BCR-ABL mRNA Levels Research Articles

Isoalantolactone mediates the degradation of BCR-ABL protein in imatinib-resistant CML cells by down-regulating survivin

ABSTRACT Isoalantolactone (Iso) is a bioactive lactone isolated from the root of Inula helenium L, which has been reported to have many pharmacological effects. To investigate the role and mechanism of isoalantolactone in chronic myeloid leukemia (CML), we first investigated isoalantolactone’s anti-proliferative effects on imatinib-sensitive and imatinib-resistant CML cells by CCK8. Flow cytometry was used to detect isoalantolactone-induced cell apoptosis. Survivin was overexpressed in KBM5 and KBM5T315I cells using the lentivirus vector pSIN-3×flag-PURO. In KBM5 and KBM5T315I cells, shRNA was used to knockdown survivin. Cellular Thermal Shift Assay (CETSA) was used to detect the interaction between isoalantolactone and survivin. The ubiquitin of survivin induced by isoalantolactone was detected through immunoprecipitation. Quantitative polymerase-chain reaction (Q-PCR) and western blotting were used to detect the levels of mRNA and protein. Isoalantolactone inhibits the proliferation and promotes apoptosis of imatinib-resistant CML cells. Although isoalantolactone inhibits the proteins of BCR-ABL and survivin, it cannot inhibit survivin and BCR-ABL mRNA levels. Simultaneously, it was shown that isoalantolactone can degrade survivin protein by increasing ubiquitination. It was demonstrated that isoalantolactone-induced survivin mediated downregulation of BCR-ABL protein. It was also revealed that isoalantolactone triggered BCR-ABL protein degradation via caspase-3. Altogether, isoalantolactone inhibits survivin through the ubiquitin proteasome pathway, and mediates BCR-ABL downregulation in a caspase-3 dependent manner. These data suggest that isoalantolactone is a natural compound, which can be used as a potential drug to treat TKI-resistant CML.

Nilotinib Vs. Dasatinib in Achieving MR4.5 for Newly Diagnosed Chronic Myeloid Leukemia: Results of the Prospective Randomized Phase 3 Study, JALSG CML212

Nilotinib Vs. Dasatinib in Achieving MR4.5 for Newly Diagnosed Chronic Myeloid Leukemia: Results of the Prospective Randomized Phase 3 Study, JALSG CML212

Cross-talk between Bcr-abl and the Thioredoxin System in Chronic Myeloid Leukaemia: Implications for CML Treatment.

Chronic myeloid leukaemia (CML) is currently treated with inhibitors of the CML specific oncoprotein, bcr-abl. While this strategy is initially successful, drug resistance can become a problem. Therefore, new targets need to be identified to ensure the disease can be appropriately managed. The thioredoxin (Trx) system, comprised of Trx, thioredoxin reductase (TrxR), and NADPH, is an antioxidant system previously identified as a target for therapies aimed at overcoming drug resistance in other cancers. We assessed the effectiveness of TrxR inhibitors on drug resistant CML cells and examined links between TrxR and the bcr-abl cell-signalling pathway. Two TrxR inhibitors, auranofin and [Au(d2pype)2]Cl, increased intracellular ROS levels and elicited apoptosis in both sensitive and imatinib resistant CML cells. Inhibition of TrxR activity by these pharmacological inhibitors, or by specific siRNA, also resulted in decreased bcr-abl mRNA and protein levels, and lower bcr-abl downstream signalling activity, potentially enhancing the effectiveness of TrxR inhibitors as CML therapies. In addition, imatinib resistant CML cell lines showed upregulated expression of the Trx system. Furthermore, analysis of datasets showed that CML patients who did not respond to imatinib had higher Trx mRNA levels than patients who responded to treatment. Our study demonstrates a link between the Trx system and the bcr-abl protein and highlights the therapeutic potential of targeting the Trx system to improve CML patients’ outcomes.

Overexpression of miR-4433 by suberoylanilide hydroxamic acid suppresses growth of CML cells and induces apoptosis through targeting Bcr-Abl.

Background: Targeting Bcr-Abl is the key for the treatment of CML. Although great progress has been achieved for the treatment of CML patients in chronic stage, effective drugs with good safety are not available for those in advanced stages of CML patients. In present study, a histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), was used to screen for microRNA that can target Bcr-Abl.Methods: RT-qPCR was used to determine Bcr-Abl and miR-4433 transcription level in CML cells. In CML cells, Proteins including PARP, caspase-3, acetyl-histone 3, histone 3 and Bcr-Abl, as well as Bcr-Abl downstream proteins were detected using western blot. Cell viability and apoptosis were monitored respectively by MTS assay and flow cytometry. The correlation between miR-4433 and Bcr-Abl was determined by luciferase reporter assay. The anti-tumor effect of miR-4433 to K562 cells was evaluated by nude mouse xenograft model in vivo.Results: SAHA up-regulated the acetylation level of histone 3, and effectively inhibited Bcr-Abl mRNA level and its downstream signal transduction pathway, while inhibiting the growth of CML cells and inducing apoptosis. Furthermore, bioinformatics tools predicted that miR-4433 is a putative microRNA targeting Bcr-Abl and that the expression level of miR-4433 was significantly increased after SAHA treatment in K562 cells. Luciferase activity analysis revealed that miR-4433 directly targets Bcr-Abl. Additionally, transient expression of miR-4433 abrogated Bcr-Abl activity and its downstream signaling pathways while inducing apoptosis in K562 cells. Moreover, stable expression of miR-4433 suppressed Bcr-Abl and its downstream signaling pathway, and inhibited the growth of K562 cells in vitro and the growth of K562-xenografts in nude mice.Conclusion: miR-4433 was identified as a microRNA targeting Bcr-Abl, which may be subject to epigenetic regulation of SAHA, a histone deacetylase inhibitor that has been approved by the US FDA for the treatment of cutaneous T-cell lymphoma. The findings of this study provide a molecular basis from another angle for the use of SAHA in the treatment of CML.

