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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

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.

Epidermal growth factor receptor (EGFR) is a major driver of head and neck cancer, a devastating malignancy with a major sub-site in the oral cavity manifesting as oral squamous cell carcinoma (OSCC). EGFR is a glycoprotein receptor tyrosine kinase (RTK) whose activity is upregulated in >80% OSCC. Current anti-EGFR therapy relies on the use of cetuximab, a monoclonal antibody against EGFR, although it has had only a limited response in patients. Here, we uncover a novel mechanism regulating EGFR activity, identifying a role of the nuclear branch of the Wnt/β-catenin signaling pathway, the β-catenin/CBP axis, in control of post-translational modification of N-glycans on the EGFR. Genomic and structural analyses reveal that β-catenin/CBP signaling represses fucosylation on the antennae of N-linked glycans on EGFR. By employing nUPLC-MS/MS, we determined that malignant human OSCC cells harbor EGFR with a paucity of N-glycan antennary fucosylation, while indolent cells display higher levels of fucosylation at sites N420 and N579. Additionally, treatment with either ICG-001 or E7386, which are both small molecule inhibitors of β-catenin/CBP signaling, leads to increased transcriptional expression of fucosyltransferases FUT2 and FUT3, with a concomitant increase in EGFR N-glycan antennary fucosylation. In order to discover which fucosylated glycan epitopes are involved in the observed effect, we performed in-depth characterization of multiply-fucosylated N-glycans via tandem mass spectrometry analysis of the EGFR tryptic glycopeptides. Data are available via ProteomeXchange with identifier PXD017060. We propose that β-catenin/CBP signaling promotes EGFR oncogenic activity in OSCC by inhibiting its N-glycan antennary fucosylation through transcriptional repression of FUT2 and FUT3.

Dysregulated activation of the WNT/β-catenin signaling pathway is essential for the initiation and development of various cancers. E7386, a small-molecule compound, attenuates WNT signaling by blocking the interaction between β-catenin and CREB-binding protein (CBP); hence, it is regarded as a therapeutic candidate for cancers with activated WNT signaling. In the present study, we evaluated the biological characteristics associated with E7386 sensitivity by using a panel of patient-derived colon cancer spheroids. An integrative approach that combined E7386 sensitivity and gene expression profiles revealed that the resistance of the cancer spheroids to E7386 was associated with the activation of the NF-κB pathway. NF-κB pathway inhibitors acted synergistically with E7386 to block proliferation and induce cell cycle arrest in E7386-resistant spheroids. These findings suggest a possibility that a combination of E7386 and NF-κB inhibition may effectively block the proliferation of a subset of colon cancer cells.

Despite their outstanding antitumour activity in mice, the limited supply from the natural sources has prevented drug discovery/development based on intact halichondrins. We achieved a total synthesis of C52-halichondrin-B amine (E7130) on a >10 g scale with >99.8% purity under GMP conditions. Interestingly, E7130 not only is a novel microtubule dynamics inhibitor but can also increase intratumoural CD31-positive endothelial cells and reduce α-SMA-positive cancer-associated fibroblasts at pharmacologically relevant compound concentrations. According to these unique effects, E7130 significantly augment the effect of antitumour treatments in mouse models and is currently in a clinical trial. Overall, our work demonstrates that a total synthesis can address the issue of limited material supply in drug discovery/development even for the cases of complex natural products.

BACKGROUND Indisulam possesses anticancer properties through down-regulation of various cell-cycle checkpoint molecules, thereby blocking the phosphorylation of retinoblastoma protein and inducing p53 and p21. Indisulam exhibits synergy with nucleoside analogs and topoisomerase inhibitors. METHODS The authors designed a phase 2 study of indisulam in combination with idarubicin and cytarabine in patients who had relapsed/refractory acute myeloid leukemia AML and high-risk myelodysplastic syndrome. In stage 1, patients received intravenous indisulam at 400 mg/m2 on days 1 and 8 of a 28-day cycle. If they had no response, then patients received same dose schedule of indisulam followed by intravenous idarubicin 8 mg/m2 daily for 3 days and cytarabine 1.0 g/m2 over 24 hours daily on days 9 through 12 (for those aged 60 years) of a 28-day cycle. Primary endpoints included the overall response rate, and secondary objectives included overall survival. RESULTS Forty patients were enrolled. Of the 37 evaluable patients, 31 received indisulam with chemotherapy. Of these, 11 (35%) responded for a median duration of 5.3 months. The estimated 1-year overall survival rate was 51% for responders compared with 8 % for nonresponders (P

RNA-binding proteins (RBPs) are essential modulators of transcription and translation frequently dysregulated in cancer. We systematically interrogated RBP dependencies in human cancers using a comprehensive CRISPR/Cas9 domain-focused screen targeting RNA-binding domains of 490 classical RBPs. This uncovered a network of physically interacting RBPs upregulated in acute myeloid leukemia (AML) and crucial for maintaining RNA splicing and AML survival. Genetic or pharmacologic targeting of one key member of this network, RBM39, repressed cassette exon inclusion and promoted intron retention within mRNAs encoding HOXA9 targets as well as in other RBPs preferentially required in AML. The effects of RBM39 loss on splicing further resulted in preferential lethality of spliceosomal mutant AML, providing a strategy for treatment of AML bearing RBP splicing mutations.

Head and neck squamous cell carcinoma (HNSCC) is a pernicious malignancy that arises from populations of cancer stem cells (CSCs). We and others have shown that the Wnt/β-catenin signaling pathway drives CSC gene expression mediated, in part, by epigenetic alterations directed by interactions between nuclear β-catenin and the cAMP-responsive element binding (CREB)-binding protein (CBP). In HNSCC, the β-catenin/CBP complex recruits the histone methyltransferase, MLL1, to drive trimethylation of H3K4me3 to induce an open chromatin structure and expression of CSC genes. Further, β-catenin/CBP signaling is highly correlated with the activity of the paralogous transcriptional regulators YAP and TAZ (YAP/TAZ), which are pro-tumorigenic factors in HNSCC. We reported that a small molecule inhibitor of the β-catenin-CBP interaction, ICG-001, blocks oncogenic phenotypes in cellular, zebrafish, and murine models of HNSCC, concomitant with the reduction of CSC traits. Recently, a novel β-catenin/CBP modulator, E7386, has been shown to be effective against a number of neoplasms in preclinical studies. Here, we compared anti-cancer properties of E7386 with ICG-001 to define its molecular mechanisms and validate the β-catenin/CBP axis as a bona fide therapeutic target in HNSCC. Anti-HNSCC activity of E7386 was evaluated in four human HNSCC cell lines using genomic, molecular, biochemical and functional approaches, including global ChIP-seq for H3K4me3. The set of transcripts significantly down-regulated by E7386 in HNSCC cells was projected onto a TCGA RNA-seq data (n=318) using ASSIGN, where samples were scored based on the coordinated expression of the gene signature which, in turn, reflected the level of E7386 inhibition per sample. The E7386 inhibition score was then tested for its association with survival by stratifying TCGA patients (n=318) into high- and low-score groups. Results showed that E7386 had highly overlapping activity signatures with ICG-001 (R = 0.997) with ~50 - 100-fold lower EC50. Similar to ICG-001, treatment with E7386 blocked association between β-catenin and CBP with a concomitant reduction in CBP and MLL1 abundance and global H3K4 trimethylation. E7386 repressed an oncogenic gene expression signature regulated by YAP1/TAZ and impeded HNSCC cell proliferation, promoting E-cadherin adhesion and junctional localization of β-catenin. Importantly, E7386 inhibition-associated transcriptional signatures tracked with tumor grade and poor human HNSCC patient survival. In conclusion, inhibiting β-catenin/CBP activity with E7386 represents a novel approach aimed at targeting epigenetically driven changes in the chromatin structure in HNSCC. Citation Format: Huamei Yang, Vinay Kartha, Khalid A. Alamoud, Anthony Federico, Andrew Tilston-Lunel, Bach-Cuc Nguyen, Takashi Owa, Kenichi Nomoto, Xaralabos Varelas, Stefano Monti, Maria A. Kukuruzinska. Inhibition of β-catenin/CBP signaling with E7386 targets epigenetic changes associated with cancer stem cells in head and neck cancer [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 2453.

