The small molecule ephrin receptor inhibitor, GLPG1790, Reduces renewal capabilities of cancer stem cells, showing anti-tumour efficacy on preclinical glioblastoma models

Therapies against glioblastoma (GBM) show a high percentage of failure associated with the survival of glioma stem cells (GSCs) that repopulate treated tumours. Forced differentiation of GSCs is a promising new approach in cancer treatment. Erythropoietin-producing hepatocellular (Eph) receptors drive tumourigenicity and stemness in GBM. We tested GLPG1790, a first small molecule with inhibition activity versus inhibitor of various Eph receptor kinases, in preclinical GBM models using in vitro and in vivo assays. GLPG1790 rapidly and persistently inhibited Ephrin-A1-mediated phosphorylation of Tyr588 and Ser897, completely blocking EphA2 receptor signalling. Similarly, this compound blocks the ephrin B2-mediated EphA3 and EphB4 tyrosine phosphorylation. This resulted in anti-glioma effects. GLPG1790 down-modulated the expression of mesenchymal markers CD44, Sox2, nestin, octamer-binding transcription factor 3/4 (Oct3/4), Nanog, CD90, and CD105, and up-regulated that of glial fibrillary acidic protein (GFAP) and pro-neural/neuronal markers, βIII tubulin, and neurofilaments. GLPG1790 reduced tumour growth in vivo. These effects were larger compared to radiation therapy (RT; U251 and T98G xenografts) and smaller than those of temozolomide (TMZ; U251 and U87MG cell models). By contrast, GLPG1790 showed effects that were higher than Radiotherapy (RT) and similar to Temozolomide (TMZ) in orthotopic U87MG and CSCs-5 models in terms of disease-free survival (DFS) and overall survival (OS). Further experiments were necessary to study possible interactions with radio- and chemotherapy. GLPG1790 demonstrated anti-tumor effects regulating both the differentiative status of Glioma Initiating Cells (GICs) and the quality of tumor microenvironment, translating into efficacy in aggressive GBM mouse models. Significant common molecular targets to radio and chemo therapy supported the combination use of GLPG1790 in ameliorative antiglioma therapy

Targeting of αv-Integrins in Stem/Progenitor Cells and Supportive Microenvironment Impairs Bone Metastasis in Human Prostate Cancer12

Acquisition of an invasive phenotype by cancer cells is a requirement for bone metastasis. Transformed epithelial cells can switch to a motile, mesenchymal phenotype by epithelial-mesenchymal transition (EMT). Recently, it has been shown that EMT is functionally linked to prostate cancer stem cells, which are not only critically involved in prostate cancer maintenance but also in bone metastasis. We showed that treatment with the non-peptide αv-integrin antagonist GLPG0187 dose-dependently increased the E-cadherin/vimentin ratio, rendering the cells a more epithelial, sessile phenotype. In addition, GLPG0187 dose-dependently diminished the size of the aldehyde dehydrogenase high subpopulation of prostate cancer cells, suggesting that αv-integrin plays an important role in maintaining the prostate cancer stem/progenitor pool. Our data show that GLPG0187 is a potent inhibitor of osteoclastic bone resorption and angiogenesis in vitro and in vivo. Real-time bioluminescent imaging in preclinical models of prostate cancer demonstrated that blocking αv-integrins by GLPG0187 markedly reduced their metastatic tumor growth according to preventive and curative protocols. Bone tumor burden was significantly lower in the preventive protocol. In addition, the number of bone metastases/mouse was significantly inhibited. In the curative protocol, the progression of bone metastases and the formation of new bone metastases during the treatment period was significantly inhibited. In conclusion, we demonstrate that targeting of integrins by GLPG0187 can inhibit the de novo formation and progression of bone metastases in prostate cancer by antitumor (including inhibition of EMT and the size of the prostate cancer stem cell population), antiresorptive, and antiangiogenic mechanisms

A dose escalating phase i study of GLPG0187, a broad spectrum integrin receptor antagonist, in adult patients with progressive high-grade glioma and other advanced solid malignancies

