AKT regulation of oncogenic Notch pathway in T-cell acute lymphoblastic leukemia

Background: Notch and PI3K/AKT signaling are two key oncogenic pathways closely associated in T-cell acute lymphoblastic leukemia (T-ALL). These pathways collaborate in controlling proliferation, survival and migration of T-ALL cells and are deregulated in ~60% (Notch pathway) and 48% (PI3K/AKT) of T-ALL patients. Recent evidences indicate that in T-ALL cells, Notch and PI3K/AKT pathways collaborate through a reciprocal control. Aim of this work was to investigate the molecular mechanisms of PI3K/AKT-dependent Notch1 activity regulation. Methods: T-ALL cell line, Molt4, and HEK293T cell line were grown in RPMI-1640 and DMEM respectively, supplemented with 10% heat-inactivated FBS. 1 mg RNA isolated from cells was retro-transcribed in 20 ml by M-MuLV reverse transcriptase using random hexamer primers. RT-PCR analysis was performed using primers for Notch1, HES1, preTCRa, GAPDH. Apoptotic cells were identified by Annexin-V and propidium iodide staining. Protein expression was detected by Western blot analysis of whole cell lysates. HEK293T cells were transfected with expression plasmid encoding the dominant negative mutant form of AKT (DN-AKT) and with plasmid encoding full length Flag-tagged NOTCH1 using the DMRIE-C reagent (Invitrogen), according to manufacturer’s instructions. Molt4 cells were electroporated with 20 µg of pcDNA3.1 DN-AKT or mock pcDNA3.1 using the Neon Transfection System (Invitrogen) according to the manufacturer’s guidelines. After 72 h cells were harvested to prepare protein lysates. Immunoprecipitation of ubiquitin-conjugated proteins was performed using the UbiQapture-Q Kit (Biomol, Exeter, UK), as described by the manufacturer. Co-immunoprecipitation analysis was performed using Protein G Agarose beads, eluted immunoprecipitates were analyzed by Western blot. Immunofluorescent staining was done on HEK293T cells incubated with anti-Flag or anti-c-Cbl primary antibodies and the appropriate AlexaFluor-conjugated secondary antibodies. Images were acquired with a Leica TCS SP2 confocal microscope. A colocalization area was determined based on a 2D cytofluorogram and density analysis performed by Multicolor Analysis Leica Confocal software. Results: Both LY294002-mediated chemical inhibition of PI3K/AKT signaling and transient transfection of a DN-AKT mutant strongly reduced Notch1 protein level and activity, without affecting Notch1 transcription. We showed that downstream AKT regulator, GSK-3β, did not mediate the effect of PI3K/AKT withdrawal on Notch1 protein. We demonstrated that Notch1 protein decrease upon PI3K/AKT inhibition was due to lysosomal degradation of the Notch1 membrane-bound form. IP and Co-IP analyses revealed that PI3K/AKT withdrawal in Molt4 cells resulted in an increased tyrosine phosphorylation of Notch1 and monoubiquitination of Notch1 as detected by ubiquitin capture assay. Co-immunoprecipitation assay and co-localization analysis further showed that E3 ubiquitin ligase, c-Cbl, interacted with Notch1 in order to direct it into the lysosome for degradation. Conclusions: To our knowledge, our results provide the first evidence of mechanism by which AKT pathway controls Notch1 activity reducing the amount of protein undergoing to lysosomal degradation. Given the crucial role of Notch1 in T-ALL, our findings suggest that hyperactive AKT signaling in T-ALL may contribute to increase the oncogenic Notch signaling in T-ALL independently from mutations in Notch1. Therefore a therapeutic strategy directed to PI3K/AKT signaling in T-ALL could provide advantages to inhibit the dysregulated NOTCH signaling.