Xpert® BCR-ABL Ultra¥ Demonstrated Good Inter-Laboratory Reproducibility in Three Consecutive GBMHM External Quality Assessments

Introduction: Monitoring BCR-ABL transcript levels in peripheral whole blood of patients using real-time quantitative PCR is standard of care in the management of Chronic Myeloid Leukemia. Therefore, it is essential to ensure that variability in testing methodology is tightly controlled and compatible with accurate assessments of treatment response. Xpert® BCR-ABL Ultra¥ (Ultra) is an automated cartridge-based assay developed by Cepheid for sensitive assessment of BCR-ABL p210 transcript levels. Every Ultra assay is standardized onto the WHO International Scale (IS) during production. The GBMHM (Groupe des Biologistes Moléculaires des Hémopathies Malignes) program has previously validated a sample preparation protocol designed for Ultra External Quality Assessment (EQA). We report here results from 3 consecutive EQA rounds for Ultra.Methods: Three Ultra EQA panels have been produced at GBMHM, containing a total of 11 members. Each member was produced from two contrived cell lysates, one BCR-ABL negative and one BCR-ABL e14a2/b3a2 positive, prepared according to the two-step Ultra sample preparation protocol per the package insert, but starting from 4 mL of a cell suspension titrating 2.5x106 cells/mL instead of blood (Table 1). Typically, 30 ml of each panel member was produced and aliquoted into 1mL samples before shipping on dry-ice to the participating laboratories. Upon receipt, individual laboratories further prepared the samples for testing following the 2nd lysis procedures.Results: A total of 209 samples were sent to up to 21 different labs and tested in 15 different Ultra lots. The %BCR-ABL/ABL IS ratios in 207 of 209 samples were returned to the GBMHM EQA program. The calculated average ratios were between 0.0066% and 24% IS with coefficients of variation (CVs) ranging from 16% to 77% (Table 1.). The data showed lower CVs at higher %BCR-ABL/ABL ratios. Nighty-five percent confidence intervals (95% CIs) for distribution of the average ratios ranged from ±4.8 fold for sample UC in EQA Round 19.2 to ±1.4 fold for sample UB in Round 19.2. Inter-lab reproducibility was acceptable, with less than 50% CV in 5 out of 8 panel members. The highest CV was observed in the panel member with an average ratio less than 0.01%IS. There was a trend towards an improvement of the CVs in the last EQA round compatible with an ongoing learning process.Conclusions: Overall, inter-laboratory reproducibility data obtained here are compatible with accurate measurements of BCR-ABL mRNA levels using Ultra, demonstrating equivalent or better results compared with non-automated methods. Since 15 different Ultra lots were used, this study verified that the Ultra standardization process is fully operative. To our knowledge, this is the first assessment of Ultra inter-laboratory reproducibility.¥For Research Use Only. Not for use in diagnostic procedures. Not reviewed by any regulatory body. [Display omitted] DisclosuresDay:Cepheid: Employment. Cayuela:Cepheid: Other: financial sponsor to attend John Goldman Conference 2017.

Abstract A58: Bcr-Abl independent activation of Erk1/2 in an imatinib resistant cell line

Abstract Tyrosine kinase inhibitor (TKI)-based therapy has dramatically enhanced chronic myeloid leukemia (LMC) panorama of treatment response, disease progression, and overall survival. In spite of its effectiveness and the development of more potent TKIs, resistance to treatment is still an issue. The recurrence of patients whose resistance is unrelated to BCR-ABL mutations points out to the importance of studying other resistance mechanisms. In this context, our group developed an imatinib (IM)-resistant cell line named K-IM through culturing K562 cells in gradually increasing IM concentrations. Since this cell line harbors no BCR-ABL kinase domain mutation, it is a perfect model for the study of Bcr-Abl unrelated resistance mechanisms. K-IM cells displayed an increase in BCR-ABL mRNA levels, but neither an increase in Bcr-Abl activity nor an impaired inhibition by IM was observed. Since ABC transporters are determinants for the multidrug resistance phenotype, P-glycoprotein (Pgp) expression as well as Pgp and breast cancer resistance protein activity was evaluated; however, K-IM cells showed neither Pgp expression nor transporter activity. Another important mechanism is the deregulation of apoptosis pathways and the inhibitor of apoptosis proteins XIAP and survivin have been extensively studied as putative targets for cancer treatment. K-IM cells presented mRNA levels of XIAP similar to its parental cell line K562 and higher mRNA levels of survivin. Protein analysis confirmed an increase in survivin expression, suggesting that this protein could contribute to the cell line's resistance. The search for signaling pathways that could promote survivin overexpression led to the observation of higher phosphorylation levels of Erk 1/2 that persisted even during Bcr-Abl inhibition in the resistant cell line K-IM. This suggests that the MAPK/Erk signaling pathway participates in a Bcr-Abl-independent resistance mechanism. There was a reduction in survivin levels after IM treatment, indicating its regulation is dependent on Bcr-Abl and not MAPK/Erk. One of the proteins that could evoke MAPK/Erk activation is the Shp2 phosphatase that interacts with adaptor proteins leading to Ras activation. However, IM treatment also impaired Shp2 phosphorylation, suggesting it is not the responsible mechanism for MAPK/Erk activation. Our data suggest that different resistance mechanisms that promote resistance may occur simultaneously and that K-IM cell line is an intriguing model for the study of resistance mechanisms and putative drug targets. Citation Format: Danielle Cardoso da Silva, Miguel A. Moreira, Raquel C. Maia, Flavia C. Vasconcelos. Bcr-Abl independent activation of Erk1/2 in an imatinib resistant cell line [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A58.