Background Integrin signaling is an attractive target for anti-cancer treatment. GLPG0187 is a broad spectrum integrin receptor antagonist (IRA). GLPG0187 inhibited tumor growth and metastasis in mouse models. Methods We aimed to determine the Recommended Phase II Dose (RP2D) and to assess safety and tolerability of continuous i.v. infusion in patients with advanced malignant solid tumors. Anticipated dose levels were 20, 40, 80, 160, 320, and 400 mg/day in a modified 3 + 3 design. Plasma concentrations of GLPG0187 were assessed to characterize the pharmacokinetics (PK). C-terminal telopeptide of type I collagen (CTX) was used as pharmacodynamics marker. Results Twenty patients received GLPG0187. No dose limiting toxicities (DLTs) were observed. The highest possible and tested dose was 400 mg/day. Fatigue was the most frequently reported side effect (25 %). Recurrent Port-A-Cath-related infections and skin toxicity suggest cutaneous integrin inhibition. No dose-dependent toxicity could be established. PK analysis showed a short average distribution (0.16 h) and elimination (3.8 h) half-life. Continuous infusion resulted in dose proportional PK profiles. We observed decreases in serum CTX levels independent of the dose given, suggesting target engagement at the lowest dose level tested. Single agent treatment did not result in tumor responses. Conclusions GLPG0187 was well tolerated with a dose-proportional PK profile upon continuous infusion. No formal maximal tolerated dose could be established. GLPG0187 showed signs of target engagement with a favourable toxicity profile. However, continuous infusion of GLPG0187 failed to show signs of monotherapy efficacy

Effect of systemic administration of GLPG0187 on tumor growth and metastasis in a preventive and curative protocol.

<p>A) Schematic representation of the <i>preventive</i> protocol. Mice were treated daily with either IP administrated vehicle or GLPG0187 (100 mg/kg/day) from day -1 onwards. At day 0, 100,000 UM-UC-3luc2 cells were inoculated into the left heart ventricle and once a week BLI images were taken. B) Number of metastasis per mouse. C) Total tumor burden for the mice treated with 100 mg/kg/day GLPG0187 (closed circles) or vehicle (open circles). In the insert, the first 14 days are shown. D) Representative images of mice treated with vehicle or 100 mg/kg/day GLPG0187 taken at day 28 after inoculation. E) Schematic representation of the <i>curative</i> protocol. At day -21, 100,000 UM-UC-3luc2 cells were injected into the left heart ventricle and once a week BLI images were taken. At day 0, mice were divided into groups with equal total tumor burden. Mice were daily treated with an IP dosage of either vehicle or GLPG0187 (100 mg/kg/day) from day 0 onwards. F) Number of metastasis per mouse. G) Total tumor burden for the mice treated with 100 mg/kg/day GLPG0187 (closed circles) or vehicle (open circles). H) Representative images of mice treated with vehicle or 100 mg/kg/day GLPG0187 taken at day 15 after start of treatment.</p

Effects of α_v integrin on clonogenicity and stem cell/metastasis markers.

<p>The relative percentage and size distribution of colony-forming cells in a 96-wells plate clonogenic assay of single-cell diluted cultures after 2 weeks in the α_v kd or NT cells and cells treated with a dose range of GLPG0187 for 48 hrs and plated afterwards. The area of the colonies was measured with Image J software and divided according to size. Small colonies are between 0.5 and 1.5 mm², medium sized colonies are between 1.5 and 4 mm² and large colonies are bigger than 4 mm². Data were normalized to the NT or control conditions and are presented as mean ± SEM. Percentage of colony-forming cells in the control NT cells are depicted above the respective bars (A). UM-UC-3 cells were seeded 100 cells/cm² in an ultra-low attachment plate in serum-starved conditions. The percentage of cells with sphere forming capacity (P0) was measured after 10 days of culture (B). P0 spheres were dissociated into single cells and seeded in ultra-low attachment 96 wells. The percentage of cells with sphere forming capacity (P1) was measured after 10 days of culture (B). The area of the spheres was measured with Image J software (D). Percentage of cells with high ALDH activity (ALDH^hi) as measured with Aldefluor assay. Data are normalized to the NT or vehicle treated cells. Percentages of ALDH^hi cells in the NT and control cells are depicted above the respective bars (E). qPCR analysis of NANOG (F) qPCR analysis of BMI1 (G). Relative expression levels are shown compared to respectively NT or vehicle-treated cells. All values were normalized for GAPDH and presented as mean ± SEM.</p

Effects of α_v integrin on migration and EMT.

