Takuji Sato

CBS9106 is a novel reversible oral CRM1 inhibitor with CRM1 degrading activity

CRM1 plays an important role in the nuclear export of cargo proteins bearing nuclear exporting signal sequences. Leptomycin B (LMB), a well-known CRM1 inhibitor, possesses strong antitumor properties. However, its toxicity prevents it from being clinically useful. In this study, we demonstrate that a novel compound, CBS9106, inhibits CRM1-dependent nuclear export, causing arrest of the cell cycle and inducing apoptosis in a time- and dose-dependent manner for a broad spectrum of cancer cells, including multiple myeloma cells. CBS9106 reduces CRM1 protein levels significantly without affecting CRM1 mRNA expression. This effect could be reversed by adding bortezomib or LMB. Moreover, CBS9106-biotin allows capture of CRM1 protein by streptavidin beads in a competitive manner with LMB and vice versa. Mass spectrometric analysis shows that CBS9106 reacts with a synthetic CRM1 peptide that contains Cys528 but not with a Cys528 mutant peptide. Oral administration of CBS9106 significantly suppresses tumor growth and prolongs survival in mice bearing tumor xenograft without a significant loss in body weight. A reduced level of CRM1 protein is also observed in tumor xenografts isolated from mice treated with CBS9106. Taken together, these results indicate that CBS9106 is a novel reversible CRM1 inhibitor and a promising clinical candidate.

Cell cycle phenotype-based optimization of G2-abrogating peptides yields CBP501 with a unique mechanism of action at the G2 checkpoint

Cell cycle G2 checkpoint abrogation is an attractive strategy for sensitizing cancer cells to DNA-damaging anticancer agent without increasing adverse effects on normal cells. However, there is no single proven molecular target for this therapeutic approach. High-throughput screening for molecules inhibiting CHK1, a kinase that is essential for the G2 checkpoint, has not yet yielded therapeutic G2 checkpoint inhibitors, and the tumor suppressor phenotypes of ATM and CHK2 suggest they may not be ideal targets. Here, we optimized two G2 checkpoint-abrogating peptides, TAT-S216 and TAT-S216A, based on their ability to reduce G2 phase accumulation of DNA-damaged cells without affecting M phase accumulation of cells treated with a microtubule-disrupting compound. This approach yielded a peptide CBP501, which has a unique, focused activity against molecules that phosphorylate Ser216 of CDC25C, including MAPKAP-K2, C-Tak1, and CHK1. CBP501 is >100-fold more potent than TAT-S216A and retains its selectivity for cancer cells. CBP501 is unusually stable, enters cells rapidly, and increases the cytotoxicity of DNA-damaging anticancer drugs against cancer cells without increasing adverse effects. These findings highlight the potency of CBP501 as a G2-abrogating drug candidate. This report also shows the usefulness of the cell cycle phenotype-based protocol for identifying G2 checkpoint-abrogating compounds as well as the potential of peptide-based compounds as focused multitarget inhibitors. [Mol Cancer Ther 2007;6(1):147–53]

CBS9106-Induced CRM1 Degradation Is Mediated by Cullin Ring Ligase Activity and the Neddylation Pathway

