Identification of the Epigenetic Reader BRD4 As a Novel Potential Target in Ph+ CML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1571-1571
Author(s):  
Barbara Peter ◽  
Gregor Eisenwort ◽  
Gabriele Stefanzl ◽  
Daniela Berger ◽  
Wolfgang R Sperr ◽  
...  
Keyword(s):  
Cell Lines ◽  
Research Funding ◽  
Drug Target ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Thymidine Uptake ◽  
Dependent Manner ◽  
Potential Drug ◽  
Ku812 Cells

Abstract Chronic myelogenous leukemia (CML) is a bone marrow-derived hematopoietic neoplasm in which BCR/ABL1 acts as a major driver of proliferation, differentiation and survival of leukemic cells. In a majority of all patients with CML, leukemic cells can be kept under control by BCR/ABL1 tyrosine kinase inhibitors (TKI), including imatinib, nilotinib, dasatinib, bosutinib, and ponatinib. Nevertheless, resistance or intolerance against one or more of these TKI may occur. Therefore, current research is focusing on novel potential drug targets in CML. A promising class of targets may be epigenetic regulators of cell growth, such as members of the bromodomain and extra-terminal domain (BET) family. The epigenetic reader and BET family member BRD4 has recently been identified as a novel potential drug target in acute myeloid leukemia (AML). However, so far, little is known about the expression and function of BRD4 in CML cells. The aims of the present study were to determine the expression of BRD4 and its downstream target MYC in CML cells and to explore whether BRD4 can serve as a novel drug target in this disease. As determined by qPCR, primary CML cells (chronic phase patients, n=7) as well as the CML cell lines KU812 and K562 expressed BRD4 mRNA. In addition, both CML cell lines stained positive for BRD4 in our immunocytochemistry staining experiments. In one patient with accelerated phase CML, putative leukemic (CD34+/CD38-) stem cells were sorted to near homogeneity and found to express BRD4 mRNA by qPCR. In order to examine the functional role of BRD4 in CML cells, a BRD4-specific shRNA was applied. In these experiments, the shRNA-induced knockdown of BRD4 in KU812 cells and K562 resulted in reduced growth compared to a control shRNA. Furthermore, the BRD4-targeting drug JQ1 was found to inhibit 3H-thymidine uptake and thus proliferation in KU812 cells in a dose-dependent manner (IC50: 0.25-0.75 µM). In addition, we were able to show that JQ1 inhibits growth of primary CML cells with variable IC50 values (0.1-5 µM). However, no substantial growth-inhibitory effects of JQ1 were seen in K562 cells (IC50: >5 µM). As determined by Annexin V/PI staining, JQ1 induced apoptosis in KU812 cells whereas no apoptosis-inducing effect of JQ1 was observed in K562 cells. Nevertheless, we were able to show that both CML cell lines as well as primary CML cells express MYC mRNA, and treatment of KU812 cells or K562 cells with JQ1 resulted in a decreased expression of MYC mRNA and MYC protein. Next, we analyzed whether MYC expression in CML cells can be blocked by BCR/ABL1 TKI. We found that imatinib, nilotinib, dasatinib, and ponatinib decrease MYC mRNA- and MYC protein expression in KU812 and K562 cells. Finally, we found that JQ1 cooperates with imatinib, nilotinib, ponatinib and dasatinib in inhibiting the proliferation of KU812 and K562 cells. Together, our data show that BRD4 serves as a potential new target in CML cells, and that the BRD4 blocker JQ1 cooperates with BCR/ABL1 TKI in inducing growth-inhibition. Whether BRD4 inhibition is a pharmacologically meaningful approach in patients with TKI-resistant CML remains to be determined in clinical trials. Disclosures Sperr: Ariad: Consultancy; Celgene: Consultancy. Zuber:Mirimus Inc.: Consultancy, Other: Stock holder; Boehringer Ingelheim: Research Funding. Valent:Novartis: Consultancy, Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria.

scholarly journals BRD4 Degradation Is a Potent Approach to Block MYC Expression and to Overcome Multiple Forms of Stem Cell Resistance in Ph+ CML

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1722-1722 ◽  
Author(s):  
Barbara Peter ◽  
Gregor Eisenwort ◽  
Alexandra Keller ◽  
Karin Bauer ◽  
Daniela Berger ◽  
...  
Keyword(s):  
Stem Cell ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Drug Targets ◽  
K562 Cells ◽  
Leukemic Cells ◽  
Multiple Forms ◽  
Tki Resistance ◽  
Ku812 Cells ◽  
Induced Apoptosis

