Zucal, Chiara (2016) Molecular Effects of the Nampt Inhibitor FK866 on Leukemia Cells. PhD thesis, University of Trento.
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Aberrant activation of metabolic pathways has emerged as an hallmark of proliferating cancer cells and pharmaceutical approaches targeting cell metabolism hold great potential for cancer treatment. A critical factor in cellular metabolism is nicotinamide adenine dinucleotide (NAD+) and cancer cells highly rely on it to face increased metabolic demands and proliferation rates. Intracellular NAD+ is a key metabolite involved in several cellular processes, acting either as a coenzyme in redox reactions or as a substrate for NAD+-degrading enzymes such as poly (ADP-ribose) polymerases (PARPs), CD38, and sirtuins, regulating processes that undergo fundamental changes during malignant transformation. Although NAD+ can be generated de novo from tryptophan precursor, the major route of biosynthesis is through a nicotinamidesalvage process. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in NAD+ biosynthesis from nicotinamide in mammalian cells. A number of cancers present an increased expression of NAMPT, and high NAMPT levels have been shown to be essential to support cancer cell growth, survival and EMT transition and to correlate with adverse prognosis. NAMPT is therefore a key factor regulating tumor cell metabolism and is thus considered a promising anti-cancer target. FK866 is a specific NAMPT inhibitor that lowers NAD+ concentration in cancer cells, reducing the activity of NAD+-dependent enzymes, impacting on ATP production and promoting cell death. NAMPT inhibition was proven to be highly effective in both lymphoid and myeloid-derived hematological malignancies in preclinical studies without affecting healthy cells, such as hematopoietic stem cells. FK866 has completed a phase I trial in oncology with advanced solid tumors. Thrombocytopenia was the dose-limiting toxicity, suggesting that this drug is a good candidate for clinical applications. We investigated the mechanism of action of FK866 in T-ALL derived cell lines as well as in primary leukemia cells. FK866-induced metabolic stress and NAMPT ablation elicited a strong arrest of protein synthesis as early cell response. FK866 induced activation of the AMP-activated protein kinase (AMPK), which subsequently drove the inhibition of the mTOR/4EBP1 signaling cascade and of the major initiation factor EIF2A, impairing protein synthesis. Furthermore, FK866-induced stress reduced the levels of the anti-apoptotic protein MCL1 and impacted on the endoplasmic reticulum homeostasis. In addition, we established and characterized an FK866-resistant model derived from the T-ALL cell line Jurkat. Target-specific acquired resistance has been described after several therapies and can be modeled in vitro by growing cells in presence of increasing concentrations of drug. In our resistant cells, FK866 treatment only partially impacted on NAD+ content, whereas ATP levels were recovered and protein translation resumed. Notably, during in vitro acquisition of drug resistance, mutations in the NAMPT gene have not occurred. In the last years, many NAMPT inhibitors have been synthesized and characterized. The obtained results provide new insight into the role of the NAMPT-mediated NAD+ salvage pathway in cancer cell metabolism and the molecular mechanisms of FK866, which will be useful to formulate specific and effective combinatorial drug therapies.
|Item Type:||Doctoral Thesis (PhD)|
|Doctoral School:||Biomolecular Sciences|
|Subjects:||Area 05 - Scienze biologiche > BIO/11 BIOLOGIA MOLECOLARE|
|Repository Staff approval on:||09 Mar 2016 09:50|
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