Parental K562 Cells Research Articles

STAT3 Inhibition Synergizes with BCR-ABL1 Inhibition to Overcome Kinase-Independent TKI Resistance in Chronic Myeloid Leukemia (CML)

Abstract 31In CML, TKI resistance in the absence of BCR-ABL1 kinase mutations is mechanistically unclear. Since extrinsic signals from the bone marrow (BM) microenvironment protect CML cells from TKIs in a STAT3-dependent manner (Bewry et al. 2008; Traer et al. 2011), we hypothesized that overt resistance may occur when CML cells maintain intrinsic STAT3 activation in the absence of extrinsic signals. We also asked whether combined targeting of STAT3 and BCR-ABL1 could produce synthetic lethality to overcome TKI resistance. To model extrinsic BCR-ABL1 kinase-independent resistance, we grew CML cell lines and CMLCD34+ progenitor cells from newly diagnosed patients in the presence of conditioned medium (CM) derived from HS-5 BM stromal cells. To model intrinsic resistance, we used the imatinib-resistant K562R and AR230R cell lines. Our models of TKI resistance proliferate in 1.0–2.5 μM imatinib and exhibit pSTAT3Y705 activation despite suppression of BCR-ABL1 kinase activity.To investigate the role of pSTAT3Y705 in TKI resistance, we used shRNA-mediated knockdown (shSTAT3), a dominant-negative mutant (dnSTAT3), and pharmacologic STAT3 inhibitors developed in our laboratories to block STAT3 activity. Compared to scrambled controls in the presence of HS-5 CM, shSTAT3 reduced colony formation (33.8% reduction, p<0.008) and increased apoptosis (17.9% increase, p<0.03) of parental K562 cells following imatinib exposure, thereby abolishing the protective effect of extrinsic BM-derived factors. In contrast, shSTAT3 had no effect on cells grown in regular medium. In intrinsic resistance, shSTAT3 reduced clonogenicity of K562R cells by 58.3% (p<0.0004) and AR230R cells by 62.2% (p<0.002) in the presence of 1.0 μM imatinib, with no effect on TKI-sensitive controls. A dnSTAT3 mutant lacking the C-terminal activation domain also impaired the clonogenicity of K562R and AR230R cells by 36.1% (p<0.003) and 50.0% (p<0.004), respectively. Thus, STAT3 plays a functional role in mediating intrinsic and extrinsic TKI resistance in BCR-ABL1-expressing cell lines.Preliminary immunofluorescence data indicates that pSTAT3Y705 levels are higher in drug-resistant CML CD34+CD38− stem cells than in CD34+CD38+ progenitor cells, suggesting a potential role for pSTAT3Y705 in TKI resistance of primitive CML stem cells. To investigate whether pharmacologic STAT3 inhibition in combination with TKIs could overcome resistance, we tested a library of compounds that interfere with STAT3 dimerization. The most selective of these inhibitors, S3I-201.1066 (SF1-066), was biochemically shown to disrupt STAT3 dimerization and transcriptional activation. Compared to cells treated with 1.0 μM imatinib alone, addition of 10 μM SF1-066 impaired the clonogenicity of K562R cells by 68.6% (p<0.03) and AR230R cells by 72.5% (p<0.0004), with no effect on TKI-sensitive controls. When CMLCD34+ progenitor cells from newly diagnosed patients (n=4) were cultured in the presence of HS-5 CM and treated with the SF1-066/imatinib combination, clonogenicity following drug exposure was reduced by 45.2% (p<0.002) compared to imatinib. Therefore, pharmacologic inhibition of STAT3 and BCR-ABL1 overcomes intrinsic and extrinsic TKI resistance in both CML cell lines and CMLCD34+ progenitor cells.We next developed a series of 14 second-generation STAT3 inhibitors and tested them using a luciferase reporter harboring sequential STAT3 cis-inducible elements to measure endogenous STAT3 activity in AR230R cells. Two lead compounds, BP2-047 and BP3-163, reduced luciferase activity by 69.2% (p<3.6×10−6) and 51.8% (p<8.6×10−6), respectively, with little effect on cells expressing a scrambled reporter. Selected inhibitors then provided scaffolds for the synthesis of focused libraries with enhanced potency and selectivity. Immunofluorescence data revealed that two of these inhibitors, BP5-087 and BP5-088, resulted in cytoplasmic accumulation of pSTAT3Y705 in CMLCD34+ progenitors grown in HS-5 CM, consistent with a block of dimerization. Screening and validation of third-generation STAT3 inhibitors are underway and will be reported.Altogether, our data identifies STAT3 as a universal target in TKI-resistant CML cells, and suggests that simultaneous pharmacologic inhibition of STAT3 and BCR-ABL1 may be a suitable therapeutic strategy for treatment of patients with TKI resistance despite inhibition of BCR-ABL1 kinase activity. Disclosures:No relevant conflicts of interest to declare.

