ALBERT

Description: Introduction Acute myeloid leukemias are a group of malignant hemopathies characterized by a poor prognosis for survival. The discovery of oncogenic mutations in the FLT3 gene (eq FLT3-ITD) has led to the development of new tyrosine kinase inhibitors such as quizartinib. But complete remissions of patients remains difficult because these new TKIs are not able to completely eradicate all leukemia cells. Residual leukemia cells persist during treatment with quizartinib and lead to the rapid emergence of drug-resistant leukemia. Since mitochondrial oxidative metabolism supports the survival of leukemia cells after exposure to several anticancer drugs, we characterized the metabolism of leukemia cells that persisted within quizartinib treatment and developed metabolic strategies to eradicate them. Results First, we evaluated glycolysis activity in FLT3-ITD leukemia cell lines (MOLM13 / MOLM14 / MV4-11) under quizartinib treatment (5-10nM). Quizartinib reduced extracellular acidification rate ECAR, but this glycolytic activity is not fully inhibited (50% of untreated condition). These results obtained using the XFe24 Seahorse were in agreement with the metabolomic analysis carried out in a medium containing isotopic U-13C6 glucose. Next we evaluated mitochondrial oxidative phosphorylation in FLT3-ITD leukemia cell lines. After treatment with quizartinib, the basal and maximal oxygen consumption (OCR) of leukemia cells decreased. Metabolomic analysis using isotopic glucose U-13C6 or glutamine U-13C5 have shown that pyruvate derived from glucose was weakly oxidized in the mitochondria of untreated or quizartinib-treated cells. In contrast, a large amount of glutamine was oxidized by the tricarboxylic acid (TCA) cycle in untreated FLT3-ITD cells. Quizartinib reduced but did not abolish the complete oxidation of glutamine in leukemia cells. This result showed that even in the presence of quizartinib, FLT3-ITD cells maintained partially oxygen consumption trough glutamine oxidation. L-asparaginases (Kidrolase, Erwinase) are enzymes capable of hydrolyzing amino acids such as asparagine and glutamine. These clinical drugs have been approved for the treatment of chronic lymphocytic leukemia (CLL) and pediatric acute myeloid leukemia. We have shown that L-asparaginases weakly induced cell death in FLT3-ITD leukemia cells. Interestingly, our isobologram analysis showed that L-asparaginase acted synergistically with quizartinib to induce apoptosis. To determine whether glutamine metabolism also promoted the persistence of AML under treatment with quizartinib, we treated MOLM13 with quizartinib for several days. After long-term treatment, the percentage of surviving cells (annexin-V negative) was less than 5%. These persistent cells were characterized by an increased mitochondrial membrane potential (Δψm) and mitochondrial ROS. After treatment with the combination of L-asparaginase and quizartinib, the percentage of persistent cells decreased drastically. The combination of L-asparaginase and quizartinib was also more effective than quizartinib alone in reducing the size and number of colonies of MOLM13 in a model based on the formation of leukemia colonies growing in methylcellulose. Conclusion Persistent leukemia cells that survive after exposure to FLT3 inhibitor quizartinib can be targeted by the clinical drug L-asparaginases. This metabolic strategy could reduce the emergence of leukemic cells resistant to quizartinib. Disclosures Kluza: Daiichi-Sankyo: Research Funding.

Description: Introduction The persistence of leukemic cells after treatment limits the effectiveness of anticancer drugs and is the cause of relapse in patients with acute myeloid leukemia (AML). After exposure to chemotherapeutic drugs, the survival of leukemic cells is mainly supported by mitochondrial energy metabolism. Several preclinical studies have shown that the combination of mitochondrial oxidative phosphorylation inhibitors with various anticancer treatments constitutes an effective therapeutic combination in vitro to eradicate the surviving leukemic cells. Evaluating the mitochondrial bioenergetic activity of blasts from AML patients could therefore provide predictive information on treatment response. The basal oxygen consumption of cells varies according to hematopoietic differentiation and depends on the energy needs in the in vitro condition of measurement. But it is necessary to treat the cells with uncoupling agents (eg FCCP) to assess the maximum activity that the respiratory chain could reach to respond to energy stress. Then, the switch from a basal level of oxygen consumption to a maximum level defines the mitochondrial spare reserve capacity (SRC). In this study, we propose to determine whether spare reserve capacity of blasts is a potential biomarker of AML aggressiveness in patients and to characterize the biochemical processes involved in the control of SRC in leukemic cells. Results Using the XFe24 Seahorse fluorometric oximeter, we first determined the mitochondrial oxygen consumption and glycolytic activity in hematopoietic cells (monocytes, lymphocytes, dendritic cells) of healthy donors, in AML patient blasts at diagnosis or at relapse and in AML cell lines (HL-60, MOLM-13, THP-1, KG1, OCI-AML3, MV-4-11, U-937). All measures have been assessed from freshly collected samples of peripheral blood and of bone marrow. As expected, AMLs are characterized by low oxidative phosphorylation activity compared to normal hematopoietic cells. From all the OXPHOS values obtained we defined a SRC threshold above which the SRC is considered high. This threshold has been set at a capacity to increase basal respiration by 250%. From patients blasts, we have therefore defined two groups characterized by high (n=14) or low (n=21) mitochondrial spare reserve capacity. Blasts with high SRC exhibit high glycolytic activity suggesting a link between spare reserve capacity and glucose metabolism. Using U-13C6 glucose and pharmacological inhibitors, we have demonstrated that the utilization of the mitochondrial spare reserve capacity of leukemic cells is supported through glycolysis and that mitochondrial oxidation of pyruvate is a key element for SRC recruitment. Mitochondrial pyruvate carrier inhibitors (as UK-5099) or gene silencing of BRP44 abolish the SRC of leukemic cells highlighting the importance of pyruvate oxidation to increase oxygen consumption. Since high mutation rate is recognized as an unfavorable prognostic factor in AML, we have also sequenced 45 commonly genes mutated in AMLs characterized by high or low SRC blasts. Interestingly, DNA sequencing analysis showed that AML with low SRC blasts have a higher mutation rate than high SRC blasts and also exhibited exclusive mutations such as ASXL1 (25%), IDH2 (25%), NPM1 (25%), IDH1 (13%), JAK2 (13%) and SF3B1 (13%). Conclusion Currently, most of the clinical biomarkers used to predict AML aggressiveness are based on DNA analysis, but the emergence of mutations is not always associated with phenotypic changes. This study shows that the mitochondrial spare reserve capacity of blasts represents a new functional biomarker based on the assessment of the energetic phenotype and could help the clinicians to determine the prognosis of AML. Moreover we have showed that altering pyruvate metabolism highly decrease spare reserve capacity of blasts and then could be evaluated as metabolic strategies to improve the therapeutic response in patients with AML. Disclosures Kluza: Daiichi-Sankyo: Research Funding.