Mutational Loss of PTEN Induces Resistance to NOTCH1 Inhibition in T-ALL.

Abstract

Activating mutations in NOTCH1 are common in T-cell lymphoblastic leukemias (T-ALL), making this receptor a promising target for drugs such as gamma-secretase inhibitors (GSIs), which block NOTCH1 activation. However, enthusiasm for these therapies has been tempered by tumor resistance and the paucity of information on the oncogenic programs regulated by NOTCH1. Here, we identify the loss of the PTEN tumor suppressor gene and activation of the PI3K-AKT signaling pathway as critical factors that determine the resistance of T-ALL cells to inhibition of NOTCH1 signaling with GSIs. Mutational loss of PTEN is found in 17% of T-ALL cases and in the majority of T-ALL cell lines. Importantly, 8/8 T-ALL lines sensitive to NOTCH inhibition with GSIs retain wild type PTEN, while this tumor suppressor is lost in 8/8 GSI-resistant T-ALLs analyzed (P<0.001). Furthermore, both the expression of a constitutively active form of AKT (Myr AKT) and PTEN shRNA knockdown induced resistance to GSIs in T-ALLs and promoted cell growth, proliferation and glucose metabolism. The close association between GSI resistance and PTEN loss prompted us to analyze the interaction between NOTCH1 signaling and the PI3K-AKT pathway. Analysis of normal and leukemic T-cells demonstrated that NOTCH1 signaling regulates PTEN expression and AKT signaling. Thus, inhibition of NOTCH1 with GSIs results in transcriptional upregulation of PTEN and concomitant downregulation of PI3K/AKT signaling in T-ALL. A similar effect -transcriptional upregulation of Pten upon loss of Notch signaling- was observed in primary mouse thymocytes, which are highly dependent on Notch1 to sustain the activity of the Akt signaling pathway. ChIP-on-chip and reporter assays demonstrate that regulation of PTEN is mediated by HES1, a transcriptional repressor directly controlled by NOTCH1. In agreement with these observations, HES1 shRNA knockdown induced transcriptional upregulation of PTEN in T-ALL cells. These results were perfectly recapitulated in a Drosophila model of Notch-induced tumorigenesis. Thus, activation of Notch signaling via expression of Delta and activation of the PI3K-AKT pathway by Akt showed marked synergism in tumor formation in the fly eye. Importantly, also in Drosophila, activation of Akt reverses the growth defect phenotype induced by the loss of Notch signaling, highlighting the importance of the interaction between these two pathways for the control of cell growth. Finally, we proposed that mutational loss of PTEN could induce an oncogene addition switch that makes T-ALL cells resistant to NOTCH inhibitors but enhanced their sensitivity to AKT inhibitors. Treatment with SH-6, a phosphatidylinositol analog inhibitor of AKT, showed a strong antileukemic effect against GSI-resistant/PTEN-null T-ALLs but not against GSI-sensitive/PTEN-positive cells, confirming this hypothesis. These results demonstrate the importance of the interaction of NOTCH1 with the PI3K-AKT pathway in T-cell homeostasis and response to therapy and provide the basis for the design of new therapeutic strategies for T-ALL.