with dysregulated FGFR. Furthermore, E7090 administration significantly prolonged the survival of mice with metastasized tumors in the lung. Our results suggest that E7090 is a promising candidate as a therapeutic agent for the treatment of tumors harboring FGFR genetic abnormalities. It is currently being investigated in a phase I clinical trial. Mol Cancer Ther; 15(11); 2630–9. ©2016 AACR.

The catalytic activities of covalent and ATP-dependent chromatin remodeling are central to regulating the conformational state of chromatin and the resultant transcriptional output. The enzymes that catalyze these activities are often contained within multiprotein complexes in nature. Two such multiprotein complexes, the polycomb repressive complex 2 (PRC2) methyltransferase and the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeler have been reported to act in opposition to each other during development and homeostasis. An imbalance in their activities induced by mutations/deletions in complex members (e.g. SMARCB1) has been suggested to be a pathogenic mechanism in certain human cancers. Here we show that preclinical models of synovial sarcoma—a cancer characterized by functional SMARCB1 loss via its displacement from the SWI/SNF complex through the pathognomonic SS18-SSX fusion protein—display sensitivity to pharmacologic inhibition of EZH2, the catalytic subunit of PRC2. Treatment with tazemetostat, a clinical-stage, selective and orally bioavailable small-molecule inhibitor of EZH2 enzymatic activity reverses a subset of synovial sarcoma gene expression and results in concentration-dependent cell growth inhibition and cell death specifically in SS18-SSX fusion-positive cells in vitro. Treatment of mice bearing either a cell line or two patient-derived xenograft models of synovial sarcoma leads to dose-dependent tumor growth inhibition with correlative inhibition of trimethylation levels of the EZH2-specific substrate, lysine 27 on histone H3. These data demonstrate a dependency of SS18-SSX-positive, SMARCB1-deficient synovial sarcomas on EZH2 enzymatic activity and suggests the potential utility of EZH2-targeted drugs in these genetically defined cancers.

Pladienolide is a naturally occurring macrolide that binds to the SF3b complex to inhibit mRNA splicing. It has not been fully validated whether the splicing impairment is a relevant mechanism for the potent antitumor activity of pladienolide. We established pladienolide-resistant clones from WiDr and DLD1 colorectal cancer cells that were insensitive to the inhibitory action of pladienolide on cell proliferation and splicing. An mRNA-Seq differential analysis revealed that these two cell lines have an identical mutation at Arg1074 in the gene for SF3B1, which encodes a subunit of the SF3b complex. Reverse expression of the mutant protein transferred pladienolide resistance to WiDr cells. Furthermore, immunoprecipitation analysis using a radiolabeled probe showed that the mutation impaired the binding affinity of paldienolide to its target. These results clearly demonstrate that pladienolide exerts its potent activity by targeting SF3b and also suggest that inhibition of SF3b is a promising drug target for anticancer therapy.

Anti-vascular endothelial growth factor (VEGF) therapies have been in clinical use to treat patients with multiple types of cancers, especially in renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC). However, benefits of anti-VEGF therapies are still limited because of the intrinsic/acquired resistance to the treatment. Lenvatinib mesilate (lenvatinib) is an oral multiple receptor tyrosine kinase (RTK) inhibitor that suppresses the kinase activities of VEGF receptors (VEGFR1-3), in addition to other proangiogenic and oncogenic pathway-related RTKs including fibroblast growth factor receptors (FGFR1-4), the platelet-derived growth factor receptor (PDGFR) α, KIT, and RET. Lenvatinib has been approved as a monotherapy for the treatment of radioiodine-refractory differentiated thyroid cancer and in combination with everolimus for the treatment of patients with RCC treated with one prior anti-VEGF therapy in the US and EU. In this study, we established an anti-VEGF treatment resistant preclinical mouse tumor model. We also examined whether lenvatinib, which targeted multiple receptor tyrosine kinases, showed anti-tumor activity in this model. To establish model resistant to anti-VEGF treatment, an expression vector encoding VEGFR-Fc protein (VEGF decoy) was introduced into a mouse RCC cell line RENCA. First, we confirmed the RENCAVEGFR-Fc transfectants secreted VEGF decoy, which selectively interacted with recombinant VEGF protein but not with ligands to other RTKs in in vitro. Tumor growth in vivo of RENCAVEGFR-Fc was significantly slower than that of RENCAMock. RENCAMock transfectants took 16 days to reach a mean tumor volume (TV) of 500 mm3 whereas RENCAVEGFR-Fc transfectants required 72 days to reach same TV. Furthermore, 72 days after inoculation of RENCAVEGFR-Fc transfectants, we resected tumor tissues and confirmed the expression of VEGF decoy protein. Tumor microvessel density was decreased in resected RENCAVEGFR-Fc tumor compared to the same size of RENCAMock tumor. From the above results, we characterized RENCAVEGFR-Fc tumor was resistant to VEGF decoy. Finally, anti-tumor activity of lenvatinib was challenged against RENCAMock or RENCAVEGFR-Fc tumors. When tumor volume reached approximately 300mm3, 10 mg/kg of lenvatinib was orally administered once daily for 14 days. Treatment of lenvatinib led to almost complete inhibition of tumor growth in both RENCAMock and RENCAVEGFR-Fc models. In conclusion, we developed a mouse RENCAVEGFR-Fc model resistant to anti-VEGF treatment, and lenvatinib showed anti-tumor activity in the resistant model as with RENCAMock model. Additional preclinical studies will be investigated to elucidate the signaling pathways associated with resistance to anti-VEGF treatment, in which lenvatinib may inhibit such as FGFR1-4, PDGFRα, KIT and RET. Citation Format: Kenji Ichikawa, Saori Watanabe Miyano, Yukinori Minoshima, Junji Matsui. Antitumor activity of lenvatinib in the renal cell carcinoma cell line RENCA model resistant to a VEGF specific inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 11.

