Francois Lehembre

Macitentan, a Dual Endothelin Receptor Antagonist, in Combination with Temozolomide Leads to Glioblastoma Regression and Long-term Survival in Mice

Purpose: The objective of the study was to determine whether astrocytes and brain endothelial cells protect glioma cells from temozolomide through an endothelin-dependent signaling mechanism and to examine the therapeutic efficacy of the dual endothelin receptor antagonist, macitentan, in orthotopic models of human glioblastoma. Experimental Design: We evaluated several endothelin receptor antagonists for their ability to inhibit astrocyte- and brain endothelial cell–induced protection of glioma cells from temozolomide in chemoprotection assays. We compared survival in nude mice bearing orthotopically implanted LN-229 glioblastomas or temozolomide-resistant (LN-229Res and D54Res) glioblastomas that were treated with macitentan, temozolomide, or both. Tumor burden was monitored weekly with bioluminescence imaging. The effect of therapy on cell division, apoptosis, tumor-associated vasculature, and pathways associated with cell survival was assessed by immunofluorescent microscopy. Results: Only dual endothelin receptor antagonism abolished astrocyte- and brain endothelial cell–mediated protection of glioma cells from temozolomide. In five independent survival studies, including temozolomide-resistant glioblastomas, 46 of 48 (96%) mice treated with macitentan plus temozolomide had no evidence of disease (P < 0.0001), whereas all mice in other groups died. In another analysis, macitentan plus temozolomide therapy was stopped in 16 mice after other groups had died. Only 3 of 16 mice eventually developed recurrent disease, 2 of which responded to additional cycles of macitentan plus temozolomide. Macitentan downregulated proteins associated with cell division and survival in glioma cells and associated endothelial cells, which enhanced their sensitivity to temozolomide. Conclusions: Macitentan plus temozolomide are well tolerated, produce durable responses, and warrant clinical evaluation in glioblastoma patients. Clin Cancer Res; 21(20); 4630–41. ©2015 AACR.

Treatment of experimental human breast cancer and lung cancer brain metastases in mice by macitentan, a dual antagonist of endothelin receptors, combined with paclitaxel

BACKGROUND We recently demonstrated that brain endothelial cells and astrocytes protect cancer cells from chemotherapy through an endothelin-dependent signaling mechanism. Here, we evaluated the efficacy of macitentan, a dual endothelin receptor (ETAR and ETBR) antagonist, in the treatment of experimental breast and lung cancer brain metastases. METHODS The effect of macitentan on astrocyte- and brain endothelial cell-mediated chemoprotective properties was measured in cytotoxic assays. We compared survival of mice bearing established MDA-MB-231 breast cancer or PC-14 non-small cell lung cancer (NSCLC) brain metastases that were treated with vehicle, macitentan, paclitaxel, or macitentan plus paclitaxel. Cell division, apoptosis, tumor vasculature, and expression of survival-related proteins were assessed by immunofluorescent microscopy. RESULTS Cancer cells and tumor-associated endothelial cells expressed activated forms of AKT and MAPK in vehicle- and paclitaxel-treated groups in both metastasis models, but these proteins were downregulated in metastases of mice that received macitentan. The survival-related proteins Bcl2L1, Gsta5, and Twist1 that localized to cancer cells and tumor-associated endothelial cells in vehicle- and paclitaxel-treated tumors were suppressed by macitentan. Macitentan or paclitaxel alone had no effect on survival. However, when macitentan was combined with paclitaxel, we noted a significant reduction in cancer cell division and marked apoptosis of both cancer cells and tumor-associated endothelial cells. Moreover, macitentan plus paclitaxel therapy significantly increased overall survival by producing complete responses in 35 of 35 mice harboring brain metastases. CONCLUSIONS Dual antagonism of ETAR and ETBR signaling sensitizes experimental brain metastases to paclitaxel and may represent a new therapeutic option for patients with brain metastases.

