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.

OP449, a Novel SET Antagonist, Is Cytotoxic To Leukemia Cells and Enhances Efficacy Of Tyrosine Kinase Inhibitors In Drug-Resistant Myeloid Leukemias

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2511-2511 ◽  
Author(s):  
Anupriya Agarwal ◽  
Ryan J. Meckenzie ◽  
Raffaella Pippa ◽  
Christopher A. Eide ◽  
Jessica Oddo ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Cell Growth ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Cell Lines ◽  
Research Funding ◽  
Kinase Inhibitors ◽  
Combination Index ◽  
Leukemia Cells ◽  
Pp2a Activity

Abstract Background The SET oncoprotein, an inhibitor of the protein phosphatase 2A (PP2A), is overexpressed in leukemia cells, preventing PP2A from performing its regulatory role in deactivating signaling proteins by dephosphorylation. Restoration of PP2A activity in both chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) cells to normal levels through shRNA-mediated knockdown of SET results in reduced leukemogenesis. Given the central role of PP2A and SET in regulating various kinase-dependent and -independent downstream signaling pathways, we evaluated the efficacy of SET antagonism in CML and AML cell lines as well as primary patient cells using OP449, a novel, specific, cell-penetrating SET antagonist. Results Treatment of human and murine CML cells with OP449 resulted in dose-dependent increase in PP2A activity and selective inhibition of cell growth (IC50: 0.60 to 1.11 μM), while parental Ba/F3 cells exhibited no measurable cytotoxicity. OP449-mediated decrease in the viability of leukemia cells was significantly rescued by co-treatment with okadaic acid, a PP2A inhibitor, confirming efficacy is mediated through PP2A activation. OP449 was also 3 to 8-fold more potent than FTY720 (a known activator of PP2A) and induced dephosphorylation/degradation of BCR-ABL1, AKT, and STAT5. Importantly, OP449 demonstrated activity against the ABL1 tyrosine kinase inhibitor-resistant BCR-ABL1T315I mutant and the BCR-ABL1E255V/T315I compound mutant (IC50: 1.62 and 1.97 μM, respectively). Consistent with cell line findings, OP449 also inhibited growth of primary cells from CML blastic phase patients harboring either wildtype BCR-ABL1 or BCR-ABL1T315I while normal CD34+ cells exhibited minimal effect. Further, treatment of CML cell lines and primary CD34+ CML cells with OP449 in combination with the ABL1 tyrosine kinase inhibitors showed significantly increased cytotoxicity as compared to each compound alone. For example, treatment of primary CD34+ CML cells with 2.5 μM OP449 or 200 nM nilotinib alone each resulted in a 50% reduction in colony formation, while combination of OP449 and nilotinib at these concentrations reduced colony formation by approximately 87%, suggesting synergistic reduction of clonogenicity (combination index: 0.195). Similar to our findings in CML cells, OP449 increased PP2A activity and suppressed growth in a dose-dependent manner in AML cell lines and primary patient samples harboring various different genetic lesions including FLT3-ITD, CSF1R overexpression, NRASQ61L, and JAK3A572V. Additionally, synergistic inhibition of these cells was observed when OP449 was combined with relevant tyrosine kinase inhibitors and chemotherapy. For example, treatment of MOLM-14 cells (FLT3-ITD) with 2.5 μM OP449 or 1 nM AC220 alone reduced cell viability by 58% and 75%, respectively; combined treatment reduced cell growth 96% (combination index: 0.723). Similarly, treatment of HL-60 cells (NRASQ61L) with 1 μM OP449 or 250 nM cytarabine alone reduced cell viability by 40% and 60%, respectively, whereas combined treatment led to a 94% reduction in viability (combination index: 0.630). Mechanistically, AML patient samples showed significantly increased SET expression compared to normal CD34+ cells, and treatment of AML cells with OP449 reduced phosphorylation of downstream ERK, STAT5, AKT and S6 ribosomal protein signaling. Finally, to evaluate OP449 antitumor efficacy in vivo, we tested OP449 (5 mg/kg intraperitoneally every 3 days) in xenograft mice bearing human HL-60 cell derived tumors. OP449 significantly inhibited tumor growth measured over time and resulted in a >2-fold reduction in tumor burden at the end of the experiment compared to vehicle-treated controls (Day 18: 1.14±0.06 g vs. 0.45±0.08 g, respectively; p<0.001). These results demonstrate the in vivo efficacy of OP449 in a murine leukemia model. Conclusions SET antagonism is selectively cytotoxic to CML and AML cells harboring various genetic lesions and drug-resistant mutations. Our results demonstrate that combined targeting of SET and tyrosine kinases provides more efficient and selective inhibition of leukemia cell growth for a broad range of oncogenic lesions as compared to normal cells. Taken together, our findings suggest a novel therapeutic paradigm of SET antagonism in combination with tyrosine kinase inhibitors for the treatment of CML and AML patients with drug resistance. Disclosures: Agarwal: Oncotide Pharmaceuticals: Research Funding. Tyner:Incyte Corporation: Research Funding. Vitek:Oncotide Pharmaceuticals: Employment. Christensen:Oncotide Pharmaceuticals: Employment. Druker:Ambit Biosciences: Consultancy, PI or co-investigator on Novartis clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU., PI or co-investigator on Novartis clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU. Other; Bristol-Myers Squibb/Novartis: Currently PI or co-I on Novartis & Bristol-Myers Squibb clinical trials. His institution has contracts with these companies to pay for patient costs, nurse and data manager salaries, and institutional overhead. He does not derive salary, nor does his lab Other; Oncotide Pharmaceuticals: Research Funding, Subaward from NIH STTR, Subaward from NIH STTR Other.

