Anticancer effects of ABTL0812, a clinical stage drug inducer of autophagy-mediated cancer cell death, in glioblastoma models

Abstract

Background: Glioblastoma multiforme (GBM) is the most malignant adult brain tumor. Current standard of care treatments have very limited efficacy, being the patients´ overall survival 14 months and the 2-year survival rate less than 10%. Therefore, the treatment of GBM is an urgent unmet clinical need.

Methods: The aim of this study was to investigate in vitro and in vivo the potential of ABTL0812, an oral anticancer compound currently in phase II clinical stage, as a novel therapy for GBM.

Results: We showed that ABTL0812 inhibits cell proliferation in a wide panel of GBM cell lines and patient-derived glioblastoma stem cells (GSCs) with half maximal inhibitory concentrations (IC50s) ranging from 15.2 µM to 46.9 µM. Additionally, ABTL0812 decreased GSCs neurosphere formation. GBM cells aggressiveness is associated with a trans-differentiation process towards a less differentiated phenotype known as proneural to mesenchymal transition (PMT). ABTL0812 was shown to revert PMT and induce cell differentiation to a less malignant phenotype in GBM cell lines and GSCs, and consequently reduced cell invasion. As previously shown in other cancer types, we demonstrated that the molecular mechanism of action of ABTL0812 in glioblastoma involves the inhibition of Akt/mTORC1 axis by overexpression of TRIB3, and the activation of endoplasmic reticulum (ER) stress/unfolded protein response (UPR). Both actions converge to induce autophagy-mediated cell death. ABTL0812 anticancer efficacy was studied in vivo using subcutaneous and orthotopic intra-brain xenograft tumor models. We demonstrated that ABTL0812 impairs tumor growth and increases disease-free survival and overall survival of mice. Furthermore, the histological analysis of tumors indicated that ABTL0812 decreases angiogenesis. Finally, we investigated the combination of ABTL0812 with the standard of care treatments for GBM radiotherapy and temozolomide in an orthotopic model, detecting that ABTL0812 potentiates the efficacy of both treatments and that the strongest effect is obtained with the triple combination of ABTL0812+radiotherapy+temozolomide.

Conclusions: Overall, the present study demonstrated the anticancer efficacy of ABTL0812 as single agent and in combination with the GBM standard of care treatments in models of glioblastoma and supports the clinical investigation of ABTL0812 as a potential novel therapy for this aggressive brain tumor type.

Keywords: ABTL0812; Akt; ER stress; TRIB3; UPR; autophagy; glioblastoma; mTOR.

Figure 1

ABTL0812 inhibits proliferation of glioblastoma cells and glioblastoma stem cells. (A) ABTL0812 cell proliferation curves and IC₅₀s at 48 hours in glioblastoma cell lines, (B) at 96 hours in patient-derived glioblastoma stem cells (GSCs) and (C) at 48 hours in HBMVEC and astrocytes. (D) Images of U87MG cells treated for 48 hours with ABTL0812 showing that ABTL0812 induce cell extensions associated to type I astrocytic phenotype and cell death and detachment. Bar represents 50 µm (E) Neurosphere formation in BT12M cells treated for 96 hours with different concentrations of ABTL0812.

Figure 2

ABTL0812 induces glioblastoma and GSCs differentiation and reverts proneural to mesenchymal transition. (A) Representative FACS expression profiles of mesenchymal (CD44, Stro1 and CD90), stemness (CXCR4), neural (βIII tubulin, NFH and GAP43) and proliferation (Ki67) markers and table and histograms showing the percentage of cells expressing the markers analyzed by FACS in glioblastoma cells U87MG treated with ABTL0812 for 48 hours (B) and in glioblastoma stem cells GSCs-5 treated for 48 hours with ABTL0812 (C) Representative confocal images of BT12M cells stained with Sox2, βIII tubulin and Ki67. Cell nuclei were stained with DAPI. (D) Representative images from Boyden chamber assays showing invasive U87MG cells after a 6-hour assay that were pretreated with ABTL0812 for 48 hours (E) Quantification of invasive cells from a matrigel invasion assays performed in U87MG, U251 and A172 cells treated with ABTL0812. CTRL, control vehicle-treated cells. * p<0.01 vs vehicle basal.

Figure 3

ABTL0812 induces autophagy in glioblastoma cells. (A) Representative immunoblotting images of LC3-II from U87MG, A172 and U251 cell lines treated with ABTL0812 for 12 hours. (B) Representative immunofluorescence images of staining with acridine orange, a green fluorophore that fluoresces red in acidic vesicular organelles (AVO) in U87MG cells treated for 12 hours with ABTL0812 (40X magnification). (C) Representative electron microscopy microphotographs of U87MG in cells treated with ABTL0812 at 20 and 40 μM for 12 hours. N= nucleus, Au=autophagosomes; M= mitochondria and V= vesicles. Blue arrows indicate small or unfunctional mitochondria. (D) Representative immunoblotting images of LC3-II from U87MG cells pretreated with lysosome protease inhibitors (E64 and pepstatin A at 10 mg/ml each) for 24 hours followed by 12 hours treatment with ABTL0812. (E) Representative immunofluorescence images of the staining of LAMP1 and LC3 in U87MG cells treated with ABTL0812 (20 μM) for 12 hours with or without a pretreatment of 1 hour with chloroquine (3 μM). LAMP1 in is a marker of endosomes/lysosomes (green signal), LC3 is a marker of autophagosomes (red signal) and DAPI stains nuclei (blue signal). The colocalization of red and green signal results in orange signal and corresponds to autolysosomes. CTRL, control vehicle-treated cells.

