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Innate and adaptive resistance to cancer therapies, such as chemotherapies, molecularly targeted therapies, and immune-modulating therapies, is a major issue in clinical practice. Subpopulations of tumor cells expressing the receptor tyrosine kinase AXL become enriched after treatment with antimitotic drugs, causing tumor relapse. Elevated AXL expression is closely associated with drug resistance in clinical samples, suggesting that AXL plays a pivotal role in drug resistance. Although several molecules with AXL inhibitory activity have been developed, none have sufficient activity and selectivity to be clinically effective when administered in combination with a cancer therapy. Here, we report a novel small molecule, ER-851, which is a potent and highly selective AXL inhibitor. To investigate resistance mechanisms and identify driving molecules, we conducted a comprehensive gene expression analysis of chemoresistant tumor cells in mouse xenograft models of genetically engineered human lung cancer and human triple-negative breast cancer. Consistent with the effect of AXL knockdown, cotreatment of ER-851 and antimitotic drugs produced an antitumor effect and prolonged relapse-free survival in the mouse xenograft model of human triple-negative breast cancer. Importantly, when orally administered to BALB/c mice, this compound did not induce retinal toxicity, a known side effect of chronic MER inhibition. Together, these data strongly suggest that AXL is a therapeutic target for overcoming drug resistance and that ER-851 is a promising candidate therapeutic agent for use against AXL-expressing antimitotic-resistant tumors.

Supplementary Data Figure S1-S7

Supplementary Table S1

Supplementary Figures 1-3 from The Novel Metastasis Promoter Merm1/Wbscr22 Enhances Tumor Cell Survival in the Vasculature by Suppressing Zac1/p53-Dependent Apoptosis

Understanding metastasis is integral to curative cancer treatments. Using a mouse genetic screening model, we identified Merm1/Wbscr22 as a novel metastasis promoter that includes a methyltransferase fold in its structure. Merm1 showed high levels of expression in invasive breast cancer. Ectopic expression of Merm1 in nonmetastatic cells enhanced metastasis formation without affecting cell growth and motility. The intact methyltransferase fold of Merm1 was required for metastasis formation. Interestingly, Merm1 expression promoted cell survival after entrapment in the lung microvasculature. Consistent with these results, knockdown of endogenous Merm1 in tumor cells reduced lung retention and metastasis formation. On the basis of comparative transcriptome analysis, Merm1 expression was negatively correlated with the expression of tumor suppressor Zac1. We confirmed that Merm1 suppressed Zac1 expression with histone H3 methylation at Lys9 in the Zac1 promoter region. Zac1 can induce apoptosis through its ability to transcriptionally coactivate p53, which regulates apoptosis in the vasculature and is often downregulated in metastasis. We found that Zac1 knockdown reduced the p53-dependent apoptosis that was enhanced by Merm1 knockdown, thereby increasing lung retention of metastatic cells. Our findings show that Merm1 enhances cancer cell survival in the vasculature by suppressing Zac1/p53-dependent apoptosis, thereby enhancing metastasis. Cancer Res; 71(3); 1146–55. ©2010 AACR.

Supplementary Figure Legends 1-3 from The Novel Metastasis Promoter Merm1/Wbscr22 Enhances Tumor Cell Survival in the Vasculature by Suppressing Zac1/p53-Dependent Apoptosis

Supplementary Methods from The Novel Metastasis Promoter Merm1/Wbscr22 Enhances Tumor Cell Survival in the Vasculature by Suppressing Zac1/p53-Dependent Apoptosis

Supplementary Tables 1-2 from The Novel Metastasis Promoter Merm1/Wbscr22 Enhances Tumor Cell Survival in the Vasculature by Suppressing Zac1/p53-Dependent Apoptosis