Abstract Chronic myeloid leukemia (CML) is a hematopoietic stem cell neoplasm in which BCR-ABL1 acts as a major driver of proliferation, differentiation and survival of leukemic cells. In a majority of all patients, leukemic cells can be kept under control by BCR-ABL1 tyrosine kinase inhibitors (TKI). Nevertheless, resistance against one or more TKI may occur. Therefore, research is focusing on novel potential drug targets in CML. We have recently identified the epigenetic reader bromodomain-containing protein 4 (BRD4) as a new therapeutic target in leukemic stem cells (LSC) in acute myeloid leukemia. In the present study, we examine the expression of BRD4 and its downstream effector MYC in CML cells and asked whether BRD4 serves as a drug target in CML cells and whether BRD4-targeting drugs, including JQ1 and newly developed BRD4 degraders (dBET1 and dBET6) are able to overcome LSC resistance in CML. Primary CML cells were obtained from 22 patients with chronic phase (CP) CML and 3 with blast phase (BP) CML. As determined by qPCR and/or immunocytochemistry, the CML cell lines KU812 and K562 as well as primary CML cells expressed BRD4 and MYC. All three BRD4-targeting drugs (JQ1, dBET1 and dBET6) were found to decrease MYC expression in KU812 and K562 cells as assessed by Western blotting. In 3H-thymidine uptake experiments, JQ1 and dBET6 were found to inhibit the proliferation of KU812 in a dose-dependent manner (IC50, JQ1: 100-500 nM; dBET6: 50-100 nM) whereas dBET1 showed only little if any effects on growth of KU812 cells (IC50: 1-5 µM), and in K562 cells, only dBET6 was found to inhibit growth with a reasonable IC50 value (250-500 nM). Corresponding results were obtained when examining drug effects on survival of CML cell lines by Annexin-V/PI staining. All three BRD4-targeting drugs were found to inhibit proliferation of primary CP CML cells with varying IC50 values. As expected, growth-inhibitory effects of dBET6 were more pronounced (IC50: <100 nM) compared to effects seen with JQ1 and dBET1. dBET1 and dBET6 were also found to inhibit growth of primary CML cells obtained from patients with BP CML, whereas JQ1 was not effective. JQ1 also failed to suppress survival on CML CD34+/CD38− LSC. By contrast, dBET1 induced apoptosis in CML LSC at 1 µM and dBET6 induced apoptosis in CML LSC at 0.1 µM. dBET6 induced apoptosis in CML LSC obtained from patients with imatinib-sensitive CML as well as patients with imatinib-resistant CML harboring BCR-ABL1 T315I or BCR-ABL1 F317L. Finally, pre-incubation of CD34+ CP CML cells with dBET6 resulted in reduced leukemic engraftment in NSG mice exhibiting human membrane-bound stem cell factor, SCF [NSG-Tg(hu-mSCF)] 6 months after transplantation (engraftment with CD45+/CD33+/CD19−cells in control mice receiving DMSO-treated cells: 8.1±6.6% vs mice receiving dBET6-treated cells: 1.1±0.6%). To further explore the ability of dBET6 to interfere with LSC resistance in CML, we established a co-culture system mimicking LSC-niche interactions in the osteoblastic niche. In this model, co-culturing K562 cells, KU812 cells or primary CML LSC with the osteoblast-like osteosarcoma cell line CAL-72 resulted in resistance against nilotinib and ponatinib. In this culture system, JQ1 was found to partially restore TKI effects in K562 cells and completely restored TKI effects in KU812 cells. Interestingly, JQ1 was not able to restore TKI effects in primary CML LSC in these co-cultures. However, dBET6 was found to overcome niche cell-induced TKI-resistance of primary CML LSC. Finally, we were able to demonstrate that JQ1, dBET1 and dBET6 inhibit interferon-gamma-induced upregulation of PD-L1 expression in CML LSC. Together we show that BRD4 and MYC are potential new therapeutic drug targets in CML and that the BET-degrader dBET6 overcomes multiple forms of LSC resistance, including i) intrinsic resistance, ii) mutation-induced resistance, iii) niche induced resistance and iv) checkpoint-mediated resistance. Whether BRD4 degradation is also able to overcome TKI-resistance of BCR-ABL1+ LSC in vivo in patients with CML remains to be determined in clinical trials. Disclosures Hoermann: Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria. Wolf:BMS: Honoraria, Research Funding; Pfizer: Honoraria; Novartis: Honoraria, Research Funding; AOP Orphan: Honoraria, Research Funding. Mayer:Amgen: Research Funding; Novartis: Research Funding. Zuber:Mirimus Inc.: Consultancy, Other: Shareholder; Boehringer Ingelheim GmbH & Co KG: Research Funding. Sperr:Novartis: Honoraria; Pfizer: Honoraria; Daiichi Sankyo: Honoraria. Valent:Pfizer: Honoraria; Incyte: Honoraria; Novartis: Honoraria.