Head and neck squamous cell carcinoma (HNSCC) is associated with high morbidity, poor survival rates, and limited treatment options. We previously reported that aberrant activation of nuclear β-catenin/cAMP-responsive element binding (CREB)-binding protein (CBP) signaling in primary HNSCC tumors was associated with progression to advanced disease and poor patient survival. Further, inhibition of β-catenin-CBP interaction with the first-generation inhibitor, ICG-001, greatly attenuated aggressive properties of HNSCC cells and tumor growth and metastases in murine and zebrafish models. Our recent findings show that a novel β-catenin/CBP modulator, E7386, displays an activity profile that closely overlaps with that of ICG-001, but exhibits ~50-100-fold lower EC50 values. In this study, we used a novel method for gene regulatory network (GRN) reconstruction which analyzes publicly available high-throughput transcriptomics data from HNSCC to further elucidate the mechanisms of β-catenin-CBP inhibition by E7386. A Bayesian framework was used to reconstruct high-dimensional GRNs using constraint-based and shared structure learning from bulk RNA-seq data from TCGA. HNSCC patients were grouped into two subtypes, based on their “high” vs. “low” response to E7386 inhibition as predicted by the activation - or enrichment by Gene Set Variation Analysis (GSVA) - of the transcriptional signature we derived. GRNs for the two subtypes were built, and their comparison highlighted new interactions in the “high” responders network between the β-catenin/CBP network and genes associated with histone modifications (ASH1L) and amino acid metabolism (MTR) concomitant with loss of interactions with regulators of cell cycle (ERCC6L) and protein secretion (SCAMP1) identified in the “low” responders network. Additionally, Kaplan-Meier survival plots showed that HNSCC patients with high activation of the E7386 signature had a significantly lower probability of survival. These results suggest that our GRN-based approach represents an important novel tool for assessing changes in regulatory network interactions in HNSCC and cancer. Citation Format: Anthony Federico, Takashi Owa, Kenichi Nomoto, Xaralabos Varelas, Maria Kukuruzinksa, Stefano Monti. Defining the effects of inhibiting the beta-catenin-CBP signaling axis with the small molecule E7386 by integrative analysis of omics data [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 6566.

Background and objectives: Natural products have been a rich source of inspiration for drug discovery as demonstrated by the fact that over one-third of current therapeutic agents are natural products or compounds derived from them. Particularly, when the supply of a natural product from the natural source is limited, organic synthesis can offer a solution. However, with increasing structural complexity, this approach becomes exponentially more challenging, as a synthesis must meet various requirements, including high overall efficiency, scalability, cost-effectiveness, and product purity as well as conforming to good manufacturing practice (GMP) regulations. Despite the outstanding in vivo antitumor activity in mice, the limited supply from the natural sources has prevented drug development based on intact halichondrins. Methods: We successfully synthesized 19.5 g of C52-halichondrin-B alcohol with 99.84% purity via a total synthesis. From 15.0 g of this material, we obtained 11.5 g of C52-halichondrin-B amine (E7130) with 99.81% purity under GMP conditions. With totally synthetic E7130, a novel microtubule dynamics inhibitor, we studied the activities in both of in vitro and in vivo. The inhibitory activity of E7130 towards tubulin polymerization was analyzed in the cell-free system in which tubulin polymerization could be monitored by fluorescence enhancement due to the incorporation of a fluorescence reporter into the microtubules during polymerization. We analyzed in vivo antitumor activities using both a human cancer cell line orthotopic transplantation mouse model and subcutaneous xenograft models. The immunohistochemical analyses were performed with tumor tissues collected from mouse models to analyze histological changes after treatment with E7130. Results: E7130 is a novel microtubule dynamics inhibitor with exceedingly potent in vitro and in vivo anticancer activities. Significantly, E7130 not only is cytotoxic, but can also increase CD31-positive endothelial cells in tumors and reduce α-SMA-positive cancer-associated fibroblasts at pharmacologically relevant compound concentrations. Notably, this dual activity has been recognized for the first time through this study. According to these unique tumor microenvironment ameliorative effects, E7130 can augment the effect of other antitumor treatments in mouse models. In particular, a dose of 90 µg/kg, one-half of the maximum tolerated dose in mice, showed a prominent combinational effect with cetuximab, which suggests that E7130 has a different mechanism than other microtubule-targeted drugs. Conclusions: E7130 ameliorates the tumor microenvironment to improve cancer treatment when used in combination with other compounds. The data provide compelling evidence that E7130 is a promising molecular-targeting anticancer agent. Citation Format: Satoshi Kawano, Takanori Abe, Ken Ito, Kenzo Yahata, Kazunobu Kira, Tsuyoshi Akagi, Makoto Asano, Kentaro Iso, Yuki Sato, Fumiyoshi Matsuura, Isao Ohashi, Yasunobu Matsumoto, Minetaka Isomura, Takeo Sasaki, Takashi Fukuyama, Yusuke Miyashita, Yosuke Kaburagi, Akira Yokoi, Osamu Asano, Takashi Owa, Yoshito Kishi. E7130 derived from total synthesis of halichondrin as a novel tumor-microenvironment ameliorator [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 4179.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy in the world with oral squamous cell carcinomas (OSCC) accounting for the majority of HNSCC cases. A major driver of OSCC is the epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK) with 12 N-glycosylation sites. Fucosylated N-linked glycans on EGFR are associated with survival e.g., they suppress receptor dimerization and signaling. High levels of fucosylated glycan epitopes have been observed in OSCC, where invasive regions lose expression of linkage-specific fucosylated epitopes, suggesting that fucosylated glycans are involved in the suppression of cell growth and invasion. Previously, it was shown that inhibition of the interaction between nuclear β-catenin and CREB-binding protein (CBP) in human OSCC cells and in mouse tumor xenografts with a small molecule inhibitor ICG-001, interfered with OSCC proliferation and aggressive features in cellular, zebrafish, and murine models. E7386, a novel β-catenin/CBP modulator displays activity profile that closely overlaps with that of ICG-001 and exhibits ~50 - 100-fold lower EC50 values. Treatment with ICG-001 and E7386, increased expression of two glycosyltransferases, FUT2 and FUT3 coincident with decreased EGFR abundance that was accompanied by higher fucosylation of EGFR and upregulated expression of E-cadherin and junctional β-catenin. Further, genomic analyses showed a positive correlation between the ICG-001 and E7386 treatments and EGFR inhibition, suggesting that higher expression of antennary fucosyltransferase genes suppresses EGFR signaling. We now show using nLC-MS/MS analyses that EGFR from metastatic HSC-3 cells had low levels of fucosylated N-glycans, while EGFR from indolent CAL27 cells displayed higher levels of fucosylation at multiple EGFR N-linked glycosylation sites, and that these changes were statistically significant. In-depth characterization of multiply-fucosylated N-glycans via tandem mass spectrometry of EGFR glycopeptides revealed new insights into the identity of fucosylated glycan epitopes. Collectively, these results suggest that the β-catenin/CBP axis promotes EGFR signaling through downregulation of fucosyltransferase expression and activity. We conclude that inhibition of β-catenin/CBP signaling with a novel small molecule E7386 may serve as a therapeutic approach to downregulate EGFR pro-tumorigenic activity in HNSCC patients. Citation Format: Kevin B. Chandler, Khalid Alamoud, Bac-Cuc Nguyen, Vanessa L. Stahl, Takashi Owa, Kenichi Nomoto, Stefano Monti, Maria A. Kukuruzinska, Catherine E. Costello. Inhibition of β-catenin/CBP signaling alters EGFR fucosylation status in head and neck squamous cell carcinoma [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 5940.