Correction: MPT0B169, a New Antitubulin Agent, Inhibits Bcr-Abl Expression and Induces Mitochondrion-Mediated Apoptosis in Nonresistant and Imatinib-Resistant Chronic Myeloid Leukemia Cells.

[This corrects the article DOI: 10.1371/journal.pone.0148093.].

MPT0B169, a New Antitubulin Agent, Inhibits Bcr-Abl Expression and Induces Mitochondrion-Mediated Apoptosis in Nonresistant and Imatinib-Resistant Chronic Myeloid Leukemia Cells.

Chronic myeloid leukemia (CML) is a clonal disorder of hematopoietic stem/progenitor cells that is caused by the Bcr-Abl oncoprotein. Clinical resistance to the Bcr-Abl inhibitor imatinib is a critical problem in treating CML. This study investigated the antitumor effect and mechanism of MPT0B169, a new antitubulin agent, in K562 CML cells and their derived imatinib-resistant cells, IMR2 and IMR3. IMR2 and IMR3 cells showed complete resistance to imatinib-induced growth inhibition and apoptosis. Resistance involved ERK1/2 overactivation and MDR1 overexpression. MPT0B169 inhibited the growth of K562, IMR2, and IMR3 cells in a dose- and time-dependent manner. MPT0B169 substantially inhibited the mRNA and protein levels of Bcr-Abl, followed by its downstream pathways including Akt, ERK1/2, and STAT3 in these cells. MPT0B169 treatment resulted in a decrease in the polymer form of tubulin according to Western blot analysis. It triggered cell cycle arrest at the G2/M phase before apoptosis, which was related to the upregulation of the mitotic marker MPM2 and the cyclin B1 level, and a change in the phosphorylation of Cdk1. MPT0B169 induced apoptosis in nonresistant and imatinib-resistant cells via a mitochondrion-mediated caspase pathway. Further study showed that the agent led to a decrease in the antiapoptotic proteins Bcl-2, Bcl-xL, and Mcl-1 and an increase in the apoptotic protein Bax. Taken together, our results suggest that MPT0B169 might be a promising agent for overcoming imatinib resistance in CML cells.

Effective Treatment of Human CML By RNAi In Vivo in a Xenotransplantation Mouse Model

Background: Considering the heterogeneity of leukemic cells in patients, current treatment regimens of chemotherapy and bone marrow transplantation lack specificity and are associated with frequent relapses and severe adverse effects. Hence, there is a need to develop novel therapeutics that can target the disease by its molecular fingerprint with minimal side effects.Despite the wide potential of RNA interference (RNAi) for translational therapeutics, systemic application of siRNA is hampered by rapid renal clearance, degradation by serum nucleases or associated immune responses. Lipid nanoparticles (LNPs) containing ionizable cationic lipids, when mixed with siRNA, embody the most advanced delivery platform for systemic administration of siRNA based therapeutics. Here, we established and employed the BCR-ABL dependent K562-CML xenotransplantation model as a proof of principle to validate LNP mediated siRNA functional delivery in vivo.Methods and Results: A microfluidic mixing technology was used to obtain reproducible ionizable cationic LNPs loaded with anti-BCR-ABL or CTRL siRNA. To determine the delivery efficiency of LNP-siRNA formulations, human leukemic K562 cells were incubated with siRNA-containing LNPs at various concentrations. Almost 100% of cells had taken up siRNA containing LNPs even at the lowest concentration of 0.0625µg/ml with stable uptake kinetics. We also observed near 100% uptake of LNP-siRNA in hard to transfect primary patient cells (CML, AML, ALL and MDS). Looking at the on-target functional efficacy of LNP-siRNA formulations, we observed a time and dose dependent increase in apoptosis (annexin V assay) and decrease in cell viability (alamar blue assay) of K562 cells treated with anti-BCR-ABL siRNA but not CTRL siRNA. A robust knockdown in BCR-ABL mRNA levels (65-90%) at 72 hours and protein at 96 hours was observed which confirmed that cell death was an on-target effect. Colony-forming potential of primary human CD34+ CML cells was significantly reduced when treated with anti-BCR-ABL siRNA compared to CTRL siRNA and to CD34+ cells from healthy donors.To translate our findings in vivo, we evaluated the safety profile, delivery potential and functional efficacy of LNP-siRNA in mice. A total dose of 15mg/kg (3 injections of 5mg/kg at day 0, 1 and 2) in healthy NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice resulted in 100% LNP positive cells in peripheral blood up to day 10. The formulations were highly tolerable in vivo with no significant differences in body weight and complete blood counts between treated and control mice. Moreover, serum analysis showed no significant toxicity in mice following LNP-siRNA treatment. With a focus on hematopoietic tissues following systemic administration, NSG mice received transplants of human K562 cells (stably expressing GFP and luciferase) intrafemorally and were injected intravenously for 3 consecutive injections of LNP-siRNA (1 or 5mg/kg body weight) at 8 hours interval. Interestingly, almost 100% LNP uptake was observed in xenograft leukemic cells in bone marrow at 48 hours at both doses. The leukemic burden of luciferase expressing K562 cells in mice was quantified using in vivo imaging before and during treatment. Treatment with anti-BCR-ABL siRNA for 10 days (n=7) resulted in a 0.5 fold decrease, whereas CTRL siRNA (n=7) resulted in a net 5-fold increase of luciferase signal, thus proving the efficacy of our approach in vivo.Conclusion: We show a highly efficient and non-toxic delivery in vitro and in vivo with nearly 100% uptake of LNP-siRNA formulations in bone marrow of leukemic mice. By inhibiting BCR-ABL we show a reduction of leukemic burden in our xenotransplant model, while leukemic cells expanded in CTRL siRNA treated mice. Our study provides a proof-of-principle that the combined use of lipid nanoparticles and RNAi technology can be used to target leukemia cells in vivo with promising therapeutic implications. DisclosuresWalsh:Precision Nanosystems Inc.: Employment. Thomas:Precision Nanosystems Inc.: Employment. Ramsay:Precision Nanosystems Inc.: Employment. Heuser:Karyopharm: Research Funding.

