Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults, with an overall incidence rate of approximately 3 per 100,000 persons per year [
1]. The unique characteristics of GBM, such as high mitotic capacity, microvascular proliferation, pseudopallisading necrosis and infiltrative growth, confer a poor prognosis, with a median overall survival of approximately 15 months after diagnosis [
2]. Postoperative radiotherapy (RT) with concomitant temozolomide (TMZ) has become the standard procedure in the treatment of patients with newly diagnosed GBM, based on the results of a large European-Canadian phase III trial [
3]. Despite these encouraging results, the majority of patients still succumb from locally recurrent disease, which is due to the diffuse infiltrative growth characteristics of this tumor type and high level of resistance to radiotherapy and chemotherapy [
4]. The treatment response and prognosis are related to several (epi)genetic characteristics of glioma like methylation status of O6-methylguanine–DNA methyltransferase (MGMT) and genetic events in GBM core pathways including the phosphatidylinositide 3-kinase (PI3K) pathway [
5]. PI3K is a central upstream node related to cell survival and cell proliferation [
6]. Its primary downstream effector protein AKT plays a pivotal role in the pathway activation via phosphorylation of AKT on two critical residues, Thr308 (through PI3K) and Ser473 (mediated predominantly via mTORC2) [
6,
7]. AKT exists in three isoforms, AKT1, −2 and −3, of which AKT2 and −3 are found to be important in glioma cells [
7,
8]. Experimental data has indicated that phosphorylated AKT is required for proper DNA-damage response (DDR) during Non-Homologous end-joining (NHEJ) by binding to DNA-PKcs and promoting its auto-phosphorylation [
9,
10]. Pharmacological inhibition of AKT has therefore also been found to sensitize cancer cells to DNA damaging agents and radiotherapy [
11,
12]. In recent years many specific PI3K/AKT/mTOR pathway targeted agents have become available for preclinical studies and clinical evaluation [
13]. MK2206 is an oral allosteric AKT inhibitor which can inhibit all isoforms of AKT [
14]. Early clinical feasibility studies already demonstrated that MK2206 monotherapy is well tolerated in patients [
15]. Emerging data show MK2206 to enhance the activity of chemotherapeutic agents in various types of cancers both pre-clinical [
14,
16‐
21] and in patients [
15,
22]. Data on MK2206 additional to the current standard GBM therapy are however not available. In the present study, we investigated the effect of MK2206 alone and its ability to synergize with radiation and TMZ to inhibit glioma growth, invasion and migration using monolayer human glioma cells and multicellular glioma spheroids.