5597Background: Lenvatinib (LEN) is a multikinase inhibitor of VEGFR 1−3, FGFR 1−4, and other targets; pembrolizumab (PEM) is an anti–PD-1 antibody. We report biomarker analyses from a phase 1b/2 t...

Background: LEN is an oral multi-targeted tyrosine kinase inhibitor that selectively inhibits vascular endothelial growth factor receptor (VEGFR)1-3, fibroblast growth factor receptor (FGFR)1-4, platelet-derived growth factor receptor α, RET, and KIT. LEN was noninferior to sorafenib (SOR) in overall survival (OS) in a phase 3 study in patients with unresectable hepatocellular carcinoma (HCC) (medians: LEN, 13.6 vs SOR, 12.3 months; hazard ratio: 0.92; 95% confidence interval, 0.79-1.06) and statistically superior to SOR in progression-free survival (PFS), time to progression (TTP), and objective response rate (ORR). We previously reported that LEN showed antitumor activity in multiple HCC xenograft models including patient-derived xenograft (PDx) models. Here, we investigated the mode of action of LEN in preclinical human HCC models to examine antitumor angiogenesis activity in vivo and the effects on FGF signaling pathways both in vitro and in vivo in the preclinical HCC models. Methods: Antiproliferative activity of LEN and SOR was examined using human HCC cells (Hep 3B2.1-7, HuH-7, SNU398, and PLC/PRF/5) in vitro, and antitumor and anti-angiogenesis activity were examined using these HCC cell lines and PDx models. The effect on the FGF signaling pathway was evaluated by determining the phosphorylation of FRS2, a direct substrate of FGFRs, with immunoblotting analysis in both cultured cells and tumor xenografts. Anti-angiogenesis activity was evaluated by determining microvessel density (MVD) within tumor xenografts by immunohistochemical analysis using anti-CD31 antibody. Result: LEN decreased the phosphorylation of FRS2 in FGF19-overexpressing Hep3B2.1-7 and HuH-7 cells at 0.03 to 3 μmol/L in a concentration-dependent manner. LEN showed selective antiproliferative activity against these cells with half-maximal inhibitory concentration (IC50) values of 0.23 and 0.42 μmol/L, respectively, and those concentrations were consistent with ones for inhibitions of the phosphorylation of FRS2. In tumor xenografts, LEN also decreased the phosphorylation of FRS2 by the single administration of LEN between 3 and 30 mg/kg. SNU398 cells are known to have a relatively weak dependence on the FGF signaling pathway without FGF19 expression. LEN inhibited in vitro proliferation of SNU398 cells, and the phosphorylation of FRS2 both in vitro and in vivo at higher doses than in Hep 3B2.1-7 and HuH-7 cells. SOR did not selectively inhibit in vitro proliferation or the phosphorylation of FRS2 in HCC cells with FGF activation in vitro and in vivo. In HCC PDx and PLC/PRF/5 xenograft models, LEN significantly decreased the MVD in a dose-dependent manner between 3 and 30 mg/kg, and it was correlated with tumor growth inhibition in each models. SOR showed clear anti-angiogenesis activity at 30 mg/kg, but not at 10 mg/kg. Conclusion: These results suggest that inhibition of the FGF signaling pathway via FGFR with LEN affects proliferation of HCC cells with activation of the FGF signaling pathway, and the potent anti-angiogenic activity underlies the antitumor activity of LEN in preclinical HCC xenograft models. Citation Format: Taisuke Hoshi, Masahiro Matsuki, Yuji Yamamoto, Yukinori Minoshima, Junji Matsui, Yasuhiro Funahashi. Antitumor activity of lenvatinib mesilate (LEN) via angiogenesis inhibition and tumor FGF signaling pathway in the human hepatocellular carcinoma models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B095.