Chromosome region maintenance 1 (CRM1) mediates the nuclear export of proteins and mRNAs, and is overexpressed in various cancers. Recent studies have also reported that CRM1 protein expression is a negative prognostic factor in patients with cancer. Therefore, CRM1 is considered a potential target for anticancer therapy. Our previous study demonstrated that CBS9106, a synthetic small-molecular inhibitor of CRM1, decreases CRM1 protein through proteasomal degradation without affecting CRM1 mRNA levels. However, the mechanism by which CRM1 is degraded is not well understood. Here, we demonstrate a novel signaling pathway that plays an important role in CBS9106-induced CRM1 degradation. We found that MLN4924, a selective inhibitor of NEDD8-activating enzyme (NAE), effectively inhibits cullin neddylation and attenuates CBS9106-induced CRM1 degradation in a time- and dose-dependent manner. MLN4924 also attenuated CBS9106-induced nuclear accumulation of Ran-binding protein 1 (RanBP1), cell growth inhibition, and apoptosis. Furthermore, RNAi-mediated knockdown of neddylation pathway proteins (NEDD8 and UBA3) or cullin ring ligase (CRL) component protein (Rbx1) attenuated CRM1 protein degradation and G1 phase cell-cycle arrest by CBS9106. Knockdown of CSN5 or CAND1 also partially inhibited CBS9106-induced CRM1 degradation. These findings demonstrate that CBS9106-induced CRM1 degradation is conferred by CRL activity involving the neddylation pathway, and that this response to CBS9106 leads to cell growth inhibition and apoptosis. Mol Cancer Ther; 13(12); 3013–23. ©2014 AACR.

CBP501 suppresses macrophage induced cancer stem cell like features and metastases

CBP501 is an anti-cancer drug candidate which has been shown to increase cis-diamminedichloro-platinum (II) (CDDP) uptake into cancer cell through calmodulin (CaM) inhibition. However, the effects of CBP501 on the cells in the tumor microenvironment have not been addressed. Here, we investigated new aspects of the potential anti-tumor mechanism of action of CBP501 by examining its effects on the macrophages. Macrophages contribute to cancer-related inflammation and sequential production of cytokines such as IL-6 and TNF-α which cause various biological processes that promote tumor initiation, growth and metastasis (1). These processes include the epithelial to mesenchymal transition (EMT) and cancer stem cell (CSC) formation, which are well-known, key events for metastasis. The present work demonstrates that CBP501 suppresses lipopolysaccharide (LPS)-induced production of IL-6, IL-10 and TNF-α by macrophages. CBP501 also suppressed formation of the tumor spheroids by culturing with conditioned medium from the LPS-stimulated macrophage cell line RAW264.7. Moreover, CBP501 suppressed expression of ABCG2, a marker for CSCs, by inhibiting the interaction between cancer cells expressing VCAM-1 and macrophages expressing VLA-4. Consistently with these results, CBP501 in vivo suppressed metastases of a tumor cell line, 4T1, one which is insensitive to combination treatment of CBP501 and CDDP in vitro. Taken together, these results offer potential new, unanticipated advantages of CBP501 treatment in anti-tumor therapy through a mechanism that entails the suppression of interactions between macrophages and cancer cells with suppression of sequential CSC-like cell formation in the tumor microenvironment.CBP501 is an anti-cancer drug candidate which has been shown to increase cis-diamminedichloro-platinum (II) (CDDP) uptake into cancer cell through calmodulin (CaM) inhibition. However, the effects of CBP501 on the cells in the tumor microenvironment have not been addressed. Here, we investigated new aspects of the potential anti-tumor mechanism of action of CBP501 by examining its effects on the macrophages.Macrophages contribute to cancer-related inflammation and sequential production of cytokines such as IL-6 and TNF-α which cause various biological processes that promote tumor initiation, growth and metastasis (1). These processes include the epithelial to mesenchymal transition (EMT) and cancer stem cell (CSC) formation, which are well-known, key events for metastasis.The present work demonstrates that CBP501 suppresses lipopolysaccharide (LPS)-induced production of IL-6, IL-10 and TNF-α by macrophages. CBP501 also suppressed formation of the tumor spheroids by culturing with conditioned medium from the LPS-stimulated macrophage cell line RAW264.7. Moreover, CBP501 suppressed expression of ABCG2, a marker for CSCs, by inhibiting the interaction between cancer cells expressing VCAM-1 and macrophages expressing VLA-4. Consistently with these results, CBP501 in vivo suppressed metastases of a tumor cell line, 4T1, one which is insensitive to combination treatment of CBP501 and CDDP in vitro.Taken together, these results offer potential new, unanticipated advantages of CBP501 treatment in anti-tumor therapy through a mechanism that entails the suppression of interactions between macrophages and cancer cells with suppression of sequential CSC-like cell formation in the tumor microenvironment.