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.

Activity of the Telomerase Inhibitor GRN163L (Imetelstat) on Acute Myeloblastic Leukemia Blasts Is Enhanced By DNA Methyltransferase Inhibitors Irrespective of TERT Promoter Methylation Status

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1267-1267
Author(s):  
Caroline R Cantilena ◽  
Xin Zhao ◽  
Sachiko Kajigaya ◽  
Neil Dunavin ◽  
Xin Tian ◽  
...  
Keyword(s):  
Cell Lines ◽  
Research Funding ◽  
Dna Methyltransferase ◽  
Methylation Status ◽  
Leukemia Cell ◽  
Advisory Board ◽  
Leukemic Cells ◽  
Telomerase Inhibitor ◽  
Leukemia Cell Lines ◽  
Primary Leukemia

Abstract Introduction. The high telomerase activity in leukemia cells protects them from proliferation arrest, senescence, and apoptosis and may be driven by mutation or epigenetic alteration in the telomerase promoter. However, the mechanism of telomerase regulation and potential therapeutic application of telomerase inhibition in leukemia are not fully understood. We evaluated epigenetic methylation patterns in the telomerase promoter region in myeloid cell lines and primary acute myeloid leukemia (AML) blasts. These epigenetic patterns may serve as a biomarker for sensitivity to DNA methyltransferase (DNMT) inhibitors and have prognostic significance. We also studied whether the telomerase inhibitor GRN163L (imetelstat)can favorably combine with the DNMT inhibitor 5-Azacytidine (5-Aza) to target poor prognosis leukemias. Methods. We developed a pyrosequencing-based methylation assay to screen methylation profiles of the proximal promoter and partial exon 1 of the human telomerase reverse transcriptase (hTERT pro/Ex1) region in primary leukemic cells and various cell lines.We used a chemosensitivity assay to determine specific killing of primary leukemia and cell lines by imetelstat. An inert mismatched oligonucleotide (Geron Corporation, Menlo Park, CA, USA) was used to control for specific inhibition of the telomerase active site. Cells were cultured for 48 hours with either active imetelstat or the inert control at varying concentrations, stained with annexin-V and propidium iodide, and then analyzed by flow cytometry to measure cell viability, apoptosis, and necrosis. Results. The hTERT pro/Ex1 region was highly methylated in cell lines, relative to de novo primary leukemic cells. Primary leukemic cells showed significantly different methylation profiles and hypermethylation status correlated to poor survival of AML patients. Three commercially available leukemia cell lines (K562, Ramos, THP-1), two primary leukemia-derived cell lines (AML1, CML1), and CD34+ blasts isolated from primary leukemia in six different AML patients with varying degrees of hTERT pro/Ex1 region methylation were tested. Imetelstat showed dose dependent cytotoxicity to both myeloid leukemia cell lines and primary leukemic blasts. Cell toxicity was telomerase specific since the inert control had no or minimal toxicity at the half inhibitory concentration (IC50) of imetelstat between 10-40 µM. Higher methylation status of the hTERT pro/Ex1 region was significantly associated with increased resistance to imetelstat in leukemia cell lines (Figure 1A). However, no correlation was found in primary leukemic blasts. Pretreatment of leukemia cell lines with 5-Aza for 24 hours prior to imetelstat exposure was associated with a decrease in viability from 0.78±0.01 to 0.54±0.01 at a concentration of 10µM of imetelstat (Figure 1B). 5-Aza alone had no effect on the leukemic cell lines' viability. Conclusion. High risk primary leukemias are susceptible to killing by the telomerase inhibitor irrespective of the degree of methylation of the hTERT pro/Ex1 region. Furthermore, demethylating agents can enhance the activity of the telomerase inhibitor, imetelstat. These findings suggest that combination therapy of imetelstat and DNMT inhibitors may have synergistic anti-leukemic efficacy in high risk AML patients. Disclosures Strickland: Amgen: Other: Advisory Board Particpation; Boehringer-Ingelheim: Other: Advisory Board Particpation; Daiichi-Sankyo: Other: Advisory Board Particpation; Sunesis Pharmaceuticals: Other: Steering Committee and Advisory Board Participation; Alexion Pharmaceuticals: Other: Advisory Board Particpation. Rezvani:Pharmacyclics: Research Funding. Townsley:Novartis: Research Funding; GSK: Research Funding.

