Imatinib-resistant Chronic Myeloid Leukemia Patients Research Articles

Distribution and Viability of Peripheral Blood Mononuclear Cells from Imatinib-resistant Chronic Myeloid Leukemia Patients: an In Vitro Study

The incidence of Chronic Myeloid Leukemia (CML) in the world is approximately 1.5 cases per 100,000 individuals. The level of resistance to CML treatment, imatinib in Indonesia is relatively high compared to Europe. Culturing CML cells can be used as a model for the determination of pathogenesis of CML, drug efficacy testing, and drug resistance testing. Studies using CML patients’ cells to be cultured in vitro and the methods used are rarely varied. This study aims to examine the distribution and viability of peripheral blood mononuclear cells from imatinib-resistant CML patients, expected to be a reference for mononuclear cell cultures from CML patients. This study was conducted in June-August 2019 using quantitative descriptive methods. The sample was mononuclear cells isolated from peripheral blood of three imatinib-resistant CML patients at the Hemato-Oncology Polyclinic of Hasan Sadikin Hospital, each of which was cultured in vitro using RPMI 1640 for 28 days. Distribution were seen using Giemsa staining, while viability was calculated using trypan blue. Data is processed using Microsoft Excel 2013 and Graphpad. Cell viability decreased during culture. Cell distribution had a different development pattern. Blast cells, eosinophils and basophils had presentation of between 0-5%. The percentage of lymphocyte changed between 11-31%. The percentage of neutrophil changed between 16-41%. The percentage of immature cells decreased, whereas the percentage of monocyte increased. In conclusion, cell viability decreases during the culture. Distribution of cells similar to the initial condition lasted until the 7th day and in the final phase it was only dominated by monocytes.Keywords: myeloid leukemia, chronic, cell culture, in vitro, peripheral blood mononuclear cell, cell viability.

Quantitative Proteomic Analysis of Plasma Exosomes to Identify the Candidate Biomarker of Imatinib Resistance in Chronic Myeloid Leukemia Patients.

BackgroundImatinib (IM), a tyrosine kinase inhibitor (TKI), has markedly improved the survival and life quality of chronic myeloid leukemia (CML) patients. However, the lack of specific biomarkers for IM resistance remains a serious clinical challenge. Recently, growing evidence has suggested that exosome-harbored proteins were involved in tumor drug resistance and could be novel biomarkers for the diagnosis and drug sensitivity prediction of cancer. Therefore, we aimed to investigate the proteomic profile of plasma exosomes derived from CML patients to identify ideal biomarkers for IM resistance.MethodsWe extracted exosomes from pooled plasma samples of 9 imatinib-resistant CML patients and 9 imatinib-sensitive CML patients by ultracentrifugation. Then, we identified the expression levels of exosomal proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS) based label free quantification. Bioinformatics analyses were used to analyze the proteomic data. Finally, the western blot (WB) and parallel reaction monitoring (PRM) analyses were applied to validate the candidate proteins.ResultsA total of 2812 proteins were identified in plasma exosomes from imatinib-resistant and imatinib-sensitive CML patients, including 279 differentially expressed proteins (DEPs) with restricted criteria (fold change≥1.5 or ≤0.667, p<0.05). Compared with imatinib-sensitive CML patients, 151 proteins were up-regulated and 128 proteins were down-regulated. Bioinformatics analyses revealed that the main function of the upregulated proteins was regulation of protein synthesis, while the downregulated proteins were mainly involved in lipid metabolism. The top 20 hub genes were obtained using STRING and Cytoscape, most of which were components of ribosomes. Moreover, we found that RPL13 and RPL14 exhibited exceptional upregulation in imatinib-resistant CML patients, which were further confirmed by PRM and WB.ConclusionProteomic analysis of plasma exosomes provides new ideas and important information for the study of IM resistance in CML. Especially the exosomal proteins (RPL13 and RPL14), which may have great potential as biomarkers of IM resistance.

Discovery and Protein Modeling Studies of Novel Compound Mutations Causing Resistance to Multiple Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia.

Objective:BCR-ABL fusion oncogene is the hallmark of chronic myeloid leukemia (CML), causing genomic instability which leads to accumulation of mutations in BCR-ABL as well as other genes. BCR-ABL mutations are the cause of tyrosine kinase inhibitors (TKIs) resistance in CML. Recently, compound BCR-ABL mutations have been reported to resist all FDA approved TKIs. Therefore, finding novel compound BCR-ABL mutations can help and clinically manage CML. Therefore, our objective was to find out novel drug-resistant compound BCR-ABL mutations in CML and carry out their protein modelling studies. Methodology:Peripheral blood samples were collected from ten imatinib resistant CML patients receiving nilotinib treatment. BCR-ABL transcript mutations were investigated by employing capillary sequencing. Patient follow-up was carried out using European LeukemiaNet guidelines. Protein modeling studies were carried out for new compound mutations using PyMol to see the effects of mutations at structural level. Results:A novel compound mutation (K245N mutation along with G250W mutation) and previously known T351I utation was detected in two of the nilotinib resistance CML patients respectively while in the rest of 8 nilotinib responders, no resistant mutations were detected. Protein modelling studies indicated changes in BCR-ABL mutant protein which may have negatively impacted its binding with nilotinib leading to drug resistance. Conclusion:We report a novel nilotinib resistant BCR-ABL compound mutation (K245N along with G250W mutation) which impacts structural modification in BCR-ABL mutant protein leading to drug resistance. As compound mutations pose a new threat by causing resistance to all FDA approved tyrosine kinase inhibitors in BCR-ABL+ leukemias, our study opens a new direction for in vitro characterization of novel BCR-ABL compound mutations and their resistant to second generation and third generation TKIs.

