scholarly journals Antimetabolic Cooperativity with l-Asparaginase and Tyrosine Kinase Inhibitor Quizartinib to Eradicate Persistant FLT3-ITD Leukemic Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 31-31
Author(s):  
Quentin Fovez ◽  
Raeeka Khamari ◽  
Anne Trinh ◽  
William Laine ◽  
Bruno Quesnel ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Glutamine Metabolism ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Metabolomic Analysis ◽  
Leukemia Cell Lines ◽  
Acute Myeloid

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.

scholarly journals Arsenic Trioxide Synergizes with FLT3 Tyrosine Kinase Inhibitors to Kill FLT3-ITD+ Leukemic Cells through Depressed Production and Degradation of FLT3 Protein

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1568-1568
Author(s):  
Kozo Nagai ◽  
Lihong Hou ◽  
Li Li ◽  
Bao Nguyen ◽  
Courtney M Shirley ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Arsenic Trioxide ◽  
Tyrosine Kinase Inhibitors ◽  
Cell Lines ◽  
Research Funding ◽  
Kinase Inhibitors ◽  
Leukemia Cell ◽  
Leukemic Cells ◽  
Potential Candidate ◽  
Leukemia Cell Lines

Abstract A number of selective FLT3 tyrosine kinase inhibitors (TKIs) have been tested for treatment of FLT3-ITD+ AML. However, monotherapy with FLT3 TKIs alone has achieved only transient and limited clinical responses due to several resistance mechanisms. Arsenic trioxide (ATO) has demonstrated significant efficacy in treating acute promyelocytic leukemia (APL). ATO has also shown some activity in treating non-APL myeloid leukemias. Recent studies have demonstrated that ATO can affect the degradation of oncogenic mutant proteins including mutant p53 and NPM through the ubiquitin-proteasome pathway (UPP). Here we investigated the feasibility of combining FLT3 TKIs with ATO in the treatment of FLT3-ITD+leukemia. Anti-leukemic effects against FLT3-ITD+ patient AML samples and leukemia cell lines (Molm14, MV4;11) by FLT3 TKIs (Sorafenib, quizartinib), ATO and the combination were examined by MTT, apoptosis, cell viability and colony forming assays. Our data revealed that the combination showed synergistic growth inhibition of the FLT3-ITD+ cell lines Molm14 and MV4;11, with combinatorial index (CI) values at ED50 below 1.0 for both cell lines (CI values were 0.46 and 0.56 for ATO + sorafenib, 0.65 and 0.57 for ATO + quizartinib in Molm14 and MV4;11 cells, respectively). In contrast, there was no synergy observed for the combination in treating leukemia cell lines that do not express mutant FLT3. Synergistic effects for the combination in inducing apoptosis and inhibiting colony formation were also observed for the FLT3-ITD+ cell lines. Furthermore, when the combination was used to treat primary FLT3-ITD+ patient samples, there was also significant reduction of viability and clonogenicity. In contrast, normal BM MNCs showed very limited responses to the combination. Western blot (WB) analysis of Molm14 and FLT3-ITD+ patient samples revealed the combination of ATO and sorefenib potently reduced phosphorylation of FLT3 and its downstream targets (STAT5, MAPK, and AKT). In vivoexperiments using the combination to treat NSG mice engrafted with Molm14 cells demonstrated a significant reduction in the level of leukemic cells. We further investigated the mechanism by which ATO contributes to an anti-leukemic effect on FLT3-ITD+ cells. Morphologic and flow cytometric analysis showed that ATO promoted the differentiation of Molm14 cells. The expression of C/EBPα and PU.1, two key regulators for myeloid differentiation, was increased in ATO-treated Molm14 cells at both the mRNA and protein levels. These data suggest ATO is capable of inducing the differentiation of leukemic cells. We also found that, in FLT3-ITD+ cells, ATO decreased expression of FLT3 protein. This could result from reduced FLT3 production and/or increased protein degradation. Further quantitative PCR analysis revealed ATO decreased expression of FLT3 and its upstream regulators HoxA9 and meis1. Co-immunoprecipitation assay showed that ATO facilitated poly-Ubiquitination and degradation of FLT3 in a dose- and time-dependent fashion. These results indicate that ATO exerts its anti-leukemic effects in FLT3-ITD+AML cell lines and primary samples at least partly through reducing the level of FLT3 protein. These studies together demonstrate that ATO has a unique activity towards FLT3-ITD+ leukemia cells. Based on these findings, ATO is a potential candidate to work in combination with FLT3 TKIs to improve the outcome of FLT3-ITD+ AML patients. Disclosures Levis: Millennium: Consultancy, Research Funding; Daiichi-Sankyo: Consultancy, Honoraria; Astellas: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

