The present study implies that the molecular mechanism underlying DNA-PKcs inhibition-enhanced TMZ efficacy can be majorly related to the inhibition of AKT signaling. Constitutive activation of AKT exists in a variety of malignances including glioma, and is closely correlated with cancer development and progression [47, 48]. Specifically, hyperactivation of AKT also confers cell resistance to many chemotherapy agents [49, 50]. As we found, allosteric AKT inhibitor MK2206 mimicked the effect of KU0060648 that substantially decreased cell growth and sensitized TMZ efficacy, suggesting KU0060648 functioned mainly through suppression of AKT. Further investigations confirmed the inhibitory effect of KU0060648 on DNA-PKcs/AKT signal. More importantly, we disclosed that KU0060648 induced inhibition of AKT activation may be, at least partly, derived from its disruption of the interaction between DNA-PKcs and AKT in glioma cells. Consistently, the downstream molecules of AKT, such as c-Myc, and AKT-related anti-apoptotic, metastatic or angiogenic molecules could be efficiently suppressed by KU0060648 treatment. Many studies have shown that these substrates participate in the resistance of certain cancers to chemotherapy [51 - 55]. Taken together, these data revealed that the single agent of KU0060648 or in combination with TMZ in glioma treatment mainly depended on inhibition of AKT signaling.