Regulation of PTEN by CK2 and Notch1 in primary T-cell acute lymphoblastic leukemia: rationale for combined use of CK2- and  -secretase inhibitors

Abstract

AS and PYJ contributed equally to this work. Acknowledgments: The authors would like to thank the contribution of patients and the clinical teams involved in providing primary leukemia samples, Dr. Miguel Abecasis for providing thymic specimens, and Dr. Adolfo Ferrando for providing some of the NOTCH sequencing primers. Funding: this work was supported by grants from Fundação para a Ciência e a Tecnologia (FCT; POCI/SAU-OBS/58913 and PTDC/SAU-OBD/69974), and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; 08/10034-1), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; 401122/2005-0). AS and PYJ have FCT SFRH and FAPESP PhD scholarships, respectively. Manuscript received on May 27, 2009. Revised version arrived on September 18, 2009. Manuscript accepted on October 8, 2009. Correspondence: João T. Barata, Cancer Biology Unit, Instituto de Medicina Molecular, Lisbon University Medical School, Av. Prof. Egas Moniz, 1649 .pt The Online version of this article has a Supplementary Appendix.T-cell acute lymphoblastic leukemia (T-ALL) patients frequently display NOTCH1 activating mutations and Notch can transcriptionally down-regulate the tumor suppressor PTEN. However, it is not clear whether NOTCH1 mutations associate with decreased PTEN expression in primary T-ALL. Here, we compared patients with or without NOTCH1 mutations and report that the former presented higher MYC transcript levels and decreased PTEN mRNA expression. We recently showed that T-ALL cells frequently display CK2-mediated PTEN phosphorylation, resulting in PTEN protein stabilization and concomitant functional inactivation. Accordingly, the T-ALL samples analyzed, irrespectively of their NOTCH1 mutational status, expressed significantly higher PTEN protein levels than normal controls. To evaluate the integrated functional impact of Notch transcriptional and CK2 post-translational inactivation of PTEN, we treated T-ALL cells with both the gamma-secretase inhibitor DAPT and the CK2 inhibitors DRB/TBB. Our data suggest that combined use of gamma-secretase and CK2 inhibitors may have therapeutic potential in T-ALL. Haematologica. 2010;95:674-678. doi:10.3324/haematol.2009 This is an open-access paper. ABSTRACT 674haematologica | 2010; 95(4) © F e r r a t a S t o r t i F o u n d a t i o nIn some cases, exon 26 and exon 27 were amplified and sequenced from genomic DNA with the intronic primers Notch26F and Notch26R, and Notch27F and Ex27R743, respectively. The region spanning TAD and PEST domains of exon 34 was amplified with primers Notch34TF and Notch34PR, and the resulting 855 bp fragment was sequenced with primers Notch34TF and Ex34PestF. The PEST coding region was also amplified with primers Ex34PestF and Ex34PestR, followed by semi-nested PCR with primers Notch34PF and Ex34PestR, and sequenced with primer Ex34PestR. This strategy covered all mutational hot-spot regions previously reported for NOTCH1. 1,6,7 Primer sequences and the PCR protocol are shown in the Online Supplementary Appendix. All mutations were c...

Targeting the translational apparatus to improve leukemia therapy: roles of the PI3K/PTEN/Akt/mTOR pathway

“…PTEN oxidization has been proposed to contribute to the development of T-cell acute lymphoblastic leukemia (T-ALL), which displays abnormally high levels of ROS. 27,55 The peroxidase peroxiredoxin1 (Prdx1) gene product regulates PTEN in response to ROS. 56 In Prdx1-deficient mouse fibroblasts and mammary epithelial cells, Akt is hyperactive.…”

“…57,58 There are multiple kinases that phosphorylate PTEN, including casein kinase 2, and GSK-3b, which may occur at other residues including T366 and S370. 55,59,60 Proteins such as PICT1 and RAK were initially identified as proteins that bind PTEN. These proteins may promote PTEN phosphorylation.…”

“…It has been proposed that both casein kinase 2 and g-secretase inhibitors (which alter Notch cleavage and activation) could be of therapeutic value in T-ALL owing to their capability of affecting PTEN regulation. 55 Another negative regulator of the PI3K pathway is the pleckstrin homology domain leucine-rich repeat protein phosphatase. Pleckstrin homology domain leucine-rich repeat protein phosphatase dephosphorylates S473 on Akt, which induces apoptosis and inhibits tumor growth.…”

Protein kinase CK2 in hematologic malignancies: reliance on a pivotal cell survival regulator by oncogenic signaling pathways

“…In a follow-up work, the same group demonstrated the potential therapeutic efficacy of targeting CK2 with TBB in combination with the inhibition of Notch-dependent signaling through the use of gamma-secretase inhibitors, in T-ALL samples. 43 CK2 in chronic lymphocytic leukemia In addition to having a role in immature lymphoid precursorderived blood tumors, CK2 has recently been implicated also in the pathogenesis of B-chronic lymphocytic leukemia (B-CLL).…”

Intracellular reactive oxygen species are essential for PI3K/Akt/mTOR-dependent IL-7-mediated viability of T-cell acute lymphoblastic leukemia cells

“…21 Proteins were analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted onto a nitrocellulose membrane (Bio-Rad, Amadora, Portugal) and probed with relevant antibodies raised against: ZAP-70 (Upstate Biotechnology, Lake Placid, NY, USA), actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and p-Akt (S473), p-GSK3b (S9), p-FOXO3a (T32) and p-S6 (S235/236) (Cell Signaling, Danvers, MA, USA). Immune complexes were detected with appropriate horseradish peroxidase-conjugated secondary antibodies.…”