Background and objectives: Despite the outstanding antitumor activity of halichondrins in mice, the limited supply from the natural sources has prevented drug development using intact halichondrins. We achieved a total synthesis of C52-halichondrin-B amine (E7130) under good manufacturing practice (GMP) conditions. E7130 is not only a novel microtubule dynamics inhibitor, but also a novel tumor-microenvironment ameliorator. E7130 can increase intratumoral CD31-positive endothelial cells and reduce α-SMA-positive cancer-associated fibroblasts (CAFs) at pharmacologically relevant compound concentrations. Here, we analyzed the molecular mechanisms of the α-SMA-positive CAF reduction with E7130. Methods: The expression levels of α-SMA, ECM proteins, ER-TR7 (pan-fibroblast marker), and Ki67 were examined using E7130-treated tumors. In vitro co-culture experiments, normal human fibroblasts were co-cultured with human cancer cells. To examine the effects of E7130 on the TGF-β-induced CAF activation, normal human fibroblasts were treated with TGF-β (final concentration of 1 ng/mL) and E7130 at the several concentrations, and gene expression analysis and immunocytochemical analysis were conducted. Results: E7130 reduced α-SMA-positive CAF and malignant ECM proteins without changing total fibroblast number in tumors. The in vitro co-culture system revealed that TGF-β from cancer cells activate normal human fibroblast and increased α-SMA expression. In addition, treatment with E7130 interfered with α-SMA induction by TGF-β in fibroblasts without growth inhibitory activity. We further found that E7130 inhibited TGF-β-induced PI3K/AKT/mTOR pathway, which resulted in the reduction of α-SMA expression in fibroblasts. Moreover, TGF-β treatment enhanced β-tubulin expression and focal adhesions formation, which were diminished by co-treatment with E7130 in fibroblasts. Many signaling complexes are assembled in focal adhesion sites, and those complexes dispatch several downstream signals, including those involved in the PI3K/AKT/mTOR pathway. Conclusions: Drug discovery using halichondrins was based on their outstanding in vivo antitumor (inhibitory) activity in mice and bone metastasis in mouse melanoma model first reported in 1996. From these results, we hypothesized that halichondrins are not simple microtubule-targeted compounds in tumor cell, and have developed E7130, which harbors unique tumor microenvironment ameliorating effects. Here we proved that E7130 impedes the TGF-β-induced myofibroblast transdifferentiation process without killing the fibroblast by disrupting microtubule network formation, which is important for focal adhesion assembly and thereby the downstream activation of the PI3K/AKT/mTOR pathway. Citation Format: Takanori Abe, Satoshi Kawano, Ken Ito, Kenzo Yahata, Kazunobu Kira, Tsuyoshi Akagi, Makoto Asano, Kentaro Iso, Yuki Sato, Fumiyoshi Matsuura, Isao Ohashi, Yasunobu Matsumoto, Minetaka Isomura, Takeo Sasaki, Takashi Fukuyama, Yusuke Miyashita, Yosuke Kaburagi, Akira Yokoi, Osamu Asano, Takashi Owa, Yoshito Kishi. Mechanism of action analysis of anti-CAF activity of E7130, a novel tumor-microenvironment ameliorator [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 4183.

Single-cell RNA-seq (scRNA-seq) is an emerging platform for high-throughput profiling of individual cells in a sample and is routinely employed to investigate the transcriptional landscapes of the cellular constituents of tumors. To this end, many scRNA-seq specific clustering algorithms have emerged to analytically partition cells into modules of comparatively similar profiles. To facilitate data driven molecular subtyping of such scRNA-seq clustering results and other large-scale-omics studies, we have developed K2 Taxonomer. K2 Taxonomer is an R package built around a novel top-down hierarchical clustering algorithm, utilizing repeated perturbations of the data to generate robust taxonomical partitions of observations. The software runs additional analyses to define gene co-expression signatures of these modules, as well as to integrate user-input annotations of genes and/or observations. An interactive web portal has been generated to assist in the interrogation of the full compendium of results. We applied K2 Taxonomer to publicly available HNSCC scRNA-seq data, identifying pertinent tumor cell subtypes, distinguished by cell cycle and epithelial-to-mesenchymal transition, and with analytical projection of this signature onto TCGA-HNSCC bulk RNA-seq data exhibiting association with worse survival in TCGA-HNSCC patients. A transcriptional signature corresponding to suppression of Mtorc1 and Wnt/β-catenin signaling was also identified in a sub-population of HNSCCs, and shown to be associated with improved patient survival. Of notice, highly ranked markers of this signature are significantly associated with gene expression changes altered by E7386 - a novel β-catenin/CBP modulator with an activity profile that closely overlaps with that of ICG-001, but exhibits ~50-100-fold lower EC50 values - suggesting a role for this signaling axis in subsets of HNSCC. In conclusion, taxonomical subtyping with K2 Taxonomer provides a novel framework to expand the scope of applicability of scRNA-seq clustering results, and has revealed potentially novel HNSCC subtypes that offer directions for future studies. Citation Format: Eric Reed, Takashi Owa, Kenichi Nomoto, Xaralabos Varelas, Maria Kukuruzinska, Stefano Monti. Subtyping of HNSCC single-cell RNA-seq identifies transcriptional programs characterized by suppression of Mtorc1 and Wnt signaling pathways and better patient prognosis [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 4419.