Effect of Dasatinib on Genes Related to Mitotic Catastrophe Pathway in Chronic Myeloid Leukemia Cells.

Chronic Myeloid Leukemia (CML) is characterized by a reciprocal translocation t(9;22) and forms BCR/ABL1 fusion gene called the Philadelphia chromosome. The therapeutic targets for CML patients mediated with BCR/ABL1 oncogenic are tyrosine kinase inhibitors such as imatinib, dasatinib, and nilotinib. The latter two of which have been approved for the treatment of imatinib-resistant or intolerance CML patients. Mitotic Catastrophe (MC) is one of the non-apoptotic mechanisms initiated in types of cancer cells in response to anti-cancer therapies. Pharmacological inhibitors of G2 checkpoint members or genetic suppression of PLK1, PLK2, ATR, ATM, CHK1, and CHK2 can trigger DNA-damage-stimulated mitotic catastrophe. PLK1 and AURKA/B are anomalously expressed in CML cells, where phosphorylation and activation of PLK1 occur by AURKB at centromeres and kinetochores. The purpose of this study is to investigate the effect of dasatinib on the expression of genes in MC and apoptosis pathways in K562 cells. Total RNA was isolated from K-562 cells treated with the IC50 value of dasatinib and untreated cells as a control group. The expression of MC and apoptosis-related genes, was analyzed by the qRT-PCR system. The array-data demonstrated that dasatinib-treated K562 cells significantly caused the decrease of several genes (AURKA, AURKB, PLK, CHEK1, MYC, XPC, BCL2, and XRCC2). The evidence supplies a basis to support clinical researches for the suppression of oncogenes such as PLKs with AURKs in the treatment of types of cancer, especially chronic myeloid leukemia.

MicroRNA 30a Mediated Autophagy and Imatinib Response in Egyptian Chronic Myeloid Leukemia Patients.

Imatinib Mesylate is the drug used for targeted tyrosine kinase inhibition in the beginning of management of all Chronic Myeloid Leukemia (CML) newly diagnosed cases. However, resistance presents a considerable limit to its efficacy. Currently, it is impossible to anticipate IM resistance which makes the recognition of early flags an important treatment goal in CML. In this work we studied the connection between microRNA 30a (miR-30a) and Beclin 1 mediated autophagy and IM resistance in Egyptian CML patients. The study included newly diagnosed (group I, n = 20), imatinib responder (group II, n = 30), imatinib resistant (group III, n = 30) CML patients and a healthy demographically matched control group (group IV, n = 20). miR-30a expression was assayed by quantitative reverse transcription polymerase chain reaction. The variation in expression of miR-30a between CML cases and healthy controls was calculated using relative quantification method (2-ΔΔCT). Beclin 1 was assayed in Peripheral blood mononuclear cells by western blotting. miR-30a was over expressed and Beclin 1 was under expressed in imatinib responders compared to resistant cases median 1.21(0.55-3.02) versus median 0.65 (0.03-1.0) (p = 0.001) and median 950.0 (400.0-2410.0) versus, median 1570.0 (920.0-5430.0) (p < 0.001) respectively. Beclin 1 correlated significantly positively with miR-30a in new cases (p = 0.001) and negatively in imatinib responders (p = 0.021). Receiver Operating Curves demonstrated the performances of miR-30a and Beclin 1 to detect imatinib resistance. They showed sensitivities of 97.14% and 94.29% and specificities of 53.33% and 42.22% at the cut-off values of 1 and 940 respectively. Both miR-30a and Beclin 1 levels showed a relation with imatinib response and can therefore be put forward as valuable markers for detection of resistance and may also have promising future therapeutic implications.

Preclinical development of a novel BCR-ABL T315I inhibitor against chronic myeloid leukemia.

