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.

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.

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.

scholarly journals Apoptosis Induced by Tanshinone IIA and Cryptotanshinone Is Mediated by Distinct JAK/STAT3/5 and SHP1/2 Signaling in Chronic Myeloid Leukemia K562 Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Ji Hoon Jung ◽  
Tae-Rin Kwon ◽  
Soo-Jin Jeong ◽  
Eun-Ok Kim ◽  
Eun Jung Sohn ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Biological Effects ◽  
K562 Cells ◽  
Tanshinone Iia ◽  
Anticancer Activities ◽  
Caspase 9 ◽  
Anticancer Effects ◽  
Induced Apoptosis ◽  
Better Than

Though tanshinone IIA and cryptotanshinone possess a variety of biological effects such as anti-inflammatory, antioxidative, antimetabolic, and anticancer effects, the precise molecular targets or pathways responsible for anticancer activities of tanshinone IIA and cryptotanshinone in chronic myeloid leukemia (CML) still remain unclear. In the present study, we investigated the effect of tanshinone IIA and cryptotanshinone on the Janus activated kinase (JAK)/signal transducer and activator of transcription (STAT) signaling during apoptotic process. We found that both tanshinone IIA and cryptotanshinone induced apoptosis by activation of caspase-9/3 and Sub-G1 accumulation in K562 cells. However, they have the distinct JAK/STAT pathway, in which tanshinone IIA inhibits JAK2/STAT5 signaling, whereas cryptotanshinone targets the JAK2/STAT3. In addition, tanshinone IIA enhanced the expression of both SHP-1 and -2, while cryptotanshinone regulated the expression of only SHP-1. Both tanshinone IIA and cryptotanshinone attenuated the expression of bcl-xL, survivin, and cyclin D1. Furthermore, tanshinone IIA augmented synergy with imatinib, a CML chemotherapeutic drug, better than cryptotanshinone in K562 cells. Overall, our findings suggest that the anticancer activity of tanshinone IIA and cryptotanshinone is mediated by the distinct the JAK/STAT3/5 and SHP1/2 signaling, and tanshinone IIA has the potential for combination therapy with imatinib in K562 CML cells.

CGP57148B (STI-571) induces differentiation and apoptosis and sensitizes Bcr-Abl–positive human leukemia cells to apoptosis due to antileukemic drugs

Blood ◽  
2000 ◽  
Vol 96 (6) ◽  
pp. 2246-2253 ◽  
Author(s):  
Guofu Fang ◽  
Caryn Naekyung Kim ◽  
Charles L. Perkins ◽  
Nimmanapalli Ramadevi ◽  
Elliott Winton ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Fas Ligand ◽  
Ectopic Expression ◽  
K562 Cells ◽  
Parp Cleavage ◽  
Human Leukemia ◽  
Sti 571 ◽  
Tnf Α ◽  
Cyt C ◽  
Induced Apoptosis

Abstract The differentiation and apoptosis-sensitizing effects of the Bcr-Abl–specific tyrosine kinase inhibitor CGP57148B, also known as STI-571, were determined in human Bcr-Abl–positive HL-60/Bcr-Abl and K562 cells. First, the results demonstrate that the ectopic expression of the p185 Bcr-Abl fusion protein induced hemoglobin in the acute myeloid leukemia (AML) HL-60 cells. Exposure to low-dose cytosine arabinoside (Ara-C; 10 nmol/L) increased hemoglobin levels in HL-60/Bcr-Abl and in the chronic myeloid leukemia (CML) blast crisis K562 cells, which express the p210 Bcr-Abl protein. As compared with HL-60/neo, HL-60/Bcr-Abl and K562 cells were resistant to apoptosis induced by Ara-C, doxorubicin, or tumor necrosis factor-α (TNF-α), which was associated with reduced processing of caspase-8 and Bid protein and decreased cytosolic accumulation of cytochrome c (cyt c). Exposure to CGP57148B alone increased hemoglobin levels and CD11b expression and induced apoptosis of HL-60/Bcr-Abl and K562 cells. CGP57148B treatment down-regulated antiapoptotic XIAP, cIAP1, and Bcl-xL, without affecting Bcl-2, Bax, Apaf-1, Fas (CD95), Fas ligand, Abl, and Bcr-Abl levels. CGP57148B also inhibited constitutively active Akt kinase and NFκB in Bcr-Abl–positive cells. Attenuation of NFκB activity by ectopic expression of transdominant repressor of IκB sensitized HL-60/Bcr-Abl and K562 cells to TNF-α but not to apoptosis induced by Ara-C or doxorubicin. Importantly, cotreatment with CGP57148B significantly increased Ara-C– or doxorubicin-induced apoptosis of HL-60/Bcr-Abl and K562 cells. This was associated with greater cytosolic accumulation of cyt c and PARP cleavage activity of caspase-3. These in vitro data indicate that combinations of CGP57148B and antileukemic drugs such as Ara-C may have improved in vivo efficacy against Bcr-Abl–positive acute leukemia.