Summary Background Activating enhancer of zeste homolog 2 (EZH2) mutations or aberrations of the switch/sucrose non-fermentable (SWI/SNF) complex (eg, mutations or deletions of the subunits INI1 or SMARCA4) can lead to aberrant histone methylation, oncogenic transformation, and a proliferative dependency on EZH2 activity. In this first-in-human study, we aimed to investigate the safety, clinical activity, pharmacokinetics, and pharmacodynamics of tazemetostat, a first-in-class selective inhibitor of EZH2. Methods We did an open-label, multicentre, dose-escalation, phase 1 study using a 3 + 3 design with planned cohort expansion at the two highest doses below the maximally tolerated dose. The study was done at two centres in France: Institut Gustave Roussy (Villejuif, Val de Marne) and Institut Bergonie (Bordeaux, Gironde). Eligible patients had relapsed or refractory B-cell non-Hodgkin lymphoma or an advanced solid tumour and were older than 18 years, with Eastern Cooperative Oncology Group performance status of 0 or 1, and adequate end-organ function. Tazemetostat was administered orally from 100 mg twice daily to 1600 mg twice daily in 28-day cycles. The primary endpoint was to establish the maximum tolerated dose or recommended phase 2 dose of tazemetostat, as determined by dose-limiting toxicities, laboratory values, and other safety or pharmacokinetic measures in cycle one according to local investigator assessment. Safety was assessed in patients who received at least one dose of tazemetostat; antitumour activity was assessed in the intention-to-treat population. This study is registered with ClinicalTrials.gov, number NCT01897571. The phase 1 part of the study is complete, and phase 2 is ongoing. Findings Between June 13, 2013, and Sept 21, 2016, 64 patients (21 with B-cell non-Hodgkin lymphoma, and 43 with advanced solid tumours) received doses of tazemetostat. The most common treatment-related adverse events, regardless of attribution, were asthenia (21 [33%] of 64 treatment-related events), anaemia (nine [14%]), anorexia (four [6%]), muscle spasms (nine [14%]), nausea (13 [20%]), and vomiting (six [9%]), usually grade 1 or 2 in severity. A single dose-limiting toxicity of grade 4 thrombocytopenia was identified at the highest dose of 1600 mg twice daily. No treatment-related deaths occurred; seven (11%) patients had non-treatment-related deaths (one at 200 mg twice daily, four at 400 mg twice daily, and two at 1600 mg twice daily). The recommended phase 2 dose was determined to be 800 mg twice daily. Durable objective responses, including complete responses, were observed in eight (38%) of 21 patients with B-cell non-Hodgkin lymphoma and two (5%) of 43 patients with solid tumours. Interpretation Tazemetostat showed a favourable safety profile and antitumour activity in patients with refractory B-cell non-Hodgkin lymphoma and advanced solid tumours, including epithelioid sarcoma. Further clinical investigation of tazemetostat monotherapy is ongoing in phase 2 studies in adults and a phase 1 study for children, which are currently enrolling patients who have B-cell non-Hodgkin lymphoma and INI1-negative or SMARCA4-negative tumours. Funding Epizyme and Eisai.

A novel beta-catenin/CBP binding modulator E7386 is expected to affect colorectal carcinoma (CRC) cells particularly those with aberrant activation of Wnt/beta-catenin signaling pathway. The Wnt signaling pathway is recently reported to play important roles for activation of not only CRC cells but also cancer-associated fibroblasts (CAFs). Here, we established an in vitro model of CRC-derived organoids as well as a co-culture method of the CRC organoids with CAFs, and effects of E7386 on the growth of the organoids were examined. Fragments of CRC tissues from a total of 45 patients who underwent surgery were obtained, dissociated into single cells, which were seeded on polymerized Matrigel (Corning), and cultured with serum-free media containing EGF and Noggin optimized for organoids using 12-well plates. Fourteen CRC organoids from 45 cases were successfully established. For 2D culture of CAFs from CRC tissues of each patient, RPMI medium containing 10% FBS was used. A co-culture method of the CRC organoids with CAFs was applied using a double chamber system with transwell inserts in 24-well plates. Cell viabilities of the organoids and CAFs were measured using CellTiter-Glo Luminescent Cell Viability Assay reagent (Promega). Gene aberrations in original CRC tissues were analyzed using NCC Oncopanel, and gene expression profiles in established CRC organoids and CAFs were analyzed using SurePrint G3 Human GE microarray (Agilent). [Effects of CAFs on cell viabilities and gene expressions of organoids] Cell viabilities of organoids and CAFs appeared to increase at 72 hours after beginning of the culture by 2 to 8 and 1 to 2-fold, respectively. When organoids were co-cultured with CAFs, cell viabilities of organoids were increased and epithelial-mesenchymal transition-related gene expressions were dramatically changed in several cases, but not in all cases. The organoids co-cultured with CAFs appeared to be more sensitive to E7386 as compared to those cultured alone. [Possible relationship between gene mutations in CRCs and activity of E7386 in CRC organoids] Cell viabilities of organoids without APC mutations (n=1) and organoids with APC and TP53 mutations alone (n=2) were less than those of organoids with other oncogene mutations, e.g., KRAS or PIK3CA (n=4). Efficacy of E7386 on the cell viabilities of organoids with APC and TP53 mutations alone was found at lower concentrations at 30-100 nM, as compared to those of organoids without APC mutations. From the results, CRC organoids were established from 14 patients, and activity of E7386 appeared to be related to gene mutation profiles in the established CRC organoids. Citation Format: Toshio Imai, Mie Naruse, Masako Ochiai, Yoichi Ozawa, Takashi Owa, Atsushi Ochiai. A possible relationship between gene mutation profiles and activity of E7386 in in vitro models of colorectal cancer organoids [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 275.

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.