Expression of miR-128 in patients with acute lymphoblastic leukemia

Objective To investigate the expression and clinical significance of miR-128 in acute lymphoblastic leukemia(ALL). Methods The relative expression levels of miR-128 from bone marrow cells were detected by real time PCR in 62 patients with ALL and 20 normal subjects, and the correlations between miR-128 expression and its clinical characteristics were analyzed. Results The relative expression level of miR-128 in ALL was higher than that in normal subjects(P 0.05), and no significant correlation was found between miR-128 expression level and bcr-abl positive rates or bcr-abl mRNA level in ALL(P > 0.05). Conclusion miR-128 is up-regulated in ALL patients, which may provide a novel ALL diagnostic marker. There was no obvious influence of bcr-abl fusion gene on miR-128 expression in ALL patients. Key words: Acute lymphoblastic leukemia; miRNA; bcr-abl

Odk-1201, One-Step RT-qPCR Major BCR-ABL/ABL mRNA Kit for the International Scale, with High Sensitivity to Detect Deeper MR

Abstract Background: Molecular monitoring by the measurement of major BCR-ABL mRNA levels is important for the evaluation of the therapeutic efficacy of TKI in CML patients. However, there is no commercial kit easily available in each laboratory or hospital, which can estimate deeper responses below MR4.5that is 4.5-log reduction by the international scale (IS) 0.0032%. Methods: An ODK-1201 kit (Otsuka Pharmaceutical Co. Japan) is based on a HawkZ05 Fast One-Step RT-PCR Kit (Roche, Indianapolis, IN, USA). In this kit, reverse transcription and quantitative PCR (RT-qPCR) are performed in one tube, of which products are measured by ABI 7500 fast Dx (Life Technologies Japan, Japan). To isolate enough amount of RNA for the evaluation of molecular responses below MR4.5in CML patients, 7 ml of peripheral blood is required for this kit. To get the conversion factor (CF) for the IS, the panel of WHO International Standard was obtained from NIBSC, which comprises four ampoules each containing freeze-dried BCR-ABL-positive and -negative cells at various ratios from 0.01% to 10%. RNAs were isolated from each ampoule with a QIAamp RNA Blood Mini Kit (QIAGEN) and were subjected to the measurement of major BCR-ABL and ABL mRNAs with an ODK-1201 kit. Since %BCR-ABL/ABL in the WHO standard panel was already defined, CF was calculated by comparing the values of %BCR-ABL/ABL obtained by ODK-1201 with those of WHO standard panel. To validate the results obtained by ODK-1201, we collected 267 peripheral blood samples from CML patients as a multicenter study, and 120 samples in these were measured by ODK-1201 and in the international reference laboratory (Adelaide Lab), respectively. In addition, we conducted another multicenter study to compare ODK-1201 with an Ipsogen BCR-ABL1 Mbcr IS-MMR DX Kit (QIAGEN) and a One-Step qRT-PCR BCR-ABL Kit (Molecular MD), in which we measured the samples from 201 CML patients, 25 patients with non-CML hematologic malignancies and 25 healthy volunteers by these methods. All of the samples were obtained after the written informed consent was given. Results: The CF of ODK-1201 determined by the WHO standard panel was 1.12. After conversion to the IS by this CF, %BCR-ABL/ABLIS determined by ODK-1201 ranged from 0.0007% to 144.6867% in CML patients. In contrast, all of the values were below 0.0007% in the samples from patients with non-CML hematologic malignancies and healthy volunteers. The overall correlation between ODK-1201 and Adelaide Lab system was very good (94% within 3-fold bias) and the evaluation of MMR was concordant in 84% of CML samples. As for 46 CML samples with %BCR-ABL/ABL IS between 0.0032% and 0.1%, IS values by ODK-1201 were similar to the ones by Adelaide Lab system (with MR4.5; 0.0032%) with 89% within 3-fold and high correlation coefficient (r=0.89). Also, the correlation between