Elucidation of the therapeutic enhancer mechanism of poly-S-nitrosated human serum albumin against multidrug-resistant tumor in animal models

Human serum albumin (HSA) is the most abundant circulating protein and its S-nitrosated form serves as a reservoir of nitric oxide (NO). Previously, we prepared poly-S-nitrosated HSA (Poly-SNO-HSA) by incubation with Traut's Reagent and isopentyl nitrite and evaluated its potential as a novel anticancer agent through apoptosis involving the caspase-3 pathway. Recently, NO donors such as nitroglycerin were reported to revert the resistance to anticancer agents. Therefore, now we have evaluated the effect of the above type of Poly-SNO-HSA on the resistance to doxorubicin (dx) in human myelogenous leukemic cells (K562 cells). P-gp expression and dx accumulation in K562 and dx-resistant K562 cells (K562/dx cells) were quantified using Western blot and FACS analysis, respectively. Compared with parent K562 cells, higher expression of P-gp and lower accumulation of dx were shown in K562/dx cells. Poly-SNO-HSA caused increased dx accumulation in K562/dx cells by decreasing the expressions of P-gp and HIF-1α. Other experiments with the guanylate cyclase inhibitor ODQ and 8-Br-cGMP revealed that also a cGMP signaling pathway is involved in the Poly-SNO-HSA induced increase in dx accumulation. Furthermore, in vivo studies showed that co-treatment with Poly-SNO-HSA enhanced the anticancer effect of dx in K562/dx cells-bearing mice. Thus, in addition to its proapoptotic effect Poly-SNO-HSA can in an efficient manner revert drug resistance both in vitro and in vivo, and two pathways for this effect have been identified.

Overcoming multidrug-resistance in vitro and in vivo using the novel P-glycoprotein inhibitor 1416

MDR (multidrug-resistance) represents a major obstacle to successful cancer chemotherapy and is usually accomplished by overexpression of P-gp (P-glycoprotein). Much effort has been devoted to developing P-gp inhibitors to modulate MDR. However, none of the inhibitors on the market have been successful. 1416 [1-(2,6-dimethylphenoxy)-2-(3,4-dimethoxyphenylethylamino)propane hydrochloride (phenoprolamine hydrochloride)] is a new VER (verapamil) analogue with a higher IC50 for blocking calcium channel currents than VER. In the present paper, we examined the inhibition effect of 1416 on P-gp both in vitro and in vivo. 1416 significantly enhanced cytotoxicity of VBL (vinblastine) in P-gp-overexpressed human multidrug-resistant K562/ADM (adriamycin) and KBV cells, but had no such effect on the parent K562 and KB cells. The MDR-modulating function of 1416 was further confirmed by increasing intracellular Rh123 (rhodanmine123) content in MDR cells. Human K562/ADM xenograft-nude mice model verified that 1416 potentiates the antitumour activity of VBL in vivo. RT-PCR (reverse transcriptase-PCR) and FACS analysis demonstrated that the expression of MDR1/P-gp was not affected by 1416 treatment. All these observations suggest that 1416 could be a promising agent for overcoming MDR in cancer chemotherapy.

Endoplasmic reticulum stress contributes to arsenic trioxide-induced apoptosis in drug-sensitive and -resistant leukemia cells

This study characterized the role of endoplasmic reticulum stress (ERS)-related pathways in arsenic trioxide-induced apoptosis in multidrug-resistant leukemia K562/ADM cells. Arsenic trioxide exposure led to much significant induction of apoptosis in K562/ADM cells than the parental K562 cells, and the chaperone proteins glucose-regulated protein 78, CHOP/GADD153, X-box binding protein-1 and caspase-12 were activated to varying degrees. Furthermore, arsenic trioxide stimulation led to inhibition of P-glycoprotein and Bcl-2 expression. This study demonstrates a missing link between arsenic trioxide and ERS-induced apoptosis, and suggests that the greater effects obtained in drug-resistant K562/ADM cells may be mediated by downregulation of P-glycoprotein and Bcl-2 expression.

Degree of kinase inhibition achieved in vitro by imatinib and nilotinib is decreased by high levels of ABCB1 but not ABCG2

Imatinib and nilotinib interact with ABCB1 and ABCG2. However, whether they are substrates or inhibitors is a source of conjecture. Here, in vitro, Bcr–Abl kinase inhibition was used to elucidate the impact of ABCB1/ABCG2 overexpression on imatinib and nilotinib transport. High levels of ABCB1 protein in K562-Dox cells resulted in a significantly increased 50% inhibitory concentration (IC50) compared with parental K562 cells for imatinib (IC50IM; 9 µM to 19 µM, p = 0.002) and nilotinib (IC50NIL; 345 nM to 620 nM, p = 0.013). This difference was abrogated by ABCB1 inhibitors. However, overexpression of ABCG2 did not significantly increase IC50IM or IC50NIL or significantly decrease IC50 upon ABCG2 inhibition. Inhibition of ABCB1 but not ABCG2 resulted in a substantial increase in intracellular nilotinib when used at 150 nM but no increase when used at 2 µM. Imatinib and nilotinib appear to be transported by ABCB1 but do not interact strongly with ABCG2. Furthermore, ABCB1 efflux of nilotinib may be concentration-dependent with transport occurring at clinically relevant concentrations.