RNA-binding proteins (RBPs) regulate many aspects of transcription and translation in a cell- and tissue-specific manner and are frequently dysregulated in malignancy. We systematically evaluated RBPs preferentially required in acute myeloid leukemia (AML) over other forms of cancer or normal hematopoietic precursors using a CRISPR/Cas9 domain-based, loss-of-function screen targeting 490 classical RBPs with 2,900 sgRNAs (Fig. A). This screen was performed in cells lines representing AML, T-cell acute lymphoblastic leukemia (T-ALL), and lung adenocarcinoma (LUAD) and revealed multiple RBPs preferentially required for AML survival, but not for T-ALL or LUAD survival. We identified genes encoding 21 RBPs that were >3-fold depleted in AML cells and significantly overexpressed in AML patient samples versus normal adult CD34+ precursors (p-value < 0.05; Fig. B). Amongst RBPs required and upregulated in AML was RBM39, an RBP described to be involved in a number of cellular processes and to interact with key splicing proteins SF3B1 and U2AF2. Genetic ablation of Rbm39 in mouse MLL-AF9 leukemia cells dramatically delayed AML development and progression (Fig. C). In parallel, it has recently been described that a class of clinically-validated anti-cancer sulfonamide compounds (including indisulam and E7820) mediate RBM39 degradation as their dominant cellular mechanism of action. This occurs via novel interactions with the DCAF15 adapter protein of the CUL4/Ddb1 ubiquitin ligase complex with RBM39 as a neo-substrate. Treatment of MOLM-13 cells xenografted into mice with indisulam conferred significant anti-leukemic effects and improved overall survival (Fig. D). To explore the mechanism of RBM39 dependence in AML, we performed proteomic analyses of RBM39 interacting proteins in MOLM-13 cells as well as transcriptome-wide analysis of RBM39 RNA binding by enhanced UV cross-linking and immunoprecipitation (eCLIP) in the same cells. RBM39 physically interacted with an entire network of RBPs identified by our CRISPR screen as crucial for AML cell survival in addition to interacting with the core SF3b splicing complex. Further, anti-RBM39 eCLIP revealed RBM39 binding to exonic regions and most enriched at exon/intron borders at 5' and 3' splice sites of pre-mRNA (Fig. E), suggesting a prominent role of RBM39 in regulating splicing. Consistent with this, RNA-sequencing of AML cells following RBM39 deletion revealed significant effects of RBM39 loss on RNA splicing, most prominently causing increased cassette exon skipping (Fig. F). Recent studies suggest that myeloid leukemias with mutations in RNA splicing factors are sensitized to pharmacologic perturbation of RNA splicing. Analysis of the effects of RBM39 degrading compounds over a panel of 18 AML cells revealed that leukemia cells bearing splicing factor mutations or with high DCAF15 expression were the most sensitive to treatment (Fig. G). Genetic introduction of SF3B1, SRSF2, or U2AF1 hotspot mutations in K562 or NALM6 cells resulted in a 20-50% reduction in IC50 in response to sulfonamides. We next performed RNA sequencing of isogenic K562 cells with or without knockin of SF3B1K700E and SRSF2P95H mutations into the endogenous loci, and treated at the IC50 of E7820 or E7107, a small molecule that inhibits the SF3b core spliceosome complex. Treatment with either drug dramatically increased cassette exon skipping events and intron retention relative to DMSO control, with greater effects in splicing mutant cells. However, at equipotent doses, E7820 markedly increased mis-splicing compared with E7107. Furthermore, E7820 treatment resulted in mis-splicing of a number of RBP targets identified in our CRISPR screen as being required for AML survival, including SUPT6H, hnRNPH, and SRSF10, as well as RBM3 and U2AF2, consistent with previous observations (Fig. H). Here through systematic evaluation of RBPs across several cancers, we identify RBPs specifically required in AML. In so doing we identify a network of functionally and physically interacting RBPs upregulated in AML over normal precursors. Genetic or pharmacologic elimination one such RBP, RBM39, led to aberrant splicing of multiple members of this RBP network as well as of transcriptional regulators required for AML survival. These data suggest important clinical potential for anti-cancer sulfonamide treatment in splicing mutant myeloid leukemias. Disclosures Uehara: Eisai: Employment. Owa:Eisai: Employment.

Twenty-four ester analogues of renieramycin M (1m) were prepared from jorunnamycin A (3a), which was easily transformed from marine natural 1m in three steps. These analogues, along with 1m itself, cyanojorumycin (2b), and jorunnamycins A (3a) and C (3b), were evaluated in vitro for cytotoxicity by measuring IC(50) values through the 3-(4,5-dimethyltriazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay using human HCT116 colon carcinoma and MDA-MB-435 breast carcinoma cell lines. Nitrogen-containing heterocyclic ester derivatives 9a-f showed similar in vitro cytotoxicity to 1m, whereas the other derivatives were slightly less cytotoxic than 1m. 2'-Pyridinecarboxylic acid ester derivative (9c) exhibited a threefold increase in cytotoxicity relative to 1m.

Pladienolide is a naturally occurring antitumor macrolide that was discovered by using a cell-based reporter gene expression assay controlled by the human vascular endothelial growth factor promoter. Despite the unique mechanisms of action and prominent antitumor activities of pladienolides B and D in diverse in vitro and in vivo systems, their target protein has remained unclear. We used 3H-labeled, fluorescence-tagged and photoaffinity/biotin (PB)-tagged 'chemical probes' to identify a 140-kDa protein in splicing factor SF3b as the binding target of pladienolide. Immunoblotting of an enhanced green fluorescent protein fusion protein of SF3b subunit 3 (SAP130) revealed direct interaction between the PB probe and SAP130. The binding affinities of pladienolide derivatives to the SF3b complex were highly correlated with their inhibitory activities against reporter gene expression and cell proliferation. Furthermore, pladienolide B impaired in vivo splicing in a dose-dependent manner. Our results demonstrate that the SF3b complex is a pharmacologically relevant protein target of pladienolide and suggest that this splicing factor is a potential antitumor drug target.

Several sulfonamides have antitumor activities and are currently undergoing clinical evaluation for the treatment of cancer. In this study, we have elucidated the antiproliferative mechanism of action of five indole sulfonamides. The indole sulfonamides inhibited the polymerization of microtubule protein into microtubules in vitro. In addition, three representative derivatives, ER-68378 (2), ER-68384 (4) and ER-68394 (5), suppressed the dynamic instability behavior at the plus ends of individual steady-state microtubules in vitro. The analogues inhibited HeLa cell proliferation with half-maximal inhibitory concentrations in the range of 6-17 microM. The compounds blocked cell cycle progression at mitosis. At their lowest effective antimitotic concentrations, they depolymerized the spindle microtubules and disorganized the chromosomes but did not affect the microtubules in interphase cells. However, at relatively high concentrations, interphase microtubules were also depolymerized by these sulfonamides. Furthermore, all five compounds were found to induce apoptosis in the cells in association with the phosphorylation of bcl-2. The results suggest that the indole sulfonamides inhibit cell proliferation at mitosis by perturbing the assembly dynamics of spindle microtubules and that they can kill cancer cells by inducing apoptosis through the bcl-2-dependent pathway.

Sulfonamide-focused compound libraries have been synthesized in our laboratories for biological evaluation using antitumor phenotypic screens such as cancer cell proliferation assay, flow cytometric cell cycle analysis, and rat aorta tube formation assay. Among thousands of sulfonamide compounds evaluated, E7010 (a microtubule depolymerizing agent), E7070 (a G1 phase cell cycle inhibitor), and E7820 (an antiangiogenesis agent) have progressed to clinical trials, thereby demonstrating some objective responses in cancer patients so far. The sequential discovery of these drug candidates allowed us to carry out a research approach of forward chemical genetics, in which phenotypically bioactive compounds are selected from a large collection of small molecules and then utilized for understanding the functions of their protein partners and relevant biological pathways via target identification. This paper describes our attempt using oligonucleotide microarray and quantitative proteomic analyses not only for identifying drug targets and downstream pathways applicable to biomarkers but also for exploring druggable chemical space in medicinal chemistry research.