ODK-1201 and Ipsogen was r = 0.97 (n = 224) and that between ODK-1201 and Molecular MD kit was r = 0.97 (n = 221), indicating that ODK-1201 was as accurate as Ipsogen and Molecular MD kits. Together, these results indicate that ODK-1201 was capable of measuring at least MR4.5 with the lowest value detected as 0.0007% (calculated sensitivity of less than MR5.0) by the IS. Conclusions: We developed a major BCR-ABLmRNA kit, ODK-1201 with CF for the IS and confirmed its high sensitivity (0.0007%) and accuracy. The procedure is very simple and takes only 2.5 h, and it needs a small amount of blood because RT-qPCR is performed in a single tube. It will enable us to evaluate deeper molecular responses in any laboratories. Conflict of interest :received research fundinand and honoraria from Otsuka I.M. received honoraria from Otsuka during the conduct of the study. H.N. received research funding from Otsuka during the conduct of the study and received research funding and honoraria from otsuka outside the submitted work. C.Y. received research funding from Otsuka during the conduct of the study and honoraria from Novartis and Bristol Myers Squibb. S.B. received research funding and honoraria from Novartis, Bristol Myers Squibb, Otsuka and Ariad. L.F. has no conflict of interest. K.D. and T.S. are employee of Otsuka. Y.K. received honoraria from Otsuka during the conduct of the study. T.N. received grants and personal fees from Otsuka during the conduct of the study, and grants and personal fees from Otsuka outside the submitted work. Disclosures Matsumura: Otsuka: Consultancy, Honoraria. Nakamae:Otsuka : Research Funding. Yoshida:Otsuka: Research Funding. Branford:Otsuka: Research Funding. Koga:Otsuka: Employment. Sogabe:Otsuka: Employment. Kanakura:Otsuka: Consultancy. Naoe:Otsuka: Consultancy.

SHP-1 gene in the disease progression of chronic myeloid leukemia

To investigate the profile of promoter methylation and expression of SHP-1 gene in the progression of chronic myeloid leukemia (CML). The expression level of SHP-1 mRNA and protein in bone marrow or peripheral blood mononuclear cells from CML patients were detected by Western blot and SYBR Green-based qRT-PCR. The methylation status of SHP-1 were assessed by methylation-specific polymerase chain reaction (MSP) assay. K562 cells were infected with the lentiviral plasmids pEX-SHP-1-puro-Lv105 (K562-SHP-1) or pEX-EGFP-puro-Lv105 (K562-EGFP). The levels of proteins and phosphorylated proteins were detected by Western blot. qRT-PCR assay was used to test the level of BCR-ABL mRNA. The relative levels of SHP-1 mRNA were sharply decreased in advanced stages CML compared to chronic phase (CP)-CML (0.79±0.37 vs 1.18±0.64, P= 0.009). The level of SHP-1 protein was lower in advanced stages CML compared to CP-CML (0.57±0.02 vs 1.02±0.04, P=0.039). The frequency of SHP-1 gene promoter methylation at selected loci in CP-CML was 23.8% (10/42), and the methylated regions were detected in all advanced CML samples (P<0.01). SHP-1 was stably transfected into K562 cells and selected with puromycin. Overexpression of SHP-1 inhibited the proliferation and induced the apoptosis of K562 cells, meanwhile leaded to G0/G1 phase arrest. After transfection, the level of BCR-ABL mRNA was not affected in K562-SHP-1 cells (1.32±0.34) compared to K562-EGFP cells (1.18±0.20, P=0.644), but overexpression of SHP-1 caused a slight decrease in BCR-ABL protein in K562-SHP-1 cells compared to K562 -EGFP cells (0.78±0.15 vs 1.27±0.24, P=0.040). Overexpression of SHP-1 resulted in a remarkable decrease in MYC protein, phosphorylated forms of JAK2, STAT5, Akt and MAPK. However, the un-phosphorylated forms of these molecules were not significantly affected. Decreased expression of SHP-1 caused by aberrant promoter hypermethylation may play a key role in the progression of CML by dysregulation of BCR-ABL, Akt, MAPK, MYC, JAK2 and STAT5 signaling.

