TARGETING CLASS I PI3KS IN THE TREATMENT OF T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

Background: Aberrant NOTCH1 activation as well as loss of PTEN function is known to play a critical role in the pathogenesis of human T-cell acute lymphoblastic leukemia (T-ALL), an aggressive hematologic malignancy that accounts for 15% of pediatric cases. Despite the overall success rate of 80% in acute leukemia, 25% of children and adolescents with T-ALL will die. Much hope was placed on gamma-secretase inhibitors (GSIs) due to the role that NOTCH1 plays in this disease. However, the anti-tumor activity of these inhibitors in clinical trials appeared limited and was associated with severe gastrointestinal toxicity, necessitating the urgent search for additional pathways essential for T-ALL cell proliferation and survival.One such possibility is class I phosphatidylinositol 3-kinases (PI3Ks), composed of PI3Kalpha, PI3Kbeta, PI3Kgamma, and PI3Kdelta, which regulate cell growth, proliferation, and survival by virtue of their ability to activate the downstream effector molecule Akt/PKB. Interestingly, the Ferrando lab has demonstrated a direct correlation between PTEN null T-ALL and its resistance to GSI due to the unregulated activity of the PI3K pathway. PTEN is a phosphatase that inactivates the second messenger phosphatidylinositol (3,4,5) trisphosphate (PIP3), which is generated by PI3Ks in response to stimuli such as growth factors. In its absence, PIP3 accumulates and ultimately increases the activity of Akt, promoting the proliferation and survival of TALL cells even when NOTCH1 activity is blocked by GSI. Previously, the Diacovo lab has shown that mice lacking both PI3Kgamma and PI3Kdelta activity have a significant impairment in the development and survival of T cells from which T-ALL originates. Other than the obvious perturbation in immune function, these animals are viable, have no major defects in vital organ system or perturbation in glucose hemostasis. By contrast, absence of PI3Kalpha or PI3Kbeta is embryonic lethal and both are known to regulate glucose metabolism. Based on these observations, we propose that dual inhibition of PI3Kgamma and PI3Kdelta may have a profound effect on the development and survival of T-ALL with minimum toxicity.Objectives/Hypothesis: The goals of this proposal are to (1) validate PI3Kgamma and PI3Kdelta as potential therapeutic targets for the treatment of T-ALL, (2) demonstrate that it is feasible to generate a small molecule inhibitor that blocks the activity of both of these PI3Ks and (3) that such an inhibitor can be effective in reducing the extent of disease in a preclinical model of human T-ALL. We believe these objectives can be achieved, as our results indicate that genetic deletion of PI3Kgamma and/or PI3Kdelta impairs or prevents NOTCH1 or PTEN-induced T-ALL in mice. In addition, treatment of NOTCH1 activated/PTEN null human TALL cell lines with a newly developed PI3Kgamma/delta dual inhibitor dramatically reduces cell survival and Akt activation.Specific Aims:1) Determine the role of PI3Kgamma and PI3Kdelta in tumor cell maintenance in genetic models of T-ALL.2) Analyze the mechanisms that mediate the anti-leukemic effects of PI3Kgamma and PI3Kdelta inhibition in T-ALL.3) Perform preclinical testing of a dual inhibitor in primary tumor models of human T-ALL in vitro and in vivo.Study Design: To further delineate the role of PI3Kgamma and PI3Kdelta in tumor maintenance; that is, once the disease has been established, we will utilize Cre/Lox system in which these PI3Ks can be genetically deleted upon administration of tamoxifen (Rosa26Cre-ERT2). For NOTCH1-induced tumors, bone marrow will be harvested from mice in which the catalytic domains of PI3Kgamma and PI3Kdelta have been flanked by LoxP sites and then infected with a retroviral construct expressing a constitutively active form of NOTCH1. Irradiated isogeneic wild-type mice will be reconstituted with these cells and once evidence of T-ALL confirmed, the animals treated with control or tamoxifen. For PTEN-induced tumors, PI3K floxed mice will be bred on to a PTEN heterozygous background, which has been shown to induce T-ALL, and treated with tamoxifen once the disease has been established. To analyze the anti-leukemic mechanisms of PI3K inhibition, T-ALL cells from mice and human cell lines will be used to identify the role of PI3Kgamma and PI3Kdelta in regulating Akt activation and its downstream effectors involved in protein synthesis, cell metabolism, and survival. Lastly, we will utilize a novel xenograft animal model that supports the growth of primary human T-ALL cells to determine the ability of a newly developed PI3Kgamma/delta dual inhibitor to reduce tumor cell burden and prolong survival.Impact: These studies will be the first to demonstrate that (1) the initiation of T-ALL, whether through NOTCH or PTEN, relies primarily on PI3Kgamma and PI3Kdelta, (2) it is feasible to develop a dual inhibitor of these PI3Ks, and (3) such inhibitors may prove beneficial in the treatment of T-ALL.