The sulfonamides constitute an important class of drugs, with several types of pharmacological agents possessing antibacterial, anti- carbonic anhydrase, diuretic, hypoglycemic and antithyroid activity among others. A large number of structurally novel sulfonamide derivatives have ultimately been reported to show substantial antitumor activity in vitro and in vivo. Although they have a common chemical motif of aromatic/heterocyclic or amino acid sulfonamide, there are a variety of mechanisms of their antitumor action, such as carbonic anhydrase inhibition, cell cycle perturbation in the G1 phase, disruption of microtubule assembly, functional suppression of the transcriptional activator NF-Y, and angiogenesis (matrix metalloproteinase, MMP) inhibition among others. Some of these compounds selected via elaborate preclinical screenings or obtained through computer-based drug design, are currently being evaluated in clinical trials. The review summarizes recent classes of sulfonamides and related sulfonyl derivatives disclosed as effective tumor cell growth inhibitors, or for the treatment of different types of cancer. Another research line that progressed much in the last time regards different sulfonamides with remarkable antiviral activity. Thus, at least two clinically used HIV protease inhibitors possess sulfonamide moieties in their molecules, whereas a very large number of other derivatives are constantly being synthesized and evaluated in order to obtain compounds with less toxicity or activity against drug-resistant viruses. Several non nucleoside HIV reverse transcriptase or HIV integrase inhibitors containing sulfonamido groups were also reported. Another approach to inhibit the growth of retroviruses, including HIV, targets the ejection of zinc ions from critical zinc finger viral proteins, which has as a consequence the inhibition of viral replication in the absence of mutations leading to drug resistance phenotypes. Most compounds with antiviral activity possessing this mechanism of action incorporate in their molecules primary sulfonamide groups. Some small molecule chemokine antagonists acting as HIV entry inhibitors also possess sulfonamide functionalities in their scaffold.

We herein report the synthesis and antitumor activity of E7070 analogues containing a 3-pyridinesulfonamide moiety. E7070 was selected from our sulfonamide-based compound collections, currently undergoing Phase II clinical trials because of its tolerable toxicity profile and some antitumor responses in the Phase I setting. Of the analogues examined, ER-35745, a 6-amino-3-pyridinesulfonamide derivative, demonstrated significant oral efficacy against the HCT116 human colon carcinoma xenograft in nude mice.

Recently, immunotherapeutic approaches have come to forefront in melanoma, non-small cell lung cancer, bladder cancer, and so on. However, sufficient benefits from these treatments have been limited. T cell infiltrating into tumor tissues is reported as one of the response biomarker candidates for the immune checkpoint inhibitors. Recent studies have shown that the activation of the Wnt/beta-catenin signaling pathway results in T cell exclusion and resistance to immune checkpoint inhibitor in melanoma patients. We established the Wnt1 tumor syngeneic mice model (Wnt1 model) by implantation of mammary adenocarcinomas isolated from MMTV-Wnt1 transgenic mice. Here we demonstrate whether E7386, a first-in-class orally active CBP/beta-catenin modulator, affects the recruitment of T cells into tumor tissues, leading to the enhancement of anti-PD-1 mAb in Wnt/beta-catenin signaling pathway activating tumor model. The mice were treated with E7386 (50 mg/kg, orally, BID) and/or anti-mouse PD-1 mAb (10 mg/kg, intraperitoneal, twice a week) for three weeks. Tumor diameters are measured with digital calipers, and the tumor volume in mm3 is calculated. Immunohistochemical (IHC) analysis was evaluated for tumor-infiltrating T cells. E7386 showed significant antitumor activity in Wnt1 model, but anti-PD-1 mAb did not. In contrast, E7386 with anti-PD-1 mAb indicated prominent antitumor activity compared to each single treatment. Enhancement of body weight loss in combination group was not observed. In IHC analysis, infiltration of T cells was limited in vehicle control group, in contrast T cell infiltration into tumors was clearly observed in both E7386 treatment group and combination group. As a result, antitumor activity of immune checkpoint inhibitor was limited in Wnt-driven tumor model and E7386 as a novel CBP/beta-catenin modulator enhanced the antitumor activity of anti-PD-1 mAb via induction of tumor-infiltrating T cells. Citation Format: Yusaku Hori, Kazuhiko Yamada, Yu Kato, Yoichi Ozawa, Takenao Odagami, Junji Matsui, Tomohiro Matsushima, Kenichi Nomoto, Hiroyuki Kouji, Takashi Owa. E7386, an orally active CBP/beta-catenin modulator, induces T cells infiltration into tumor and enhances antitumor activity of anti-PD-1 mAb in Wnt1 tumor syngeneic mice model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5172. doi:10.1158/1538-7445.AM2017-5172

E7386, a novel orally active CBP/beta-catenin modulator, has an impact on cancer cells with aberrant activation of Wnt/beta-catenin signaling pathway driven by adenomatous polyposis coli (APC) mutation or beta-catenin mutation and shows significant antitumor activity in xenograft models. CBP/beta-catenin transcriptional activation has an important role in not only malignancy of cancer cells but also regulation of tumor microenvironment such as fibroblast, pericyte, endothelial cells and immune cells. In this study, we investigated E7386 effect on tumor microenvironment and if it leads to enhance antitumor activity of lenvatinib. Lenvatinib is a potent anti-angiogenic inhibitor targeting vascular endothelial growth factor receptors, fibroblast growth factor receptors and other proangiogenic and oncogenic kinases. Firstly, we tested the combination of E7386 with lenvatinib in Wnt-1 tumor isogenic models, where tumors isolated from MMTV-Wnt1 transgenic mice were inoculated in mice. While E7386 and lenvatinib individually suppressed tumor growth and caused tumor dormancy, the combination resulted in approximately 75% tumor reduction. To clarify the effect on tumor microenvironment, we tested an isograft model of 4T1 murine breast cancer cells which was resistant to E7386 in vitro proliferation. E7386 and lenvatinib individual treatments showed a significant antitumor effect, but the antitumor effect of the combination was significantly superior to that of each mono-treatment. Therefore, we compared effects of E7386 and lenvatinib on tumor microenvironment in immunohistochemical analysis using CD31 Ab as an endothelial marker and alpha-SMA Ab as a pericyte and cancerous fibroblast marker. E7386 significantly decreased alpha-SMA positive cells and microvessel density in tumors. In addition, remaining tumor vessels were not covered with pericytes. In lenvatinib treated tumors, greater reduction of microvessel density was observed than in E7386 treated tumors, but tumor vessels covered with pericytes, which are known to be resistant against VEGF inhibitors, were remained. In combination treatment, most of the tumor vessels disappeared. The enhancement of antitumor activity in the combination was also observed in SEKI human melanoma xenograft model which is relatively resistant to lenvatinib. These data suggest E7386 sensitizes tumor to lenvatinib through the modulation of the tumor microenvironment that is resistant to VEGF inhibitors. Taken together, E7386 shows not only antitumor activity via the effect on cancer cells but also through the modulation of tumor microenvironment, and the treatment of E7386 with lenvatinib is a novel combination therapy to overcome resistance to VEGF inhibitors. Citation Format: Yoichi Ozawa, Yusaku Hori, Kazuhiko Yamada, Yasuhiro Funahashi, Junji Matsui, Tomohiro Matsushima, Takashi Owa. E7386, an orally active CBP/beta-catenin modulator, effects tumor microenvironment, resulting to the enhancement of antitumor activity of lenvatinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5176. doi:10.1158/1538-7445.AM2017-5176

