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The identification of biologically active target compounds and their binding proteins is important in mechanism-of-action studies for drug development. Additionally, the newly discovered binding proteins provide unforeseen ideas for novel drug discovery and for subsequent structural transformation to improve target specificity. Based on the lead and final candidate compounds related to the type 5 phosphodiesterase (PDE5) inhibitor E4021, we designed chemical probes and identified their target proteins by the affinity chromatography approach. Aldehyde dehydrogenase family 1 member A3 (ALDH1A3), currently reported as a cancer stem cell target, was clearly isolated as a binding protein of the lead 'immature' inhibitor probe against PDE5. In the early derivatization to the closely related structure, Compound 5 (ER-001135935) was found to significantly inhibit ALDH1A3 activity. The discovery process of a novel ALDH1A3-selective inhibitor with affinity-based binder identification is described, and the impact of this identification method on novel drug discovery is discussed.

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.

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.

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

An 86-year-old woman in a wheelchair was accompanied by her husband and son as she visited our outpatient clinic due to disturbed consciousness and fever. Twenty-seven years earlier, she had been diagnosed with rheumatoid arthritis and had been treated with methotrexate (MTX) and low-dose prednisolone (PSL). She stopped taking MTX four years previously when she was diagnosed with diffuse large B cell lymphoma of the paranasal sinus. Her lymphoma went into remission after six cycles of systemic immunochemotherapy. MRI after hospitalization revealed a lesion in the splenium of the corpus callosum that was hyperintense on diffusion-weighted imaging and which had low apparent diffusion coefficient values. An analysis of the cerebrospinal fluid revealed no atypical cells. The MRI findings were atypical, but her consciousness disturbance improved, leading to the diagnosis of mild encephalitis/encephalopathy with a reversible splenial lesion, which would be associated with a transient consciousness disturbance with a good course. However, her consciousness worsened over the next 3 weeks. One month later, a contrast-enhanced MRI showed an enlarged lesion in the callosum as well as new lesions, and the diagnosis of secondary CNS lymphoma was made. Brain biopsy is often not feasible. Less invasive and highly accurate diagnostic methods are needed, such as the identification of a spinal fluid tumor marker.

Background The advantage of up-front neck dissection (UFND) followed by chemoradiotherapy (CRT) for hypopharyngeal cancer (HPC) with advanced neck involvement remains controversial. We aimed to determine the indications. Methods The data of 41 and 14 patients with stage IVA/B (T1-T3 and ≥N2a) HPC who underwent UFND followed by CRT and received CRT, respectively, were retrospectively analyzed and compared. Results The 5-year overall survival (OS) and disease-specific survival rates for the UFND and CRT groups were 61% and 52% (p = 0.1019), and 89% and 74% (p = 0.2333), respectively. Moreover, patients aged ≥70 years or those with a pulmonary disease history had a significantly poorer prognosis due to aspiration pneumonia in the UFND group. The 5-year regional control (RC) for the UFND and CRT groups were 92% and 57%, respectively (p = 0.0001). Conclusions UFND followed by CRT was feasible with satisfactory RC. To further improve OS, aspiration pneumonia prevention is essential.

This squib considers the syntax of Right Node Raising (RNR) and offers evidence for the movement analysis of RNR (e.g., Ross 1967). I argue that the shared rightmost DP in RNR survives VP-ellipsis, suggesting that it moves to a position higher than the base position. Moreover, when VP-ellipsis applies to RNR with an island, the island effect occurs. From these facts, I argue that movement is involved in RNR and demonstrate that Sabbagh’s (2007) linearization-based movement analysis cannot explain the data.

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.

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.