Metabolic Capability to Induce the Pasteur Effect Mediates Sensitivity of Human Leukemic Cells to Metformin

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2601-2601
Author(s):  
Sarah Scotland ◽  
Estelle Saland ◽  
Lindsay Peyriga ◽  
Rémi Peyraud ◽  
Elizabeth Micklow ◽  
...  
Keyword(s):  
Cell Lines ◽  
Research Funding ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Pasteur Effect ◽  
Cytotoxic Response ◽  
Leukemic Cell Lines

Abstract Abstract 2601 An emerging hallmark of cancer cells is the reprogramming of intermediary and energy metabolism these cells undergo. Several epidemiological studies have shown that metformin, widely used to treat patients with type 2 diabetes, may reduce their risk of cancer. Despite several reports of anti-neoplastic activity of metformin, the mechanisms responsible for this activity have not been fully elucidated in cancer or leukemic cells. We hypothesized that metformin elicits a metabolic reprogramming driven by alterations in mitochondrial function and signaling, which induces apoptosis in leukemic cells, and that metabolic flexibility determines the variation(s) of the cytotoxic response to metformin among different leukemic cell lines. We first demonstrated that metformin markedly decreased oxygen consumption of six leukemic cell lines in a concentration-dependent manner. We also observed that the cytotoxic effect of metformin varies between cell lines reflecting their energetic capacity to compensate for the mitochondrial inhibition induced by metformin (eg. to induce the Pasteur effect). Importantly, metformin-insensitive leukemic cells did not exhibit a Pasteur effect in response to metformin. All leukemic cells exhibited high basal conversion of glucose to lactate (eg. aerobic glycolysis) and specific expression of key metabolic genes as compared to normal mononuclear cells. Despite dependence on glucose catabolism, metformin sensitivity was associated with relative resistance to glucose starvation. Metformin effects in drug-resistant cells were potentiated by the addition of a glycolytic inhibitor, but not by inhibitors of the pentose phosphate pathway or glutaminolysis. Leukemic cells with broad metabolic capacities to utilize other energetic substrates in response to diverse nutrient starvation showed insensitivity to metformin. Metformin induced a significant decrease in metabolites of the upper segment of glycolysis and the oxidative branch of the pentose phosphate pathway as well as a clear increase of PRPP and IMP biosynthesis. Energy charge, the nucleotide phosphate pool and lactate/glucose ratio remained stable after metformin treatment. Furthermore, our results showed that basal glucose uptake/consumption and the activity of the lower segment of the glycolytic pathway are key determinants of a cytotoxic response to metformin. In addition, high glutathione, malate, IMP and orotate content were observed in metformin-insensitive leukemic cells. Moreover, the cytotoxic effect of metformin was independent of AMPK/LKB1 status of the leukemic cells while p53 expression abrogated this effect. The presence of wild-type p53 appears to partially protect tumor cells from glucose starvation and metformin cytotoxicity and prevents the induction of the Pasteur effect. Finally, we demonstrated that metformin increased the cytotoxicity of chemotherapy agent, cytarabine, on all leukemic cell lines in vitro and significantly reduced leukemic colony-forming units (CFU-L) from six primary AML patient samples in a concentration-dependent manner. Additional experiments on metabolic and signaling pathways as well as in vivo studies are in progress to better understand the cytotoxic response of metformin in both AML cell lines and primary AML patient specimens that impact the therapeutic potential of metformin in vivo. Disclosures: Carroll: Agios Pharmaceuticals: Research Funding; TetraLogic Pharmaceuticals: Research Funding; Sanofi Aventis Corporation: Research Funding; Glaxo Smith Kline, Inc.: Research Funding.

