Activating mutations in NOTCH1 are common in T-cell acute lymphoblastic leukemia (TALL). Here we identify glutaminolysis as a critical pathway for leukemia cell growth downstream of NOTCH1 and a key determinant of clinical response to anti-NOTCH1 therapies. Mechanistically, inhibition of NOTCH1 signaling in T-ALL induces a metabolic shutdown with prominent inhibition of glutaminolysis and triggers autophagy as a salvage pathway supporting leukemia cell metabolism. Consequently, both inhibition of glutaminolysis and inhibition of autophagy strongly and synergistically enhance the antileukemic effects of anti-NOTCH1 therapies. Moreover, we demonstrate that Pten loss induces increased glycolysis and consequently rescues leukemic cell metabolism abrogating the antileukemic effects of NOTCH1 inhibition. Overall, these results identify glutaminolysis as a major node in cancer metabolism controlled by NOTCH1 and as therapeutic target for the treatment of T-ALL.

Oxidative stress can be defined as the imbalance between cellular oxidant species production and antioxidant capability. Reactive oxygen species (ROS) are involved in a variety of different cellular processes ranging from apoptosis and necrosis to cell proliferation and carcinogenesis. In fact, molecular events, such as induction of cell proliferation, decreased apoptosis, and oxidative DNA damage have been proposed to be critically involved in carcinogenesis. Carcinogenicity and aging are characterized by a set of complex endpoints, which appear as a series of molecular reactions. ROS can modify many intracellular signaling pathways including protein phosphatases, protein kinases, and transcription factors, suggesting that the majority of the effects of ROS are through their actions on signaling pathways rather than via non-specific damage of macromolecules; however, exact mechanisms by which redox status induces cells to proliferate or to die, and how oxidative stress can lead to processes evoking tumor formation are still under investigation.

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Cancer cells require a robust supply of reduced nitrogen to produce nucleotides, non-essential amino acids and a high cellular redox activity. Glutamine provides a major substrate for respiration as well as nitrogen for the production of proteins, hexosamines, and macromolecules. Therefore, glutamine is one of key molecules in cancer metabolism during cell proliferation. The notion of targeting glutamine metabolism in cancer, originally rationalized by the number of pathways fed by this nutrient, has been reinforced by more recent studies demonstrating that its metabolism is regulated by oncogenes. Glutamine can exert its effects by modulating redox homeostasis, bioenergetics, nitrogen balance or other functions, including by being a precursor of glutathione, the major nonenzymatic cellular antioxidant. Glutaminase (GA) is the first enzyme that converts glutamine to glutamate, which is in turn converted to alpha-ketoglutarate for further metabolism in the tricarboxylic acid cycle. Different GA isoforms in mammals are encoded by two genes, Gls and Gls2. As each enzymatic form of GA has distinct kinetic and molecular characteristics, it has been speculated that the differential regulation of GA isoforms may reflect distinct functions or requirements in different tissues or cell states. GA encoded by Gls gene (GLS) has been demonstrated to be regulated by oncogenes and to support tumor cell growth. GA encoded by Gls2 gene (GLS2) reduces cellular sensitivity to reactive oxygen species associated apoptosis possibly through glutathione-dependent antioxidant defense, and therefore to behave more like a tumor suppressor. Thus, modulation of GA function may be a new therapeutic target for cancer treatment.

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In mammals, there are two different genes encoding for glutaminase isoforms, named liver (LGA) and kidney (KGA) types. LGA has long been believed to be present only in liver mitochondria from adult animals. However, we have recently reported the presence of LGA mRNA in human brain. We now describe the expression of LGA mRNA in the brain of other mammals (cow, mouse, rabbit, and rat) and in different areas of human brain as assessed by Northern blot analysis. The presence of mRNA encoding for this isoform in rat brain was further confirmed by reverse transcriptase-PCR cloning and sequencing. Although it has been well accepted that glutaminase is a mitochondrial enzyme, using newly generated isoform-specific antibodies, we have found a differential intracellular immunolocalization of both glutaminase isoforms in rat and monkey brain. In both species, KGA protein was present in mitochondria, whereas LGA protein was localized in nuclei. Furthermore, subcellular fractionation and Western blot analysis revealed that brain LGA was enriched in nuclei where it was catalytically active. Nuclear glutaminase exhibited a kinetic behavior that resembles that of the liver-type enzyme with regard to the low phosphate concentration requirement; however, nuclear glutaminase was susceptible to glutamate inhibition, a property that is absent in the rat liver enzyme.