Metabolic Re-Programming in Notch-Activated T-ALL By mTOR Inhibitor AZD2014 Combined with L-Asparaginase
Authors: Tabe Yoko , Harada Masako , Miyamae Yuka , Fujimura Tsutomu , Kazuno Saiko , Ueno Takashi , Miida Takashi , Andreeff Michael , Konopleva Marina 
Published: October 2019

Abstract
Adult T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis associated with resistance and rapid relapses, necessitating novel therapeutic strategies. Malignant T cells require high concentrations of nutrients to sustain their increased rates of proliferation, and frequent activation of mTOR signaling, a central regulator of cell metabolism, has been shown in a high-risk subgroup of T-ALL with constitutive Notch activation (Chan, Blood, 2007;110,278).
In this study, we analyzed the metabolic response mechanisms of Notch-activated aggressive T-ALL to the ATP-competitive mTOR inhibitor AZD2014 and to the combination of AZD2014 with a conventional chemotherapeutic agent, L-asparaginase (ASNase). To investigate the molecular modifications of key cellular metabolic processes induced by these agents, we undertook a comprehensive and quantitative analysis of charged metabolites by capillary electrophoresis mass spectrometry (CE-MS, Agilent Technologies) for metabolic profiling.
We first assessed the anti-proliferative effects of the combination of AZD2014 and ASNase in CUTLL1, a T-ALL cell line with a Notch gain-of-function mutation. The medianinhibitory concentrations of AZD-2014 and ASNase were 250 nM and 0.005 U/mL, respectively (MTT assay, 48 hours). The combination of AZD2014 (100 nM) and ASNase (0.001 U/mL) caused apoptosis induction with increase in the % of sub-G1 fraction (Control: 5.5±1.8, AZD-2014: 9.5±2.1, ASNase: 10.9 ±1.5, AZD-2014/ASNase: 17.9 ±4.9; p<0.05). Inhibition of mTOR kinase activity by AZD2014 resulted in downregulation of mTORC1 substrates phospho-(p-)S6K (Ser240-244) and p-4EBP1 (Thr37/46) and mTORC2 target p-Akt (Ser473) and induced autophagy, as shown by the conversion of the autophagosomal marker LC3-I to LC-II. AZD2014 further impaired the expression of oncogenic transcription factor c-Myc, an intermediary between Notch and mTOR signaling. In turn, ASNase downregulated c-Myc expression without inhibiting mTOR signaling.
We next investigated the metabolic changes in CUTLL1 cells after AZD2014 (100 nM) and/or ASNase (0.001 U/mL) treatment (24 hours). CE-MS analysis demonstrated differences in 66 polar metabolites (p<0.05). As expected, ASNase downregulated L-asparagine monohydrate levels. Although AZD2014 did not change the level of L-asparagine monohydrate by itself, it significantly enhanced its depletion by ASNase. The AZD2014 and ASNase combination also impaired aspartate synthesis. In the TCA cycle, the AZD2014 / ASNase combination significantly downregulated gluconeogenesis by decreasing malate and fructose-6-phosphate and inhibited fatty acid synthesis with citrate depletion. In nucleotide metabolism, the combination treatment suppressed guanine and hypoxanthine levels, which led to decreases in uric acid. Finally, glutamate metabolism was markedly downregulated by depleting intracellular glutamine. All these changes were less prominent in CUTLL1 cells treated with single-agent AZD2014 or ASNase.
These results indicate that AZD2014 effectively facilitates the ASNase-induced blockade of glycolysis/gluconeogenesis and fatty acid synthesis and represses asparate and glutamate metabolism in Notch-activated T-ALL cells. The multiple molecular alterations resulting from mTOR blockade by AZD2014, such as repression of translation initiation through potent inhibition of S6K and 4EBP1, induction of autophagy, and inhibition of c-Myc, may further impair the cellular metabolism altered by ASNase in aggressive T-ALL. In conclusion, metabolic re-programming by the combination of AZD2014 and L-asparaginase in Notch-activated T-ALL provides avenues for novel rationally designed combination regimens in ALL.

Disclosures
No relevant conflicts of interest to declare.