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Acute myeloid leukemia (AML) is one of the most frequently occurring and fatal types of leukemia. Initiated by genetic alterations in hematopoietic stem and progenitor cells, rapidly proliferating cancer cells (leukemic blasts) infiltrate the bone marrow and damage healthy hematopoiesis. Subgroups of AML are defined by underlying molecular and cytogenetic abnormalities, which are decisive for treatment and prognosis. For AML patients that can be intensively treated, the first line treatment remains a combination of cytarabine and anthracycline, which was developed in the 1970s. While this treatment regimen clears the disease and reinstates normal hematopoiesis (complete remission, CR) in 60% to 80% of patients below the age of 60, CR rates in patients above the age of 60 are only 40% to 50%. Relapse and refractory disease are the major cause of death of AML patients, despite large efforts to improve risk-adjusted post-remission therapy with further chemotherapy cycles and, if possible, allogeneic bone marrow transplantation. Elderly patients are particularly difficult to treat because of age-related comorbidities and because their disease tends to relapse more often than the disease of younger patients. Thus, the cure rates of AML vary with age, with 5-year survival rates of about 50% in young patients, and less than 20% in patients above the age of 65 years. With the median age of AML patients being 68 years, the need for novel therapeutic options is immense. The recent approval of eight new agents (venetoclax, midostaurin, gilteritinib, glasdegib, ivosidenib, enasidenib, gemtuzumab ozogamicin and CPX-351 (liposomal cytarabine and daunorubicin)) has added considerably to the therapeutic armamentarium of AML and has increased cure rates in specific subgroups of AML. However, the high heterogeneity among patients, clonal evolution and commonly occurring drug resistance, which cause the high relapse rates, remain a substantial problem in the treatment of AML. Therefore, a better understanding of currently used therapeutics and further development of novel therapeutics is urgently needed.
In recent years, attention has increasingly focused on therapeutic strategies to interfere with the metabolic requirements of cancer cells. The last three decades have provided extensive insights into the diversity and flexibility of AML metabolism. AML cells use different sources of nutrients compared to normal hematopoietic progenitor cells and reprogram their metabolic pathways to fulfill their exquisite anabolic and energetic needs. As a result, they develop high metabolic plasticity that enables them to thrive in the bone marrow microenvironment, where oxygen and nutrient availability are subject to constant change.
Cancer cells, specifically AML cells, have a strong dependency for the amino acid glutamine. Glutamine serves in energy production, redox control, cell signaling as well as an important nitrogen source. The only enzyme capable of de novo glutamine synthesis is glutamine synthetase (GS). GS catalyzes glutamine production from glutamate and ammonium. In AML, the metabolic role and dependency of GS is poorly understood. Here, we investigated the effects of GS deletion on AML growth, and its functional relevance in AML metabolism. Genetic deletion of GS resulted in a significant decrease of cell growth in vitro, and impaired leukemia progression in vivo in a xenotransplantation mouse model. Interestingly, the dependency of AML cell growth on GS was shown to be independent of its functional role in glutamine synthesis. Glutamine starvation did not increase the dependency of the AML cells on GS, nor did increased glutamine availability rescue the GS-knockout-associated growth disadvantage. Instead, functional studies revealed the role of GS in the detoxification of ammonium. GS-deficient cells showed elevated ammonium secretion as well as a higher sensitivity towards the toxic metabolite. Exogenous provision of 15N-labeled ammonium was detoxified by GS-driven incorporation into glutamine. Studies on cells that had gained resistance to GS-knockout-mediated growth inhibition indicated enzymes involved in the urea cycle and the arginine biogenesis pathway to compensate for a loss of GS. Together, these findings unveiled GS as an important ammonium scavenger in AML.
Clinical studies on AML patients revealed increased ammonium concentrations in the blast-infiltrated bone marrow compared to peripheral blood. In line with this finding, proteome and transcriptome analysis of AML blasts showed a significant upregulation of GS in AML compared to healthy progenitors, further indicating its importance in ammonium detoxification.