Chronic Myeloid Leukemia (CML) is a myeloproliferative neoplasm primarily due to the presence of the BCR-ABL fusion gene that produces the constitutively active protein, BCR-ABL. Imatinib, a BCR-ABL-targeted drug, is a first-line drug for the treatment of CML. Resistance to imatinib occurs as a result of mutations in the BCR-ABL kinase domains. In this study, we evaluated S116836, a novel BCR-ABL inhibitor, for its anti-cancer efficacy in the wild-type (WT) and T315I mutant BCR-ABL. S116836 was efficacious in BaF3 cells with WT or T315I mutated BCR-ABL genotypes. S116836 inhibits the phosphorylation of BCR-ABL and its downstream signaling in BaF3/WT and BaF3/T315I cells. Mechanistically, S116836 arrests the cells in the G0/G1 phase of cell cycle, induces apoptosis, increases ROS production, and decreases GSH production in BaF3/WT and BaF3/T315I cells. Moreover, in mouse tumor xenografts, S116836 significantly inhibits the growth and volume of tumors expressing the WT or T315I mutant BCR-ABL without causing significant cardiotoxicity. Overall, our results indicate that S116836 significantly inhibits the imatinib-resistant T315I BCR-ABL mutation and could be a novel drug candidate for treating imatinib-resistant CML patients.

Knockdown of ribonucleotide reductase regulatory subunit M2 increases the drug sensitivity of chronic myeloid leukemia to imatinib‑based therapy.

Imatinib-based targeted treatment is the standard therapy for chronic myeloid leukemia (CML); however, drug resistance is an inevitable issue for imatinib-based CML treatment. Imatinib resistance can be ascribed to Bcr-Abl-dependent and independent resistance. In the present study, peripheral blood samples were collected from imatinib-sensitive (IS) and imatinib-resistant (IR) CML patients and transcriptome sequencing was carried out. From the RNA-seq data, a significantly altered IR-related gene (IRG), ribonucleotide reductase regulatory subunit M2 (RRM2) was identified. Using real-time quantitative fluorescence PCR (qF-PCR), we found that RRM2 was elevated in both IR CML patients and an IR cell line. Using reverse-transcription PCR (RT-PCR) and western blot analysis, we indicated that imatinib can increase RRM2 level in a dose-dependent manner in IR cells. We also demonstrated that RRM2 is involved in the Bcl-2/caspase cell apoptotic pathway and in the Akt cell signaling pathway, and therefore affects the cell survival following imatinib therapy. The present study, for the first time, indicates that RRM2 is responsible for drug resistance in imatinib-based therapy. Therefore, RRM2 gene can be considered as a potential therapeutic target in the clinical treatment of CML.

PB1920 BCR‐ABL GENE ABL KINASE DOMAIN MUTATIONS IN IMATINIB RESISTANT CHRONIC MYELOID LEUKEMIA PATIENTS FROM AZERBAIJAN

Background:While chronic myeloid leukemia (CML) can successfully be treated with tyrosine kinase inhibitors, point mutations within ABL kinase domain of the BCR‐ABL gene are the most common cause of resistance to Imatinib Mesylate. More than 100 BCR‐ABL1 kinase domain point mutations with various frequencies of incidence, domain positions and implications on tyrosine kinase inhibitors (TKI) response in CML are associated with TKI resistance.Aims:To assess the frequency and type of ABL kinase domain mutations in Imatinib resistant CML patients.Methods:115 Imatinib resistant CML patients having been observed during the period from 2014 to 2018. 90 patients at the time of diagnosis were in the chronic phase (78.3%), 19 patients in the acceleration phase (16.5%) and 6 patients in the blast crisis phase (5.2%). By risk groups by Socal et al., the patients were distributed as: low risk ‐ 35 patients; the average risk is 67 patients; high risk ‐ 13 patients. Total RNA was isolated from whole blood and reverse‐transcribed. The resulting cDNA was subjected to an initial round of RT‐PCR across the BCR‐ABL1 breakpoint followed by a second round to amplify the sequence. The final PCR product was pyrosequenced. A total of 14 mutations were identified. Statistical analysis was performed using SPSS 25.0 (IBM, USA) with Chi‐Square. Critical p‐value was set to 0.05.Results:115 Imatinib resistant CML patients were analyzed, 44.3% men (n = 51) and 55.7% women (n = 64), average age – 45.3 ± 14.8 (from 16 to 80). BCR‐ABL1 mutations were found in 16.5% (19/115) CML patients. 7 different mutation variants were identified in 19 patients (Y253H – 2, E255K – 1, F317L (TTG) – 2, F317L (TTA) – 1, T315I – 7, F359 V – 3, F359C ‐ 3). Mutations Y253F, E255 V, V299L(CTG), V299L(TTG), F317 V, F317L(CTC), T315A didn’t identified. Mutation associated resistance rate was higher in men than in women (24.5% against 7.8%, p = 0,025). Also these mutations were analyzed between the group of patients aged ≥50 and &lt;50. The results were 20.45 % and 14.08 %, respectively, p = 0.371. In men and women, T315I mutation prevailed, which occurred in 9.8% and 3.1% of cases, respectively, p = 0.136. In addition, the mutations identified in men were Y253H ‐ 2 (3.92%), F317L (TTG) ‐ 2 (3.92%), F359 V ‐ 2 (3.92%), F359C ‐ 2 (3.92%), E255K ‐ 1 (1.96%), and in women, F317L (TTG) ‐ 1 (1.56%), F359 V ‐ 1 (1.56%), F359C ‐ 1 (1.56%). Studying the frequencies of mutations in the different phases of the disease allowed us to establish that mutations were detected in 10 patients in the chronic phase, which accounted for 11.1% of the total number of patients in this phase, and they were detected in 5 patients in the acceleration phase (26.3%), and in 4 patients in the blast crisis phase (66.7%), p = 0.0008. Studying the distribution of mutations by risk groups showed that mutations were detected in 2 patients in the high‐risk group, which accounted for 15.4% of the total number of patients in this group, in 12 patients in the medium‐risk group, (17.9%), and in 5 patients in the low‐risk group (14.3%), p = 0.890.Summary/Conclusion:Since different mutations of the kinase domain of BCR‐ABL1 are associated with different types of resistance to TKI, identifying trends in the distribution of mutations in CML patients receiving TKI treatment is very important for choosing treatment strategy and early prediction of therapy resistance.

