The allosteric AKT inhibitor MK2206 shows a synergistic interaction with chemotherapy and radiotherapy in glioblastoma spheroid cultures

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.

We investigated the efficacy of MK2206 at attenuating U87 glioma cell proliferation and found it to be effective at 1 μM and higher (Fig. 1a). Protein analysis showed complete AKT dephosphorylation at 1 μM (Fig. 1c). In the combination of MK2206 with 4 Gy irradiation or 5 μM TMZ no synergistic interaction was found. Synergy calculations show a combination index (CI) > 1.2, indicating a slight antagonism (Fig. 1a). Next we tested the combination with irradiation on clonogenic cell survival using U251 glioma cells. None of the different MK2206 treatment schedules showed a reduction in clonogenic survival (Fig. 1b). In line with these findings, the expression of γH2A.X phosphorylation was not increased at 24 h after 4 Gy irradiation in cells treated with MK2206 (Fig. 1c). The data show that in monolayer growing cell cultures MK2206 was not able to induce sensitization to DNA damaging therapies and actually showed antagonistic tendencies.

In the present study, we investigated the effect of AKT inhibition by MK2206 on adherent growing human glioma cells and on multicellular spheroids. The most important finding is that low dose MK2206 is able to synergistically sensitize glioma spheroids to the current standard treatment modalities in GBM therapy, i.e. irradiation and TMZ. This is in contrast to adherent growing which showed at best modest additivity for the combinatorial treatments. This has not described earlier in the context of radiosensitization since the golden standard for radiosensitization has always been monolayer clonogenic assays. Hence, studies for evaluation of radiosensitizing agents should take in to account the biological and physiological limitations of monolayer cultures. Cells growing in multicellular organoid structures more faithfully resemble the context of real life tumors. Cells in this context deal with complex interactions due to heterotypic cell-to-cell contact, signaling, extracellular matrix deposition and intracellular structure. This micro milieu results in gradients of oxygen, nutrients and biomolecules can result in hypoxia/oxidative stress [26, 27], which has been shown to culminate to an increase of the autophagic flux near the core of spheroids [28]. Cheng and colleagues have previously shown that the treatment of glioma cells with MK2206 preferentially leads to an increased autophagic flux and that this could be increased synergistically with gefitinib leading to autophagic cell death [29, 30]. In our study RT and TMZ were used to induce genotoxic stress (DNA-damage) which is a well-known inducer of autophagy [31, 32]. We therefore hypothesize that AKT inhibition together with RT/TMZ in spheroids tips the autophagic equilibrium towards autophagic cell death which cannot be achieved in adherent cells. The presence of these mechanisms and the concomitant different phenotype makes spheroids a preferred model over adherent monolayer growing cells for studying radiosensitizing potential of targeted agents [33]. Recently, a study has shown similar results where the authors show that AKT inhibition does not radiosensitize U87 monolayer cells but does sensitize primary glioma stem-like cell cultures to irradiation [34]. U87 cells grown as spheroids have been shown to have elevated levels of the stem cell marker CD133 [35], indicating another mechanism of how the structural organization of cells influence internal signaling and drug response.

The ability of glioma cells to invade into the brain and migrate to different regions of the brain is one of the most important physical means of therapy resistance. AKT is thought to stimulate cell migration through signaling routes [36]. Cellular invasiveness can be inhibited by irradiation, which has been reported before [37]. However, our data shows a modest attenuation of glioma cell mobility through AKT inhibition and shows synergy in only one of the four cell lines tested (Figure 3). Nevertheless, this modest inhibition together with the synergistic effect the combination therapy shows on growth inhibition warrant further investigation in an orthotopic in vivo glioma model. Cheng and colleagues have shown MK2206 alone to have inhibitory effects on in vivo glioma growth [30].

Taken together, low dose MK2206 enhanced the effect of radiotherapy and TMZ on brain tumor spheroids in vitro which was not seen in adherent growing cell lines. Furthermore, High dose MK2206 inhibited migration and invasion of glioma cells and also synergized with irradiation in a primary GBM line. Our findings indicate the AKT pathway to be a promising target to be combined with the current standard of care for GBM therapy.