Mechanisms of regulation of the PI#K/AKT pathway downstream of NOTCH1 in T-ALL.

Background

Aberrant activation of NOTCH signaling due to the presence of activating mutations in the NOTCH1 receptor plays a critical role in the pathogenesis of human T-cell acute lymphoblastic leukemia (T-ALL). The identification of NOTCH1 mutations in more than 50% of T-ALL cases has prompted the initiation of clinical trials to test the effectiveness of blocking NOTCH1 signaling with gamma-secretase inhibitors (GSIs) in this disease. However, GSIs are active only in a small fraction of T-ALL cell lines with NOTCH1 mutations, suggesting that mechanisms responsible for GSI resistance are readily present in most human T-ALL cell lines and may limit the effectiveness of GSIs as antileukemic agents.

Our preliminary data shows that inhibition of NOTCH1 signaling with GSIs is associated with upregulation of the PTEN tumor suppressor gene and decrease activity of the PI3K-AKT signaling pathway suggesting that PTEN regulation downstream of NOTCH1 may mediate some of the leukemogenic effects of oncogenic NOTCH1. Furthermore, we have found mutational loss of PTEN with concomitant overactivation of the PI3K-AKT signaling pathway in 5/5 GSI resistant T-ALL cell lines, which despite harboring prototypical NOTCH1 mutations and high levels of NOTCH1 signaling fail to undergo cell cycle arrest and apoptosis upon inhibition of NOTCH1 activation with GSIs.

Project Goal:

Based on these results, we postulate that the activity of the PI3K-AKT pathway mediates, at least in part, the oncogenic effects of aberrant NOTCH1 signaling and that mutational loss of PTEN and constitutive activation of PI3K-AKT promotes cell growth, proliferation and survival, rendering T-ALL cells insensitive to NOTCH1 inhibition.

However important questions remain to be solved: What is the mechanism of PTEN regulation downstream of NOTCH1? How does constitutive activation of PI3K-AKT bypass the requirement for continuous NOTCH1 signaling to maintain cell growth, metabolism and survival in T-ALL? Does inhibition of constitutive active PI3K-AKT signaling pathway restore sensitivity of PTEN-null T-ALL cells to GSIs? To resolve these important mechanistic and therapeutic questions we propose the following specific aims:

• Analyze the mechanism(s) downstream of NOTCH1 that regulate the expression of PTEN in T-ALL.
• Analyze the functional consequences in cell growth, glucose metabolism, proliferation and survival of activation of the PI3K-Akt signaling due to the loss of the PTEN in the NOTCH1 induced T-ALL.
• Analyze the interaction between NOTCH and AKT signaling in the response of T-ALL cell lines to molecularly targeted drugs

The elucidation of the mechanisms that mediate the interaction of NOTCH1 and the PI3K-AKT pathway and the specific pathways involved in GSI resistance in T-ALL will provide the background to develop novel and more effective therapies targeting NOTCH1 in the treatment of T-ALL.
 

Project Update 2024:

Acute leukemias represent the most frequent type of cancer (~30%) in children and young adults. The lack of robust systems for in vitro culture of primary leukemia samples is a significant barrier for the development of effective genetic and chemical screens for novel therapeutic targets in pediatric leukemia. The work funded by the Alex’s Lemonade Foundation has allowed us to establish an advanced human, patient-specific in vitro model of leukemic bone marrow that could potentially be used for evaluating therapeutic intervention. Our team has developed a highly relevant novel multicellular model of engineered bone marrow (eBM) by including stromal components into a 3D bone scaffold along with healthy hematopoietic stems cells. We have demonstrated that this “bone-marrow on a chip” model creates biologically meaningful conditions that support infiltration and engraftment maintenance of phenotype of malignant blasts. More importantly, throughout this project we have demonstrated that (i) the combination of all supporting populations in BM is crucial in supporting difficult-to-culture acute lymphoblastic leukemia (ALL) primary cells; (ii) eBM outperforms animal xenografts and gold-standard monolayer cultures; and (iii) eBM niche protects ALL cells from therapeutic treatment of BCL-2 inhibitor ABT-199, as compared to monolayers, highlighting the role of incorporating stromal components for modeling targeted therapies in vitro. Overall, our eBM model will advance studies of the human BM during malignant transformation and support the development of personalized therapeutics in pediatric leukemia.