Superior activity of a new histone deacetylase inhibitor (ZYJ-34c) in inhibiting growth of human leukemia cells by inducing p21 WAF1 expression and cell cycle arrest

Histone deacetylase inhibitors are a new class of anticancer agents that inhibit cancer cell proliferation and induce apoptosis and cell cycle arrest in various cancer cells. Recently, we identified ZYJ-34c, a modified histone deacetylase inhibitor that showed significantly higher antitumor activity in vivo than the FDA-approved drug suberoylanilide hydroxamic acid. We aimed to investigate its exact mechanism of action. Activity of ZY-34c against human erythroleukemic (K562) cells, human myeloid leukemia (HL-60) cells, and primary leukemic cells were investigated in vitro using proliferation assays, cell cycle assays, apoptosis assays, RNA interference, promoter acetylation assays, and assays of transcription of related molecules. ZYJ-34c strongly inhibited the proliferation of leukemia cells compared with suberoylanilide hydroxamic acid. Primary leukemic cells isolated from patients with acute myeloid leukemia were more sensitive to ZYJ-34c. The 50% growth-inhibitory concentrations of ZYJ-34c and suberoylanilide hydroxamic acid were 2.95±0.75 and 4.45±0.29 μmol/l for K562 cells treated for 48 h, respectively. We found that ZYJ-34c caused more significant G1 cell cycle arrest than suberoylanilide hydroxamic acid, in a time-dependent manner. ZYJ-34c-induced hyperacetylation of histone H3 and H4 around the promoter region of p21. P21 RNA interference markedly impaired ZYJ-34c-induced or suberoylanilide hydroxamic acid-induced G1 cell cycle arrest. In addition, levels of bcr-abl mRNA and protein in K562 cells decreased after treatment with either suberoylanilide hydroxamic acid or ZYJ-34c; moreover, ZYJ-34c had a higher inhibition activity. ZYJ-34c could represent a novel pharmacological agent with potential benefit for patients with leukemia.

Single-cell analysis of K562 cells: An imatinib-resistant subpopulation is adherent and has upregulated expression of BCR-ABL mRNA and protein

In chronic myeloid leukemia (CML) cells from different stages of maturation may have differential expression of BCR-ABL at both messenger RNA (mRNA) and protein level. However, the significance of such differential expression to clinical disease behavior is unknown. Using the CML-derived, BCR-ABL expressing cell line, K562, distinct plastic-adherent (K562/Adh) and nonadherent (K562/NonAdh) subpopulations were established and then analyzed both as single cells and as bulk cell populations. BCR-ABL mRNA was upregulated in K562/Adh compared with K562/NonAdh cells in both single cell and bulk population analyses (p < 0.0001). Similarly, phosphorylation of BCR protein was upregulated in K562/Adh, compared with K562/NonAdh cells (63.42% vs. 23.1%; p = 0.007), and these two K562 subpopulations were found to express significantly different microRNA species. Furthermore, treatment with the BCR-ABL tyrosine kinase inhibitor, imatinib, reduced cell viability more rapidly in K562/NonAdh compared with K562/Adh cells (p < 0.005) both at single and bulk cell levels. This discovery of an adherent subpopulation of K562 cells with increased BCR-ABL mRNA, increased phosphorylated BCR protein expression, differential microRNA expression, and increased imatinib resistance suggests that a similar subpopulation of cells can also mediate clinical resistance to imatinib during treatment of patients with CML.

CD34+CD38-CD58- Candidate Leukemia-Initiating Cells Are Clinically Relevant With The Unfavorable Prognosis In Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia

▪ BackgroundThe prognosis of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) has been greatly improved in the modern era of imatinib. Nevertheless, relapse is still a major cause of treatment failure in human Ph+ALL. Leukemia-initiating cells (LICs) are presumed to be responsible for relapse in leukemia. Therefore, we conducted a study to identify the candidate LICs that are responsible for disease progression and its clinical significance in patients with Ph+ALL. AimsTo investigate the leukemia-initiating and self-renewal capacities of CD34+CD38-CD58- cells and determine the prognostic significance of CD34+CD38-CD58- phenotype in patients with Ph+ALL treated in Peking University Institute of Hematology. MethodsThe leukemia-initiating potential and self-renewal capacity of the sorted CD34+CD38-CD58-, CD34+CD38-CD58+,CD34+CD38+CD58- and CD34+CD38+CD58+ compartments were investigated in vivo using sublethally irradiated and anti-mouse CD122 monoclonal antibody conditioned NOD/SCID mice by intra-bone marrow–injection. Furthermore, we prospectively analyzed whether the identified CD34+CD38-CD58- compartment at diagnosis correlates with minimal residual disease (MRD) after therapy and clinical outcomes in 63 adult patients (18-60 years) with de novo Ph+ALL. ResultsXenotransplantation of the sorted CD34+CD38-CD58- cells led to a repopulation of human B-ALL in primary and secondary recipient mice, which were phenotypically and clonally derived from the original Ph+ALL patients analyzed by flow cytometry, as well as quantitative real-time RT-PCR and fluorescence in situ hybridization for leukemia-specific cytogenetic abnormalities. Furthermore, the candidate CD34+CD38-CD58- LICs phenotype at diagnosis (n=16) significantly correlated with a lower complete remission rate and higher MRD frequency monitored by BCR-ABL mRNA levels in BM of Ph+ALL patients. Additionally, it directly correlated with higher cumulative incidence of relapse (CIR, 60% ± 1.97% vs. 15.51% ± 0.30%, P=0.002) and unfavorable disease-free survival (DFS, 33.75%±12.64% vs. 71.31%±7.17%, P=0.009) at 3-year. The CD34+CD38-CD58- group exhibited a higher rate of BCR-ABL mutations conferring higher level imatinib resistance than the other group (43.75% vs. 17.02%, P=0.04). Multivariate analyses revealed that CD34+CD38-CD58- phenotype at diagnosis was an independent risk factor for relapse (HR=4.35, P=0.009) and DFS (HR=3.38, P=0.008) in adult Ph+ALL. Summary/ConclusionBoth the xenotransplantation data as well as the clinical correlation studies show that CD34+CD38-CD58- compartment enrich for leukemia-initiating cells in adult Ph+ALL. CD34+CD38-CD58- phenotype at diagnosis independently correlates with an adverse prognosis, which promises to be an efficient tool for relapse prediction and risk-stratification treatment in adult Ph+ALL patients. AcknowledgmentsThis work was supported by grants from National Natural Science Foundation of China (grants no. 30800483&81230013) and Beijing Municipal Science and Technology Program (grant no.Z111107067311070). Disclosures:No relevant conflicts of interest to declare.