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.

scholarly journals The pan-Bcl-2 inhibitor obatoclax promotes differentiation and apoptosis of acute myeloid leukemia cells

2020 ◽  
Vol 38 (6) ◽  
pp. 1664-1676
Author(s):  
Małgorzata Opydo-Chanek ◽  
Iwona Cichoń ◽  
Agnieszka Rak ◽  
Elżbieta Kołaczkowska ◽  
Lidia Mazur
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Treatment Strategies ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Apoptotic Pathway ◽  
Acute Myeloid Leukemia Cells ◽  
Induced Apoptosis ◽  
Acute Myeloid

Summary One of the key features of acute myeloid leukemia (AML) is the arrest of differentiation at the early progenitor stage of myelopoiesis. Therefore, the identification of new agents that could overcome this differentiation block and force leukemic cells to enter the apoptotic pathway is essential for the development of new treatment strategies in AML. Regarding this, herein we report the pro-differentiation activity of the pan-Bcl-2 inhibitor, obatoclax. Obatoclax promoted differentiation of human AML HL-60 cells and triggered their apoptosis in a dose- and time-dependent manner. Importantly, obatoclax-induced apoptosis was associated with leukemic cell differentiation. Moreover, decreased expression of Bcl-2 protein was observed in obatoclax-treated HL-60 cells. Furthermore, differentiation of these cells was accompanied by the loss of their proliferative capacity, as shown by G0/G1 cell cycle arrest. Taken together, these findings indicate that the anti-AML effects of obatoclax involve not only the induction of apoptosis but also differentiation of leukemic cells. Therefore, obatoclax represents a promising treatment for AML that warrants further exploration.

Bcr-Abl-Mediated Suppression of Normal Hematopoiesis in Chronic Myeloid Leukemia: Involvement of a Modified Form of NGAL.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2869-2869
Author(s):  
Hui Lin ◽  
Xiaohong Leng ◽  
Tong Sun ◽  
Giuseppe Monaco ◽  
Clifton Stephens ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Hematopoietic Cells ◽  
K562 Cells ◽  
Soft Agar ◽  
Leukemia Cells ◽  
Active Process ◽  
Culture Studies ◽  
Induced Apoptosis