CBP501 induces immunogenic tumor cell death and CD8 T cell infiltration into tumors in combination with platinum, and increases the efficacy of immune checkpoint inhibitors against tumors in mice

CBP501, a calmodulin-binding peptide, is an anti-cancer drug candidate and functions as an enhancer of platinum uptake into cancer cells. Here we show that CBP501 promotes immunogenic cell death (ICD) in combination with platinum agents. CBP501 enhanced a clinically relevant low dose of cisplatin (CDDP) in vitro as evidenced by upregulation of ICD markers, including cell surface calreticulin exposure and release of high-mobility group protein box-1. Synergistic induction of ICD by CDDP plus CBP501 as compared to CDDP alone was confirmed in the well-established vaccination assay. Furthermore, cotreatment of CDDP plus CBP501 significantly reduced the tumor growth and upregulated the percentage of tumor infiltrating CD8+ T cell in vivo. Importantly, the antitumor effect of CDDP plus CBP501 was significantly reduced by anti-CD8 antibody treatment. Based on this novel effect of CBP501, we analyzed the combination treatment with immune checkpoint inhibitors in vivo. Mice treated with CBP501 in combination with CDDP and anti-PD-1 or anti-PD-L1 showed an additive antitumor effect. These results support the conclusion that CBP501 enhances CDDP-induced ICD in vitro and in vivo. The findings also support the further clinical development of the CBP501 for enhancing the antitumor activity of immune checkpoint inhibitors in combination with CDDP.

Abstract A37: CBP501 induces tumor immunogenic cell death and CD8 T cell infiltration into tumor in combination with platinum, thereby increasing the efficacy of immune checkpoint inhibitors against tumors in mice

Introduction: CBP501, a calmodulin-binding peptide, is an anti-cancer drug candidate that has completed two Phase II clinical trials for patients with malignant pleural mesothelioma and non-small cell lung carcinoma (NSCLC). CBP501 enhances the uptake of platinum agents by cancer cells specifically and induces immunogenic cell death within the clinically achievable dose levels of both CBP501 and cisplatin (CDDP) (EORTC 2015 abstract#C106). Here we treated tumor bearing mice with the combination of CBP501, platinum agents (CDDP and carboplatin), and immune checkpoint inhibitors and analyzed the tumor growth and behaviors of immune cells. Methods: Immuno-competent BALB/c mice were subcutaneously inoculated with CT26WT (5 x105 cells/mouse). A week later, mice were apportioned into 8 groups (6 mice/group) and treated with 2 dosing cycles for 3 anti-cancer agents, alone or in different combinations [CDDP: 4 mg/kg x 1/week, CBP501: 6.0 mg/kg x 1/week, anti-mPD1 mAb (RMP1-14) or anti-PD-L1 mAb (10F.9G2): 200 ug/mice x 2/week]. Results: In BALB/c mice bearing CT26WT tumor, CDDP- or immune checkpoint inhibitor-treated mice showed a minimal anti-tumor effect compared to the vehicle-treated mice. CDDP/immune checkpoint inhibitor-treated mice showed an additive anti-tumor effect. CBP501 significantly enhanced the anti-tumor effect of CDDP/immune checkpoint inhibitor combination. Flow cytometry analysis of single suspension cells from the tumor tissue showed that treatment of CDDP/CBP501 or CDDP/CBP501/anti-mPD1 mAb but not CDDP or anti-mPD1 mAb increased the percentage of tumor-infiltrating CD8+ T cell. Conclusion: An increase of tumor-infiltrating CD8+ T cells in CDDP/CBP501-treated mice supported the conclusion that CDDP/CBP501 treatment induced immunogenic cell death not only in vitro but also in vivo. It also supported the notion that CBP501 would not increase cytotoxicity of platinum agents to non-tumor cells. The fact that CBP501 enhanced anti-tumor activity of immune checkpoint inhibitors in combination with platinum agents (CDDP and carboplatin) would support further clinical development of CBP501. Citation Format: Keiichi Sakakibara, Takuji Sato, Donald W. Kufe, Daniel D. VonHoff, Takumi Kawabe. CBP501 induces tumor immunogenic cell death and CD8 T cell infiltration into tumor in combination with platinum, thereby increasing the efficacy of immune checkpoint inhibitors against tumors in mice. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A37.