HDAC1,2 Knock-Out and HDACi Induced Cell Apoptosis in Imatinib-Resistant K562 Cells.

Since imatinib (Glivec or Gleevec) has been used to target the BCR-ABL fusion protein, chronic myeloid leukemia (CML) has become a manageable chronic disease with long-term survival. However, 15%–20% of CML patients ultimately develop resistance to imatinib and then progress to an accelerated phase and eventually to a blast crisis, limiting treatment options and resulting in a poor survival rate. Thus, we investigated whether histone deacetylase inhibitors (HDACis) could be used as a potential anticancer therapy for imatinib-resistant CML (IR-CML) patients. By applying a noninvasive apoptosis detection sensor (NIADS), we found that panobinostat significantly enhanced cell apoptosis in K562 cells. A further investigation showed that panobinostat induced apoptosis in both K562 and imatinib-resistant K562 (IR-K562) cells mainly via H3 and H4 histone acetylation, whereas panobinostat targeted cancer stem cells (CSCs) in IR-K562 cells. Using CRISPR/Cas9 genomic editing, we found that HDAC1 and HDAC2 knockout cells significantly induced cell apoptosis, indicating that the regulation of HDAC1 and HDAC2 is extremely important in maintaining K562 cell survival. All information in this study indicates that regulating HDAC activity provides therapeutic benefits against CML and IR-CML in the clinic.

Discovery of the Oncogenic Parp1, a Target of bcr-abl and a Potential Therapeutic, in mir-181a/PPFIA1 Signaling Pathway

miR-181a is downregulated in leukemia and affects its progression, drug resistance, and prognosis. However, the exact mechanism of its targets in leukemia, particularly in chronic myelogenous leukemia (CML), has not previously been established. Here, we use a multi-omics approach to demonstrate that protein tyrosine phosphatase, receptor type, f polypeptide, leukocyte common antigen (LAR) interacting protein (liprin), alpha 1 (PPFIA1) is a direct target for miR-181a in CML. Phospho-array assay shows that multiple phosphorylated proteins, particularly KIT signaling molecules, were downregulated in PPFIA1 inhibition. Additionally, PPFIA1 bound PARP1, a common molecule downstream of both PPFIA1 and BCR/ABL, to upregulate KIT protein through activation of nuclear factor kappa B (NF-κB)-P65 expression. Targeted inhibition of PPFIA1 and PARP1 downregulated c-KIT level, inhibited CML cell growth, and prolonged mouse survival. Overall, we report a critical regulatory miR-181a/PPFIA1/PARP1/NF-κB-P65/KIT axis in CML, and our preclinical study supports that targeted PPFIA1 and PARP1 may serve as a potential CML therapy.

Novel AF1q/MLLT11 favorably affects imatinib resistance and cell survival in chronic myeloid leukemia

Tyrosine kinase inhibitor treatment of chronic myeloid leukemia (CML) has demonstrated beneficial effects. However, resistance to tyrosine kinase inhibitors and disease relapse are still a challenge for CML therapy. In this study, we analyzed bone marrow samples from 149 CML patients and 15 control donors, and investigated the affect of AF1q on CML cell survival and engraftment in vitro and in vivo. We found that AF1q/MLLT11 expression was significantly upregulated in CML patients, especially in CD34+ CML cells. Elevated AF1q expression was associated with disease progression. Knockdown of AF1q enhanced imatinib sensitivity, induced apoptosis, and suppressed growth in CML cells. Moreover, AF1q deficiency sensitized CD34+ CML cells to imatinib. In contrast, upregulation of AF1q promoted cell survival, protected CML cells from imatinib-induced apoptosis, and increased engraftment of CML cells in vivo. We further identified a positive correlation between AF1q and CD44 expression in chronic phase CML patients and CD34+ CML cells. Importantly, AF1q contributes to imatinib-resistance in CML by regulating the expression of CD44. These findings reveal a novel BCR-ABL-independent pathway, AF1q/CD44, involves imatinib resistance in CML, thus representing a potential therapeutic target for imatinib-resistant CML patients.