Abstract The BCR-ABL oncogene plays an essential role in chronic myeloid leukemia (CML). In NOD/scid mice injected with soft agar clones of a human CML cell line (K562), we observed a leukemia syndrome involving not only leukemia but also a severe reduction of normal mouse hematopoiesis (Lin et al., Oncogene, 2001). Some of these mice died of a wasting syndrome that involved suppression of hematopoiesis without extensive tumor cell invasion of the spleen and marrow. In CML patients, since normal hematopoietic cells in marrow and spleen are replaced with proliferating leukemic blasts, we postulate that this is an active process mediated by the leukemia cells. The lipocalin 24p3 is secreted by mouse hematopoietic cells deprived of IL-3, resulting in apoptosis induction in a variety of hematopoietic cells including bone marrow cells (Devireddy et al., Science, 2001). We found that BCR-ABL+ mouse hematopoietic cells induce a persistent secretion of a modified form of 24p3 (21 kDa). Co-culture studies show that BCR-ABL+ cells induced apoptosis in BCR-ABL negative cells. Importantly, BCR-ABL+ hematopoietic cells are resistant to apoptosis under the same conditions. Conditioned medium (CM) from BCR-ABL+ cells expressing anti-sense/siRNA 24p3 or CM mixed with 24p3 antibody have reduced apoptotic activity for target cells. We also found that the expression of the Bcr-Abl oncoprotein and its tyrosine kinase are required for induction of 24p3 expression. Leukemic mice induced by BCR-ABL+ cells expressing anti-sense/siRNA 24p3 have increased levels of normal hematopoiesis (marrow and spleen erythropoiesis and blood platelet levels) and reduced invasion of leukemia cells in marrow and spleen tissues, but the leukemia cells readily invade liver and the abdomen as ascites (Lin et al, Oncogene, 2005). These findings indicate that suppression of normal hematopoiesis in BCR-ABL induced leukemia is an active process involving the apoptotic factor 24p3, raising the possibility that similar factors are involved in BCR-ABL+ CML patients. We have found that the K562 clones (Lin et al. 2001) have enhanced expression of NGAL (neutrophil gelatinase-associated lipocalin, human homologue of 24p3) transcripts compared to uncloned K562 cells. We generated additional soft agar K562 clones, each with different expression levels of NGAL transcripts. NOD/scid mice injected with the clone (C5) of K562 cell line expressing a high level of NGAL had severe depression of hematopoiesis and significantly shorter survival time as compared with mice injected with parental K562 cells and a clone (C6) expressing a low level of NGAL. Co-culture studies showed that the C5 K562 clone also induced apoptosis in BCR-ABL negative cells. We detected two glycosylated forms of NGAL/24p3 migrating at 24 kDa and 21 kDa on SDS-PAGE. The 21 kDa form is the major form in CM from mouse BCR-ABL+ cells and K562 clones. Our preliminary data with CML patient samples showed that levels of 21 kDa NGAL protein in bone marrow fluid correlated with BCR-ABL/ABL ratio. Further studies with more patient samples are ongoing to confirm the role of NGAL in suppressing normal hematopoiesis in CML patients and to determine the structural change(s) that leads to the modified form of 24p3/NGAL secreted by CML cells.

Relationship between the As2O3 Induced Expression of NF-κB and Drug Resistance in K562/ADR Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4414-4414
Author(s):  
Xiao-hong Zhang ◽  
Li-da Su ◽  
Xiao-ying Zhao ◽  
Qing-hua Lv
Keyword(s):  
Flow Cytometry ◽  
Drug Resistance ◽  
K562 Cells ◽  
Suppressive Effect ◽  
Resistance Mechanisms ◽  
Leukemic Cells ◽  
Ic50 Values ◽  
Induced Apoptosis ◽  
The Relationship ◽  
Cells Cultured

Abstract Summery: Anti-apoptosis is one of drug resistance mechanisms in leukemic cells. It was found in our early study that As2O3 can induce apoptosis of K562 cells, and this effect involve the degradation of IκB-αand consequently the activation of NF-κB. The relationship between drug resistance of leukemic cells and the expression of both IκB-αand NF-κB associated with apoptosis induced by arsenic trioxide(As2O3) was studied in K562 and K562/ADR cells. Methods: Apoptosis was induced in K562 and K562/ADR cells cultured with As2O3 in different concentrations. Western blot was used to analyze the expression of NF-κB in nuclear and IκB-α in cytoplasm of these cells. Apoptosis and degradation of IκB-αprotein were also observed by flow cytometry. Results: The suppressive effect of As2O3 on proliferation of K562/ADR was lower than that in K562 cell, IC50 values were 19.07μmol/L and 5.26μmol/L, respectively. After exposure to As2O3, the ratio of apoptosis cells increased with the concentration of As2O3 in K562 cells, from(13.25±1.83)% to (50.56±8.62)% with variation of As2O3 from 1μmol/L to 4μmol/L(P<0.05). The ratio of apoptosis cells in K562/ADR cultured with 4μmol/L As2O3 was significantly lower than that in K562 cells, (8.00±1.47)% vs. (50.56±8.62)%, (P<0.05). The level of IκB-α in K562 cytoplasm was down-regulated from (88.07±0.99)% to (49.21±0.95)%, (P<0.01) after As2O3 stimulation, while NF-κB in nuclear was up-regulated, that was not found in K562/ADR cells. Conclusion: As2O3 could induce apoptosis of K562 cells, associated with the degradation of IκB-αand the activation of NF-κB. There were resistance to As2O3 induced apoptosis and an abnormal regulation of NF-κB expression by As2O3 in K562/ADR cells.