Abstract A112: Observed separation of survival curves by WBC in CBP501-treated patients leads to experimental identification of macrophage functions suppressed by CBP501

The CBP501 calmodulin-binding peptide is an anti-cancer drug candidate that has completed a Phase II clinical trial for patients with non-small cell lung carcinoma (NSCLC). CBP501 was also previously identified as a unique G2 checkpoint-directed agent and as an enhancer of cisplatin (CDDP) uptake. In a post-trial analysis of the NSCLC patients, it was found that the survival was statistically significantly different between subpopulations with normal white blood cell counts (WBC) (WBC 8000). Notably, treatment by CDDP with or without CBP501 had no effect on the neutrophil oxidative burst or phagocytosis Here, we show that CBP501 inhibits several functions of macrophages. The LPS-stimulated production of several cytokines (IL-6. TNF-alpha and IL-10) by macrophages were suppressed by CBP501 treatment in vitro. The population of potential cancer stem-like cells was reduced by CBP501 in co-cultures of macrophages and tumor cell lines. In human macrophages isolated from peripheral blood-derived monocytes or THP-1 monocytic cells, phagocytic activity induced by IgG-conjugated beads was suppressed by in vitro CBP501 treatment. The neutrophil extracellular traps (NETs), which are supposed to be cleared by macrophages, were increased by CBP501 in response to leukocyte stimulation. Thrombotic tendency was increased in LPS- and CBP501-treated mice. These results suggest that the inhibition of macrophages by CBP501 could explain the observed dramatic separation of survival curves by WBC in patients with NSCLC. Many reports (Roy Noy and Jeffrey W. Pollard Immunity. 2014) indicate that tumor associated macrophages (TAMs) promote metastasis. To see if CBP501 might affect this relationship between macrophage activity and metastasis, we performed in vivo mouse study of mice bearing subcutaneous Lewis lung carcinomas. Results showed reduced metastasis to the lung upon CBP501 administration. Further investigation is underway to clarify the role of CBP501 in modulating the earlier characterized TAM-promoted metastasis. Citation Format: NAOKI MINE, Sayaka Yamamoto, Naoya Saito, Takuji Sato, Keiichi Sakakibara, Donald W. Kufe, Daniel D. Von Hoff, Takumi Kawabe. Observed separation of survival curves by WBC in CBP501-treated patients leads to experimental identification of macrophage functions suppressed by CBP501. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A112.

Abstract C106: CBP501 potentiates the appearance of cisplatin-induced indicators of immunogenic cell death and promotes anti-tumor effects in an immuno-competent mouse model