Effects of Bosutinib (SKI-606) in CML: Kinase Target Profile, Effects on BCR/ABL Mutants, and Synergism with Dasatinib in T315I+ Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3195-3195
Author(s):  
Karoline Veronika Gleixner ◽  
Lily L Remsing Rix ◽  
Christian Baumgartner ◽  
Uwe Rix ◽  
Alexander Gruze ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Synergistic Effects ◽  
K562 Cells ◽  
Leukemic Cells ◽  
Kinase Assay ◽  
Thymidine Uptake ◽  
Dependent Manner ◽  
Growth Inhibitory ◽  
Imatinib Resistant

Abstract Chronic myeloid leukemia (CML) is a stem cell disease characterized by the BCR/ABL oncoprotein. The ABL kinase inhibitor imatinib is effective in most patients and considered standard first line therapy. However, not all patients show a long-lasting response. Treatment failure is usually associated with the occurrence of imatinib-resistant mutants of BCR/ABL. For these patients, novel multi-kinase inhibitors such as dasatinib represent alternative treatment options. Still, however, not all patients respond to these drugs, especially when leukemic cells bear the BCR/ABL mutant T315I that confers resistance against most kinase-blockers. Bosutinib is a novel multi-kinase inhibitor that has been described to act growth-inhibitory in ABL-transformed leukemias. In the current study, we examined the effects of bosutinib alone and in combination with dasatinib on growth and survival of CML cells. Bosutinib was found to inhibit 3H-thymidine uptake and thus proliferation in imatinib-sensitive and imatinib-resistant K562 cells in a dose-dependent manner, with identical IC50 values (10–100 nM). Moreover, bosutinib was found to inhibit the growth of primary CML cells and Ba/F3 cells bearing various imatinibresistant mutants of BCR/ABL, except the T315I mutant (IC50&gt;1 μM). The growth-inhibitory effects of bosutinib were found to be associated with signs of apoptosis. Dasatinib showed similar effects on CML cells, and again did not block the growth of subclones bearing BCR/ABL T315I. Unexpectedly, however, we found that bosutinib and dasatinib synergize with each other in producing growth inhibition in primary CML cells exhibiting BCR/ABL T315I at pharmacologic concentrations (0.01–1 μM). Clear synergistic effects were also observed in imatinib-sensitive and imatinib-resistant K562 cells as well as in Ba/F3 cells bearing BCR/ABL T315I. In parallel, we performed multiplexed kinase assays as well as chemical proteomics analysis and mass spectrometry using K562 cells and primary CML cells and coupleable dasatinib and bosutinib analogues. In these experiments, dasatinib and bosutinib were found to express an overlapping, but non-identical profile of target kinases. As expected, both drugs were found to bind to wt ABL, SRC kinases, and TEC-family kinases including BTK. Specific targets preferentially bound and inhibited by bosutinib were STE20s, the FES/FER family, CAMKIIG, PYK2 and TBK1. We were also able to confirm that the dasatinib-targets KIT and PDGFRA are not recognized by bosutinib. Interestingly, whereas wt ABL (IC50&lt;0.5 nM) and most of the ABL mutants tested (H396P, M351T, Q252H, and Y253F) were all completely inhibited by both drugs at 1 μM in the kinase assay, the ABL T315I mutant was inhibited by bosutinib (IC50=26 nM) almost 70 times more potently than by dasatinib. Together, these data show that bosutinib and dasatinib synergize with each other in producing antileukemic effects on CML cells including BCR/ABL T315I+ subclones. These synergistic effects may be explained by differential target kinase profiles and by the fact that bosutinib retains some activity against the BCR/ABL T315I mutant kinase.

Identification of Basophils as Source of Hepatocyte Growth Factor (HGF) In CML: a Potential Trigger of Disease Acceleration.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1202-1202
Author(s):  
Sabine Cerny-Reiterer ◽  
Karl J. Aichberger ◽  
Harald Herrmann ◽  
Gregor Hoermann ◽  
Barbara Peter ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Hepatocyte Growth Factor ◽  
Cell Line ◽  
Research Funding ◽  
K562 Cells ◽  
Leukemic Cells ◽  
Mrna Levels ◽  
Ku812 Cells ◽  
Hepatocyte Growth