Induction of apoptosis in imatinib sensitive and resistant chronic myeloid leukemia cells by efficient disruption of bcr-abl oncogene with zinc finger nucleases

BackgroundThe bcr-abl fusion gene is the pathological origin of chronic myeloid leukemia (CML) and plays a critical role in the resistance of imatinib. Thus, bcr-abl disruption-based novel therapeutic strategy may warrant exploration. In our study, we were surprised to find that the characteristics of bcr-abl sequences met the design requirements of zinc finger nucleases (ZFNs).MethodsWe constructed the ZFNs targeting bcr-abl with high specificity through simple modular assembly approach. Western blotting was conducted to detect the expression of BCR-ABL and phosphorylation of its downstream STAT5, ERK and CRKL in CML cells. CCK8 assay, colony-forming assay and flow cytometry (FCM) were used to evaluate the effect of the ZFNs on the viablity and apoptosis of CML cells and CML CD34+ cells. Moreover, mice model was used to determine the ability of ZFNs in disrupting the leukemogenesis of bcr-abl in vivo.ResultsThe ZFNs skillfully mediated 8-base NotI enzyme cutting site addition in bcr-abl gene of imatinib sensitive and resistant CML cells by homology-directed repair (HDR), which led to a stop codon and terminated the translation of BCR-ABL protein. As expected, the disruption of bcr-abl gene induced cell apoptosis and inhibited cell proliferation. Notably, we obtained similar result in CD34+ cells from CML patients. Moreover, the ZFNs significantly reduced the oncogenicity of CML cells in mice.ConclusionThese results reveal that the bcr-abl gene disruption based on ZFNs may provide a treatment choice for imatinib resistant or intolerant CML patients.

Cytogenetic and molecular genetic studies of number of chronic mylogenous leukemia in Erbil province

Chronic myeloid leukemia (CML) is amyeloprolirative disorder characterized by the Philadelphia chromosome originates from the translocation of chromosome 9 and 22 t (9;22) (q34;q11), creating a BCR-ABL fusion gene which results in the constitutive activation BCR-ABL tyrosine kinase. Patients of BCR-ABL fusion gene positive in (CML) are treated by an effective therapy called Imatinib (Gleevec). Point mutations alter the conformation of the ATP binding site that disturbs the binding of therapy to its target which leading to imatinib resistance. Fifty CML patients were collected from Nanakali Hospital in Erbil city which were diagnosed by physicians. The age of the patients from 9 to 80 years, 62% were male and 39% were female, median age of the patients were 38 years. The present study deals with two different aspects; conventional cytogenetic (G-banding) analysis to diagnose the Philadelphia chromosome as a (CML) marker and using Allele Specific Oligonucleotide Polymerase Chain Reaction (ASO-PCR) assay for screening the mutant allele at the three codons 351, 311 and 315 of the BCR-ABL ATP binding domain in Imatinib resistant CML patients. The phenotype of fifteen CML patients were successfully analyzed, thirteen patients were in complete cytogenetic response state (Philadelphia 0%) only two of those patients showed the Philadelphia chromosome. Three point mutations T1052C, T932C and C944T were identified by allele specific oligonucleotide polymerase chain reaction (ASO-PCR). One patient showed T1052C mutation, T932C was detected in three patients and two patients showed the C944T ATP binding domain mutation. In conclusion, conventional cytogenetic and molecular genetic tests are complementary techniques to show the exact types of abnormalities, which allow better evaluation of the genomic aberration involved in CML patients, specifically for kinase domain mutation that plays an important role in different diagnosis, prognosis, therapy treatment and drug resistant management of chronic myeloid leukemia.

MicroRNAs that affect the Fanconi Anemia/BRCA pathway are downregulated in imatinib-resistant chronic myeloid leukemia patients without detectable BCR-ABL kinase domain mutations

Chronic myeloid leukemia (CML) patients who do not achieve landmark responses following treatment with imatinib mesylate (IM) are considered IM-resistant. Although IM-resistance can be due to BCR-ABL kinase domain (KD) mutations, many IM-resistant patients do not have detectable BCR-ABL KD mutations. MicroRNAs (miRNAs) are short non-coding RNAs that control gene expression. To investigate the role of miRNAs in IM-resistance, we recruited 8 chronic phase CML patients with IM-resistance who tested negative for BCR-ABL KD mutations and 2 healthy normal controls. Using miRNA sequencing, we identified 54 differentially expressed miRNAs; 43 of them downregulated. The 3 most differentially downregulated miRNAs were miR-146a-5p, miR-99b-5p and miR-151a-5p. Using real-time quantitative reverse transcriptase-polymerase chain reaction, the expression patterns of the 3 miRNAs were validated on the same cohort of 8 patients in addition to 3 other IM-resistant CML patients. In-silico analysis showed that the predicted gene targets are ATRIP, ATR, WDR48, RAD51C and FANCA genes which are involved in the Fanconi Anemia/BRCA pathway. This pathway regulates DNA damage response (DDR) and influences disease response to chemotherapy. Thus it is conceivable that DDR constitutes a key component in IM-resistance. Further research is needed to elucidate miRNA modulation of the predicted gene targets.