Bcr-Abl Amplification Plays a Major Role in Resistance to Tyrosine Kinase Inhibitors in K-562 Cell Line

An emerging problem in patients with chronic myeloid leukemia (CML) is increasing resistance to tyrosine kinase inhibitors (TKIs). To determine genetic and cellular mechanisms involved in the development of resistance to TKIs, nine imatinib-resistant cell lines were derived from K- 562 cell line followed by testing of drug sensitivity, multidrug resistance proteins and cytogenetic studies. In imatinib-resistant cell lines cross-resistance to daunorubicin, etoposide and cytarabine were observed whereas sensitivity to dasatinib, nilotinib, cyclophpsphamide, bortezomib and busulfan was preserved. Treatment with imatinib decreased PGP and LRP expression, however it did not significantly influence MRP1 expression. Amount of signals in FISH analysis from ABL, BCR and from fusion genes (BCR-ABL or ABL-BCR) was mostly higher in imatinib-resistant cell lines in comparison to parental K-562 cell line. We concluded that BCR-ABL amplification but not cellular sensitivity is the major mechanisms of resistance in K-562 cell line.

Abstract 814: P-glycoprotein transport of the active imatinib metabolite, CGP74588, in chronic myeloid leukemia cells

Abstract The tyrosine kinase inhibitor (TKI) imatinib is, despite the introduction of second generation TKI's, the standard first-line therapy in chronic myeloid leukemia (CML). Early response to therapy is correlated to long-term therapeutic effect in CML. To be able to switch to second generation TKI's in an early stage of CML, it would be advantageous to identify predictive markers for failure on imatinib. Imatinib is metabolized by hepatic CYP3A4 and CYP3A5, forming approximately 30 metabolites. The main metabolite, CGP74588, is pharmacologically active with similar potency to that of imatinib. CGP74588 is present in ∼20% of imatinib plasma concentrations but with a large inter-individual variation. We have previously reported that high CYP3A activity is associated with a better therapeutic outcome of imatinib, indicating a clinical significance of imatinib metabolites (Gréen et al. 2010). Furthermore, imatinib is a substrate for the efflux transporter P-glycoprotein which is the product of the ABCB1 gene. Several ABCB1 polymorphisms have been described and some have been shown to influence the therapeutic response to imatinib in CML patients. The aim of this study was to investigate the effects of ABCB1 over-expression on the in vitro resistance to imatinib and its CYP3A metabolite CGP74588. ABCB1 wild type human cDNA was infected to the CML cell line K562 using a retroviral system. Co-expression of ABCB1 and the reporter gene for enhanced yellow fluorescent protein (EYFP) was used for the assessment of ABCB1 expression by the analysis of EYFP in FACS. The influence of ABCB1 expression on the cytotoxic effects of imatinib and CGP74588 was assessed using MTT assays. FACS analysis of EYFP confirmed an over-expression of ABCB1 in infected K562 cells with a mean fluorescence intensity of 23.5 compared to 0.55 in parental K562 cells. ABCB1-expressing cells (K562/ABCB1) were slightly, but not significantly, more resistance to imatinib than K562 cells (IC50 K562 = 0.41µM, K562/ABCB1 = 0.50µM). However, ABCB1 expression induced a 12-fold increase in resistance when cells were treated with the imatinib metabolite CGP74588 (IC50 K562 = 0.72µM, K562/ABCB1 = 8.53µM, p = &amp;lt;0.000). Recent reports have shown that CGP74588 accumulates in cell lines with acquired multi-drug resistance and high P-gp expression. Our studies on cells with ABCB1 expression as a single resistance mechanism proves that P-gp is indeed confer resistance to CGP74588 in CML cells. Furthermore, CGP74588 cytotoxicity is affected by ABCB1 expression to a greater extent than imatinib itself, indicating that ABCB1 activity could be important for CGP74588 plasma and target cell concentrations in vivo and might contribute to the large intra-individual variation seen in patient plasma concentrations. Future studies will be needed to determine the clinical significance of P-gp activity and CGP74588 pharmacokinetics in relation to TKI therapy of CML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 814. doi:1538-7445.AM2012-814

Identification of DNA methylation biomarkers in imatinib-resistant chronic myeloid leukemia cells

Drug resistance is an obstacle to successful cancer treatment. In hematological neoplasms, such as chronic myelogenous leukemia (CML), drug resistance is often associated with gene hypermethylation and loss of function. An imatinib-resistant subclone of CML-like K562 (K562-R) was used as a model to study the role of hypermethylation in drug resistance. K562-R was selected by culturing cells with increasing concentrations of imatinib, ranging from 0.2–5 μM. A DNA methylation microarray was performed on the K562-R and parental K562 cells, and selected gene expression levels were confirmed using real-time polymerase chain reaction. The methylation level was significantly increased and the gene expression level significantly was decreased for MLH1, RPRM, FEM1B, and THAP2 in K562-R cells compared with parental K562 cells. Exposing K562-R cells to methylation inhibitors, such as 5-azacytidine (AzaC) and trichostatin A (TSA), reduced imatinib resistance. Our approach of using a drug-limiting dilution model followed by the use of a methylation microarray was able to identify methylation biomarkers for drug resistance. Specifically, MLH1, RPRM, FEM1B, and THAP2 might be potential epigenetic targets of imatinib resistance. Further understanding the methylation domain and epigenetic regulation machinery of these biomarkers will help researchers find potential effective therapeutic strategies that could be used to overcome drug resistance in CML patients.