106 Background: E7386 is a novel oral anticancer agent that inhibits the binding of β-catenin to its transcriptional co-activator, CBP, thereby modulating Wnt/β-catenin signaling. In preclinical studies, E7386 showed promising activity (eg, modulation of tumor angiogenesis, alteration of the immune microenvironment, and inhibition of tumor growth). At a prior cutoff point for the dose-escalation part of a phase 1 study of E7386 in pts with advanced solid tumors, we reported 2 dose-limiting toxicities (DLTs; both grade 3 decreased appetite) with E7386 160 mg twice daily (BID). Here we describe an update of the dose-escalation part of this phase 1 study—we mainly report updated safety, tolerability, and preliminary efficacy and biomarker results, which led to the inclusion of an additional dose level for investigation in the expansion part of this study. Methods: E7386 was administered orally in escalating doses on a BID continuous schedule in 28-day cycles. Adverse events (AEs) were graded using CTCAE v5.0. Tolerability was judged by DLTs during cycle 1 (C1). Tumor response was assessed every 8 weeks from C1 day (D) 1, or sooner if clinically indicated, and at the end of treatment by investigators using RECIST v1.1. Samples for pharmacokinetic analyses were collected on C1D1 and C1D8; samples for biomarker analyses were collected at protocol-defined time points. To determine the appropriate dose(s) for the expansion part of this study, additional investigation of the dose-escalation part was conducted. Results: As of the data cutoff (DCO) date (30 June 2022), 36 pts (24 men, 12 women; median age, 61.5 y) were enrolled in E7386 dose cohorts (10 to 160 mg BID). Doses were tolerable up to 120 mg BID. The most common treatment-related AEs (all grades; > 10%) were nausea (80.6%), vomiting (58.3%), aspartate aminotransferase increased (16.7%), and alanine aminotransferase increased, decreased appetite, and diarrhea (13.9% each). Nausea and vomiting were well-controlled with antiemetics such as 5HT3 antagonists, except in the 160 mg BID cohort. Two pts with a Wnt-related adenomatous polyposis coli (APC)- mutation (small bowel adenocarcinoma and desmoid tumor) showed a partial response. As of the DCO date, 2 pts (who continued on-treatment as of that date) had received over 1.5 y of E7386 treatment. The preliminary PK analysis showed plasma exposure of E7386 generally increased with an increasing dose over the assessed dose range. FGF21 levels in plasma increased following E7386 administration. Conclusions: E7386 120 mg BID was tolerated and determined as the recommended dose for the expansion part. Based on additional analyses of the dose-escalation part of this study, further investigation of safety, preliminary efficacy, PK, and biomarker analyses of E7386 is ongoing using 2 dose levels (100 and 120 mg BID) in the expansion part. Clinical trial information: NCT03833700 .

Characterizing RNA-protein interactions remains an important endeavor, complicated by the difficulty in obtaining the relevant structures. Evaluating model structures via statistical potentials is in principle straight-forward and effective. However, given the relatively small size of the existing learning set of RNA-protein complexes optimization of such potentials continues to be problematic. Notably, interaction-based statistical potentials have problems in addressing large RNA-protein complexes. In this study, we adopted a novel strategy with covariance matrix adaptation (CMA-ES) to calculate statistical potentials, successfully identifying native docking poses.

Covalent inhibitors have emerged as an important drug class in recent years, largely due to their many unique advantages as compared to noncovalent inhibitors, including longer duration of action, lower prolonged systemic exposure, higher potency, and selectivity. However, the potential off-target toxicity of covalent inhibitors, particularly of irreversible covalent inhibitors, represents a great challenge in covalent drug development. Therefore, accurate calculation of protein covalent inhibitor reaction kinetics to guide the design of selective inhibitors would greatly benefit covalent drug discovery efforts. In the present paper, we present a computational method to calculate the relative reaction kinetics between congeneric irreversible covalent inhibitors and their protein receptors. The method combines density functional theory calculations of the transition state barrier height of the rate-limiting step for reaction between the warhead of the inhibitor and a single protein residue, and molecular-mechanics-based free energy calculations to account for the interactions between the ligand in the transition state and the protein environment. The method was tested on four pharmaceutically interesting irreversible covalent binding systems involving 28 ligands; the mean unsigned error (MUE) of the relative reaction rate for all pairs of ligands between the predictions and experimental results for these tested systems is 0.79 log unit. This is to our knowledge the first time where the reaction kinetics of protein irreversible covalent inhibition have been directly calculated with physics-based free energy calculation methods and transition state theory. We anticipate the outstanding accuracy demonstrated here across a broad range of target classes will have a strong impact on the design of selective covalent inhibitors.