Inhibition of the NAMPT-Triggered Deacetylation of HCLS1 Protein: A New Therapeutic Option in Chronic Myeloid Leukemia

G-CSFR-triggered phosphorylation and activation of the hematopoietic-specific lyn-substrate 1 (HCLS1) protein is important for the granulocytic differentiation of hematopoietic stem cells (HSCs) (Skokowa J. et al., Nat Med 2012). G-CSF also activates protein deacetylation via Nicotinamide phosphoribosyltransferase (NAMPT)/NAD+/sirtuin 1 (SIRT1) pathway (Skokowa J. et al., Nat Med 2009). SIRT1 is NAD+-dependent protein deacetylase playing an important role in the regulation of gene transcription. Interestingly, having strong pro-differentiation effects on myeloid progenitors, both HCLS1 and NAMPT are markedly hyper-activated in myeloid leukemias, inducing proliferation and survival of undifferentiated blasts.Knowing this, we investigated whether HCLS1 could be activated by NAMPT/NAD+/SIRT1 pathway via deacetylation in HSCs. Indeed, we found that HCLS1 is deacetylated in HSCs by NAMPT/SIRT1 and that G-CSF treatment of these cells induced deacetylation and phosphorylation of HCLS1 via NAMPT/SIRT1. Moreover, we found that deacetylation of HCLS1 is important for myeloid differentiation and proliferation of HSCs.We further demonstrated that HCLS1 protein was activated by deacetylation in blasts of chronic myeloid leukemia (CML) patients, as compared to HSCs of healthy individuals. This was in line with elevated mRNA and protein levels of NAMPT and HCLS1. We further investigated, whether inhibition of NAMPT-triggered deacetylation of HCLS1 has any effect on the survivial and differentiation of CML blasts in vitro. We found that treatment of CML blasts with the specific NAMPT inhibitor FK866 resulted in increased acetylation of HCLS1. In addition, treatment of CML blasts with the FK866 in a dose of 10nM resulted in marked inhibition of proliferation and elevated cell death, which was consistent between different patients and was comparable to the effects of Imatinib treatment. In CFU assay in the presence of FK866 (10nM) no CFU colonies were grown. Even at a dose of 3nM FK866 drastically suppressed total colony counts. This inhibitory effect was further amplified by combination of FK866 with Imatinib. Addition of 10nM of FK866 to CFU culture medium was not toxic for CD34+ cells of healthy donors.Taken together, we demonstrated that NAMPT and HCLS1 have dose-dependent effects on hematopoietic cells and that NAMPT activates HCLS1 functions by SIRT1-triggered deacetylation. Inhibition of NAMPT in CML blasts markedly reduced their survival, proliferation and differentiation. In conclusion, we suggest a possible therapeutic application of NAMPT inhibition in the treatment of CML patients via deactivation of HCLS1 function by acetylation. This therapy could be applied in combination with Imatinib, or in Imatinib-resistant CML patients. DisclosuresNo relevant conflicts of interest to declare.

Characteristics of the TCR Vβ repertoire in imatinib-resistant chronic myeloid leukemia patients with ABL mutations.

Diversity in the T cell receptor (TCR) repertoire provides a miniature defense ability for the T cell immune system that may be related to tumor initiation and progression. Understanding the T cell immune status of leukemia patients is critical for establishing specific immunotherapies. Previous studies have reported abnormal TCR repertoires and clonally expanded TCR Vβ T cells in chronic myeloid leukemia in chronic phase (CP-CML). In this study, we investigated the distribution and clonality of the TCR Vβ repertoire in 4 cases with imatinib-resistant CML in blast crisis (BC-CML) with abelson murine leukemia viral oncogene homolog 1 (ABL1) kinase domain mutations (KDMs). Examination of TCR V expression and clonality was performed by reverse transcription-polymerase chain reaction (RT-PCR) and GeneScan analysis. Significantly skewed TCR Vβ repertoires were observed in BC-CML patients with different KDMs, and 4 to 8 oligoclonally expanded TCR Vβ subfamilies could be identified in each sample. Intriguingly, a relatively highly expanded Vβ9 clone with the same length as complementarity- determining region 3 (CDR3) (139 bp) was found in all three CML patients in lymphoid blast crisis (LBC-CML) who had different KDMs, but the clone was not detected in the only CML patient in myeloid blast crisis (MBC-CML). In conclusion, restricted TCR Vβ repertoire expression and decreased clone complexity was a general phenomenon observed in the BC-CML patients with different KDMs, indicating the T-cell immunodeficiency of these patients. In addition, clonally expanded Vβ9 T cell clones may indicate a specific immune response to leukemia-associated antigens in LBC-CML patients.