Abstract Abstract 1202 Chronic myeloid leukemia (CML) is a myeloproliferative disorder in which BCR/ABL leads to enhanced survival of leukemic cells. Several different angiogenic molecules, including vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), have been implicated in the pathogenesis of CML. Enhanced production of these pro-angiogenic molecules in CML may be associated with disease acceleration. However, little is known so far about the exact origin of these growth factors and about mechanisms underlying their production and secretion in CML cells. In the current study, we analyzed the cellular distribution of HGF and its receptor c-MET in CML cells, and explored the mechanism of expression of HGF in BCR/ABL-transformed cells. As assessed by immunostaining of bone marrow sections and isolated blood and bone marrow samples, the HGF protein was found to be expressed in a subset of leukemic cells in all patients tested. In addition, CML cells were found to express HGF mRNA and c-MET mRNA. In consecutive experiments, we were able to show that basophils are the primary source of HGF in CML. In particular, highly enriched sorted CD203c+ CML basophils were found to express substantial amounts of HGF mRNA as well as the HGF protein. Correspondingly, leukemic cell samples obtained from patients with accelerated phase CML were found to contain higher HGF mRNA levels compared to cells obtained from patients in chronic phase or blast phase CML. Finally, HGF mRNA and the HGF protein were detectable in the basophil-committed CML cell line KU812, but not in the more immature Ph+ cell line K562. We next asked whether expression of HGF in CML cells depends on BCR/ABL. To address this question, Ba/F3 cells with doxycycline-inducible expression of BCR/ABL were employed. However, BCR/ABL failed to induce expression of HGF mRNA or the HGF protein in Ba/F3 cells. Correspondingly, the BCR/ABL-blocker imatinib was found to inhibit expression of VEGF mRNA, but did not inhibit HGF mRNA expression in KU812 cells. Next, we examined the expression of c-MET in CML cells. c-MET mRNA was found to be expressed in KU812 and K562 cells, in highly enriched CD34+/CD38- CML stem cells, and less abundantly in more mature CD34+/CD38+ CML cells and CML basophils. The c-MET inhibitor MSC-2156119J-15 (Merck-Serono Darmstadt, Germany) was found to counteract growth of primary CML cells, K562 cells, and KU812 cells with comparable IC50 values (0.5-1.0 μM). In summary, our data suggest that HGF is a BCR/ABL-independent basophil-derived mediator in CML. Basophils and basophil-derived mediators may play a more active role in CML-acceleration as has been considered previously. Whether targeting of HGF or/and c-MET is an effective approach to block acceleration in CML remains to be elucidated. Disclosures: Valent: Novartis: Honoraria, Research Funding; Merck-Serono: Research Funding.

Investigation of the Roles of Non-neuronal Acetylcholine in Chronic Myeloid Leukemic Cells and their Erythroid or Megakaryocytic Differentiated Lines

2019 ◽  
Vol 18 (10) ◽  
pp. 1440-1447 ◽  
Author(s):  
Banu Aydın ◽  
Hülya Cabadak ◽  
M. Zafer Gören
Keyword(s):  
Cell Lines ◽  
K562 Cell ◽  
Ache Activity ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
Choline Uptake ◽  
Dependent Manner ◽  
Uptake Inhibition ◽  
Cell Counts

Background: Many studies suggested that Acetylcholine (ACh) might serve as an autocrine/ paracrine growth factor in several types of tumors or tumor cell lines. High levels of Acetylcholinesterase (AChE) activity have been reported in primary brain tumors, ovarian, colon and lung tumors. Objectives: The role of cholinergic signaling needs to be clarified in in leukemia. Method: K562 cells were derived from a chronic myelogenous leukemia patient during blast crisis serving as pluripotent hematopoietic stem cells. K562 cells were incubated with various cholinergic agonists or antagonists to investigate the role of ACh in different differentiated cell lines. Results: Our experiments showed that AChE activity was increased in response to ACh in undifferentiated K562 cells, but in the erythroid differentiated K562 cells a high concentration of ACh (1 mM) decreased the AChE activity. ACh failed to elevate the AChE activity in the megakaryocytic differentiated K562 cells. An AChE inhibitor, eserine, also suppressed the AChE activity in a concentration-dependent manner. Choline uptake inhibition by hemicholinium did increase the AChE activity but not in the erythroid differentiated K562 cell line. Likewise, megakaryocytic differentiated K562 cells also displayed a similar pattern. Vesamicole, a vesicular choline uptake inhibitor, produced similar results. Curare, a nicotinic antagonist, elevated the cell counts of the megakaryocytic differentiated cells. Conclusion: Our findings may suggest excess extracellular ACh will decrease the cell growth in undifferentiated and megakaryocytic differentiated K562 cell lines through nicotinic type cholinoceptors.