Which method better evaluates the molecular response in newly diagnosed chronic phase chronic myeloid leukemia patients with imatinib treatment, BCR-ABLIS or log reduction from the baseline level?

The molecular response of chronic myeloid leukemia (CML) patients to tyrosine kinase inhibitor treatment can be evaluated either by BCR-ABL mRNA levels on international scale (IS) or by log reduction from the baseline level of the laboratory. Both methods were compared in 248 newly diagnosed chronic phase CML patients treated with imatinib. The major molecular responses (MMR) obtained by both methods predict progression-free survival (PFS, all P<0.0001). Thirty-six patients, who were identified as MMR patients by the IS method but as non-MMR patients by the log reduction method, had the same PFS as MMR patients identified by both methods. The molecular responses of patients at 3 and 6 months, as evaluated by the two methods, have similar predictive values on their cytogenetic responses at 12 months and on their molecular responses at 18 months. Both ≤ 10%(IS) and ≥ 1 log reduction at 3 months and ≤ 1%(IS) at 6 months were significantly associated with PFS (P=0.0011, 0.0090, and 0.0064). The percentages of patients with BCR-ABL(IS) of ≤ 1%, >1-10%, and of >10% at 3 months and 6 months in the German CML Study IV were similar with those with corresponding BCR-ABL(IS) in our center, but was significantly different with those evaluated by the log reduction method. Therefore, the molecular response evaluated by BCR-ABL(IS) has similar trends in PFS and in response prediction, but can better differentiate patients than that by the log reduction method. Furthermore, the IS method allows comparison among molecular response results from different laboratories.

The Musashi 2 mRNA Expression in the Course of CML.

Abstract 2552The Musashi (MSI) gene family members, MSI1 and MSI2, represent an evolutionarily conserved family of RNA-binding proteins that regulate mRNA translation through binding in their N-termini. High levels of MSI2 protein are associated with increased cell proliferation, decreased cell maturation, more aggressive hematologic malignancy diseases and worse clinical prognosis. Recently obtained data pointed to MSI2 playing an important role in acute myeloid leukemia (AML) and in deadly blast crisis of chronic myeloid leukemia (CML) (Ito et al. 2010 Nature 5; 466).In this study we screened the level of MSI2 mRNA in 49 patients in different phases of CML and with different response to therapy – 18 patients at diagnosis (DG), 5 in major molecular response (MMR), 4 in complete molecular response (CMR), 2 after bone marrow transplantation (BMT), 10 in hematology relaps (HR), 6 in accelerrated phase (AP), and 4 in blast crisis (BC), and in 6 healthy donors. The level of MSI2 mRNA was quantified by real-time reverse-transcriptase-polymerase chain reaction using in-house designed specific primers and TaqMan probe and normalized to B2M endogenous control. Expression ratios were calculated by ΔΔCt method, and the differences between groups were statistically evaluated using Mann Whitney test.We detected MSI2 expression in all samples. The median expression of mRNA MSI2 in patients at DG was 1,43 (0,33–3,28), in MMR 0,52 (0,20–0,62), in CMR 0,37 (0,30–0,63), after BMT 1,28 (1,02–1,54), in HR 0,41 (0,16–0,58), in AP 3,78 (1,94–13,69), in BC 15,17 (2,61–28,15). MSI2 expression was statistically up-regulated in patients in advanced phases of CML (AP, BC) when compared with patients in CP (P<0.0001). The difference between patients in DG and remaining patients in CP was also statistically significant (P= 0,0006). No correlation of MSI2 expression level in DG patients with their responsiveness to treatment, BCR-ABL transcript level or survival was found. No significant differences were observed among groups of patients in MMR, CMR, HR, and after BMT.In addition, in order to check whether MSI2 expression level can serve as a marker of CML progression we also retrospectively screened kinetics of MSI2 transcript in 5 CML patients monitored on average 27 months (18–48). During this period, 3 patients developed HR, 1 patient AP and 1 BC. In BC patient the MSI2 transcript level increased with progression of CML in accordance with the increase of leucocytes and BCR-ABL transcript level. In 1 patient with a rising AP BCR-ABL levels remained constant compared to sevenfold increase of the MSI2 transcript level. On the other hand in HR patients we detected a constant or even decreasing level of MSI2 transcript regardless of the increase of leucocytes and BCR-ABL.In summary, our results confirm the association of high MSI2 mRNA level with advanced phases of CML and indicate that increase of MSI2 mRNA level may serve as a valuable marker of advanced phases of CML. In particular for CML patients with constantly high level of BCR-ABL mRNA the monitoring of MSI2 level can be important tool for early recognition of CML progression. Potential contributions of MSI2 to leukemic pathogenesis and its regulation in CML progression remain unknown.Grant support: NT/12392-4 IGA MZ-CR. Disclosures:No relevant conflicts of interest to declare.