scholarly journals AXL/MERTK inhibitor ONO-7475 potently synergizes with venetoclax and overcomes venetoclax resistance to kill FLT3-ITD acute myeloid leukemia

Haematologica ◽  
2021 ◽  
Author(s):  
Sean M. Post ◽  
Huaxian Ma ◽  
Prerna Malaney ◽  
Xiaorui Zhang ◽  
Marisa J.L. Aitken ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cell Lines ◽  
Acute Myeloid ◽  
Myeloid Leukemia Cell ◽  
Resistant Cells

FMS-like Tyrosine Kinase 3 (FLT3) mutation is associated with poor survival in AML. The specific Anexelekto/MER Tyrosine Kinase (AXL) inhibitor ONO-7475 kills FLT3-mutant acute myeloid leukemia cells with targets including Extracellular-signal Regulated Kinase (ERK) and Myeloid Cell Leukemia 1 (MCL1). ERK and MCL1 are known resistance factors for Venetoclax (ABT-199), a popular drug for AML therapy, prompting the investigation of the efficacy of ONO-7475 in combination with ABT-199 in vitro and in vivo. ONO-7475 synergizes with ABT-199 to potently kill FLT3-mutant acute myeloid leukemia cell lines and primary cells. ONO-7475 is effective against ABT-199-resistant cells including cells that overexpress MCL1. Proteomic analyses revealed that ABT-199-resistant cells expressed elevated levels of pro-growth and anti-apoptotic proteins compared to parental cells, and that ONO-7475 reduced the expression of these proteins in both the parental and ABT-199-resistant cells. ONO-7475 treatment significantly extended survival as a single agent in vivo using acute myeloid leukemia cell lines and PDX models. Compared to ONO-7474 monotherapy, the combination of ONO- 7475/ABT-199 was even more potent in reducing leukemic burden and prolonging survival of mice in both model systems. These results suggest the ONO-7475/ABT-199 combination may be effective for acute myeloid leukemia therapy.

scholarly journals Mitochondria in human acute myeloid leukemia cell lines have ultrastructural alterations linked to deregulation of their respiratory profiles

2020 ◽  
Author(s):  
Julie Mondet ◽  
Caroline Lo Presti ◽  
Simon Chevalier ◽  
Anne Bertrand ◽  
Sylvie Tondeur ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Calcium Homeostasis ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Leukemic Cells ◽  
Functional Assay ◽  
Ultrastructural Alterations ◽  
Leukemia Cell Lines ◽  
Acute Myeloid