Overexpression of P-glycoprotein induces acquired resistance to imatinib in chronic myelogenous leukemia cells

Imatinib, a breakpoint cluster region (BCR)-Abelson murine leukemia (ABL) tyrosine kinase inhibitor (TKI), has revolutionized the treatment of chronic myelogenous leukemia (CML). However, development of multidrug resistance (MDR) limits the use of imatinib. In the present study, we aimed to investigate the mechanisms of cellular resistance to imatinib in CML. Therefore, we established an imatinib-resistant human CML cell line (K562-imatinib) through a stepwise selection process. While characterizing the phenotype of these cells, we found that K562-imatinib cells were 124.6-fold more resistant to imatinib than parental K562 cells. In addition, these cells were cross-resistant to second- and third-generation BCR-ABL TKIs. Western blot analysis and reverse transcription-polymerase chain reaction(RT-PCR) demonstrated that P-glycoprotein (P-gp) and MDR1 mRNA levels were increased in K562-imatinib cells. In addition, accumulation of [14C]6-mercaptopurine (6-MP) was decreased, whereas the ATP-dependent efflux of [14C] 6-MP and [3H]methotrexate transport were increased in K562-imatinib cells. These data suggest that the overexpression of P-gp may play a crucial role in acquired resistance to imatinib in CML K562-imatinib cells.

Intrinsic and Extrinsic Survival Signals Converge on STAT3 As a Critical Mediator of BCR-ABL-Independent Tyrosine Kinase Inhibitor Resistance,

Abstract 3742In Philadelphia chromosome positive leukemia, mutations in the BCR-ABL kinase domain are a well established mechanism of resistance to tyrosine kinase inhibitors (TKIs), but fail to explain many cases of primary and acquired resistance. Extrinsic survival signals from the bone marrow microenvironment can protect CML cells from the effects of TKIs in a STAT3-dependent manner. To define the role of pSTAT3 in extrinsic versus intrinsic TKI resistance, we used BCR-ABL+ cell lines exhibiting ‘extrinsic resistance’ through culture in conditioned media (CM) from human HS-5 bone marrow stromal cells (Re), or derivative lines expressing native BCR-ABL with ‘intrinsic resistance’ adapted for growth in the presence of 1.0–2.5 μM imatinib (Ri).Immunoblot analysis showed near-complete suppression of BCR-ABL tyrosine kinase activity upon exposure to imatinib in all Re and Ri cells tested, suggesting BCR-ABL-independent resistance in both cases. K562-Ri and AR230-Ri cells proliferating in 1.0 μM imatinib showed upregulation of pAKTS473, pJAK2Y1007/1008, pSTAT3, and pSTAT5 compared to sensitive cells under the same conditions. When imatinib was increased (2.5 μM), pJAK2Y1007/1008 and pSTAT5 were markedly reduced, suggesting regulation by BCR-ABL kinase activity. In contrast, levels of pSTAT3 and pAKTS473 were further increased. Similar to Ri cells, extrinsically resistant cells (K562-Re, LAMA84-Re, KBM5-Re) showed increased pSTAT3 when cultured in HS-5 CM; however, upregulation was not observed for pAKTS473. Of key importance, pSTAT3 levels were also increased by HS-5 CM in CD34+ cells from newly diagnosed CML patients.To further investigate the role of pSTAT3 in TKI resistance, we used lentiviral shRNA to knockdown STAT3 (shSTAT3) in sensitive and resistant BCR-ABL+ cells. Cells were kept in culture for 24 hours with and without 1.0 μM imatinib in regular medium or CM, followed by trypan blue exclusion and/or plating in semisolid medium. Compared to cells expressing a scrambled control (shSCR), shSTAT3 reduced the in vitro growth of intrinsically resistant K562-Ri and AR230-Ri cells by 54.6% and 33.3% in the presence of imatinib (1.0 μM), respectively. Consistent with these observations, shSTAT3 impaired the clonogenic potential of K562-Ri cells by 65.0% following culture in 1.0 μM imatinib, and similar results were obtained for AR230-Ri cells, indicating that STAT3 plays a functional role in mediating intrinsic TKI resistance in multiple BCR-ABL-expressing cell lines. In extrinsic resistance, 3–12 hours of culture with CM from HS-5 stromal cells enhanced the clonogenic potential of K562-Re and LAMA84-Re cells by 25–40% in the presence of imatinib compared to controls cultured in regular medium. However, the protective effects of CM were abrogated by introduction of shSTAT3. In contrast, shSTAT3 had no effect on colony formation following growth in regular medium, consistent with the low level of pSTAT3 in parental K562 and LAMA-84 cells. These data suggest that STAT3 activation is a prominent feature of both extrinsic and intrinsic BCR-ABL-independent imatinib resistance. Additionally, preliminary experiments implicate pSTAT3 in intrinsic BCR-ABL-independent resistance to the third-generation TKI, ponatinib (AP24534), suggesting that activated STAT3 may have a role in resistance to other TKIs.Altogether, our data suggest that intrinsic and extrinsic pathways converge on STAT3 as a critical mediator of BCR-ABL-independent TKI resistance, and implicate this pathway as a potential therapeutic target for the treatment of patients with TKI resistance despite BCR-ABL inactivation. We posit that microenvironmental cues activate pathways that initially support survival of leukemia cells despite BCR-ABL inhibition, and that overt resistance develops when these cells establish an intrinsic mechanism to maintain activation of the same pathways. Disclosures:No relevant conflicts of interest to declare.