Response and Adverse Effects of Nilotinib in Imatinib-resistant Chronic Myeloid Leukemia Patients: Data From a Developing Country

PurposeThe purpose of this study was to determine the frequency of major cytogenetic response (MCyR) and adverse events with nilotinib in adults with imatinib-resistant Philadelphia chromosome–positive chronic myeloid leukemia (CML). MethodsThis is a descriptive cross-sectional study conducted at The Aga Khan University Hospital in Karachi from October 17, 2010, to October 17, 2011. A cytogenetic assessment using fluorescent in situ hybridization was performed on peripheral blood before initiation of treatment and 6 months after treatment with nilotinib. The frequency of adverse effects was assessed at 6 months, and the patient overall survival was calculated after 3 years. FindingsThis study enrolled 82 imatinib-resistant patients. The mean (SD) patient age was 38.9 (12.2) years. There were 62 patients (75.6%) in chronic phase, 15 patients (18.3%) in accelerated phase, and 5 patients (6%) in blast crisis phase. At 6 months cytogenetic studies were available for 40 (52%) of 77 patients, and MCyR was observed in 31 (77.5%) of 77 patients. The patients in chronic phase had the highest frequency of MCyR (n = 27 [87%] of 31). We observed 6 deaths (7.3%), and the overall survival at 3 years was 92.7%. There was isolated thrombocytopenia in 12 patients (15.6%). The most frequent nonhematologic adverse events were myalgia and headache. ImplicationsThe nilotinib response rates were higher in chronic phase patients, and the most common adverse events were thrombocytopenia, myalgia, and headache.

DJ-1 Plays an Important Role in Homoharringtonine-Induced Apoptosis of Imatinib-Resistant Chronic Myeloid Leukemia Cells

Background and Objective: Although the treatment of chronic myeloid leukemia (CML) has improved since the introduction of tyrosine kinase inhibitors (TKI), cases of resistance have been reported and resulted in challenges to the treatment. Recent studies have suggested that Homoharringtonine (HHT), a cephalotaxine ester, has demonstrated a clinical activity in imatinib-resistant CML patients, however, the molecular mechanisms underlying this phenomenon are unknown. Our previous study found that treatment with HHT significantly increased apoptosis of K562 cells. Moreover, the protein DJ-1, identified by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry, was demonstrated to decrease after HHT treatment. Therefore, we performed the experiment to address the hypothesis that DJ-1 might play an important role in Homoharringtonine-induced apoptosis of Imatinib-resistant chronic myeloid leukemia cellsMethods and Results: To find the pivotal protein by HHT, Imatinib-resistant K562 cells were treated with HHT (10 ug/ml) for 5 h, 12 h, 24 h respectively and the control group without HHT were harvested to assess apoptosis with Annexin V-FITC and propidium iodide per the manufacturer’s protocol and analyzed by flow cytometry. The data indicated a time dependent induction of apoptosis by HHT, with the number of apoptotic cells (FITC-Annexin-V and PI double-positive cells) significantly increasing from 2.2± 1.5 % in control to 35.9 ± 6.7% in cells treated with HHT for 24 h (P<0.01). The protein DJ-1 expression change upon HHT treatment which were analyzed with western blot, found that the protein level of DJ-1 had significantly decreased after the treatment of HHT for 24 h.Furthermore, primary cells from six CML patients and three healthy donors were obtained with informed consent and divided into three groups: the CML-CP group (three newly diagnosed patients in the chronic phase), the imatinib-resistant CML group(three imatinib-resistant patients in the blastic phase) and the control group. Mononuclear cells were all cultured in vitro in the absence and presence of 10 mg/ml HHT for 5 h and 24 h. The results showed that DJ-1 expression in primary leukemia cells (both CML-CP group and imatinib-resistant CML group) were found to be decreased after HHT treatment and the expression level of DJ-1 seemed lower in the healthy donor as compared to primary CML cells, moreover, the protein changes induced by HHT were significantly different among three groups and the protein changes were not as significant in CML-CP cells as in imatinib-resistant CML cells (P<0.05).Conclusions: These findings indicated that DJ-1 might play an important role in Homoharringtonine-induced apoptosis of Imatinib-resistant chronic myeloid leukemia cells. Further study may help to assess a promising potential of this protein to be used as a target for a molecular therapy. DisclosuresNo relevant conflicts of interest to declare.

Glutathionylation of NF-Kb and Procaspase-3 Regulates Inducible Nitric Oxide Synthase Expression and Apoptosis of Chronic Myeloid Leukemia Cells