Growth-inhibiting effects of arsenic trioxide plus epigenetic therapeutic agents on leukemia cell lines

2009 ◽  
Vol 51 (2) ◽  
pp. 297-303 ◽  
Author(s):  
Chun-Yan Peng ◽  
Jikai Jiang ◽  
Hai-Tao Zheng ◽  
Xiao-Shan Liu
Keyword(s):  
Arsenic Trioxide ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Therapeutic Agents ◽  
Leukemia Cell Lines ◽  
Growth Inhibiting

Arsenic Trioxide and Melarsoprol Induce Programmed Cell Death in Myeloid Leukemia Cell Lines and Function in a PML and PML-RAR Independent Manner

Blood ◽  
1998 ◽  
Vol 92 (5) ◽  
pp. 1497-1504 ◽  
Author(s):  
Zhu-Gang Wang ◽  
Roberta Rivi ◽  
Laurent Delva ◽  
Andrea König ◽  
David A. Scheinberg ◽  
...  
Keyword(s):  
Cell Line ◽  
Arsenic Trioxide ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Nuclear Bodies ◽  
Independent Manner ◽  
Leukemia Cell Lines ◽  
Induced Apoptosis ◽  
Myeloid Leukemia Cell

Abstract Inorganic arsenic trioxide (As2O3) and the organic arsenical, melarsoprol, were recently shown to inhibit growth and induce apoptosis in NB4 acute promyelocytic leukemia (APL) and chronic B-cell leukemia cell lines, respectively. As2O3 has been proposed to principally target PML and PML-RAR proteins in APL cells. We investigated the activity of As2O3 and melarsoprol in a broader context encompassing various myeloid leukemia cell lines, including the APL cell line NB4-306 (a retinoic acid–resistant cell line derived from NB4 that no longer expresses the intact PML-RAR fusion protein), HL60, KG-1, and the myelomonocytic cell line U937. To examine the role of PML in mediating arsenical activity, we also tested these agents using murine embryonic fibroblasts (MEFs) and bone marrow (BM) progenitors in which the PML gene had been inactivated by homologous recombination. Unexpectedly, we found that both compounds inhibited cell growth, induced apoptosis, and downregulated bcl-2 protein in all cell lines tested. Melarsoprol was more potent than As2O3 at equimolar concentrations ranging from 10−7 to 10−5 mol/L. As2O3 relocalized PML and PML-RAR onto nuclear bodies, which was followed by PML degradation in NB4 as well as in HL60 and U937 cell lines. Although melarsoprol was more potent in inhibiting growth and inducing apoptosis, it did not affect PML and/or PML-RAR nuclear localization. Moreover, both As2O3 and melarsoprol comparably inhibited growth and induced apoptosis of PML+/+ and PML−/− MEFs, and inhibited colony-forming unit erythroid (CFU-E) and CFU granulocyte-monocyte formation in BM cultures of PML+/+ and PML−/− progenitors. Together, these results show that As2O3 and melarsoprol inhibit growth and induce apoptosis independent of both PML and PML-RAR expression in a variety of myeloid leukemia cell lines, and suggest that these agents may be more broadly used for treatment of leukemias other than APL. © 1998 by The American Society of Hematology.

scholarly journals Tyrosine kinase inhibitors and interferon‐α increase tunneling nanotube (TNT) formation and cell adhesion in chronic myeloid leukemia (CML) cell lines

2020 ◽  
Vol 34 (3) ◽  
pp. 3773-3791 ◽  
Author(s):  
Maria Omsland ◽  
Vibeke Andresen ◽  
Stein‐Erik Gullaksen ◽  
Pilar Ayuda‐Durán ◽  
Mihaela Popa ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Interferon Α ◽  
Tunneling Nanotube

Tyrosine kinase inhibitor resistance in chronic myeloid leukemia cell lines: investigating resistance pathways

2011 ◽  
Vol 52 (11) ◽  
pp. 2139-2147 ◽  
Author(s):  
Carine Tang ◽  
Lisa Schafranek ◽  
Dale B. Watkins ◽  
Wendy T. Parker ◽  
Sarah Moore ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Leukemia Cell ◽  
Leukemia Cell Lines ◽  
Inhibitor Resistance ◽  
Myeloid Leukemia Cell
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