3D proteome-wide scale screening and activity evaluation of a new ALKBH5 inhibitor in U87 glioblastoma cell line

Abstract

The imidazobenzoxazin-5-thione MV1035, synthesized as a new sodium channel blocker, has been tested on tumoral cells that differ for origin and for expressed Na_V pool (U87-MG, H460 and A549). In this paper we focus on the effect of MV1035 in reducing U87 glioblastoma cell line migration and invasiveness. Since the effect of this compound on U87-MG cells seemed not dependent on its sodium channel blocking capability, alternative off-target interaction for MV1035 have been identified using SPILLO-PBSS software. This software performs a structure-based in silico screening on a proteome-wide scale, that allows to identify off-target interactions. Among the top-ranked off-targets of MV1035, we focused on the RNA demethylase ALKBH5 enzyme, known for playing a key role in cancer. In order to prove the effect of MV1035 on ALKBH5 in vitro coincubation of MV1035 and ALKBH5 has been performed demonstrating a consequent increase of N6-methyladenosine (m6A) RNA. To further validate the pathway involving ALKBH5 inhibition by MV1035 in U87-MG reduced migration and invasiveness, we evaluated CD73 as possible downstream protein. CD73 is an extrinsic protein involved in the generation of adenosine and is overexpressed in several tumors including glioblastoma. We have demonstrated that treating U87-MG with MV1035, CD73 protein expression was reduced without altering CD73 transcription. Our results show that MV1035 is able to significantly reduce U87 cell line migration and invasiveness inhibiting ALKBH5, an RNA demethylase that can be considered an interesting target in fighting glioblastoma aggressiveness. Our data encourage to further investigate the MV1035 inhibitory effect on glioblastoma.

Introduction

Glioblastoma (GBM, grade IV glioma) represents the most aggressive brain neoplasm and patients with GBM have a poor prognosis. Despite various combinations of surgery, chemotherapy and radiotherapy, GBM is characterized by frequent recurrence and very high lethality. In fact, patients with GBM show a median survival of only 15 months after diagnosis.¹ The greatest challenge in developing a therapy able to arrest GBM invasiveness is represented by glioblastoma stem cells (GSCs) that are very heterogeneous and are responsible for GBM chemoresistance and radio resistance as well as for the ability of GBM to infiltrate neighbouring healthy brain tissue. Moreover drug delivery to GBM is difficult due to the complex structure of the blood brain tumor barrier (BBTB).2, 3, 4 Until now surgical resection followed by radiotherapy and temozolomide (TMZ) treatment represent the standard strategy for GBM.5, 6 However almost all patients manifest resistance and are subject to relapses more aggressive than the primary tumors.¹

Among the various strategies that could be followed to limit the aggressiveness of GBM, we figured out if targeting sodium channels could have been a suitable approach. Our hypothesis was based on the fact that several studies reported the role of Voltage-Gated Sodium Channels (VGSCs or Na_V) in cancer cells, where they seem associated with cell migration and invasiveness.7, 8, 9

In particular Na_V are macromolecular protein complexes that are present in both excitable and non-excitable cells. They are involved in the generation of action potential, but also in different physiological processes.10, 11, 12

Moreover, Na_V are widely expressed in various cancers, including small-cell lung cancer, non-small-cell lung cancer, prostate cancer, melanoma, breast cancer, neuroblastoma, mesothelioma, cervical cancer, ovarian cancer and gliomas.13, 14, 15, 16, 17, 18

Literature data bring evidence regarding the implication of ion channels in GBM.19, 20, 21 Since Na_V1.1, Na_V1.2, Na_V1.3 and Na_V1.6 are the isoforms mainly expressed in the CNS,²² we have investigated the effects of some sodium channel blockers, previously synthesized by us, with a documented blocking capability against Na_V, on GBM cell proliferation, migration and invasion.

Among the compounds tested (data not shown) the 2-methyl-3-propyl-5H-imidazo[1,2-c][1,3]benzoxazin-5-thione (MV1035)²³ showed a peculiar activity on glioblastoma cells that, unexpectedly, seemed not related with the sodium channel blockade (see results). In order to identify a molecular mechanism able to justify MV1035 effect we used SPILLO potential binding sites searcher (SPILLO-PBSS)24, 25 to perform an in silico screening of the human structural proteome publicly available in the RCSB Protein Data Bank.26, 27 SPILLO-PBSS is an innovative software designed to identify target proteins of any small molecule on a proteome-wide scale. Their putative binding sites are successfully recognized even within really strongly distorted protein conformations.24, 25

Among the top-ranked potential targets for MV1035 provided by the in silico analysis, we focused our attention on the RNA demethylase ALKBH5 (α-ketoglutarate-dependent dioxygenase alkB homolog 5), which catalyses the conversion of N6-methyladenosine (m6A) to adenosine in mRNA, in the presence of 2-oxoglutarate, molecular oxygen, and iron(II).²⁸ It is widely demonstrated that m6A RNA methylation represents a post transcriptional modification crucial for modulating expression of proteins involved in tumor initiation and progression.²⁹ Interestingly, Zhang et al. have demonstrated that ALKBH5 is overexpressed in glioblastoma stem-like cells (GSCs) being fundamental to maintain GSCs tumorigenicity. Moreover investigating the ALKBH5 target mRNA, they have identified the gene encoding for CD73.³⁰

Ecto-5′-nucleotidase (ecto-5′-NT, NT5E or CD73) is an extrinsic protein linked to the extracellular surface of the plasma membrane through glycosylphosphatidylinositol (GPI). Besides its enzymatic activity CD73 acts as adhesive and signalling molecule influencing invasiveness and metastatic properties of cancer cells.³¹ Furthermore it has been demonstrated that extracellular adenosine influences glioma proliferation³² and CD73 promotes glioma cell line growth.33, 34

Here we show, integrating cellular, molecular and in silico techniques, that MV1035 reduces GBM cell line U87 migration and invasiveness, targeting the SPILLO-PBSS recognized RNA N6-methyladenosine (m6A) demethylase ALKBH5 activity and ultimately inducing a decrease in CD73 expression.