Innate and adaptive resistance to cancer therapies, such as chemotherapies, molecularly targeted therapies, and immune-modulating therapies, is a major issue in clinical practice. Subpopulations of tumor cells expressing the receptor tyrosine kinase AXL become enriched after treatment with anti-mitotic drugs, causing tumor relapse. Elevated AXL expression is closely associated with drug resistance in clinical samples, suggesting that AXL plays a pivotal role in drug resistance. Although several molecules with AXL inhibitory activity have been developed, none have sufficient activity and selectivity to be clinically effective when administered in combination with a cancer therapy. Here, we report a novel small molecule, ER-001259851-000, which is a potent and highly selective AXL inhibitor. To investigate resistance mechanisms and identify driving molecules, we conducted a comprehensive gene expression analysis of chemo-resistant tumor cells in mouse xenograft models of genetically engineered human lung cancer and human triple-negative breast cancer. Consistent with the effect of AXL knockdown, co-treatment of ER-001259851-000 and antimitotic drugs produced an anti-tumor effect and prolonged relapse-free survival in the mouse xenograft model of human triple-negative breast cancer. Importantly, when orally administered to BALB/c mice, this compound did not induce retinal toxicity, a known side effect of chronic MER inhibition. Together, these data strongly suggest that AXL is a therapeutic target for overcoming drug resistance and that ER-001259851-000 is a promising candidate therapeutic agent for use against AXL-expressing anti-mitotic-resistant tumors.

Recent studies suggest a functional involvement of Epithelial-Mesenchymal Transition (EMT) in tumor chemoresistance. Specifically, EMT is associated with chemoresistance and poor prognosis in triple-negative breast cancer. However, no effective therapy targeting EMT has been developed. Here, we report that periostin, an extracellular matrix protein, was induced upon chemotherapy and tightly correlated with the EMT gene signature and poor prognosis in breast cancer. In triple-negative breast cancer xenografts, chemotherapy upregulated periostin expression in tumor cells, triggered expansion of mesenchymal tumor cells and promoted invasion in residual tumors. Knockdown of periostin inhibited outgrowth and invasion of mesenchymal tumor cells upon chemotherapy. Furthermore, chemotherapy upregulated cancer-specific variants of periostin and application of a blocking antibody specifically targeting those variants overcame chemoresistance and halted disease progression without toxicity. Together, these data indicate that periostin plays a key role in EMT-dependent chemoresistance and is a promising target to overcome chemoresistance in triple-negative breast cancer.

Notch4 is selectively expressed on tumor endothelial cells and hypothesized to contribute to cancer malignancy through mechanisms including tumor angiogenesis regulation and promoting drug resistance. We developed E7011, a humanized IgG2κ antibody targeting Notch4 receptor signaling. To obtain anti-human Notch4 monoclonal antibody, human Notch4 3EGFR (epidermal growth factor repeat) domain - NRR (negative regulatory region) - SEAP (secreted alkaline phosphatase) - His protein was immunized to mice and 6698 hybridomas were obtained. By screening these clones with the Notch4 binding assay and Notch4-specific gal4-fusion luciferase reporter assay, mouse anti-human Notch4 mAb clone (6-3-A6) was selected. The CDRs (complementarity determining regions) of 6-3-A6 were grafted into the variable regions of human IgG2κ, and E7011, novel humanized anti-human Notch4 monoclonal antibody, was developed by screening in assays above. The biomolecular interaction analysis showed that E7011 is able to bind to human and mouse Notch4-NRR domain-SEAP-His, and inhibit human Notch4 receptor signaling in an antibody-dose dependent manner as well as 6-3-A6. IHC analysis showed that Notch4 was expressed on CD31 positive endothelial cells in Calu6 human non-small cell lung carcinoma xenograft tumor. Intravenous administration of E7011 at 1, 3, 10 mg/kg in twice a week significantly decreased tumor growth compared with control IgG treatment without severe body weight loss. We also investigated the functional tumor angiogenesis by hoechest dye-perfusion and staining with anti-CD31 antibody in the Calu6 xenograft model. E7011 treatment significantly decreased hoechst-positive perfusion area compared with vehicle treatment. In conclusion, E7011 is a novel antitumor agent that is a specific signal inhibitor targeting the Notch4 receptor and suppresses tumor growth through decreasing tumor vessel function in based on preclinical studies. Citation Format: Yusuke Adachi, Yoshimasa Sakamoto, Youya Nakazawa, Sho Tachino, Ken Ito, Takanori Abe, Yukinori Minoshima, Kana Hoshino-Negishi, Hideaki Ogasawara, Tomomi Kawakatsu, Miyuki Nishimura, Masashi Shimizu, Kazuhiro Tahara, Toshitaka Sato, Katsuhisa Suzuki, Kishan Agarwala, Masao Iwata, Yasuhiro Funahashi, Kenichi Nomoto, Yoichi Ozawa, Junji Matsui, Toshio Imai, Yu Kato. E7011, a novel anti-angiogenesis inhibitor targeting Notch4 receptor signal pathway [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 1485.