As a result of substantial advances in recent cancer biology, cell cycle regulation in the G1 phase has attracted a great deal of attention as a promising target for the research and treatment of cancer. Many of the important genes associated with G1 regulation have been shown to play a key role in proliferation, differentiation and oncogenic transformation and programmed cell death (apoptosis). Currently, a variety of "cytostatic" agents that affects G1 progression and/or G1/S transition are being evaluated in clinical trials. Flavopiridol is a potent inhibitor of cyclin-dependent kinases (CDKs). UCN-01 was originally found to be a PKC-selective protein kinase antagonist. More recent studies have revealed that this agent can also inhibit several CDKs and the checkpoint kinase CHK1. FR901228, MS-27-275 and SAHA are histone deacetylase inhibitors that induce changes in the transcription of specific genes via the hyperacetylation of histones. The proteasome inhibitor PS-341 disrupts the degradation process of intracellular proteins, including cell cycle regulatory proteins such as cyclins. R115777, SCH66336 and BMS-214662 are non-peptidic farnesyl transferase inhibitors that prevent p21 ras oncogene activation. Rapamycin derivative CCI-779 downregulates signals through S6 kinase and FRAP (FKBP-rapamycin associating protein), affecting the expression levels of mRNAs important for progression from G1 to S phase. 17-Allylaminogeldanamycin targets the Hsp-90 (heat shock protein-90) family of cellular chaperones regulating the function of signaling proteins. TNP-470 (AGM-1470), a fumagillin derivative shows antiangiogenic action through binding to MetAP-2 (methionine aminopeptidase-2). The antitumor sulfonamide E7070, causing a cellular accumulation in the G1 phase, has been shown to suppress the activation of CDK2 and cyclin E expression in HCT116 colorectal cancer cell line highly sensitive to the drug. With respect to several growth factor receptors such as EGFR, PDGFR, bFGFR and VEGFR, potent and specific inhibitors of receptor tyrosine kinases have been also examined as hopeful drug candidates. In this report, we review the current status of extensive efforts directed towards the discovery and development of new chemotherapeutic anticancer agents targeting cell cycle regulation in the G1 phase, with particular focus on the compounds undergoing clinical investigations.

E7070 (N-(3-Chloro-7-indolyl)-1,4-benzenedisulphonamide) was selected from our sulphonamide compound collections via antitumour screening and flow cytometric analysis. Following treatment with E7070, the cell cycle progression of P388 murine leukaemia cells was disturbed in the G1 phase. The cell-killing effect on human colon cancer HCT116 cells was found to be time-dependent. In the panel of 42 human tumour cell lines, E7070 showed an antitumour spectrum that was distinct from those of other anticancer drugs used in clinic. Animal tests using human tumour xenograft models demonstrated that E7070 could cause not only tumour growth suppression, but also tumour regression in three of five colorectal and two of two lung cancers. In the HCT116 xenograft model, E7070 was shown to be superior to 5-FU, MMC and CPT-11 (irinotecan). Furthermore, complete regression of advanced LX-1 tumours was observed in 80% of E7070-treated mice. All of these observations have promoted this drug to clinical evaluation.

The sulphonamides constitute an important class of therapeutic agents in current medicinal science. After the discovery by Gerhard Domagk, of sulphamidochrysoidine (prontosil) as the first antibiotic sulpha-drug an active metabolite of the drug, sulphanilamide, was further derivatised in order to find compounds exhibiting superior antibacterial activity or different pharmacological effects. Diversification of the sulphanilamide structure led to the serial development of improved antibiotics, insulin-releasing hypoglycaemic drugs, carbonic anhydrase- (CA) inhibitory diuretics, anti-hypertensive drugs etc. It is of particular interest that various structurally novel sulphonamide derivatives have recently been reported to show substantial anti-tumour activity in vitro and/or in vivo. Although they have a common chemical motif of an aromatic/heterocyclic sulphonamide, there are a variety of mechanisms for their anti-tumour action, such as disruption of microtubule assembly, cell cycle arrest in the G1 phase, ...

A series of compounds containing an N-(7-indolyl)benzenesulfonamide pharmacophore was synthesized and evaluated as a potential antitumor agent. Cell cycle analysis with P388 murine leukemia cells revealed that there were two different classes of potent cell cycle inhibitors; one disrupted mitosis and the other caused G1 accumulation. Herein described is the SAR summary of the substituent patterns on this pharmacophore template.

Described herein is the discovery of a novel series of antitumor sulfonamides targeting G1 phase of the cell cycle. Cell cycle control in G1 phase has attracted considerable attention in recent cancer research, because many of the important proteins involved in G1 progression or G1/S transition have been found to play a crucial role in proliferation, differentiation, transformation, and programmed cell death (apoptosis). We previously reported our first antitumor sulfonamide E7010 as a novel tubulin polymerization inhibitor. Interestingly enough, continuous research on structurally related compounds led us to the finding of another class of antitumor sulfonamides that block cell cycle progression of P388 murine leukemia cells in G1 phase, but not in M phase. Of the compounds examined, N-(3-chloro-7-indolyl)-1,4-benzenedisulfonamide (E7070) showed significant antitumor activity against HCT116 human colon carcinoma both in vitro (IC(50) 0.11 microg/mL in cell proliferation assay) and in vivo (not only growth suppression but also a marked reduction of tumor size in nude mice). Because of its promising efficacy against human tumor xenografts and its unique mode of action, E7070 is currently undergoing phase I clinical trials in European countries.

The objective of this study is to report on long-term survival of a patient with metastatic melanoma treated with indisulam showing a distinct genetic pattern of repression of subsets of genes involved in mitochondrial energy metabolism. Gene expression profiling was performed with oligonucleotide microarray analysis. A 45-year-old patient with metastatic malignant melanoma was treated in third-line with indisulam (goal, E7070), a new chloroindolyl-sulphonamide cell-cycle inhibitor. The patient was treated weekly with a dose of 40 mg/m2 within a phase I study. On the basis of an amendment, the dose was escalated to 320 mg/m2 at maximum and de-escalated to 160 mg/m2 for long-term application in this individual patient. At the start of treatment the tumour burden consisted of two-in-transit-metastases, two further skin lesions, two cervical lymph nodes and four pulmonary metastases. Under a 2.5-year treatment with indisulam the tumour shrunk markedly although the objective response only reached stable disease. Lymph node biopsy revealed absence of vital melanoma cells. Therapy was stopped upon request of the patient. The gene expression profile indicated a profound transcriptional repression of subsets of genes involved in mitochondrial energy metabolism; namely NDUFB8, NDUFS1, NDUFV1, ACADVL and Homo sapiens clone 24408. The survival of this patient with metastatic melanoma lasted now 9 years, the progression-free interval 105 months. It can be assumed that this treatment effect is attributed to the down-regulating effect of indisulam on metabolic genes involved in energy production. Thus, knowledge on individual's tumour gene regulation may predict sensitivity and resistance to antitumoural agents.