Abstract Mitochondria are not only essential for cell metabolism and energy supply but they are also engaged in calcium homeostasis, reactive oxygen species generation and play a key role in apoptosis. As a consequence, functional mitochondria disorders are involved in many human cancers including acute myeloid leukemia (AML). However, very little data are available about the deregulation of their number and/or shape in leukemic cells, despite the evident link between ultrastructure and function. In this context, we analyzed the ultrastructural mitochondrial parameters (number per cell, mitochondria area, number of cristae/mitochondria, cristae thickness) in five leukemia cell lines (HEL, HL60, K562, KG1 and OCI-AML3) together with the functional assay of their respiratory profile. First of all, we show significant differences within basal respiration, maximal respiration, ATP production and spare respiratory capacity between our cell lines, confirming the various respiratory profiles between leukemia subtypes. Second, we highlight that these variations were obviously associated with significant inter-leukemia heterogeneity of the number and/or shape of mitochondria. For instance, KG1 characterized by the lowest number of mitochondria together with reduced cristae diameter displayed a very particularly deficient respiratory profile. In comparison, HEL and K562, both cell lines with high respiratory profiles, harbored the highest number of mitochondria/cells with high cristae diameters. We show the leukemia lines present ultrastructural alterations of their mitochondria likely to impact the regulatory pathways of cell mortality, such as the process of mitophagy or calcium homeostasis. Indeed, a significant disparity in the presence of Mitochondrial-derived vesicles (MDVs) precursors among AML cell lines, suggesting that leukemic cells displayed alteration of mitophagy, is also shown. For instance, few MDV precursors were observed in K562, carrying ASXL1mutation. Moreover, HL60 carried high levels of matrix granules and Mitochondria-associated Endoplasmic Reticulum membranes (MAMs) both implicated in calcium-dependent apoptosis. In conclusion, this study offers new and original data on mitochondria heterogeneity linked to the deregulation of respiration profiles in AMLs, suggesting that modifications of mitochondria shape and/or number in leukemic cells could be a targeted mechanism to regulate their proliferative potential.

Bortezomib Interacts Synergistically with Belinostat to Induce Apoptosis In Human Acute Myeloid and Lymphoid Leukemia Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3266-3266
Author(s):  
Yun Dai ◽  
Shuang Chen ◽  
Li Wang ◽  
Xin-Yan Pei ◽  
Lora Kramer ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Dose Effect ◽  
Leukemia Cells ◽  
Parp Cleavage ◽  
Lymphoid Leukemia ◽  
Acute Leukemias ◽  
Leukemia Cell Lines ◽  
Acute Myeloid

Abstract Abstract 3266 Previous studies have demonstrated interactions between histone deacetylase (HDAC) and proteasome inhibitors (PIs) in multiple myeloma, NHL, and CLL. However, exploration of this strategy in acute leukemias has been more limited. In this context, we have previously demonstrated that HDACIs activate the cytoprotective NF-κB pathway in acute myeloid leukemia (AML) cells, and that interruption of this process dramatically increases lethality. Such findings raise the possibility that PIs, which block degradation of the NF-κB-inhibitory protein IκBα, may act via an analogous mechanism in acute leukemias. Consequently, interactions between the clinically relevant pan-HDAC inhibitor belinostat (PXD-101) and the FDA-approved proteasome inhibitor bortezomib were evaluated in both continuously cultured cell lines and primary AML and acute lymphoid leukemia (ALL) samples. First, whereas each agent individually displayed only modest toxicity, co-treatment for 24 hr or 48 hr with low concentrations of bortezomib (3 - 5 nM) and belinostat (50 - 300 nM) led to pronounced increases in apoptosis in diverse human acute leukemia cell lines (e.g., AML, U937, HL-60, MV-4-11/Flt3-ITD; T-cell ALL, Jurkat; B-cell ALL, SEM). Interactions between these agents were determined to be synergistic by Median Dose Effect analysis. Significantly, equivalent interactions were observed in multiple primary AML (n = 4) and ALL (n = 3) blast specimens, while largely sparing normal CD34+ hematopoietic cells isolated from umbilical cord blood (n = 4), as determined by annexin V/PI, DiOC6, and/or 7-AAD uptake by flow cytometry. Western blot analysis demonstrated that co-exposure of primary leukemia blasts to bortezomib and belinostat resulted in marked increase in PARP cleavage, compared with each agent administrated alone. In addition, cell morphology exhibited classical features of apoptosis in primary acute leukemia blasts, but not in normal CD34+ cells, following combination treatment. Second, in both cell lines and primary blasts, administration of bortezomib resulted in accumulation of the phosphorylated (S32/S36) form of IκBα, accompanied by diminished belinostat-mediated hyperacetylation (K310) of RelA/p65. Bortezomib also blocked processing of the precursor p100 into the active p52, an event enhanced by co-treatment with belinostat. These results indicate that a regimen combining bortezomib and belinostat interrupts both canonical and non-canonical NF-κB signaling pathways in acute leukemia cells. Moreover, co-exposure to these agents diminished expression of NF-κB-dependent pro-survival proteins including Bcl-xL, XIAP, and SOD2, but not NF-κB-independent anti-apoptotic proteins such as survivin. Third, because the BH3-only Bcl-2 family pro-apoptotic protein Bim plays an important role in the lethality of PIs or HDACIs as single agents, the expression and functional role of Bim in bortezomib/belinostat interactions was examined. Notably, whereas treatment with marginally toxic concentrations of either agent alone clearly increased Bim protein levels, co-exposure of either leukemia cell lines or primary blasts to bortezomib and belinostat led to sharply increased Bim expression (particularly the BimEL isoform). Importantly, shRNA knock-down of Bim substantially attenuated lethality mediated by co-treatment with bortezomib and belinostat in both AML (U937) and ALL (Jurkat) cells, supporting the notion that up-regulation of Bim plays a critical role in anti-leukemic activity of the combination regimen. Lastly, exposure of cultured leukemia cells and primary blasts to belinostat ± bortezomib induced hyperacetylation of a-tubulin, indicating inhibition of HDAC6, a microtubule-associated deacetylase that regulates aggresome formation and cell survival in response to misfolded protein-induced stress. Together, these findings indicate that the regimen combining belinostat and bortezomib is highly active against human AML and ALL cells, including primary leukemic blasts, in association with perturbation in the balance between pro-survival (NF-κB-dependent) and pro-death (e.g., Bim) signals. They also suggest that this strategy warrants further attention in acute leukemias. Accordingly, a Phase I trial of belinostat and bortezomib in patients with refractory acute leukemia or MDS has recently been initiated. Disclosures: Off Label Use: Investigational use of belinostat and bortezomib.