Almost all cancers show intrinsic and/or evasive resistance to vascular endothelial growth factor (VEGF) inhibitors by multiple mechanisms. Serum angiopoietin-2 (Ang2) level has been proposed as a potential biomarker of VEGF inhibitor response in several cancers. From these clinical observations, the Ang2 and Tie2 (its receptor) axis has been focused on as a promising target. Here, we show a novel strategy to circumvent the resistance by combining multi-tyrosine kinase inhibitors lenvatinib (VEGF receptor, fibroblast growth factor receptor, and RET inhibitor) and golvatinib (E7050; c-Met, Tie2, and EphB4 inhibitor). Tie2 identifies a highly pro-angiogenic macrophage subset, Tie2-expressing macrophages (TEM). Angi-Tie2 and EphB4-EphrinB2 signaling plays critical roles in pericyte-mediated vessel stabilization. In vitro analyses suggested that golvatinib combined with lenvatinib inhibited pericyte-mediated vessel stabilization and TEM differentiation. In thyroid and endometrial cancer models, golvatinib and lenvatinib inhibited pericyte network development and TEM infiltration, resulting in severe perfusion disorder and massive apoptosis. Body weight loss was tolerable, and no macroscopic change was observed. These preclinical studies suggest that modulation of the tumor microenvironment by a strategic and well-tolerated combination of multi-targeting tyrosine kinase inhibitors may sensitize cancer to VEGF inhibitors.

Vascular endothelial growth factor receptor (VEGFR) inhibitors are approved for the treatment of several tumor types; however, some tumors show intrinsic resistance to VEGFR inhibitors, and some patients develop acquired resistance to these inhibitors. Therefore, a strategy to overcome VEGFR inhibitor resistance is urgently required. Recent reports suggest that activation of the hepatocyte growth factor (HGF) pathway through its cognate receptor, Met, contributes to VEGFR inhibitor resistance. Here, we explored the effect of the HGF/Met signaling pathway and its inhibitors on resistance to lenvatinib, a VEGFR inhibitor. In in vitro experiments, addition of VEGF plus HGF enhanced cell growth and tube formation of HUVECs when compared with stimulation by either factor alone. Lenvatinib potently inhibited the growth of HUVECs induced by VEGF alone, but cells induced by VEGF plus HGF showed lenvatinib resistance. This HGF-induced resistance was cancelled when the Met inhibitor, golvatinib, was added with lenvatinib. Conditioned medium from tumor cells producing high amounts of HGF also conferred resistance to inhibition by lenvatinib. In s.c. xenograft models based on various tumor cell lines with high HGF expression, treatment with lenvatinib alone showed weak antitumor effects, but treatment with lenvatinib plus golvatinib showed synergistic antitumor effects, accompanied by decreased tumor vessel density. These results suggest that HGF from tumor cells confers resistance to tumor endothelial cells against VEGFR inhibitors, and that combination therapy using VEGFR inhibitors with Met inhibitors may be effective for overcoming resistance to VEGFR inhibitors. Further evaluation in clinical trials is warranted.

