Yoshihiko Kotake

Biological validation that SF3b is a target of the antitumor macrolide pladienolide

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.

Selective degradation of splicing factor CAPER[alpha] by anticancer sulfonamides

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.

Apratoxin A Shows Novel Pancreas-Targeting Activity through the Binding of Sec 61.

Apratoxin A is a natural product with potent antiproliferative activity against many human cancer cell lines. However, we and other investigators observed that it has a narrow therapeutic window in vivo. Previous mechanistic studies have suggested its involvement in the secretory pathway as well as the process of chaperone-mediated autophagy. Still the link between the biologic activities of apratoxin A and its in vivo toxicity has remained largely unknown. A better understanding of this relationship is critically important for any further development of apratoxin A as an anticancer drug. Here, we describe a detailed pathologic analysis that revealed a specific pancreas-targeting activity of apratoxin A, such that severe pancreatic atrophy was observed in apratoxin A–treated animals. Follow-up tissue distribution studies further uncovered a unique drug distribution profile for apratoxin A, showing high drug exposure in pancreas and salivary gland. It has been shown previously that apratoxin A inhibits the protein secretory pathway by preventing cotranslational translocation. However, the molecule targeted by apratoxin A in this pathway has not been well defined. By using a 3H-labeled apratoxin A probe and specific Sec 61α/β antibodies, we identified that the Sec 61 complex is the molecular target of apratoxin A. We conclude that apratoxin A in vivo toxicity is likely caused by pancreas atrophy due to high apratoxin A exposure. Mol Cancer Ther; 15(6); 1208–16. ©2016 AACR.

Small molecule schweinfurthins selectively inhibit cancer cell proliferation and mTOR/AKT signaling by interfering with trans-Golgi-network trafficking

Natural compound schweinfurthins are of considerable interest for novel therapy development because of their selective anti-proliferative activity against human cancer cells. We previously reported the isolation of highly active schweinfurthins E-H, and in the present study, mechanisms of the potent and selective anti-proliferation were investigated. We found that schweinfurthins preferentially inhibited the proliferation of PTEN deficient cancer cells by indirect inhibition of AKT phosphorylation. Mechanistically, schweinfurthins and their analogs arrested trans-Golgi-network trafficking, an intracellular vesicular trafficking system, resulting in the induction of endoplasmic reticulum stress and the suppression of both lipid raft-mediated PI3K activation and mTOR/RheB complex formation, which collectively led to an effective inhibition of mTOR/AKT signaling. The trans-Golgi-network traffic arresting effect of schweinfurthins was associated with their in vitro binding activity to oxysterol-binding proteins that are known to regulate intracellular vesicular trafficking. Moreover, schweinfurthins were found to be highly toxic toward PTEN-deficient B cell lymphoma cells, and displayed 2 orders of magnitude lower activity toward normal human peripheral blood mononuclear cells and primary fibroblasts in vitro. These results revealed a previously unrecognized role of schweinfurthins in regulating trans-Golgi-network trafficking, and linked mechanistically this cellular effect with mTOR/AKT signaling and with cancer cell survival and growth. Our findings suggest the schweinfurthin class of compounds as a novel approach to modulate oncogenic mTOR/AKT signaling for cancer treatment.

A novel carbazole topoisomerase II poison, ER-37328: potent tumoricidal activity against human solid tumors in vitro and in vivo

We have discovered a novel topoisomerase II (topo II) poison, ER‐37328 (12,13‐dihydro‐5‐[2‐(dimethylamino)ethyl]‐4H‐benzo[c]py‐rimido[5,6,1‐jk]carbazole‐4,6,10(5H, 11H)‐trione hydrochloride), which shows potent tumor regression activity against Colon 38 cancer inoculated s.c. Here, we describe studies on the cell‐killing activity against a panel of human cancer cell lines and the antitumor activity of ER‐37328 against human tumor xenografts. In a cell‐killing assay involving 1‐h drug treatment, ER‐37328 showed more potent cell‐killing activity (50% lethal concentrations (LC50s) ranging from 2.9 to 20 μM) than etoposide (LC50s>60 μM) against a panel of human cancer cell lines. ER‐37328 induced double‐stranded DNA cleavage, an indicator of topo II‐DNA cleavable complex formation, within 1 h in MX‐1 cells, and the extent of cleavage showed a bell‐shaped relationship to drug concentration, with the maximum at 2.5 μM. After removal of the drug (2.5 μM) at 1 h, incubation was continued in drug‐free medium, and the amount of cleaved DNA decreased. However, at 10 μM, which is close to the LC50 against MX‐1 cells, DNA cleavage was not detected immediately after 1‐h treatment, but appeared and increased after drug removal. This result may explain the potent cell‐killing activity of ER37328 in the 1‐h treatment. In vivo, ER‐37328 showed potent tumor regression activity against MX‐1 and NS‐3 tumors. Moreover, ER‐37328 had a different antitumor spectrum from irinotecan or cisplatin against human tumor xenografts. In conclusion, ER‐37328 is a promising topo II poison with strong cell killing activity in vitro and tumor regression activity in vivo, and is a candidate for the clinical treatment of malignant solid tumors. (Cancer Sci 2003; 94: 119–124)

Microregional antitumor activity of a small-molecule hypoxia-inducible factor 1 inhibitor.