Deactivation of Signal Transducer and Activator of Transcription 3 Reverses Chemotherapeutics Resistance of Leukemia Cells via Down-Regulating P-gp

Multidrug resistance (MDR) caused by overexpression of p-glycoprotein is a major obstacle in chemotherapy of malignant cancer, which usually is characterized by constitutive activation of signal transducer and activator of transcription 3 (STAT3), but their relation between MDR and STAT3 remains unclear. Here, we showed that STAT3 was overexpressed and highly activated in adriamycin-resistant K562/A02 cells compared with its parental K562 cells. Blockade of activation of STAT3 by STAT3 decoy oligodeoxynucleotide (ODN) promoted the accumulation and increased their sensitivity to adriamycin by down-regulating transcription of mdr1 and expression of P-gp, which were further confirmed by using STAT3-specific inhibitor JSI-124. Inhibition of STAT3 could also decrease mdr1 promoter mediated luciferase expression by using mdr1 promoter luciferase reporter construct. Otherwise, activation of STAT3 by STAT3C improved mdr1 transcription and P-gp expression. The ChIP results demonstrated that STAT3 could bind to the potential promoter region of mdr1, and STAT3 decoy depressed the binding. Further mutation assay show +64∼+72 region could be the STAT3 binding site. Our data demonstrate a role of STAT3 in regulation of mdr1 gene expression in myeloid leukemia and suggest that STAT3 may be a promising therapeutic target for overcoming MDR resistance in myeloid leukemia.

Up-regulation of P-glycoprotein confers acquired resistance to 6-mercaptopurine in human chronic myeloid leukemia cells

To investigate the mechanisms of cellular resistance to 6-mercaptopurine (6-MP) in chronic myeloid leukemia (CML), a 6-MP resistant cell line (K562-MP5) was established by stepwise selection of the CML cell line (K562). The results of the drug sensitivity analysis of the K562-MP5 cell line revealed the cells to be 339-fold more resistant to 6-MP compared with the parental K562 cells. K562-MP5 cells exhibited decreased accumulation and increased efflux of [(14)C]6-MP and its metabolites. In addition, K562-MP5 cells showed increased [(3)H]MTX transport. K562-MP5 cells over-expressed P-glycoprotein (P-gp) and up-regulated MDR1 mRNA levels. Taken together, these results suggest that the up-regulation of P-gp, which contributes to the decreased accumulation by increasing the efflux of 6-MP and its metabolites, underlies the mechanism of 6-MP resistance in K562 cells.

Effects of 5-Bromotetrandrine and Daunorubicin on the Expression of Survivin In K562/AO2 Cells

AbstractAbstract 4947 Objective:The aim of this study was to investigate the expression of survivin and the apoptosis induced by DNR and BrTet in the leukemic cells K562/A02. Methods:In a typical experiment, the K562/AO2 cells were treated with daunorubicin (DNR), 5-bromotetrandrine (BrTet), or DNR and BrTet for 48 hours, and the cells treated without any drugs were used as control group. Cell proliferation was analyzed by MTT assay. Cells apoptosis and the concentration of DNR within the cells were measured by Flow cytometry (FCM). The expressions of mRNA and protein of survivin were determined by semi-quantitative reverse transcription PCR (RT-PCR) and Western blot, respectively. Results:The results of MTT assay indicated that DNR and BrTet were both able to inhibit the proliferation of K562/AO2 cells in dose-dependent manner. The fresh evidence from flow cytometry showed that a higher apoptosis rate could be induced and a higer concentration of DNR could be detected in K562/AO2 cells by DNR and BrTet as compared with those by DNR or BrTet in the same concentrations(P<0.01). RT-PCR revealed that the expression of survivin mRNA, a higer expression in K562/AO2 cells with acquired resistance to adriamycin than that in parent K-562 cells, decreased in the DNR and BrTet group (P<0.05), but there was no obvious change in other groups(P>0.05). Western bolt demonstrated that the expression of survivin protein was much lower in the DNR and BrTet group(P<0.05). Conclusion:BrTet could increase the concentration of DNR and reverse the multidrug resistance(MDR) in the K562/AO2 cells. Survivin may play an important role in apoptosis induced by DNR. Survivin could be a target for the treatment of MDR in haematopoietic malignancies. Disclosures:No relevant conflicts of interest to declare.

