Abstract 2603: Inhibition of Notch and mTOR by nelfinavir as a novel approach for T cell acute lymphoblastic leukemia (T-ALL)

Abstract

Abstract Background: T-ALL accounts for 10-15% of childhood ALL and 25% of adult ALL. Although intensive chemotherapy improves disease free survival, patients who relapse or progress have a dismal prognosis. Notch1 is a promising target in T-ALL because it is commonly activated, and inhibitors of γ-secretase (GSI), an aspartic protease that processes Notch1, are in clinical trials. Nelfinavir is an HIV protease inhibitor with anti-cancer activity through its inhibition of the Akt/mTOR pathway as well as unknown mechanisms. Because the HIV protease is an aspartic protease, we hypothesized that a target for nelfinavir in cancer cells could be γ-secretase. Methods: Nelfinavir was compared to compound E, a commercially available GSI, in in vitro studies. Notch1 and mTOR signalling was evaluated in human and murine T-ALL cell lines by immunoblotting. γ-secretase activity was assessed in cell-free in vitro assays. Cell death was measured by flow cytometry after annexin V and propidium iodide staining. Nelfinavir was also tested in a transgenic model of T-ALL (established by overexpression of SCL and LMO1 in the mouse thymus) using microCT scans, flow cytometry and IHC. Results: Among 7 HIV protease inhibitors evaluated, only nelfinavir inhibited expression of the Notch1 transmembrane and intracellular domains as well as Hes1 and survivin, well-established downstream targets of Notch1. At clinically achievable concentrations, nelfinavir rapidly inhibited Notch1, which was sustained to 16 hr when death was evident. Cell-free assays showed that although compound E was a more potent inhibitor of γ-secretase, the maximal enzymatic inhibition by nelfinavir was equivalent at higher doses. Nelfinavir was far more effective than compound E in reducing expression of Notch targets such as phospho-Rb, c-Myc, and Hrb, Hes1, and survivin. Nelfinavir induced apoptosis to levels equivalent to 20 µM etoposide, but compound E was only cytostatic. To explore other mechanisms that could explain the decreased protein expression and higher levels of cell death with nelfinavir, we assessed the Akt/mTOR pathway. Nelfinavir rapidly induced AMPK activation and mTOR inhibition, coincident with Notch1 inhibition. In SCL-LMO1 double transgenic mice, nelfinavir decreased thymic size and markedly increased apoptotic cells in the thymus. It also decreased the CD3+ populations in spleen and bone marrow and decreased CD4+CD8+ double positive leukemic cells. Conclusion: These studies show that nelfinavir is superior to a GSI and highly active in preclinical studies of T-ALL. Clinical testing of nelfinavir in T-ALL patients is planned. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2603. doi:10.1158/1538-7445.AM2011-2603