Chronic myeloid leukemia (CML), a myeloproliferative disorder, characterized by sustained neutrophilia and constitutive BCR-ABL tyrosine kinase activity. Constitutive expression of the BCR-ABL kinase and elevated reactive oxygen species (ROS) levels through mitochondria and NADPH oxidase 4 (NOX4) activation leads to genomic instability and enhanced cell survival. Nitric oxide (NO), a signaling molecule has been associated with hematopoesis and suppression of NOS activity may induce profound changes in hematopoietic stem cells/progenitor cells. NO addition or iNOS transfection in K562 cells (BCR-ABL+) altered genes expression involved in the iron metabolism, inhibited cell proliferation and enhanced apoptosis, which were reversed by addition of exogenous iron. Moreover, anti-cancer effect of farnesyltransferase inhibitor in these cells was also mediated by NO production/iNOS induction. The present study investigates status and regulation of NO/iNOS in neutrophils from CML patients.The present study was undertaken to explore NO generation/iNOS expression and its regulation in circulating neutrophils so as to access the role NO/iNOS in CML pathology.All CML patients (Drug/treatment naïve, n=70; imatinib responders, n=62; imatinib resistant, n=25) included in this study were diagnosed in chronic phase. The study protocol was approved by the ethical committees of CSIR-CDRI and KGMU, Lucknow and was conducted in accordance with the declaration of Helsinki. Total nitrite level in neutrophils (PMNs) was assessed by Griess reagent. Nitric oxide, Superoxide, ROS/RNS and mitochondrial ROS generation was assessed by DAF-2DA, DHE, DCF-DA and Mitosox Red respectively. H2O2was measured by Amplex red assay kit. Expression of iNOS gene was evaluated by a SYBR green real-time RT-PCR and Western blot. Binding of NF-kB (p50 and p65 subunit) to iNOS promoter was analysed by CHIP assay. NF-kB (p50 and p65 subunit) and procaspase-3 glutathionylation was assessed by Immunoprecipitation followed by Western blot. Findings in CML patients were further validated using in vitro experiments on K562 cells. Statistical analysis were performed by one way ANOVA test followed by Newman-Keul’s post hoc analysis using the Graph Pad prism software.PMNs total nitrite, NO level and iNOS expression in drug naïve and imatinib resistant patients were significantly less as compared to healthy subjects. However, significant recovery in all the parameters was observed in imatinib responsive patients. Superoxide, ROS/RNS, mitochondrial ROS generation as well as H2O2 level was significantly more in drug naïve and imatinib resistant patients and it was attenuated significantly in imatinib responsive patient’s PMNs. In vitro treatment of K562 cells with Imatinib (2µM) also showed augmented NO generation and iNOS expression, while superoxide, ROS/RNS and mitochondrial ROS generation was decreased. To decipher the molecular mechanisms underlying the modulation of iNOS in BCR-ABL+ cells, we examined binding of NF-κB to iNOS promoter/enhancer and protein S-glutathionylation. Binding of NF-κB (p50 and p65 subunits) to iNOS promoter/enhancer was less in BCR-ABL positive cells, while it was augmented following treatment with imatinib. Moreover, glutathionylation of p50, p65 and procaspase-3 was more in drug naïve as well as in imatinib resistant CML patients PMNs, while it was comparable to healthy subjects in imatinib responders CML patients PMNs. Glutathionylation of NF-κB (p50 and p65 subunit) and procaspase-3 was also attenuated in imatinib treated K562 cells.The results obtained suggest that reduced NO generation/iNOS expression in BCR-ABL positive cells was due to the S-glutathionylation of NF-κB, which decrease it’s binding to iNOS promoter. S-glutathionylation of procaspase-3 in CML however, inhibited apoptosis of BCR-ABL positive cells. The study thus highlights importance of S-glutathionylation as key regulators involved in the proliferation and apoptosis of BCR-ABL positive cells. DisclosuresNo relevant conflicts of interest to declare.

Methylation analysis of the DAPK1 gene in imatinib-resistant chronic myeloid leukemia patients.

Death-associated protein kinase-1 (DAPK1) is a pro-apoptotic gene that induces cellular apoptosis in response to internal and external apoptotic stimulants. The silencing of DAPK1 can result in uncontrolled cell proliferation, indicating that it may have a role in tumor suppression. DAPK1 activity can be inhibited by the cytosine methylation that occurs in its promoter region. These methylation changes in the promoter region of DAPK1 have been reported in a range of solid and hematological malignancies. In the present study, DAPK1 methylation was investigated in chronic myeloid leukemia patients (n=43) using bisulfite conversion followed by methylation-specific polymerase chain reaction. The present study included a number of patients who were identified to be resistant to the common chemotherapeutic agent imatinib (STI571, Gleevec®, Glivec®), exhibiting at least one mutation in the breakpoint cluster region-Abelson murine leukemia (BCR-ABL) gene. Thus, the patients in the present study were divided into two groups according to their response to imatinib therapy: Non-resistant (n=26) and resistant (n=17) to imatinib. Resistant patients were characterized by the presence of single or multiple mutations of the BCR-ABL gene: i) T315I, ii) M351T, iii) E255K, iv) T315I and M351T or v) T315I, M351T and E255K. The present study identified that: i) The incidence of DAPK1 methylation was significantly higher in the resistant patients compared with the non-resistant patients; ii) the extent of resistance varied between mutation types; and iii) there was no DAPK1 methylation in any of the healthy controls. These findings indicate that DAPK1 methylation may be associated with a signaling pathway for imatinib resistance in chronic myeloid leukemia.