Antitumor Compound Testing in Glioblastoma Organotypic Brain Cultures

Glioblastoma multiforme (GBM) is the most common and most aggressive type of primary brain tumor. Identification of new therapeutic regimens is urgently needed. A major challenge remains the development of a relevant in vitro model system with the necessary capacity and flexibility to profile compounds. The authors have developed and characterized a 3D culture system of brain cells (brain Hi-Spot) where GBM-derived cells can be incorporated (GBM/brain Hi-Spot). Immuno-fluorescence and electrophysiological recordings demonstrate that brain Hi-Spots recapitulate many features of brain tissue. Within this tissue, GBM-derived cell growth is monitored using a fluorescence assay. GBM-derived cells growing in Hi-Spots form tumor nodules that display properties of GBM such as 5-Ala positive staining, an acidic environment, and tumor-surrounding astrocyte activation. Temozolomide inhibits GBM growth in brain Hi-Spots, but it is not effective in 2D cultures. Other chemotherapeutics that have proven to be inefficient in GBM treatment display low activity against GBM-derived cells growing in brain Hi-Spots in comparison to their activity against GBM 2D cultures. These findings suggest that GBM/brain Hi-Spots represent a simple system to culture cells derived from brain tumors in an orthotopic environment in vitro and that the system is reliable to test GBM targeting compounds.

Abstract 601: Macitentan (ACT-064992), a tissue-targeting endothelin receptor antagonist enhances therapeutic efficacy of paclitaxel by modulating survival pathways in orthotopic models of metastatic human ovarian cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: In 2009, ovarian cancer was the leading cause of death from gynecologic cancer in the United States. Efforts to develop tools for early detection and therapeutic regimens to overcome drug resistance of the ovarian cancer have not made a significant achievement to prolong the median survial of the patients. Potential treatments for ovarian cancers that have become resistant to standard chemotherapies include modulators of tumor cell survival, such as endothelin receptor (ETR) antagonist. We investigated the therapeutic efficacy of the dual ETR antagonist, macitentan, on human ovarian cancer cells, SKOV3ip1 and IGROV1, growing orthotopically in nude mice. Materials and Methods: Mice were injected with one million cells of SKOV3ip1 or IGROV1. Ten days later, mice with established disease were randomized into vehicle (control), paclitaxel (weekly, intraperitoneal injections), macitentan (daily oral administrations) or a combination of paclitaxel and macitentan treatment groups. After 4 weeks of treatment, mice were necropsied and tumor incidence, tumor weight, and incidence of ascites were recorded. Tumor tissues were processed for immunohistochemical analyses. Results: Treatment with paclitaxel decreased tumor weight and volume of ascites. Combination therapy with macitentan and paclitaxel reduced tumor incidence and further reduced tumor weight and volume of ascites when compared with paclitaxel alone. Macitentan alone occasionally reduced tumor weight but alone had no effect on tumor incidence or ascites. Immunohistochemical analyses revealed that treatment with macitentan and macitentan plus paclitaxel inhibited the phosphorylation of ETRs and suppressed the survival pathways of tumor cells by decreasing the levels of pVEGFR2, pAkt, and pMAPK. The dose of macitentan necessary for inhibition of phosphorylation correlated with the dose required to increase antitumor efficacy of paclitaxel. Conclusion: Treatment with macitentan enhanced the cytotoxicity mediated by paclitaxel as measured by the degree of apoptosis in tumor cells and tumor-associated endothelial cells. Collectively, these results show that administration of macitentan in combination with paclitaxel prevents the progression of ovarian cancer in the peritoneal cavity of nude mice in part by inhibiting survival pathways of both tumor cells and tumor-associated endothelial cells. 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 601. doi:10.1158/1538-7445.AM2011-601

Metastatic disease: a drug discovery perspective.