DIGE-Based Proteomic Analysis Identifies NPM/B23 and Nucleolin C23 as Overexpressed Proteins In Relapsed /Refractory Acute Leukemia

AbstractAbstract 1711Resistance to chemotherapy is a challenge in treatment of acute leukemia. Although the classic multidrug resistance (MDR) phenotype is often characterized by expression of drug efflux pump P-glycoprotein or by multidrug resistance-associated proteins, precise molecular mechanisms are largely unknown. To investigate novel protein changes involved in resistance mechanism, protein expression profiles between human myeloid leukemia HL-60 cell lines and adriamycin- resistant HL-60 cell lines (HL-60/ADR) was compared, which was based on a differential proteomic approach — 2 dimensional difference in gel Electrophoresis(2D-DIGE) followed by mass spectrometry (MALDI-TOF-MS) and complemented by western blot validation. 16 protein spots were identified as being differentially expressed (> 1.2 fold change and p≤ 0.05) between above two cell lines, among which 13 protein spots were identified as up-regulated and 3 as down-regulated in the HL-60/ ADR cell line. Proteins found to have higher abundance levels in the resistant HL-60/ADR cells included enzymes, proteins and oncogenes related to signal transduction, protein synthesis, cell growth regulation and metabolism. 3 lower abundance proteins are related to transcription. From 16 proteins, 2 proteins, nucleophosmin B23 (NPM B23)and nucleolin C23, were selected and verified in leukemia cell lines and primary leukemia samples by western blot. Compared to healthy control samples, which showed no expressions of these 2 proteins, leukemic cell lines revealed an obvious up-regulation of B23 and C23. Moreover, significantly higher expressions of B23 and C23 were found in 3 resistant leukemic cell lines, HL-60/ADR, K562/ADR and KG01 cells, compared to the parent HL-60 and K562 cells, and other leukemic cell lines. In de novo leukemia samples, 43.8%(35/80) expressed B23 and C23 proteins, 37.9% (22/58) AML and 59.1% (13/22) ALL respectively. Meanwhile, concomitant expression of B23 and C23, both positive or negative, was noted in 97%(79/80)patients. Over-expressions of B23 and C23 were observed in 68.8% relapased/refractory leukemia patients. With regard to treatment outcome,among those patients who achieved ongoing CR, fewer patients expressed 2 proteins, only 13.35% (7/52) AML and 46%(7/15) ALL respectively. It implicated that B23 and C23 may be involved in drug resistance and be useful in assessing treatment outcome and prognosis of leukemia. To a conclusion, these results provide a novel clue for the molecular mechanism of MDR and suggest that B23 and C23 are prognostic indicators for leukemia. Disclosures:No relevant conflicts of interest to declare.

Expression profile of Notch‐related genes in multidrug resistant K562/A02 cells compared with parental K562 cells

Drug resistance is the major setback of acute myeloid leukemia (AML) therapy. Notch proteins have demonstrated functional regulation in cell proliferation, differentiation, and apoptosis and thus may affect drug resistance. Our study aimed to identify the Notch-related gene profile in drug-resistant AML cells and provide potential strategies for resistant AML therapy. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was conducted to detect the cytotoxicity of adriamycin toward K562 and drug-resistant K562/A02. Intracellular mean fluorescence intensity was monitored to reflect the intake of adriamycin by confocal microscopy. cDNA microarray was used to test the expression of 113 Notch signaling pathway-related genes in K562/A02 and K562. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) and western blot were used to validate the results from microarray. K562/A02 cells showed a 65-fold higher IC(50) to adriamycin and less intracellular accumulation of adriamycin than K562. cDNA microarray showed marked increases in binding of collagen and cell proliferation-related genes (CD44, DLL3, IL17B, NUMB, and NUMBL) and decreases in signal transduction and transcription factor activity related genes (FZD9, GBP2, GLI1, GLI2, IFNG, KRT5, Notch2, and Notch3). The change of gene expression was further validated by real-time RT-PCR and western blot. Notch signaling pathway-related genes may contribute to the drug resistance of AML.

Reduction of doxorubicin resistance in P-glycoprotein overexpressing cells by hybrid cell-penetrating and drug-binding peptide

Drug efflux by the membrane transporter P-glycoprotein (P-gp) plays a key role in multidrug resistance (MDR). In order to bypass P-gp, thus overcoming MDR, a hybrid peptide comprising a cell penetrating peptide (Tat) and a drug binding motif (DBM) has been developed to noncovalently bind and deliver doxorubicin (Dox) into MDR cells. The uptake of Dox into the leukemia cell line K562 and its P-gp overexpressing subline KD30 increased in the presence of DBM-Tat peptide. Confocal microscopy indicated that DBM-Tat associated Dox was directed to a perinuclear area of KD30 cells, while this was not observed in parent K562 cells. When KD30 cells were pretreated with the endosomotropic agent chloroquine (CLQ), peptide associated Dox redistributed into the cytosol, indicating that endocytosis was the predominant uptake route. Altered drug uptake kinetics observed by cellular accumulation assay also supported an endocytic uptake. In the presence of CLQ, DBM-Tat was able to enhance the cytotoxicity of Dox by 68.4% at 5 µM peptide concentration in KD30 cells but there were only minor effects on Dox cytotoxicity in K562 cells even in the presence of CLQ. Thus, combining Dox with DBM-Tat reduces P-gp mediated drug efflux, without a requirement for drug modification or inhibiting P-gp function.