Depudecin is a fungal metabolite that reverts the rounded phenotype of NIH 3T3 fibroblasts transformed with v- ras and v- src oncogenes to the flattened phenotype of the nontransformed parental cells. The mechanism of detransformation induced by this agent had not been determined. Here, we demonstrate that depudecin inhibits histone deacetylase (HDAC) activity effectively both in vivo and in vitro . Depudecin induces similar morphological reversion in v- ras transformed NIH 3T3 cells as do other naturally occurring HDAC inhibitors such as trichostatin A or trapoxin. It competitively inhibits the binding of [ 3 H]trapoxin in vitro and the nuclear binding of a trapoxin–coumarin fluorophore in vivo , suggesting that depudecin shares a nuclear binding protein and site on that protein with trapoxin. Furthermore, depudecin induces hyperacetylation of histones in a dose-dependent manner and at concentrations comparable with that required for detransformation. An in vitro histone deacetylase assay, using purified recombinant HDAC1, reveals that depudecin inhibits 50% of the enzyme activity at a concentration of 4.7 μM. These results demonstrate that depudecin is a novel HDAC inhibitor and suggest that its ability to induce morphological reversion of transformed cells is the result of its HDAC inhibitory activity.

Indisulam (N-(-3-chloro-7-indolyl)-1,4-benzenedisulfonamide; E7070) is an experimental anticancer agent. Microarray analysis indicates that indisulam downregulates several genes involved in drug resistance, and this finding led us to test the effect of combining indisulam with other anticancer drugs. We investigated the antitumor effect and mechanism of synergism when indisulam was administered in combination with CPT-11. In vitro cytotoxic activity was examined using a cell counter kit, and the combination effect was determined by isobologram analysis. The level of topoisomerase IIα was measured by Western blotting. The in vivo antitumor effect was assessed in mice inoculated with human colorectal cancer SW620 cells. Isobologram analysis indicated that a 24-h exposure to indisulam and SN-38, an active metabolite of CPT-11, had a synergistic effect in HCT116 and SW620 cells and an additive effect in HCT15 and WiDr cells. Prolongation of exposure to 48 h resulted in a synergistic effect in HCT15 and WiDr cells. Treatment with SN-38 alone increased the amount of intracellular topoisomerase IIα in all cell lines tested. Co-treatment with indisulam suppressed the SN-38-induced upregulation of topoisomerase IIα after 24 h of exposure in HCT116 and SW620 cells and after 48 h of exposure in HCT15 and WiDr cells. This apparent association between a synergistic effect and suppression of SN-38-mediated upregulation of topoisomerase IIα suggests that indisulam enhances SN-38 cytotoxicity by suppressing topoisomerase IIα upregulation to compensate for topoisomerase I inhibition by SN-38. Synergy was also observed in xenografted tumors and was accompanied by complete suppression of topoisomerase IIα upregulation induced by CPT-11 treatment. These observations prompted the clinical evaluation of indisulam and CPT-11 combination therapy.

Indoles are selectively acylated at the 3-position in high yields on treatment with a wide variety of acyl chlorides in CH2Cl2 in the presence of diethylaluminum chloride or dimethylaluminum chloride. The reaction proceeds under mild conditions and is applicable to indoles bearing various functional groups without NH protection.

We herein report the synthesis and antitumor activity of E7070 analogues containing a 3-pyridinesulfonamide moiety. E7070 was selected from our sulfonamide-based compound collections, currently undergoing Phase II clinical trials because of its tolerable toxicity profile and some antitumor responses in the Phase I setting. Of the analogues examined, ER-35745, a 6-amino-3-pyridinesulfonamide derivative, demonstrated significant oral efficacy against the HCT116 human colon carcinoma xenograft in nude mice.

Comparison of the DNA cleavage activity of man-designed bleomycins demonstrates that belomycins are small enzymes comprised of a catalytic site and a binding site. The linker moiety is shown to be significant for DNA binding, and inversion of its stereochemistry results in a dramatic decrease in the DNA-cleaving efficiency. One of the man-designed BLMs shows excellent cytotoxicity against L1210.

[reaction--see text] Indoles are selectively acylated at the 3-position in high yields on treatment with a wide variety of acyl chlorides in CH(2)Cl(2) in the presence of diethylaluminum chloride or dimethylaluminum chloride. The reaction proceeds under mild conditions and is applicable to indoles bearing various functional groups without NH protection.

Renieramycin M and jorunnamycin C, two isoquinolinequinone compounds differing only at the C-22 ester side chain, were evaluated for their cytotoxic effects on human colon (HCT116) and breast (MDA-MB-435) cancer cell lines. These two compounds displayed potent cancer cell growth inhibition, their IC(50) values reaching nanomolar order. To examine their effects on transcription, we carried out oligonucleotide microarray analysis with focus on the similarities and differences between the two compounds in terms of transcriptional profiles. We found that the down-regulation of PTPRK (protein tyrosine phosphatase receptor type K) can be considered as a biomarker responsive to the cytotoxic effects of this class of antitumor marine natural products.

Bleomycins (BLMs) are antitumor antibiotics of unusual glycopeptide structure. The potent activity of BLM is attributed to the oxygen activation and the DNA cleavage by the formation of iron-chelate of the peptide moiety. erythro-β-Hydroxy-L-histidine, a pivotal amino acid for the oxygen activation, is prepared enantioselectively by aldol reaction of (R) -3-bromoacetyl-4-isopropyl-1, 3-oxazolidin-2-one with 1-triphenylmethylimidazole-4-carbaldehyde. Model ligands for the metal binding site of BLM with 4-methoxypyridine (PYML-6) and 4-dimethylaminopyridine (PYML-8) show oxygen activation up to 97% and 125% of that of BLM, respectively. cis-β-Methylstyrene is oxidized either with Fe (III) -H2O2 or Fe (II) -O2 complex systems of BLM and PYML-6 to give the corresponding optically active epoxide. The DNA binding region of BLM is combined with PYML-6 to give the first man-designed BLM, PYML (6) -bleomycin, which shows nucleotide cleavage mode remarkably similar to that of BLM. On the other hand, PYML-6 moiety and distamycin are coupled to afford PYML (6) - (4R-APA) -distamycin which shows dramatically altered AT specific mode in the DNA scission.

In order to clarify the mechanism of the self-inactivation of bleomycin, a synthetic model for the metal binding site of bleomycin is treated with Fe(III)H2O2 or Fe(II)O2, and the degradation products are isolated and characterized. A breakdown at the amino acid side chain of the metal binding site has been demonstrated.