Disseminated tumor cells are present in many patients at diagnosis. At a time when the disseminated disease becomes prominent, patients have already been treated with many cycles of therapy to which their metastases were also exposed. These metastases have genetically evolved from primary tumors. Furthermore, their interaction with the tissue microenvironment plays an important role in all phases of disease development. These facts have only partially been taken into consideration when profiling anti-cancer compounds foreseen to treat patients with disseminated metastatic disease. In this perspective, we discuss the unique features of metastatic disease and review the model systems available for drug profiling. Based on an analysis of how compounds are profiled today in pre-clinical models of metastatic disease and what would be desirable and possible with the present know-how, we recommend a refined profiling process to validate drugs with potential to treat patients with overt metastatic disease.

Abstract 3779: Treatment of experimental brain metastasis of human breast cancer by macitentan, a dual antagonist of endothelin receptors combined with paclitaxel

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Purpose: Breast cancer is a leading cause of cancer-related mortality among women in the United States. The major cause of death is due to metastasis that is resistant to conventional therapy. The median survival of women with brain metastases is measured in months due to resistance to all standard therapies. Thus, the development of effective treatment options for these patients is mandatory. We have reported that astrocytes can protect tumor cells from chemotherapeutic drugs by a mechanism involving the phosphorylation of endothelin receptors on tumor cells (Kim et al, AACR, 2012). Moreover, we observed a heterogeneous expression of endothelin receptors A and B in clinical samples of breast cancer brain metastasis. We therefore examined whether blockade of the endothelin axis by using macitentan, a dual endothelin antagonist, combined with paclitaxel can produce therapeutic effects in an orthotopic model of human breast cancer brain metastasis. Materials and Methods: Twenty thousand MDA-MB-231 human breast cancer cells were injected into the internal carotid artery of female nude mice to produce orthotopic brain metastasis. Two weeks later, the mice were randomized into 4 groups (n=10): (1) control group - mice receiving vehicle (p.o., daily; i.p. injection once per week); (2) paclitaxel group - mice receiving paclitaxel (8 mg/kg; i.p. injection once per week) and vehicle (p.o.; daily); (3) macitentan group - mice receiving macitentan (10 mg/kg; p.o.; daily) and vehicle (i.p. injection, once per week); and (4) paclitaxel plus macitentan group. Moribund mice were euthanized, days of survival were recorded, and the brains were harvested and processed for histology and immunohistochemical analyses. The study was repeated with luciferase-labeled MDA-MB-231 cells and produced similar therapeutic results supported by IVIS imaging. Results: After 130-140 days of treatment, the survival was significantly higher in mice treated with the combination of paclitaxel and macitentan (80%) as compared with control (20%), paclitaxel (20%), and macitentan alone (40%) (p<0.0001, log-rank test). Immunohistochemical analyses revealed that the phosphorylation of endothelin receptors A and B on tumor cells and tumor-associated endothelial cells were inhibited by macitentan. Treatment with macitentan also suppressed the expression of survival proteins such as pAKT, pMAPK, BCL2L1, TWIST1, and GSTA5 in tumor cells. More importantly, only the combination therapy induced significant apoptosis of tumor cells and tumor-associated endothelial cells and reduced the number of KI67 positive tumor cells. IVIS imaging confirmed regression of brain lesions. Conclusion: Treatment with macitentan, a dual endothelin receptor antagonist, combined with paclitaxel leads to regression of experimental breast cancer brain metastasis and therefore should be considered for clinical development. Citation Format: Ho Jeong Lee, Sun Jin Kim, Seung Wook Kim, Junqin He, Qiuyu Wu, Erica J. Lawson, Francois Lehembre, Urs Regenass, Isaiah J. Fidler. Treatment of experimental brain metastasis of human breast cancer by macitentan, a dual antagonist of endothelin receptors combined with paclitaxel. [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 3779. doi:10.1158/1538-7445.AM2014-3779

