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

Abstract

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.

Figure 1.

NOTCH1 mutations associate with decreased PTEN expression in primary T-ALL. Diagnostic T-ALL specimens classified as NOTCH1 mutated (NOTCH-Mut; n=9) or wild-type (NOTCH-WT; n=9, except for PTEN analysis n=8) were compared by Q-PCR for mRNA expression of (A) HES1 (P=0.436), (B) MYC (P=0.004), and (C) PTEN (P=0.021). Transcript copy numbers were normalized with respect to the number of ABL transcripts and transformed into logarithmic values. (D) NOTCH-WT and NOTCH-Mut samples were analyzed by Western blot for PTEN protein expression. Actin was used as a loading control. Densitometry analysis values of PTEN normalized to Actin are shown for each case. (E) PTEN protein levels in NOTCH-WT T-ALL (n=3), NOTCH-mut T-ALL (n=5) and normal thymocytes (n = 6) were evaluated by densitometry analysis after Western Blot and normalized to the Actin loading control. ** P<0.01; *** P<0.0001. (F) Primary-like leukemia TAIL7 cells were cultured with or without 1 or 5μM DAPT. At the indicated time points, cells were lysed and evaluated by Western blot for the expression of cleaved (activated) Notch (Val1744), PTEN, and P-PTEN(S380). ZAP-70 was used as a loading control. Data are representative of two independent experiments.

Figure 2.

GSI and CK2 inhibitors collaborate in decreasing T-ALL cell proliferation. PTEN-positive T-ALL cell lines HPB-ALL (A and C) and TAIL7 (B), and primary T-ALL cells (D), were used to test the cooperative impact of combined treatment with the GSI DAPT and the CK2 inhibitors DRB or TBB, at the indicated concentrations (see Design and Methods section). (A) Cells were cultured for 72h and evaluated for alterations in cell size by forward scatter flow cytometry analysis. (B and C) After seven days of culture, total cell counts (B) were calculated by trypan blue exclusion using a hemocytometer, and proliferation (C) was evaluated by assessing DNA synthesis by ³H-thymidine incorporation. *P<0.05; **P<0.01. (D) Primary T-ALL cells from 3 different patients were assessed for cell viability by flow cytometry at 72h of culture. P=0.020; One-way ANOVA.