Figure 4

ABTL0812 induces mitochondrial depolarization and apoptosis in glioblastoma cells. (A) Representative immunofluorescence images of staining with JC-1, a dye indicator of mitochondrial membrane potential in U251MG cells treated for 16 hours with ABTL0812 (200X magnification). Red staining indicates polarized mitochondria and green indicates depolarized mitochondria. (B) Apoptosis TUNEL assay performed on U87MG cells treated with ABTL0812 for 16 hours. (C) Table showing the percentage of cell populations as determined by FACS analysis of annexin V/propidium iodide staining of U87MG cells treated for 16 hours with ABTL0812. (D) Representative images of immunoblotting detection of caspase 3, 8 and 9 pro-enzymes and cleaved isoforms in U87MG cells treated with ABTL0812 for 16 hours. (E) Quantification of caspases 8, 9 and 3 activities by using caspase-specific chromogenic substrates in U87MG, A172 and U251 cells treated for 16 hours with ABTL0812. The mTORC1 inhibitor everolimus was used as a comparator to ABTL0812 *p<0.05 and ** p<0.01 vs vehicle treated cells. (F) Quantization of caspase 3 activity in U87MG cells treated for 16 hours with ABTL0812 and with or without a 1 hour chloroquine pretreatment. *p<0.05 and ** p<0.01 vs vehicle basal; ^#p<0.05 and ^## p<0.01 vs vehicle+chloroquine.

Figure 5

ABTL0812 inhibits Akt/mTORC1 axisA and induces ER stress (A) Representative immunoblotting images of Akt/mTORC1 axis markers TRIB3, Akt and p70S6K from U81MG cells treated with ABTL0812 for 16 hours. (B) ELISA quantification of p-Akt Ser473, and p-Akt Thr308 in U81MG, U251, A172 and GSCs-5 cells treated with ABTL0812 for 16 hours (C) Representative immunoblotting images of p-eIF2α, ATF4, CHOP and LC3-II from U81MG cells treated with ABTL0812 for 16 hours The ER stress inducer Brefeldin A was used as a positive control for ER stress response (D) Representative immunoblotting images of PERK and phase G1-S cell cycle regulators from U81MG cells treated with ABTL0812 for 16 hours. (E) Diagrams showing percentages of cell population in each cell cycle phase in U87MG and U251 cells treated with ABTL0812 for 16 hours determined by flow citometry analysis of propidium iodide-stained cells.

Figure 6

ABTL0812 impairs tumor growth in glioblastoma subcutaneous xenograft models. U87MG and T98G cells were injected subcutaneously in athymic female cd1 nu/nu mice (N=10 each group). Mice were treated daily with vehicle or ABTL0812 at 120 or 240 mg/Kg by oral administration. The mTORC inhibitor everolimus (ve) was used as a comparator for antitumor activity and was administered orally at a dose of 5 mg/kg/2 days per week (A, B) Weight of U87MG and T98G cells-derived xenograft tumors removed from nude mice. (C, D) Representative immunohistochemistry images from U87MG (C) and T98G (D) xenograft tumors stained with Akt-mTORC axis markers (TRIB3, p-Akt Ser473 and p-Akt Thr308); the cell proliferation marker Ki67; the endothelial cell marker CD34; the apoptosis marker caspase3; the hypoxia marker HIF-1α; and TUNEL staining to measure apoptosis (Magnification 400X). Statistical significance levels: *p<0.05, **p<0.01 and n =10. CTRL, control vehicle-treated cells.

Figure 7

ABTL0182 inhibits tumor growth in an orthotopic model of glioblastoma in mice that reproduces the clinical setting after surgical removal of tumors. (A) Diagram showing the experimental design of the treatment of glioblastoma orthotopic models using U87MG and GSCs-5 luciferase-tagged cells ortotopically inoculated into the brain of athymic nude mice and treated with ABTL0812 (120 and 240 mg/Kg) and Everolimus (5 mg/Kg). Tumors were monitored by bioluminescence detection which was performed every 7 days in order to determine Disease Free Survival (DFS) and Overall Survival (OS). (B) DFS of mice bearing orthotopic tumor of U87MG-Luc cells. (C) Overall survival of mice bearing U87MG-Luc cells orthotopic tumors. (D) DFS of mice bearing orthotopic tumor of GSC-5 -Luc cells. (E) Overall survival of mice bearing GSC-5 -Luc cells orthotopic tumors. Statistical significance levels: *p<0.05, **p<0.01, ***p<0.01 n=10 in each group. CTRL= control vehicle-treated cells.

Figure 8

ABTL0182 potentiates the anticancer effect of the standard of care treatments radiotherapy and temozolomide in an orthotopic model of glioblastoma in mice (A) Diagram showing the experimental design of the treatment of glioblastoma orthotopic models using U87MG luciferase-tagged cells orthotopically inoculated into the brain of athymic nude mice and treated with ABTL0812 (120 mg/Kg), radiotherapy (4 Gy), temozolomide (32mg/kg) and combinatorial treatments. Tumors were monitored by bioluminescence detection which was performed every 7 days in order to determine Overall Survival (OS). (B) Overall survival of mice bearing U87MG-Luc cells orthotopic tumors. n=10 in each group.