Abstract Introduction. The PI3K/AKT (phosphatidylinositol 3-kinase / protein kinase B) pathway is one of the most frequently mutated and deregulated pathways in cancer, including non-Hodgkin lymphomas. Particularly frequent in germinal center B-cell-like diffuse large B-cell lymphoma (GCB-DLBCL) is deletion of the PI3K/AKT pathway negative regulator PTEN (phosphatase and tensin homolog), a phosphatase acting on phosphoinositol-3,4,5-triphosphate. PI3K/AKT signaling regulates multiple critical cellular processes including cellular proliferation and metabolism. However, it is not known whether there is specific regulation of AKT activity during the cell cycle, and how it may be affected by oncogenic AKT hyperactivation. Methods. We used a spectrum of GCB-DLBCL lines, some modified to allow assessment of PTEN deletion. Knock in (KI) and knock out (KO) were done by CRISPR-Cas9 technology. Overexpression used the Sleeping Beauty transposon system. AKT activity was measured using a genetically encoded Förster resonance energy transfer (FRET)-based reporter by flow cytometry. Cell cycle was evaluated by Vibrant DyeCycle Ruby staining, FxCycle propidium iodide staining, or genetically encoded FastFUCCI biosensor. Cell cycle synchronization was done by double thymidine block. Cellular metabolism was evaluated by untargeted liquid chromatography mass spectrometry (LC-MS)-based metabolomics and by Seahorse instrument-based measurement of live cells. Results. Simultaneous flow cytometry-based evaluation of AKT activity and cell cycle allowed us to identify an increase of AKT activity at the beginning of the S phase of the cell cycle in GCB-DLBCL cell lines. An S phase-specific increase of AKT activity was verified in cell cycle-synchronized cells over several cell cycles and was necessary for cell cycle progression. AKT inhibition blocked the cells in G1 phase. Additionally, after cell cycle synchronization and release, immunoblotting confirmed increased pAKT (Ser473, a surrogate marker for AKT activity) during S phase, as determined by cyclin A and E dynamics. Given the importance of cyclin dependent kinases (CDK) for cell cycle regulation, we next evaluated to what degree CDKs could drive AKT activity. Remarkably, AZD-5438 (CDK1/2/9 inhibitor) and JNJ-7706621 (pan-CDK inhibitor) fully eliminated AKT activity. In contrast, palbociclib (CDK4/6 inhibitor) did not have any significant AKT inhibitory effect. This prompted us to focus specifically on CDK2, a major kinase involved in G1-S transition. Induced expression of CDK2 resulted in a strong and specific increase of AKT activity. Importantly, we hypothesized that the CDK2 mediated increase of AKT activity is at least partially responsible for S phase specific metabolic increase necessary for synthesis of all cellular components before cell division. AKT activation by PTEN deletion, or expression of a constitutively active form of AKT, was associated with increased general metabolic activity (glycolysis and respiratory capacity, assessed by Seahorse) as well as in specific metabolic pathways (assessed by LC-MS). The degree of AKT activity reduction after CDK2 inhibition and AKT activity increase in response to CDK2 overexpression prompted us to investigate additional potential functional interactions of CDK2 with AKT, apart from cell cycle-specific regulation. The PTEN inhibitor HOpic (PTENi) is able to increase AKT activity within minutes, but the PTENi-induced increase of AKT activity was completely eliminated by CDK2 inhibition (AZD-5438 - CDK1/2/9 inhibitor). This suggests that CDK2 acts on AKT within a feed-forward loop that is cell cycle-independent. Further details of interaction between CDK2 and AKT will be presented. Lastly, oncogenic AKT activation by PTEN deletion not only increased AKT activity in general, but also eliminated cell cycle dependent dynamics of AKT activity. Conclusion. We have shown cell cycle- and CDK2-dependent dynamics of AKT activity in GCB-DLBCL, responsible for stimulation of metabolism at the beginning of the S phase of the cell cycle. Additionally, CDK2 seems to be involved in a critical feed-forward AKT activation loop. PTEN deletion leads to easier G1-S transition on the background of increased AKT and metabolic activity. Supported by MHCR (DRO - VFN00064165), National Institute for Cancer Research (EXCELES - LX22NPO5102), and MEYSCR (Cooperatio, SVV 260637).
Rajmonová et al. (Mon,) studied this question.