Mutational loss of PTEN induces resistance to NOTCH1 inhibition in T-cell leukemia

Abstract

Gain-of-function mutations in NOTCH1 are common in T-cell lymphoblastic leukemias and lymphomas (T-ALL), making this receptor a promising target for drugs such as gamma-secretase inhibitors, which block a proteolytic cleavage required for NOTCH1 activation. However, the enthusiasm for these therapies has been tempered by tumor resistance and the paucity of information on the oncogenic programs regulated by oncogenic NOTCH1. Here we show that NOTCH1 regulates the expression of PTEN (encoding phosphatase and tensin homolog) and the activity of the phosphoinositol-3 kinase (PI3K)-AKT signaling pathway in normal and leukemic T cells. Notch signaling and the PI3K-AKT pathway synergize in vivo in a Drosophila melanogaster model of Notch-induced tumorigenesis, and mutational loss of PTEN is associated with human T-ALL resistance to pharmacological inhibition of NOTCH1. Overall, these findings identify transcriptional control of PTEN and regulation of the PI3K-AKT pathway as key elements of the leukemogenic program activated by NOTCH1 and provide the basis for the design of new therapeutic strategies for T-ALL.

Fig. 1. PTEN loss and AKT activation in GSI-resistant T-ALLs

(a) Nearest Neighbor analysis of genes associated with GSI sensitivity and resistance in T-ALL cell lines. Relative gene expression levels are color coded with red (higher levels of expression) and blue (lower levels of gene expression). (b) Western blot analysis of PTEN and p-AKT (Ser473) in T-ALL cell lines. AKT and α-tubulin are shown as loading controls. (c) Representative images of PTEN immunostaining in T-cell lymphoblastic tumors. PTEN positive cell are immunostained in brown. The panel on the top shows negative staining of T-ALL lymphoblasts with scattered non lymphomatous positive cells (arrowheads). The panel on the bottom shows diffuse cytoplasmic immunostaining in a PTEN positive sample. (d) Schematic representation of PTEN mutations identified in T-ALL samples. Scale bars represent 100μm.

Fig. 2. PTEN loss and AKT activation induce GSI resistance in T-ALL

(a and b) Decreased cell size (a; FSC-H) and decreased cell growth (b) induced by GSI treatment (CompE 100 nM for 4 days) are rescued by retroviral expression of a constitutive active AKT (Myr-AKT) in CUTLL1 cells. (c and d) shRNA knock-down of PTEN restores cell size defects (c) and reduced cell growth (d) of DND41 cells treated with GSI (CompE 100 nM for 4 days) compared to that of vehicle (DMSO) treated controls. No protective effect was observed by expression of a control shRNA targeting the luciferase gene (shRNA LUC). Mean FSC-H values for GSI and vehicle only treatment controls are indicated. Bar graphs represent means ± standard deviation of triplicate samples. P values were derived from Student's t-test.

Fig. 3. NOTCH1 regulates PTEN expression, AKT signaling and glucose metabolism

(a) Real-time PCR analysis of PTEN transcript levels upon NOTCH1 inhibition by GSI in CUTLL1 and HPB-ALL relative to (DMSO) controls. GAPDH levels were used as reference control. (b) Western blot analysis of PTEN and p-AKT (Ser473) in GSI sensitive T-ALL cell lines treated with CompE. AKT and α-Tubulin are shown as loading controls. (c) Real-time PCR analysis of Hes1 and Pten expression in mouse DN3 thymocytes cocultured with stromal cells (OP9) or stromal cells expressing the NOTCH1 ligand Delta-like-1 (OP9-DL1). Data are means +/- s.d. of duplicate (day 1) and triplicate (day 2) experiments. (d) Glucose uptake analysis in HPB-ALL and P12-ICHIKAWA T-ALL cell lines in basal conditions (vehicle treatment only). (e) Glucose oxidation analysis in HPB-ALL and P12-ICHIKAWA T-ALL cell lines in basal conditions (vehicle treatment only). (f) Effects of GSI treatment in glucose uptake in HPB-ALL and P12-ICHIKAWA T-ALL cells. (g) Effects of GSI treatment in glucose oxidation in HPBALL and P12-ICHIKAWA T-ALL cells. Data shown in d-g are means ± standard deviation of triplicates. P values in a, c-g were derived from Student's t-test.

Fig. 4. HES1 and MYC regulate PTEN expression downstream of NOTCH1

(a) Quantitative ChIP analysis of HES1 binding to PTEN promoter sequences. (b) Quantitative ChIP analysis of c-MYC binding to PTEN promoter sequences. Data are means ± standard deviation of triplicates. TIS: transcription initiation site. (c) Effects of HES1 and MYC expression in PTEN promoter activity. Luciferase reporter assays were performed in 293T cells with a 2,666 bp PTEN promoter construct (pGL3 PTEN HindIII-NotI). Data are means +/- s.d. of triplicates. (d) Lentiviral shRNA knock-down of HES1 in CUTLL1 cells induces transcriptional upregulation of PTEN. Expression of a control shRNA targeting the luciferase gene (shRNA LUC) was used as control.

Fig. 5. Interaction of Notch and Pten/PI3K/Akt signaling in growth control and tumorigenesis in Drosophila

(a-h) The size and morphology of female adult eyes from identically reared animals [at 25°C] were scored in six different genotypes: (a) wild type; (b) ey-Gal4>Dl; (c-d) ey-Gal4>Dl/+; GS1D233C (Akt1)/+; (e) ey-Gal4>Dl/+ treated with 1mM DAPT. (f) ey-Gal4>Dl/+; UAS-Pten/+ (g) ey-Gal4>UAS-fng (h) ey-Gal4>UAS-fng/+; GS1D233C (Akt1)/+. (b) Generalized expression of Delta by the eye-specific driver eyeless (ey)-Gal4 results in mild eye overgrowth (130% eye size compared with wild type). (c and d) Co-overexpression of Delta and Akt1 in the developing eye results in massive eye overgrowth (190-230% eye size) in 100% of the flies analyzed (n>200) (c) and metastases in distant tissues within the thorax (7.14% of mutant flies, n=232) (d, white arrow). (e) Inhibition of Notch receptor proteolysis by nonlethal doses (1mM) of the GSI DAPT inhibits Delta-induced overgrowth and results in flies with reduced eyes (eye size of 72% compared with wild type size) and wings smaller than wild type (see Supplementary Fig. 9 online). (f) Gain of Pten results in strong suppression of Delta-mediated eye overgrowth (eye size of 46% compared with siblings ey-Gal4>Dl) (n>160). (g) Overexpression of fringe (UAS-fng), a Notch pathway modulator, results in Notch inhibition in the eye and hence a small eye defect (5-13% eye size). (h) Gain of expression of Akt1 gene using the GS1D233C P-element fully rescued growth defect (95-106% eye size) caused by reducing Notch pathway activation (see also Supplementary Figs. 6 and 7).

Fig. 6. Transcriptional networks downstream of NOTCH1 in T-ALL and effects of pharmacologic inhibition of AKT in T-ALL cells

a. Schematic representation of the transcriptional regulatory networks controlling cell growth downstream of NOTCH1 in PTEN-positive/GSI-sensitive and PTEN-null/GSI-resistant T-ALL cells. The dashed arrow indicates a weak positive effect of MYC on PTEN expression compared with the strong negative transcriptional effects of HES1 in the promoter of this gene. b. Relative cell growth of GSI-sensitive/PTEN-positive and GSI-resistant/PTEN-null T-ALL cell lines treated with the SH6 AKT inhibitor at 10 μM concentration for 72 hours. Data are means ± standard deviation of triplicates.