Monotherapy efficacy of blood–brain barrier permeable small molecule reactivators of protein phosphatase 2A in glioblastoma

Abstract

Glioblastoma is a fatal disease in which most targeted therapies have clinically failed. However, pharmacological reactivation of tumour suppressors has not been thoroughly studied as yet as a glioblastoma therapeutic strategy. Tumour suppressor protein phosphatase 2A is inhibited by non-genetic mechanisms in glioblastoma, and thus, it would be potentially amendable for therapeutic reactivation. Here, we demonstrate that small molecule activators of protein phosphatase 2A, NZ-8-061 and DBK-1154, effectively cross the in vitro model of blood–brain barrier, and in vivo partition to mouse brain tissue after oral dosing. In vitro, small molecule activators of protein phosphatase 2A exhibit robust cell-killing activity against five established glioblastoma cell lines, and nine patient-derived primary glioma cell lines. Collectively, these cell lines have heterogeneous genetic background, kinase inhibitor resistance profile and stemness properties; and they represent different clinical glioblastoma subtypes. Moreover, small molecule activators of protein phosphatase 2A were found to be superior to a range of kinase inhibitors in their capacity to kill patient-derived primary glioma cells. Oral dosing of either of the small molecule activators of protein phosphatase 2A significantly reduced growth of infiltrative intracranial glioblastoma tumours. DBK-1154, with both higher degree of brain/blood distribution, and more potent in vitro activity against all tested glioblastoma cell lines, also significantly increased survival of mice bearing orthotopic glioblastoma xenografts. In summary, this report presents a proof-of-principle data for blood–brain barrier—permeable tumour suppressor reactivation therapy for glioblastoma cells of heterogenous molecular background. These results also provide the first indications that protein phosphatase 2A reactivation might be able to challenge the current paradigm in glioblastoma therapies which has been strongly focused on targeting specific genetically altered cancer drivers with highly specific inhibitors. Based on demonstrated role for protein phosphatase 2A inhibition in glioblastoma cell drug resistance, small molecule activators of protein phosphatase 2A may prove to be beneficial in future glioblastoma combination therapies.

Development of Actionable Targets of Multi-kinase Inhibitors (AToMI) screening platform to dissect kinase targets of staurosporines in glioblastoma cells

“…S4A). Consistently with high intrinsic kinase inhibitor resistance of GB cells (25,39), none of the kinase inhibitors as monotherapies, and used at doses that effectively inhibited their intended targets (Fig. S2D, E), did induce cytotoxic response (Fig.…”

“…Finally, in intracranial model the triplet therapy was tested on luciferase-expressing E98 cells that carry characteristics of GSCs and has very infiltrative growth pattern in vivo (25). In addition to these faithful human GB characteristics, E98 cells displayed indistinguishable triplet therapy response as compared to patient derived GSC cell lines in vitro (Fig.…”

“…All cell cultures were maintained in a humified atmosphere of 5% CO2 at 37°C. The patient-derived GSCs BT3-CD133 + and BT12 (Kuopio University Hospital, Kuopio, Finland) (25,38) were cultured in DMEM/F12 (Gibco) and supplemented with 2 mM L-glutamine, 2% B27-supplement (Gibco), penicillin (50 U/mL) / streptomycin (50 μg/mL), 0.01 μg/mL hFGF-β (Peprotech), 0.02 μg/mL hEGF (Peprotech) and 15 mM HEPES-buffer (Gibco). For assays requiring adherent cell, such as colony growth, GSC populations were cultured on Matrigel…”

“…PP2A regulates several kinase pathways and drug resistance mechanisms, and PP2A inhibition in cancer cells has been shown to drive broad-range kinase inhibitor resistance (24). PP2A is frequently inactivated in GB by non-genetic mechanisms including overexpression of endogenous PP2A inhibitor proteins such as CIP2A, PME-1, SET and ARPP19 (25)(26)(27). We recently published proof-of-concept data that reversal of PME-1mediated PP2A inhibition strongly sensitized GB cells to several kinase inhibitors, including clinically tested STS derivatives (28).…”

Direct Activation of Protein Phosphatase 2A (PP2A) by Tricyclic Sulfonamides Ameliorates Alzheimer's Disease Pathogenesis in Cell and Animal Models

“…These studies clearly verify PP2A as a main target for SMAPs. Relevant to the current study, the SMAPs cross the blood-brain barrier after oral administration, and are active in an intracranial mouse model of invasive glioblastoma [33]. Given the above, and the significance of diminished PP2A activity in AD, we decided to investigate PP2A reactivation by SMAPs in neurons undergoing AD-like pathogenesis in vitro and in vivo.…”