Hypoxia-inducible factor 1 (HIF-1) activates the transcription of genes that play crucial roles in the adaptation of cancer cells to hypoxia. HIF-1α overexpression has been associated with poor prognosis in patients with various types of cancer. Here, we describe ER-400583-00 as a novel HIF-1 inhibitor. ER-400583-00 suppressed the production of HIF-1α protein in response to hypoxia, with a half-maximal inhibitory concentration value of 3.7 nM in human U251 glioma cells. The oral administration of 100 mg/kg ER-400583-00 to mice bearing U251 tumor xenografts resulted in a rapid suppression of HIF-1α that persisted for 24 h. Immunohistochemical analysis revealed that ER-400583-00 suppressed the proliferation of cancer cells most prominently in areas distal to the region of blood perfusion, where HIF-1α-expressing hypoxic cancer cells were located. These hypoxic cancer cells were resistant to radiation therapy. ER-400583-00 showed a synergistic interaction with radiation therapy in terms of antitumor activity. These data suggest that HIF-1 blockade by small compounds may have therapeutic value in cancer, especially in combination with radiation therapy.

Synthesis and structure-activity relationships of novel, potent, orally active hypoxia-inducible factor-1 inhibitors.

Hypoxia-inducible factor-1 (HIF-1) is the chief transcription factor regulating hypoxia-driven gene expression. HIF-1 overexpression is associated with poor prognosis in several cancers and therefore represents an attractive target for novel antitumor agents. We explored small molecule inhibitors of the HIF-1 pathway. Using high-throughput-screening, we identified benzanilide compound 1 (IC50=560 nM) as a seed. Subsequent extensive derivatization led to the discovery of compounds 43a and 51d, with anti-HIF-1 activities in vitro (IC50=21 and 0.47 nM, respectively), and in vivo. Additionally, 43a (12.5-100mg/kg) also displayed in vivo anti-tumor efficacy, without influencing body weight.

Abstract 1377: Chemical probe-based approach clarifies binding of receptor tyrosine kinases (RTKs) to lenvatinib in preclinical models

Background: Lenvatinib mesilate (lenvatinib) is the selective inhibitor of VEGFR1-3, and 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 inhibits angiogenesis through the inhibition of VEGFR2 and FGFR1 and tumor proliferation through the inhibition of FGFR1 and RET in preclinical thyroid cancer models. Chemical probe-based binding protein isolation is a powerful tool to demonstrate the direct compound-protein interactions even in complicated biological materials such as intact cells and their robust protein extracts. The purpose of this study is to assess the interaction of lenvatinib with RTKs by using lenvatinib derivative probe as an analysis of mode of action in preclinical models. Methods: Lenvatinib chemical probe was designed based on a result of co-crystal structure of lenvatinib and VEGFR2. A linker moiety was introduced onto free space of quinoline core (position-7) and its inhibitory activities against RTKs were confirmed by cell-free enzyme assay system. Lenvatinib chemical probe was immobilized onto the monolithic affinity matrices and the binding proteins were isolated from the extracts of the human umbilical vein endothelial cells (HUVEC), the human differentiated thyroid cancer cell lines, RO82-W-1 and TPC-1. SDS-PAGE and western blotting (WB) with drug competition assay were performed to assess the binding RTKs of lenvatinib. Results: The chemical probe showed acceptable inhibitory activities against RTKs for affinity isolation. In the isolated protein sample from HUVEC, VEGFR2 and FGFR1 were detected by WB and each protein band was clearly reduced by lenvatinib competition in a dose-dependent manner. It indicates lenvatinib specifically interacts with VEGFR2 and FGFR1 even in such complicated protein mixture. In the sample from RO82-W-1, WB analysis showed that lenvatinib chemical probe efficiently isolated PDGFRα and FGFR1. Those protein bands were dramatically decreased by drug competition, similar to the experiment with HUVEC. The chemical probe-based approach was subjected to the protein extract from TPC-1, which expresses aberrant RET fusion kinase derived from CCDC6-RET gene rearrangement. WB analysis showed that CCDC6-RET was detected as binding protein of lenvatinib probe. Conclusions: The chemical probe-based approach proved the interaction of lenvatinib with VEGFR2, FGFR1, PDGFRα and RET fusion kinase in the complicated biological materials from HUVEC and thyroid cancer cell lines. These results supported that lenvatinib is likely to possess a unique anti-tumor activity, in addition to the anti-angiogenic activity, through the interaction with these RTKs. Citation Format: Takayuki Kimura, Noboru Yamamoto, Hiroshi Kamiyama, Megumi Ikemori Kawada, Akihiko Yamamoto, Yoshihiko Kotake, Yasutaka Takase, Yasuhiro Funahashi, Yoshiya Oda. Chemical probe-based approach clarifies binding of receptor tyrosine kinases (RTKs) to lenvatinib in preclinical models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1377. doi:10.1158/1538-7445.AM2015-1377