Chlorogenic acid inhibits Bcr-Abl tyrosine kinase and triggers p38 mitogen-activated protein kinase–dependent apoptosis in chronic myelogenous leukemic cells

Blood ◽  
2004 ◽  
Vol 104 (8) ◽  
pp. 2514-2522 ◽  
Author(s):  
Gautam Bandyopadhyay ◽  
Tanusree Biswas ◽  
Keshab C. Roy ◽  
Swapan Mandal ◽  
Chhabinath Mandal ◽  
...  
Keyword(s):  
Chlorogenic Acid ◽  
Cell Lines ◽  
Salivary Gland Tumor ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
Mitogen Activated Protein Kinase ◽  
Leukemic Cells ◽  
Abl Kinase ◽  
Mitogen Activated Protein

Abstract We report that chlorogenic acid (Chl) induces apoptosis of several Bcr-Abl–positive chronic myelogenous leukemia (CML) cell lines and primary cells from CML patients in vitro and destroys Bcr-Abl–positive K562 cells in vivo. In contrast, this compound has no effect on the growth and viability of Bcr-Abl–negative lymphocytic and myeloid cell lines and primary CML cells. Sodium chlorogenate (NaChl) exhibits 2-fold higher efficiency in killing K562 cells compared with Chl. NaChl also induces growth inhibition of squamous cell carcinoma (HSC-2) and salivary gland tumor cells (HSG), although at 50-fold higher concentration. NaChl inhibits autophosphorylation of p210Bcr-Abl fusion protein rapidly. We demonstrate that p38 phosphorylation is increased in Bcr-Abl–positive cells after treatment with NaChl and closely paralleled the inhibition of Bcr-Abl phosphorylation. NaChl did not increase phosphorylation of p38 in Bcr-Abl–negative cells including HSC-2 and HSG that are responsive to this compound, indicating that p38 activation by NaChl is dependent on Bcr-Abl kinase inhibition. Inhibition of p38 activity by SB203580 significantly reduced NaChl-induced apoptosis of K562 cells, whereas activation of p38 by anisomycin augmented the apoptosis. These findings indicate that inhibition of Bcr-Abl kinase leading to activation of p38 mitogen-activated protein (MAP) kinase may play an important role in the anti-CML activity of Chl.

scholarly journals The ABCC6 Transporter Plays a Significant Role in the Efflux of Nilotinib and Dasatinib, and May Contribute to Tyrosine Kinase Inhibitor Resistance

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4237-4237
Author(s):  
Laura N Eadie ◽  
Jarrad M Goyne ◽  
Timothy P. Hughes ◽  
Deborah L White
Keyword(s):  
Mrna Expression ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
K562 Cells ◽  
Mrna Levels ◽  
Advisory Committees ◽  
Ku812 Cells