Abstract 5481: Therapy of human LN229 glioblastoma growing in the brain of nude mice by Macitentan, a dual endothelin receptor A and B antagonist, combined with Temozolomide.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Purpose: We determined whether the blockade of endothelin receptor pathways can reverse the chemoprotection mediated by astrocytes and produce therapy of Human LN 229 Glioblastoma cells growing in the brain of nude mice treated with the dual endothelin receptor antagonist, Macitentan and Temozolomide (TMZ). Materials and Methods: In the first set of experiments, murine astrocytes were cultured with tumor cells in the presence or absence of Macitentan and TMZ. For therapy studies the leuciferas labeled LN229 cells were implanted (stereotactically) into the brains of nude mice. The mice were ear tagged and imaged by IVIS spectrometer at multiple times. On day 24 the mice were randomized to receive vehicle, TMZ (p.o., 7.5mg/kg, one week on and two weeks off), Macitentan (p.o., 10mg/kg, daily) and Macitentan plus TMZ. Survival of mice was compared by Kaplan-Meyer curve (log-rank test) and immunohistochemical analyses were carried out for multiple markers. Results: Simultaneous blocking of endothelin receptor A and B pathways reversed the in culture protection induced by astrocytes against TMZ (p<0.05). Therapy with Macitentan and TMZ significantly prolonged the survival of mice (p<0.0001) .The blockade of endothelin receptor A and B significantly decreased the level of expression of phosphorylated AKT and MAPK, and down-regulated the expression of BCL2L1, TWIST1 and GSTA5 in LN 229 cells growing in the brain. Proliferation of tumor cells and tumor-associated endothelial cells was significantly decreased. Moreover the apoptosis of LN229 cells was significantly increased in the brain of the combination treatment group. Conclusion: Blockade of endothelin receptor A and B pathways by Macitentan combined with TMZ provides a new approach for the treatment of patients with Glioblastoma. Citation Format: Sun Jin Kim, Seung Wook Kim, Hyun Jin Choi, Ho Jeong Lee, Francois Lehembre, Junqin He, Urs Regenass, Charles A Conrad, W.K. Yung, Isaiah J. Fidler. Therapy of human LN229 glioblastoma growing in the brain of nude mice by Macitentan, a dual endothelin receptor A and B antagonist, combined with Temozolomide. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5481. doi:10.1158/1538-7445.AM2013-5481

Abstract 4274: Characterization and drug sensitivity testing of HTS compatible cancer microtissues

The tumor microenvironment strongly effects the drug response. Although the advantages of organotypic 3D-cell-culture models are known for years, complex production and readout processes impede the implementation in an industrial setting. This study, presents for the first time a novel multi-well platform in a standard 96-well format to produce organotypic, scaffold-free cancer microtissues models and its implementation in an automated screening process. This technology allows to produce either homotypic or heterotypic tumor microtissues in a hanging-drop culture by liquid top-loading similar to standard 96-well plates. The core piece of the well is a microfluidic channel connecting an inlet funnel at the top and an outlet funnel at the bottom of the plate, where a hanging drop is formed and stabilized by a combination of capillary and surface-tension forces. The use of this technology enables the automated production of microtissues with a very narrow size distribution below 10% on plate and batch-to-batch. 3 cancer microtissue models were developed from two colon cancer and one breast cancer cell lines and characterized in terms of their proliferative capacity and growth characteristics. Process parameters for media exchange, addition of compounds, microtissue-size variances and implementation of cell-based viability assays (ATP, LDH and size) were established. As a proof of concept, a whole screening process using four reference compounds was performed in comparison to classical monolayer cultures underlining the different behavior of both cell-culture models. Finally, four novel compounds that inhibit tumor cell proliferation in monolayer cultures, were tested for their potential in a 3-D system. Overall, we could demonstrate that organotypic microtissues can be implemented into an automated screening process using standard liquid-handling robotics. The use of this system in drug development may strengthen the predictive force of the in-vitro evaluation of novel drug candidates and reduce drug failure in clinics. 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 4274. doi:10.1158/1538-7445.AM2011-4274