scholarly journals The U2AF homology motif kinase 1 (UHMK1) is upregulated upon hematopoietic cell differentiation

2017 ◽  
Author(s):  
Isabella Barbutti ◽  
João Agostinho Machado-Neto ◽  
Vanessa Cristina Arfelli ◽  
Paula de Melo Campos ◽  
Fabiola Traina ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Hematopoietic Cell ◽  
Leukemic Cells ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Induced Differentiation ◽  
Aberrant Expression ◽  
Leukemia Cell Lines

AbstractUHMK1 (KIS) is a nuclear serine/threonine kinase that possesses a U2AF homology motif and phosphorylates and regulates the activity of the splicing factors SF1 and SF3b155. Mutations in these components of the spliceosome machinery have been recently implicated in leukemogenesis. The fact that UHMK1 regulates these factors suggests that UHMK1 might be involved in RNA processing and perhaps leukemogenesis. Here we analyzed UHMK1 expression in normal hematopoietic and leukemic cells as well as its function in leukemia cell line.In the normal hematopoietic compartment, markedly higher levels of transcripts were observed in differentiated lymphocytes (CD4+, CD8+ and CD19+) compared to the progenitor enriched subpopulation (CD34+) or leukemia cell lines. UHMK1 expression was upregulated in megakaryocytic-, monocytic-and granulocytic-induced differentiation of established leukemia cell lines and in erythrocytic-induced differentiation of CD34+ cells. No aberrant expression was observed in patient samples of myelodysplastic syndrome (MDS), acute myeloid (AML) or lymphoblastic (ALL) leukemia. Nonetheless, in MDS patients, increased levels of UHMK1 expression positively impacted event free and overall survival.Lentivirus mediated UHMK1 knockdown did not affect proliferation, cell cycle progression, apoptosis or migration of U937 leukemia cells, although UHMK1 silencing strikingly increased clonogenicity of these cells. Thus, our results suggest that UHMK1 plays a role in hematopoietic cell differentiation and suppression of autonomous clonal growth of leukemia cells.

scholarly journals Ampelopsin Inhibits Cell Proliferation and Induces Apoptosis in HL60 and K562 Leukemia Cells by Downregulating AKT and NF-κB Signaling Pathways