Abstract Efflux transporters ABCB1 and ABCG2 interact with tyrosine kinase inhibitors (TKIs) and mediate drug resistance, however, evidence of the interaction of other potentially relevant drug transporters with TKIs is lacking. We investigated the involvement of the closely related transporter ABCC6, in imatinib (IM), nilotinib (NIL) and dasatinib (DAS) transport and also the role of ABCC6 in NIL resistance. The impact of short-term (overnight) exposure to NIL on mRNA expression of ABC transporters in three BCR-ABL1+ cell lines was assessed by Taqman transporter array: K562, K562-Dox and KU812 cells. Several transporters of interest were identified, including ABCC6, based on alterations in mRNA expression. In order to elucidate the importance of ABCC6 in the development of NIL resistance, ABCC6 mRNA levels were determined by RT-PCR in K562 and K562-Dox NIL-resistant lines generated in vitro and compared with ABCC6 mRNA levels in respective parental control cells. ABCC6 protein expression was confirmed by western blot. p-Crkl dependent IC50 experiments in the absence and presence of three ABCC6 inhibitors (indomethacin, INDO; probenecid, PRO; pantoprazole, PP) were performed in patient mononuclear cells (MNCs) and BCR-ABL1+ cell lines to assess the role of ABCC6 in NIL, IM and DAS transport. A marked increase in ABCC6 mRNA expression in response to short-term in vitro NIL exposure occurred: in K562 and KU812 cells ABCC6 mRNA levels increased 9.5- and 9.7-fold in response to overnight NIL exposure respectively. Increased expression of ABCC6 was also observed in cells subjected to long-term NIL exposure during development of NIL resistance in vitro. NIL-resistant K562 cells demonstrated up to 57-fold higher levels of ABCC6 mRNA compared with control cells (p=0.002). Analogous results were observed in NIL-resistant K562-Dox cells (up to 33-fold higher levels of ABCC6 mRNA p=0.002). In order to determine the relevance of ABCC6 in patient cells, p-Crkl dependent IC50 experiments were performed in MNCs from de novo CML patients in the absence and presence of ABCC6 inhibition. Results demonstrated a significant reduction in IC50NIL in the presence of all three ABCC6 inhibitors compared with IC50NIL in the absence of inhibitors. Similar results were observed for IC50DAS but not IC50IM. Experiments in three parental BCR-ABL1+ cell lines confirmed these findings (Table 1). Notably, comparison of IC50 values in the absence of ABCC6 inhibition in KU812 vs. K562 cells revealed that KU812 cells demonstrated increased IC50NIL (307 vs. 257 nM, p=0.0493) and IC50DAS (14 vs 8 nM, p=0.0005). This was unexpected given both cell lines demonstrate negligible expression of ABCB1 (a transporter known to interact with both NIL and DAS). However, assessment of ABCC6 protein levels by western blotting revealed KU812 cells have greater levels of ABCC6 when compared with K562 cells: 53% in KU812 vs. 24% in K562 (ABCC6 normalised to β-actin). A greater %reduction in IC50NIL and IC50DAS in the presence of ABCC6 inhibition was also observed in KU812 cells compared with K562 cells confirming the role of ABCC6 in the transport of NIL and DAS. Combined, these studies highlight the importance of ABCC6 in the export of NIL and DAS from patient MNCs and BCR-ABL1+ cell lines. This is the first report of ABCC6 involvement in TKI transport and results suggest ABCC6 overexpression may also contribute to NIL resistance. The addition of ABCC6 inhibitors to NIL and DAS therapy may enhance the efficacy of these TKIs in the treatment of CML. Disclosures Hughes: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Australasian Leukaemia and Lymphoma Group (ALLG): Other: Chair of the CML/MPN Disease Group. White:Ariad: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Regulation of the Erk2-Elk1 signaling pathway and megakaryocytic differentiation of Bcr-Abl+ K562 leukemic cells by Gab2

Blood ◽  
2002 ◽  
Vol 99 (4) ◽  
pp. 1388-1397 ◽  
Author(s):  
Jay F. Dorsey ◽  
Jess M. Cunnick ◽  
Shrikant M. Mane ◽  
Jie Wu
Keyword(s):  
Map Kinase ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Blast Crisis ◽  
Terminal Differentiation ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Megakaryocytic Differentiation

In the blast crisis phase of chronic myelogenous leukemia (CML), Bcr-Abl+ myeloblasts fail to undergo terminal maturation. The extracellular signal–regulated kinase (Erk) mitogen-activated protein (MAP) kinase has been shown to mediate terminal differentiation of myeloid cells. Interestingly, Bcr-Abl+ CML cell lines established from blast crisis were found to have low Erk MAP kinase activity. In this study, we analyzed the role of the Gab2 docking protein in regulation of the Erk MAP kinase in Bcr-Abl+K562 human CML cells. Overexpression of Gab2 in K562 cells resulted in transcriptional activation of the c-fos serum response element (SRE) promoter, whereas overexpression of SHP2, Grb2, and CrkL had no effect. Activation of the c-fos SRE transcriptional activity by Gab2 required tyrosine 604, which is a SHP2 docking site on Gab2, and the SHP2 tyrosine phosphatase activity. Elk1, c-Jun, and CHOPtrans-reporting assays indicated that overexpression of Gab2 selectively activated the Erk2-Elk1 signaling pathway. To determine cellular consequences of elevating the Gab2 level in K562 cells, stable cell lines for doxycycline-inducible expression of the wild-type Gab2 (Gab2WT) and an SHP2-binding defective Gab2 (Gab2Tyr604Phe) were established. Analysis of these cell lines indicated that induction of Gab2WT expression, but not Gab2Tyr604Phe expression, led to Erk activation, growth arrest, cell spreading, and enlargement; expression of megakaryocyte/platelet lineage–specific integrins αIIb/β3 (CD41/CD61); and upregulation of RNA for megakaryocyte/platelet proteins. All of these changes are characteristics of megakaryocytic differentiation. Together, these results reveal Gab2 as a limiting signaling component for Erk MAP kinase activation and terminal differentiation of K562 CML cells.