Analyzing pathways that contribute to ammonium production revealed protein uptake followed by amino acid catabolism as a yet not identified mechanism supporting AML growth. Protein endocytosis and subsequent proteolytic degradation were shown to rescue AML cells from otherwise growth-inhibiting glucose or amino acid depletion. Furthermore, protein metabolization led to the reactivation of the mammalian target of rapamycin (mTOR) signaling pathway, which was deactivated upon leucine and glutamine depletion, revealing protein consumption as an important alternative source of amino acids in AML.
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Cancer cells, including leukemic cells, can react to therapeutic treatment by altering their metabolic phenotype (“metabolic reprogramming”) to keep their accelerated proliferative state, eventually becoming resistant to the treatment. There is an increasing amount of evidence indicating that metabolic reprogramming is one of the key mechanisms of acquisition of drug resistance by cancer cells. In agreement, several metabolic studies targeting leukaemia and specifically acute myeloid leukaemia (AML) and chronic myeloid leukaemia (CML), have been conducted over the last decades. However, there is still a lack of understanding the metabolic features of both AML and CML leukaemia specially in the acquisition of drug resistance, that is needed for unveiling novel and effective treatments for resistant and non-resistant patients. Therefore, the main objective of this thesis was to investigate the rewiring of cell metabolism occurring in the process of acquisition of resistance to conventional therapeutic treatments in AML and CML malignancies. Next, by revealing this metabolic rewiring, we intended to highlight potential metabolic and non-metabolic targets that could be exploited to overcome resistance to treatments. To this end, we have performed a comprehensive and comparative multi-OMIC study to analyse the links between the metabolic reprogramming and the resistance acquisition of THP-1 and HL-60 AML cell models sensitive or resistant to cytarabine (AraC) and doxorubicin (Dox), and of KU812 CML cell model sensitive or resistant to imatinib, all under normoxic (21% O2) and hypoxic (1% O2) conditions. The results of this thesis are divided into two chapters. On the one hand, in Chapter 1, the multi-OMIC study performed in AML parental and resistant cells unveiled that the acquisition of AraC resistance causes the reprogramming of the glucose metabolism of THP-1 and HL-60 cells by increasing the glycolytic flux whereas it is not associated with an alteration in the mitochondrial respiration. Moreover, our results also exhibited a possible disfunction of ETC complex I as well as alterations in glutamine and serine-glycine-1C metabolism in AML cells that display a more active mitochondrial metabolism. Moreover, we have also identified that the acquisition of Dox resistance causes alterations in the glucose and amino acid metabolism. Importantly, we have observed an important loss of mitochondrial respiration capacity of AML cells resistant to Dox chemotherapeutic drug, which constitutes a potential metabolic vulnerability that can be exploited for the treatment of AML patients resistant to Dox. On the other hand, in Chapter 2 is shown that the acquisition of imatinib resistance causes the reprogramming of glucose metabolism by enhancing the glycolytic flux, PPP, and glycogen metabolism, thus highlighting these metabolic pathways as potential metabolic weaknesses of KU812 cells resistant to imatinib. Moreover, we have observed a high metabolic plasticity of KU812 cells resistant to imatinib which includes the orchestration of many metabolic routes associated with the amino acid metabolism. Importantly, the CML multi-OMIC study has also unveiled an enhanced mitochondrial respiration capacity, which constitutes another potential vulnerability that can be exploited to overcome imatinib resistance. Finally, both AML and CML multi-OMIC studies have allowed us to propose and/or validate different metabolic and non-metabolic targets. In this regard, in this thesis we have identified and validated a battery of single-hit inhibitions that were able to reduce the cell viability of both parental and resistant AML and CML cells. Finally, we have confirmed that the repurposing of Dox chemotherapeutic drug counteracts the imatinib resistance in the KU812 cells resistant to imatinib.