Involvement of Annexin A1 in Multidrug Resistance of K562/ADR Cells Identified by the Proteomic Study

Multidrug resistance (MDR) to chemotherapy is a significant barrier to the effective treatment of chromic myeloid leukemia (CML). In an attempt to identify more factors associated with MDR for an understanding of the mechanism, we first established an adriamycin (ADR)-resistant human erythroleukemia cell line K562/ADR by stepwise selection in vitro using ADR. Besides the elevated resistance to ADR, the K562/ADR cells also showed significantly increased crossed-resistance to vincristin and Gleevec, compared to the parental K562 cells. Then we compared the global protein profiles between K562 and K562/ADR cells. Following two-dimensional gel electrophoresis and image analysis, some of the proteins with different levels between the two cell lines were identified by MALDI TOF/TOF mass spectrometry and Western blot analysis. The differentially expressed proteins were classified into groups based on their functions: calcium-binding proteins, chaperones, metabolic enzymes, proteins related to protein synthesis or DNA synthesis, and proteins related to signal transduction. In particular, ANXA1, a protein that was downregulated in K562/ADR, was analyzed further for its involvement in MDR by transfection and subsequent assays. The functional validation showed that the downregulated ANXA1 expression contributes considerably to the observed drug resistance in K562/ADR cells. These data will be valuable for further study of the mechanisms of MDR and may reveal a potential new diagnostic marker to chemotherapy.

Inhibition of Wnt/β-Catenin Signaling by AV65 Treatment Caused Cell Cycle Arrest and Induced Caspase-Independent or -Dependent Apoptosis in CML Cells.

Abstract 2184Poster Board II-161Imatinib has dramatically improved the management of CML, but cases of imatinib resistance have been reported. The second-generation ABL tyrosine kinase inhibitors (TKIs) such as dasatinib and nilotinib overcome imatinib-resistant CML.These agents, however, are ineffective in CML cells harboring T315I mutation and in CML stem cells. Recently, loss of β-catenin has been reported to impair the renewal of CML stem cells (Chao et al, Cancer Cell 2007) and an in vivo study has showed that β-catenin is essential for survival of leukemic stem cells (Hu et al, Leukemia 2009). Thus, we hypothesized that the inhibition of β-catenin signaling may be efficacious in the treatment of CML. We have previously reported that a novel β-catenin inhibitor, AV65, suppresses the growth of imatinib resistant CML cell lines harboring Abl kinase domain mutations including T315I and hypoxia-adaptation (Nagao et al, ASH 2008). We herein examine the cell cycle and apoptotic effects of AV65 on CML cell lines and its therapeutic possibility for CML stem/progenitor cells.We observed that expression of β-catenin is increased 20 to 45-fold in K562, BV173, KT-1, and MYL CML cell lines compared with total bone marrow cells from healthy volunteers. We have previously demonstrated that AV65 induced apoptosis of CML cells. To investigate how AV65 inhibits β-catenin, we next analyzed the effects of AV65 using Western blotting and real time PCR. AV65 suppressed the expression of β-catenin in K562 in a time- and a dose-dependent manner in nuclear and cytosolic fractions as well as whole cell lysates. AV65 did not diminish the transcripts of β-catenin in K562 indicating the depletion of β-catenin due to an inhibition of its accumulation in CML cells. Next we examined the effects of AV65 on cell cycle. The fractions of G1 phase to S phase increased by AV65 treatment. TUNEL/PI staining showed that both K562 and BV173 began to be nicked by AV65 at 30 nM for 12 hours, resulting in the induciton of apoptosis from G1 phase to S phase 24 hours after AV65 treatment (Figure). In real-time PCR analysis, the transcripts of p21, p27, and p57 in CML cell lines increased by AV65 treatment, however, those of p53 were not altered. Taken together, it is suggested that CML cells first arrested from G1 phase to S phase and then induced apoptosis after AV65 treatment. Next we examined the mechanisms of apoptosis by AV65 treatment. AV65 treatment in the presence of Z-VAD did not induce cell death in BV173, indicating that AV65 induced caspase-dependent apoptosis in BV173. In K562 cells however, AV65 induced apoptosis with or without Z-VAD suggesting that AV65 induces apoptosis in CML cell lines in caspase-dependent or -independent pathways. Lastly, we investigated the effects on hypoxia-adapted CML cells. We established hypoxia-adapted K562 cell lines (K562/HA). This cell line shows characteristics of more primitive CML cells, including resistance to serial Abl TKIs and a higher transplantation efficacy compared to the parental K562 cells (Takeuchi, et al. ASH 2008). In Western blotting analysis, K562/HA cell line expressed more β-catenin than its parental K562 cell line. AV65 inhibited the growth of K562/HA at the similar concentration to K562. Taken together, AV65 is effective for primitive CML cells which overexpress β-catenin. This suggests that AV65 has a potential to eradicate CML stem/progenitor cells. In conclusion, AV65 inhibits the accumulation of β-catenin in CML cells and this causes cell cycle arrest from G1 to S phase, resulting in induction of caspase-independent or -dependent apoptosis in CML cells. The inhibition of Wnt/β-catenin signaling has great potential as a novel and attractive therapy for CML. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

A Novel AHI-1-BCR-ABL-JAK2 Interaction Complex Mediates Cellular Resistance to Tyrosine Kinase Inhibitors in CML .