The platelet aggregation-inducing factor, Aggrus (also known as podoplanin), is reported to contribute to cancer metastasis by mediating cancer cell-platelet interaction. Aggrus has been shown to be upregulated in many different types of cancers. Thus, not only the functional inhibition of Aggrus, but also its application as a cancer-specific antigen has therapeutic potential. Among a series of anti-Aggrus mAb established previously, no mouse anti-human Aggrus mAb exists that possesses the ability to neutralize platelet aggregation. For precise preclinical examinations of mouse and monkey models, the establishment of Aggrus-neutralizing mouse mAb and their chimeric Abs is needed. In this study, we established two mouse anti-human Aggrus mAb, P2-0 and HAG-3. A precise analysis of their epitopes revealed that P2-0 recognized the conformation near the bioactive O-glycosylation site at the Thr(52) residue. In contrast, HAG-3 recognized the amino-terminus side at a short distance from the conformation recognized by P2-0. We observed that only P2-0 attenuated Aggrus-induced platelet aggregation and Aggrus binding to its platelet receptor, that is, the C-type lectin-like receptor-2. Consistent with these data, only P2-0 prevented the experimental metastasis of human Aggrus-overexpressing CHO cells. Subsequently, we cloned the complementary determining region of P2-0 and produced the murine/human chimeric P2-0 antibody. This chimeric antibody maintained its inhibitory activity of Aggrus-induced platelet aggregation and experimental metastasis. Thus, P2-0 and its chimeric antibody are expected to aid the development of preclinical and clinical examinations of Aggrus-targeted cancer therapy.

Understanding metastasis is integral to curative cancer treatments. Using a mouse genetic screening model, we identified Merm1/Wbscr22 as a novel metastasis promoter that includes a methyltransferase fold in its structure. Merm1 showed high levels of expression in invasive breast cancer. Ectopic expression of Merm1 in nonmetastatic cells enhanced metastasis formation without affecting cell growth and motility. The intact methyltransferase fold of Merm1 was required for metastasis formation. Interestingly, Merm1 expression promoted cell survival after entrapment in the lung microvasculature. Consistent with these results, knockdown of endogenous Merm1 in tumor cells reduced lung retention and metastasis formation. On the basis of comparative transcriptome analysis, Merm1 expression was negatively correlated with the expression of tumor suppressor Zac1. We confirmed that Merm1 suppressed Zac1 expression with histone H3 methylation at Lys9 in the Zac1 promoter region. Zac1 can induce apoptosis through its ability to transcriptionally coactivate p53, which regulates apoptosis in the vasculature and is often downregulated in metastasis. We found that Zac1 knockdown reduced the p53-dependent apoptosis that was enhanced by Merm1 knockdown, thereby increasing lung retention of metastatic cells. Our findings show that Merm1 enhances cancer cell survival in the vasculature by suppressing Zac1/p53-dependent apoptosis, thereby enhancing metastasis. Cancer Res; 71(3); 1146–55. ©2010 AACR.

CD9 has been reported to play a role in tumor metastasis suppression. However, it is not fully understood how CD9 affects the hematogenous spread of tumor cells. To clarify a new mechanism (or mechanisms), we generated HT1080 cells that had been transfected with a CD9-expressing plasmid. Ectopic expression of CD9 in HT1080 cells actually reduced their metastatic ability. CD9 expression reduced lung retention and platelet ag-gregation activity of the transfectants. Because HT1080 cells express the metastasis-promoting, platelet aggregation-inducing factor Aggrus/podoplanin on their surface, we examined the relationship between CD9 and Aggrus. We discovered that CD9 formed a complex with Aggrus via transmembrane domains 1 and 2 (TM1 and TM2) of CD9. Investigation of the interaction revealed that each CD9 and Aggrus interacted homophilically, and that they colocalized in low-density membrane fractions. Deleting TM1 and TM2 attenuated the ability of CD9 to interact homophilically or to localize in low-density membrane fractions. The expression of CD9–wild-type (WT), but not CD9 lacking TM1 and TM2, attenuated the platelet aggregation and metastasis induced by forced expression of Aggrus in CHO cells. Therefore, CD9 may act as a metastasis suppressor, at least in part, by neutralizing Aggrus-mediated platelet aggregation.