Introduction: The CBP501 calmodulin-binding peptide is an anti-cancer drug candidate that has completed two Phase II clinical trials for patients with malignant pleural mesothelioma and non-small cell lung carcinoma (NSCLC). CBP501 was previously described as a unique G2 checkpoint-directed agent and as an enhancer of cisplatin (CDDP) uptake. In a post-trial analysis of patients with NSCLC, it was found that overall survival (OS) was prolonged in a subpopulation of patients with normal white blood cell counts (WBC) (WBC Methods: CT26WT, a strain of CBP501 sensitive cells, were treated with clinically achievable dose levels of CDDP (10 or 20 uM) and 0.5 uM CBP501 for 0.75 h in vitro. After exchange with fresh drug-free medium, cells were incubated for 24, 48, or 72 h for IB analysis of phospho-eIF2-alpha, for calreticulin analysis by FACS, or for HMGB1 ELISA. The in vivo mouse study examined the effects of 3 dosing cycles for 3 anti-cancer agents, alone or in different combinations [CDDP: 5 mg/kg x 1/week, CBP501: 7.5 mg/kg x 3/week, anti-mPD1 antibody (RMP1-14): 200 ug x 1/week] using immuno-competent BALB/c mice bearing subcutaneous inoculated CT26 WT cells. Results: In vitro, CDDP in combination with CBP501 elicited increased death in CT26WT cells, as well as an increase in different indicators of immunogenic cell death. These indicators include induction of phospho-eIF2-alpha, increase in cell surface-exposed calreticulin, and extracellular release of HMGB1. In an in vivo BALB/c mouse model, CDDP-treated mice showed a reduction of tumor growth by 52.7% as compared to vehicle-treated mice. CBP501 + CDDP showed an additional reduction of tumor growth by 63.1% as compared to vehicle-treated mice. Treatment with anti-mPD-1 antibody alone showed a slight reduction of tumor growth by 25.2% as compared to vehicle-treated mice. However, combined treatment with anti-mPD-1 + CDDP or anti-mPD-1 + CDDP + CBP501 showed significant reductions in the tumor growth in comparison to the vehicle-treated mice by 69.3% and 78.7%, respectively. Conclusion: These results suggest that the anti-tumor activity of combined CBP501 + CDDP treatment is potentiated by inducing immunogenic cell death. This novel effect might contribute to the prolonged OS found in the phase II clinical trials. Combined treatments that include the anti-mPD-1 immune-checkpoint inhibitor were effective and shall be examined further. Further, immunohistochemical analyses of the effects of this combined treatment on tumor microenvironment are under way. Citation Format: Keiichi Sakakibara, Takuji Sato, Donald W. Kufe, Daniel D. Von Hoff, Takumi Kawabe. CBP501 potentiates the appearance of cisplatin-induced indicators of immunogenic cell death and promotes anti-tumor effects in an immuno-competent mouse model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C106.

Abstract 658: CBS9106 is a novel low toxic reversible oral CRM1 inhibitor with CRM1 degrading activity

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL CRM1 plays an important role in the nuclear export of cargo proteins bearing Nuclear Exporting Signal sequences. Leptomycin B (LMB), a well-known irreversible inhibitor of CRM1, possesses strong anti-tumor properties, however, its toxicity prevents it from being clinically useful. In this study, we show that a novel compound, CBS9106, inhibits CRM1-dependent nuclear export and induces G1-arrest and apoptosis in time- and dose-dependent manner in a broad-spectrum of cancer cells including multiple myeloma cells. IC50s of growth inhibition by CBS9106 (48h) are in the range from 46.7 to 366 nM in multiple myeloma cell lines (MM.1R, MM.1S, ARH-77 and RPMI-8226). In contrast with LMB, CBS9106 acts reversibly and reduces CRM1 protein without affecting the mRNA expression level. CBS9106-dependent reduction of CRM1 is abolished by MG132, a proteasome inhibitor. Pretreatment of cells with LMB suppresses CBS9106-induced CRM1 degradation. Moreover, CBS9106-biotin captured CRM1 in a pull down analysis and the amount of CRM1 captured by CBS9106-biotin was reduced by LMB treatment and vice versa. These results suggest that CBS9106 acts on the domain of CRM1 that includes LMBs covalent binding site at Cys-528, leading to proteasome-dependent CRM1 degradation. Oral administration of CBS9106 significantly suppressed tumor growth and prolonged survival duration in xenograft model mice. It caused prolonged reduction of CRM1 protein in tumor xenografts without showing significant body weight loss. Taken together, the results indicate that CBS9106 is a novel CRM1 inhibitor and a promising clinical candidate. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 658. doi:10.1158/1538-7445.AM2011-658