2021 ◽  
Vol 22 (8) ◽  
pp. 4265
Author(s):  
Jang Mi Han ◽  
Hong Lae Kim ◽  
Hye Jin Jung
Keyword(s):  
Signaling Pathways ◽  
Cell Lines ◽  
White Blood Cells ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Ros Generation ◽  
Anticancer Activities ◽  
Nuclear Condensation ◽  
Leukemia Cell Lines ◽  
Antileukemic Effect

Leukemia is a type of blood cancer caused by the rapid proliferation of abnormal white blood cells. Currently, several treatment options, including chemotherapy, radiation therapy, and bone marrow transplantation, are used to treat leukemia, but the morbidity and mortality rates of patients with leukemia are still high. Therefore, there is still a need to develop more selective and less toxic drugs for the effective treatment of leukemia. Ampelopsin, also known as dihydromyricetin, is a plant-derived flavonoid that possesses multiple pharmacological functions, including antibacterial, anti-inflammatory, antioxidative, antiangiogenic, and anticancer activities. However, the anticancer effect and mechanism of action of ampelopsin in leukemia remain unclear. In this study, we evaluated the antileukemic effect of ampelopsin against acute promyelocytic HL60 and chronic myelogenous K562 leukemia cells. Ampelopsin significantly inhibited the proliferation of both leukemia cell lines at concentrations that did not affect normal cell viability. Ampelopsin induced cell cycle arrest at the sub-G1 phase in HL60 cells but the S phase in K562 cells. In addition, ampelopsin regulated the expression of cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors differently in each leukemia cell. Ampelopsin also induced apoptosis in both leukemia cell lines through nuclear condensation, loss of mitochondrial membrane potential, increase in reactive oxygen species (ROS) generation, activation of caspase-9, caspase-3, and poly ADP-ribose polymerase (PARP), and regulation of Bcl-2 family members. Furthermore, the antileukemic effect of ampelopsin was associated with the downregulation of AKT and NF-κB signaling pathways. Moreover, ampelopsin suppressed the expression levels of leukemia stemness markers, such as Oct4, Sox2, CD44, and CD133. Taken together, our findings suggest that ampelopsin may be an attractive chemotherapeutic agent against leukemia.

Aurora-A kinase: a novel target of cellular immunotherapy for leukemia

Blood ◽  
2009 ◽  
Vol 113 (1) ◽  
pp. 66-74 ◽  
Author(s):  
Toshiki Ochi ◽  
Hiroshi Fujiwara ◽  
Koichiro Suemori ◽  
Taichi Azuma ◽  
Yoshihiro Yakushijin ◽  
...  
Keyword(s):  
Cell Lines ◽  
Leukemia Cell ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Cellular Immunotherapy ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Hematopoietic Stem ◽  
Specific Ctls ◽  
Leukemia Cell Lines

Abstract Aurora-A kinase (Aur-A) is a member of the serine/threonine kinase family that regulates the cell division process, and has recently been implicated in tumorigenesis. In this study, we identified an antigenic 9–amino-acid epitope (Aur-A207-215: YLILEYAPL) derived from Aur-A capable of generating leukemia-reactive cytotoxic T lymphocytes (CTLs) in the context of HLA-A*0201. The synthetic peptide of this epitope appeared to be capable of binding to HLA-A*2402 as well as HLA-A*0201 molecules. Leukemia cell lines and freshly isolated leukemia cells, particularly chronic myelogenous leukemia (CML) cells, appeared to express Aur-A abundantly. Aur-A–specific CTLs were able to lyse human leukemia cell lines and freshly isolated leukemia cells, but not normal cells, in an HLA-A*0201–restricted manner. Importantly, Aur-A–specific CTLs were able to lyse CD34+ CML progenitor cells but did not show any cytotoxicity against normal CD34+ hematopoietic stem cells. The tetramer assay revealed that the Aur-A207-215 epitope–specific CTL precursors are present in peripheral blood of HLA-A*0201–positive and HLA-A*2402–positive patients with leukemia, but not in healthy individuals. Our results indicate that cellular immunotherapy targeting Aur-A is a promising strategy for treatment of leukemia.

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