Myeloperoxidase Is Required for the Combined Apoptotic Effects of Arsenic Trioxide with Ascorbic Acid or Epigallocatechin-3-Gallate in Myeloid Leukemia Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3474-3474
Author(s):  
Lijuan Xia ◽  
Min Lu ◽  
Duo Chen ◽  
Samuel Waxman ◽  
Yongkui Jing
Keyword(s):  
Ascorbic Acid ◽  
Nadph Oxidase ◽  
Arsenic Trioxide ◽  
Myeloid Leukemia ◽  
K562 Cells ◽  
H2o2 Production ◽  
Nb4 Cells ◽  
Combination Treatments ◽  
Induced Apoptosis ◽  
High Level

Abstract Arsenic trioxide (As2O3) is a drug used world-wide that selectively causes the death of acute promyelocytic leukemia (APL) cells by novel mechanisms and induces complete clinical remission in 90% of patients without significant toxicity. The outstanding success of As2O3 therapy in relapsed APL patients has not been replicated by an equivalent success in other types of acute myeloid leukemia (AML). We have studied As2O3 mechanism(s) of action and identified agents to use in combination to improve the use of As2O3 as a treatment for other types of AML. As2O3 produces higher levels of H2O2 and apoptosis in APL NB4 cells than in other AML cells at therapeutic concentrations of 1-2 uM. As2O3 does not induce apoptosis in HL-60 cells but is synergistic with ascorbic acid (AA) or epigallocatechin-3-gallate (EGCG). Both AA and EGCG produce H2O2 which is augmented by the addition of As2O3. Apoptosis induction by As2O3 in combination with AA or EGCG is inhibited by catalase and the antioxidant N-acetylcysteine. Myeloperoxidase (MPO), a major neutrophil enzyme, augments H2O2-induced apoptosis by converting it into more potent reactive oxygen species. HL-60, NB4, SKNO-1 and PLB985 cells which express high level of MPO, but not U937 and K562 cells without expression of MPO, are responsive to As2O3 plus AA or EGCG-induced apoptosis. HP-100 cells, a subclone of HL-60 cells without MPO expression, are resistant to both combination treatments. MPO stable transfection sensitizes K562 cells to apoptosis following treatment with As2O3 and AA or EGCG. NADPH oxidase is an enzyme complex which generates O2− and H2O2 in neutrophils. In APL NB4 cells, As2O3 induces expression of NADPH oxidase members and is thought to participate in H2O2 production required for As2O3 response. X-CGD cells, a subclone of PLB-985 with targeted disruption of the gp91phox gene (Zhen et al, PNAS90:9832, 1993) and loss of NADPH oxidase activity but with high levels of MPO activity, are responsive to treatment with As2O3 and AA or EGCG. These results suggest that AA and EGCG enhance As2O3-induced apoptosis through a synergistic production of H2O2 which is independent NADPH oxidase activation in non-APL AML cells. MPO augments the apoptotic effect of H2O2 produced by As2O3 plus AA or EGCG and it could be used as a marker to predict the sensitivity of AML cells to both combination treatments.

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