Introduction: Wnt/β-catenin pathway regulates many biological processes such as cell proliferation, stem cell renewal and tissue differentiation. Activation of Wnt/β-catenin pathway has been reported in many cancers. Tankyrase, one of the positive regulators of Wnt/β-catenin pathway, induces degradation of Axin, a negative regulator of Wnt/β-catenin pathway. Therefore, tankyrase inhibition stabilizes Axin protein and inhibits Wnt/β-catenin pathway, leading to exert antitumor effect. Recently, the role of Wnt/β-catenin pathway in immunotherapy has also gained attention; aberrant activation of Wnt/β-catenin pathway causes inactivation of dendritic cells and suppresses chemokine production resulting in paucity of CD8 T+ cells in tumor tissue. Although immune checkpoint inhibitors (ICIs), such as anti-CTLA-4 and anti-PD-1/PD-L1 antibodies have been successfully approved in some cancer therapies, some patients are still resistant to ICIs. One of the main causes is considered to be immune-desert tumor microenvironment, characterized by the absence of infiltrating lymphocytes including CD8+ T cells, cytotoxic effector cells for immunotherapy. Thus, Wnt/β-catenin pathway inhibition may overcome the resistance of anti-PD-1/PD-L1 therapy by attracting CD8+ T cells into tumor. K-476 is a highly potent and selective tankyrase inhibitor synthesized in house. In this study, we investigated whether K-476 enhanced the antitumor effect of anti-PD-L1 antibody. Furthermore, since tankyrase inhibitors are reported to have a potential gastrointestinal toxicity, we also evaluated the toxicity in mice administrated with K-476. Methods and results: Antitumor effect of K-476 combined with anti-PD-L1 antibody was evaluated in B16-derived melanoma bearing mice. Although K-476 monotherapy did not show antitumor effect, a combination with anti-PD-L1 antibody demonstrated a significant antitumor effect at doses from 50 to 200 mg/kg compared to anti-PD-L1 antibody alone, and the magnitude of the effect was comparable among the doses tested. Axin stabilization was observed in tumors after administration of K-476, suggesting that the effect was exerted through inhibition of Wnt/β-catenin pathway. The expression of chemokines, Ccl3 and Ccl4 which attract CD8+ T cells, was also upregulated. In addition, significant increase of CD8+ T cells was observed in the tumor. Gastrointestinal toxicity was not observed up to 200 mg/kg of K-476 in C57BL/6J mice. These results suggested that K-476 enhanced antitumor effect of anti-PD-L1 antibody through upregulation of Ccl3 and Ccl4 production and induction of CD8+ T cells without obvious toxicity. Conclusion: K-476 could be an attractive therapeutic agent that enhances the efficacy of anti-PD-L1 antibody. Citation Format: Haruka Kinosada, Kana Kunieda, Ryoko Okada, Minami Suzuki-Imaizumi, Motoya Mie, Toshihiko Ishii, Ryuichiro Nakai. Dual pocket binding novel tankyrase inhibitor, K-476, enhances the efficacy of immune checkpoint inhibitor by attracting CD8+ T cells into tumor [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 2200.

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We searched for compounds that affect the cyclin D1/retinoblastoma protein pathway from the in-house natural product library using a recombinant adenovirus with the Cre/loxP-regulated cyclin D1 overexpression system, and we found that oligomycin inhibited cell growth more effectively in cyclin D1-overexpressing SW480 cells than in control SW480 cells. We also found that oligomycin reduced the expression levels of cyclin D1 protein and that this reduction is, at least in part, mediated by Thr-286 phosphorylation-dependent proteasomal degradation. The Journal of Antibiotics (2009) Keywords: cell growth inhibition; chemosensitivity; cyclin D1; pRB; E2F-1 INTRODUCTIONThe transition from G1 to S phase is regulated by cyclins, cyclindependent kinases (CDK) and their inhibitor proteins. The protooncogenic function of cyclin D1 has been attributed in part to its role in promoting cell cycle progression. Cyclin D1 is a key cell cycle regulator of the G1 to S phase progression. The binding of cyclin D1 to cyclin-dependent kinase (cdk4 or cdk6) leads to the phosphorylation of retinoblastoma protein (pRB) subsequently triggering the release of E2F transcription factors to allow the transcription of genes required for the G1 to S phase progression of the cell cycle. In several types of human cancer, there is abundant evidence that disturbances of specific cyclins, CDKs or CDK inhibitory proteins enhance tumor cell growth. [1][2][3] In particular, disruption of the regulation of the cyclin D1/pRB/E2F pathway, overexpression of cylin D1, loss of pRB function and amplification of E2F-1 are often observed in several types of cancer. 2,4,5 Although recent reports have shown that some antitumor drugs may affect the cyclin D1/pRB/E2F pathway, [6][7][8] it is unclear which types of antitumor drugs induced the disruption of this pathway. In this study, we searched for compounds that inhibit cell growth selectively in cyclin D1-overexpressing human colorectal carcinoma cell lines (SW480 and LoVo) using a recombinant adenovirus with the Cre/loxP-regulated expression system.