<p>Effects of knockdown of ITGAV and 48 hrs of GLPG0187 treatment on migratory capacity of UM-UC-3luc2 and RT-4 cells as determined by Transwell Boyden chamber migration assays. Mean numbers of migrated cells per area were measured. Mean number of migrated cells of the control NT cells are depicted above the respective bars (A). Effects of ITGAV knockdown and 48 hrs of GLPG0187 treatment on the CDH1/CDH2 ratio (B). Data were normalized to the NT or control conditions (n = 3) and are presented as mean ± SEM. qPCR (C) and protein analysis of SNAI1 (D). qPCR analysis (E) and protein analysis of SNAI2 (F). qPCR analysis (G) and protein analysis of ZEB1 (H). qPCR analysis (I) and protein analysis of ZEB2 (J). Relative expression levels are shown compared to respectively NT or vehicle treated cells. All qPCR values were normalized for GAPDH and presented as mean ± SEM.</p

Effect of α_v integrin on expression levels of CD24 and urothelial differentiation markers and on senescence.

<p>Relative expression levels of CD24 (A) and CD227 (B). Relative expression levels (% of positive cells * Mean fluorescence intensity) were measured by flow cytometry and normalized to the NT or vehicle treated cells. Data are represented as mean ± SEM. Percentages of positive cells are depicted above the respective bars. qPCR analysis of KRT20. Relative expression levels are shown compared to respectively NT or non-treated cells. All values were normalized for GAPDH and presented as mean ± SEM (C). UM-UC-3 luc2 and RT-4 cells were seeded into a six-well plate and exposed to a concentration series of GLPG0187 (0–500 ng/ml). 48 h after incubation, cells were harvested and senescence associated acid β-galactosidase activity was measured. Data are represented as fold change in fluorescence intensity of the signal (D).</p

ITGAV knockdown in UM-UC-3luc2 cells affects intra-bone growth in a preclinical model.

<p>A) Percentage of mice with tumors after intra-bone inoculation of either α_v-kd-UM-UC-3luc2 or NT-UM-UC-3luc2 cells B) Total tumor burden of the mice injected with α_v-kd-UM-UC-3luc2 (<i>closed circles</i>) or NT-UM-UC-3luc2 cells (<i>open circles</i>). C) Representative images of mice intra-osseously inoculated with either αv-kd-UM-UC-3luc2 or NT-UM-UC-3luc2 cells 7 days after inoculation (<i>n</i> = 10/group *<i>P</i><0.05).</p

Effects of GLPG0187 and ITGAV knockdown on adherence to tissue culture plastic.

<p>Representative images of cells treated for 24 hours with a concentration series of GLPG0187 (dosage between 0–500 ng/ml, indicated underneath the images). Treatment resulted in a dose-dependent loss of adherence to tissue culture plastic in both UM-UC-3luc2 cells (A) and RT-4 cells (C). After 48 hours of GLPG0187 treatment, cells cultured for 4 days in GLPG0187-free medium regained their adherence to the tissue culture plastic in UM-UC-3luc2 cells (B) and RT-4 cells (D). Loss of adherence was also observed in UM-UC-3luc2 and RT4 cells stably transduced with a short hairpin targeted against ITGAV (respectively F–G for UMUC3luc2 sh ITGAV clones 1 and 2 and J–K for RT4 shITGAV clones 1 and 2). As a control, cells stably transduced with a non-targeting short hairpin (NT) were used (UMUC3 (E) and RT4 (I)). Flow cytometric analysis of relative ITGAV expression levels in UM-UC-3luc2 (H) and RT4 (L) cells (% of positive cells * mean fluorescence intensity). Data are presented as mean ± SEM, n = 3, the percentage of ITGAV positive cells is indicated above the bars.</p