Podoplanin, a mucin-like transmembrane sialoglycoprotein, promotes platelet aggregation and may be involved in cancer cell migration, invasion, metastasis, and malignant progression. Podoplanin/aggrus is highly expressed in testicular seminoma, suggesting that it may be a sensitive marker for testicular seminomas. Here we investigated the expression of podoplanin in central nervous system (CNS) germ cell tumors (GCTs) by immunohistochemical staining of tumor samples from 62 patients. In 40 of 41 (98%) germinomas (including germinomatous components in mixed GCTs), podoplanin was diffusely expressed on the surface of germinoma cells; lymphocytes, interstitial cells, and syncytiotrophoblastic giant cells were negative for podoplanin. Except for immature teratomas (12/17; 71%), podoplanin expression was absent in non-germinomatous GCTs, including seven teratomas, seven embryonal carcinomas, seven yolk sac tumors, and seven choriocarcinomas. In immature teratomas, focal podoplanin staining was observed in fewer than 10% of immature squamous and columnar epithelial cells. Thus, podoplanin expression may be a sensitive immunohistochemical marker for germinoma in CNS GCTs. As such, it may be useful for diagnosis, for monitoring the efficacy of treatment, and as a potential target for antibody-based therapy.

Although vascular endothelial growth factor (VEGF) inhibitors provide significant clinical benefit, they often require dose reductions or even withdrawals due to their severe toxicities. Furthermore, almost all cancers show intrinsic and/or evasive resistance to VEGF inhibitors by multiple mechanisms. Serum angiopoietin-2 (Ang2) level has been proposed as a potential biomarker of VEGF inhibitor response in several cancers. Response to lenvatinib (E7080; VEGFR1-3 inhibitor) is also reported to correlate with low Ang2 level in differentiated thyroid cancer and endometrial cancer. From these clinical observations, Ang2 and its receptor Tie2 has been focused as promising targets. Here, we demonstrated mechanisms of resistance induced by Ang2 and a novel strategy to circumvent the resistance by combination of multi-tyrosine kinase inhibitors (TKIs), lenvatinib and golvatinib (E7050; c-Met, Tie2, EphB4 inhibitor). Tie2 defines a highly pro-angiogenic macrophage subset, Tie2-expressing macrophage (TEM). Ang-Tie2 and EphB4-EphrinB2 signaling play critical roles in pericyte-mediated vessel stabilization. Ectopic expression of Ang2 in thyroid cancer conferred resistance to lenvatinib and enhanced pericyte-associated endothelial network development and TEM infiltration. In vitro analyses suggested that golvatinib/lenvatinib combination inhibited pericyte-mediated vessel stabilization and TEM differentiation. In thyroid and endometrial cancer models, golvatinib/lenvatinib combination inhibited pericyte network development and TEM infiltration, resulting in severe perfusion disorder and massive apoptosis. Body weight loss was tolerable in mice, and no macroscopic change was observed. These results suggest that modulation of tumor microenvironment by strategic and well-tolerated combination of multi-targeting TKIs sensitizes cancer to VEGF inhibitors, which warrants further clinical investigation to determine the clinical benefit of anti-angiogenesis cancer therapy. Citation Format: Youya Nakazawa, Satoshi Kawano, Junji Matsui, Yasuhiro Funahashi, Osamu Tohyama, Hiroki Muto, Takayuki Nakagawa, Tomohiro Matsushima. Maximizing the efficacy of anti-angiogenesis cancer therapy: A multi-targeting strategy by tyrosine kinase inhibitors. [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 2980. doi:10.1158/1538-7445.AM2014-2980