Synthesis and Antitumor Activity of Combretastatin D-4. -Combretastatin D-4 (IV) is synthesized using the electrochemical dimerization of phenol (I) as the key step. Synthetic combretastatin D-4 shows potent inhibitory activity against cellular proliferation of human HT-29 colon carcinoma cells although the natural product has been reported to exhibit no remarkable biological activity. -(UNO, K.; TANABE, T.; OGAMINO, T.; OKADA, R.; IMOTO, M.; NISHIYAMA*, S.; Heterocycles 75 (2008) 2, 291-296; Dep. Chem., Fac. Sci. Technol., Keio Univ., Kohoku, Yokohama 223, Japan; Eng.) -C. Cyrus

Background: Wnt/β-catenin pathway controls many biological processes including cell proliferation and tissue development. In Wnt pathway, β-catenin is a key downstream factor which interacts with transcription factor T-cell factor (TCF) in the nucleus and induces expression of TCF responsive genes. β-catenin degradation is promoted by a cytoplasmic complex of APC, Axin and GSK3β. In many cancers, constitutive activation of Wnt/β-catenin pathway is observed due to mutations of the related genes and overexpression of Wnt ligands and receptors. For example, truncating mutation of the tumor suppressor APC are the most prevalent in colon cancer. APC deficiency leads to accumulation of nuclear β-catenin and promotes the transcription of downstream target genes. Hence, Wnt/β-catenin pathway inhibitor can be an attractive therapeutic agent for cancer patients. Results: To screen for Wnt/β-catenin pathway inhibitor, a reporter-based screen using a colon cancer cell line harboring APC mutation was performed. We identified a Wnt/β-catenin pathway inhibitor K-756 with an IC50 of 110 nmol/L, which was a distinct chemical structure from previously reported Wnt/β-catenin pathway inhibitors. From target identification study, it became clear that K-756 was a selective tankyrase inhibitor (AACR-NCI-EORTC 2013). Further development of the derivatives of K-756 using a structure-based drug design approach led to synthesis of K-476. K-476 inhibited Wnt/β-catenin pathway in APC mutant DLD-1 cell line with an IC50 of 0.3 nmol/L. K-476 stabilized Axin proteins and decreased active β-catenin. Wnt downstream target gene inhibition was also observed by K-476. PARP family profile assay was performed to evaluate the selectivity of K-476. K-476 inhibited tankyrase 1 and 2 and did not inhibit other PARP family isoforms even at 1000 nmol/L. Kinase profile assay was also performed towards 448 kinase but K-476 did not inhibit any of the kinases at 1000 nmol/L. Therefore K-476 is a highly selective tankyrase inhibitor. K-476 inhibited Wnt pathway signal and cell growth in colon cancer cell lines with an GI50 of less than 5 nmol/L. Oral dosing of K-476 resulted in inhibition of the reporter activity and Wnt downstream genes in a colon cancer xenograft mouse model. Taken together, K-476 represents a highly potent tankyrase inhibitor and is anticipated to be an important compound for further development of antitumor agent targeting the Wnt/β-catenin pathway regulation. Citation Format: Ryoko Okada, Yuichi Takahashi, Keiichi Motosawa, Yasuo Watanabe, Asae Igarashi, Ran Okada, Masahiro Ikkaku, Hikaru Miyagi, Yusuke Miura, Kazuki Asanome, Noriaki Uesaka, Jun-ichi Saito, Hiroshi Ishida, Ryuichiro Nakai. Discovery and characterization of a highly potent Wnt/ β -catenin pathway inhibitor targeting tankyrase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2715. doi:10.1158/1538-7445.AM2014-2715