Abstract 38▪▪This icon denotes an abstract that is clinically relevant.The molecular signature of chronic myeloid leukemia (CML) is the BCR-ABL fusion gene originating in a multipotent hematopoietic stem cell. The BCR-ABL oncoprotein (p210BCR-ABL) has constitutively elevated tyrosine kinase activity that perturbs several signalling cascades, including the PI3K/AKT, JAK2/STAT5, NF-kB, and RAS/MAPK pathways. The current first line treatment for CML is the tyrosine kinase inhibitor imatinib mesylate (IM) that induces clinical remission in most chronic phase CML patients. However, early relapses and IM-resistant disease have emerged and are frequently associated with mutations in the BCR-ABL kinase domain. Our recent studies indicate that CML stem cells are less responsive to IM and other tyrosine kinase inhibitors and are critical target population for IM resistance. It is therefore critical to identify other therapies that target CML stem cells to prevent acquisition of resistance. One candidate target is AHI-1 (Abelson helper integration site 1), a recently discovered oncogene that is deregulated in primary leukemic stem cells from CML patients. AHI-1 contains several domains indicative of signalling functions, including an SH3 and a WD40-repeat domain. We have recently identified a novel AHI-1-BCR-ABL-JAK2 interaction complex that modulates BCR-ABL transforming activity both in vitro and in vivo and play a key role in the IM response/resistance of primary CML stem/progenitor cells. To investigate AHI-1's involvement in mediating this cellular resistance to IM and to test the comparative ability of new ABL and JAK2 inhibitors to inhibit this complex in CML cells, AHI-1 was either stably overexpressed in K562 cells by transduction of EF1a-AHI-1-IRES-YFP lentivirus or suppressed in K562 cells using RNA interference. Interestingly, an increase in cellular proliferation and colony formation and a decrease in apoptosis were observed in the presence of 1, 5 and 10 uM of IM when AHI-1 was overexpressed. Survival of these cells was similar to IM resistant K562 cells, which are highly resistant to IM in vitro and display higher AHI-1 protein expression than parental K562 cells. Suppression of AHI-1 had the opposite effect, with cells displaying heightened sensitivity to IM at concentrations as low as 1 uM. Phosphorylation and total protein expression levels of several proteins known to be involved in BCR-ABL signalling, including JAK2, STAT5, MAPK, SRC, AKT and NF-kB (P105, P50, and P65 subunits), were quantified by Western blot analysis. Elevated phosphorylation and total protein expression levels of several of these proteins were observed when AHI-1 was overexpresessed, in particular in the JAK2/STAT5 pathway and especially in the presence of Interleukin 3. Due to the strong effects AHI-1 had on this signalling cascade, we next inhibited JAK2 activity using a selective JAK2 inhibitor, TG101209, that is highly effective against the V617F mutation and inhibits JAK2 and STAT5 activities in polycythemia vera progenitor cells. AHI-1 overexpressing cells showed reduced proliferation and colony formation when treated with IM and TG101209 in combination compared to either IM or TG101209 alone. Interestingly, treatment with IM (5 uM) or dasatinib (150 nM, DA) in combination with TG101209 (100 nM) resulted in greater inhibition (81% and 85%) of CD34+ CML stem/progenitor cells from IM nonresponders (n=4), compared to the same cells treated with a combination of IM and DA (∼60%, p<0.05), as measured by colony-forming cell assays. CFSE tracking analysis of cell division in these cells further demonstrated additive antiproliferative activity as a result of combined ABL and JAK2 inhibitors. These results suggest that targeting both BCR-ABL and JAK2 activities may be a potential therapeutic option for IM resistant patients. Disclosures:No relevant conflicts of interest to declare.

The relationship between multi-drug resistance and proportion of leukemia stem cells and expression of drug transporters in drug-resistant leukemia K562/ADM cells

To investigate the drug resistance, proportion of leukemia stem cells (LSC) and expression of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in drug-sensitive and multidrug-resistant leukemia cell population. The multidrug-resistant leukemia K562/ADM cell and its parental K562 cell were used as the model cells. The drug sensitivity was tested with a MTT assay. Flow cytometry was employed to detect the immunophenotype of stem cells and the expression of P-gp and BCRP. The self-renewal and proliferating potential were examined with methylcellulose colony-forming unit assay. K562/ADM cells were highly resistant to adriamycin, daunorubicin and etoposide. The amount of CD34+, CD123+ and CD34+ CD38- cells in K562/ADM cells was much higher than that in K562 cells, and the proportion of CD34+ CD38- CD123+ cells (LSC) in K562/ADM cells was (5.23 +/- 0.21)% versus (1.27 + 0.17)% in K562 cells, which was 4.12-fold higher than that in K562 cells. Both P-gp and BCRP were overexpressed in K562/ADM cells relative to K562 cells, and the K562/ADM cells coexpressing P-gp and BCRP were 11.25-fold higher over K562 cells. The proportion of CD34+ CD38- CD123+ BCRP+ and CD34+ CD38- P-gp+ BCRP+ cells in K562/ADM cells were (4.13 +/- 0.40)% and (5.80 +/- 1.19)% respectively, which were 3.66- and 11.37-fold higher than the same cells in K562 cells [(1.13 +/- 0.15)% and (0.51 +/- 0.01)%]. Furthermore, drug-resistant K562/ADM cells displayed 4.17-time greater colony-forming ability over the parent K562 cells, corresponding to the proportion of LSCs in K562/ADM cells. The ABC transporters-overexpressing LSC population exists in drug-resistant leukemic K562/ADM cells relative to drug-sensitive K562 cells, and the drug-resistant LSCs may be the source of chemotherapeutic resistance of leukemia.