The differences and correlations of BCR‐ABL transcripts between peripheral blood and bone marrow assays are associated with the molecular responses in the bone marrow for chronic myelogenous leukemia

Previous studies concerning BCR-ABL mRNA levels by quantitative real-time RT-PCR (Q-PCR) for chronic myelogenous leukemia (CML) have shown a significant concordance between peripheral blood (PB) and bone marrow (BM) assays. The objective of this study was to determine whether molecular monitoring using PB was comparable to using BM for CML. A comparative study was performed that analyzed the Q-PCR results of 712 simultaneous PB and BM samples from 330 patients before and during imatinib therapy. For the 78 paired pretreatment samples, the level of BCR-ABL mRNA in PB was lower than that in BM (P = 0.007). Although the overall amounts of BCR-ABL mRNA in the PB and BM were comparable (P= 0.072) and there was a strong correlation (r = 0.839, P < 0.001) with the 634 paired on-treatment samples, the depth of the molecular response in PB was lower than that in BM (P < 0.001). The level of BCR-ABL mRNA in PB was lower than that in BM where the BM BCR-ABL mRNA < 1 log reduction (P < 0.001) or ≥ 1-< 2 log-reductions (P = 0.008) from the baseline, and higher than that where the BM BCR-ABL mRNA ≥ 2 log-reductions (P < 0.001). A strong correlation (r = 0.811, P < 0.001) was only found where the BM BCR-ABL mRNA < 1 log reduction. We conclude that the differences and correlations of BCR-ABL mRNA between PB and BM assays depend on the depth of the molecular response in BM for CML during imatinib therapy.

Cyclin-Dependent Kinase 7/9 Inhibitor SNS-032 Abrogates FIP1-like-1 Platelet-Derived Growth Factor Receptor α and Bcr-Abl Oncogene Addiction in Malignant Hematologic Cells

The "gate-keeper" mutations T674I platelet-derived growth factor receptor α (PDGFRα) in hypereosinophilic syndrome (HES) and T315I Bcr-Abl in chronic myeloid leukemia (CML) are resistant to imatinib and the second-generation small-molecule tyrosine kinase inhibitors (TKI). However, to combat acquired resistance to imatinib, an alternative approach is to decrease the expression of the addicted gene to efficiently kill resistant malignant hematologic cells. The purpose of this study was to evaluate the strategy of shutting down the transcription and expression of FIP1-like-1 (FIP1L1)-PDGFRα and Bcr-Abl with SNS-032, an inhibitor of cyclin-dependent kinase 7 (CDK7) and CDK9 in phase I clinical trials. The effects of SNS-032 on PDGFRα and Bcr-Abl signaling pathways, apoptosis, and cell cycling were analyzed in TKI-resistant cells of HES and CML. The in vivo antitumor activity of SNS-032 was assessed with xenografted BaF3-T674I FIP1L1-PDGFRα and KBM5-T315I Bcr-Abl cells in nude mouse models. SNS-032 inhibited the phosphorylation on Ser5 and Ser2 of RNA polymerase II. SNS-032 decreased both the mRNA and protein levels of FIP1L1-PDGFRα and Bcr-Abl and inhibited the proliferation of malignant cells expressing FIP1L1-PDGFRα or Bcr-Abl. It also decreased the phosphorylation of downstream molecules. It induced apoptosis by triggering both the mitochondrial pathway and the death receptor pathway. This CDK7/9 inhibitor potently inhibits FIP1L1-PDGFRα-positive HES cells and Bcr-Abl-positive CML cells regardless of their sensitivity to imatinib. SNS-032 may have potential in treating hematologic malignancy by abrogating oncogene addiction.

Quantification of BCR-ABL mRNA in Plasma/Serum of Patients with Chronic Myelogenous Leukemia

Quantification of tumor-associated mRNA extracted from blood cells/tissues containing tumor cells is used for evaluation of treatment efficacy or residual tumor cell burden in tumors including leukemia. However, this method using tumor cell-containing blood/tissue is difficult to evaluate the whole tumor cell burden in the body. In order to establish an efficient method to evaluate the whole tumor cell burden in the body, we tried to quantify tumor-associated mRNA existing in plasma/serum instead of leukemia cell-containing blood cells in patients with chronic myelogenous leukemia (CML) and compared the levels of BCR-ABL mRNA between plasma/serum and peripheral blood cells. mRNA of BCR-ABL, WT1 or GAPDH (control molecule) was detected by real-time RT-PCR using RNA extracted from plasma/serum of almost all the patients with CML. Copy numbers of BCR-ABL mRNA were significantly correlated between plasma/serum and peripheral blood cells. However, levels of BCR-ABL mRNA extracted from serum were low compared with those extracted with peripheral blood cells. The present findings suggest that although real-time RT-PCR of mRNA existing in plasma/serum could be used for evaluating the whole tumor cell burden in the body, it's required to establish an efficient method to quantify plasma/serum mRNA by nature without degrading during the procedure.