scholarly journals Induction of apoptosis by Kola nut extract as a recent and promising treatment strategy for Leukemia

Bionatura ◽  
2021 ◽  
Vol 6 (2) ◽  
pp. 1725-1732
Author(s):  
Hamdah Alsaeedi ◽  
Rowaid Qahwaji ◽  
Talal Qadah
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
Time Dependent ◽  
Leukemia Cell Line ◽  
Apoptotic Cells ◽  
Dependent Manner ◽  
Anti Cancer

Kola nut extracts have recently been reported to contain chemopreventive compounds providing several pharmacological benefits. This study investigated Kola nut extracts' anti-cancer activity on human immortalized myelogenous leukemia cell line K562 through apoptosis and cell cycle arrest. Fresh Kola nuts were prepared as powder and dissolved in DMSO. Different concentrations (50, 100, 150, 200, and 250 μg/ml) of working solutions were prepared. The K562 cells were treated with the different concentrations of Kola nut extract or vehicle control (10% DMSO) followed by incubation at 37°C for 24, 48, and 72 hours, respectively. Treatment activity was investigated in K562 cells; by Resazurin, and FITC/Propidium Iodide and 7-AAD stained cells to evaluate apoptotic cells and the cell cycle's progression. Inhibition of leukemia cell proliferation was observed. The extract effectively induced cell death, early and late apoptosis by approximately 30% after 24 and 48 hours incubation, and an increase in the rate of dead cells by 50% was observed after 72 hours of incubation. Also, cell growth reduction was seen at high dose concentrations (150 and 200 µg/ml), as evident by cell count once treated with Kola nut extract. The total number of apoptotic cells increased from 5.8% of the control group to 27.4% at 250 µg/ml concentration. Moreover, Kola nut extracts' effects on K562 cells increased gradually in a dose and time-dependent manner. It was observed that Kola nut extracts could arrest the cell cycle in the G2/M phase as an increase in the number of cells by 29.8% and 14.6 % were observed from 9.8% and 5.2% after 24 and 48 hours of incubation, respectively. This increase was detected in a dose and time-dependent manner. Kola nut extracts can be used as a novel anti-cancer agent in Leukemia treatment as it has shown significant therapeutic potential and therefore provides new insights in understanding the mechanisms of its action. Keywords: Kola nut extracts, Leukemia, K562 cell line, Apoptosis, Cancer.

scholarly journals All-trans retinoic acid enhances, and a pan-RAR antagonist counteracts, the stem cell promoting activity of EVI1 in acute myeloid leukemia

2019 ◽  
Vol 10 (12) ◽  
Author(s):  
Chi Huu Nguyen ◽  
Katharina Bauer ◽  
Hubert Hackl ◽  
Angela Schlerka ◽  
Elisabeth Koller ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Retinoic Acid ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Acute Myeloid

AbstractEcotropic virus integration site 1 (EVI1), whose overexpression characterizes a particularly aggressive subtype of acute myeloid leukemia (AML), enhanced anti-leukemic activities of all-trans retinoic acid (atRA) in cell lines and patient samples. However, the drivers of leukemia formation, therapy resistance, and relapse are leukemic stem cells (LSCs), whose properties were hardly reflected in these experimental setups. The present study was designed to address the effects of, and interactions between, EVI1 and retinoids in AML LSCs. We report that Evi1 reduced the maturation of leukemic cells and promoted the abundance, quiescence, and activity of LSCs in an MLL-AF9-driven mouse model of AML. atRA further augmented these effects in an Evi1 dependent manner. EVI1 also strongly enhanced atRA regulated gene transcription in LSC enriched cells. One of their jointly regulated targets, Notch4, was an important mediator of their effects on leukemic stemness. In vitro exposure of leukemic cells to a pan-RAR antagonist caused effects opposite to those of atRA. In vivo antagonist treatment delayed leukemogenesis and reduced LSC abundance, quiescence, and activity in Evi1high AML. Key results were confirmed in human myeloid cell lines retaining some stem cell characteristics as well as in primary human AML samples. In summary, our study is the first to report the importance of EVI1 for key properties of AML LSCs. Furthermore, it shows that atRA enhances, and a pan-RAR antagonist counteracts, the effects of EVI1 on AML stemness, thus raising the possibility of using RAR antagonists in the therapy of EVI1high AML.

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