Gene In Chronic Myeloid Leukemia Research Articles

Gene Variants in Components of the microRNA Processing Pathway in Chronic Myeloid Leukemia.

Current therapy in chronic myeloid leukemia (CML) has improved patient life expectancy close to that of healthy individuals. However, molecular alterations other than BCR::ABL1 fusion gene in CML are barely known. MicroRNAs are important regulators of gene expression, and variants in some of the components of microRNA biosynthesis pathways have been associated with genetic susceptibility to different types of cancer. Thus, the aim of this study was to evaluate the association of variants located in genes involved in the biogenesis of microRNAs with susceptibility to CML. Fifteen variants in eight genes involved in the biogenesis of miRNAs were genotyped in 296 individuals with CML and 485 healthy participants using TaqMan probes. The association of gene variants with CML and clinical variables was evaluated by a Chi-square test, and odds ratios and 95% confidence intervals were estimated by logistic regression. The variant rs13078 in DICER1 was significantly higher among CML individuals than in healthy participants. In addition, the variants rs7813 and rs2740349 were significantly associated with worse prognosis, according to their Hasford scores, whereas the rs2740349 variant was also associated with a later age at diagnosis. These findings suggest that variants in components of the microRNA biogenesis pathway could be involved in CML genetic risk.

Mutations in Genes Producing Nitric Oxide and Hydrogen Sulfide and Their Connection With Apoptotic Genes in Chronic Myeloid Leukemia.

Background Despite advances in chronic myeloid leukemia (CML) genetics, the role of nitric oxide (NO) and hydrogen sulfide (H2S) gene mutations and their relationship to apoptotic genes is unclear. Therefore, this study investigated NO- and H2S-producing genes' mutations and their interactions with apoptotic genes using Sanger sequencing and next-generation sequencing (NGS). Methodology A complete blood count (CBC) was carried out to measure the total number of white blood cells, while IL-6 levels were assessed in both control and CML patients using an ELISA technique.Sanger sequencing was used to analyzemutations in the CTH and NOS3 genes, whereas NGS was applied to examine mutations on all chromosomes. Results White blood cell (WBC) and granulocyte counts were significantly higher in CML patients compared to controls (p<0.0001), and monocyte counts were similarly higher (p<0.05). Interleukin-6 (IL-6) levels were significantly elevated in CML patients thancontrols (p<0.0001), indicating a possible link to CML etiology or progression. Multiple mutations have been identified in both genes, notably in CTH exon 12 and the NOS3genes VNTR, T786C, and G894T. This study also measured IL-6 concentrations using IL-6 assays, identifying its potential as a CML prognostic diagnostic. WBC counts, granulocyte counts, and mid-range absolute counts, or MID counts, were significantly higher in CML patients than in normal control individuals. NGS identified 1643 somatic and sex chromosomal abnormalities and 439 actively expressed genes in CML patients. The findings imply a genomic landscape beyond the BCR-ABL1 mutation in CML development compared to other databases. Conclusion In conclusion, this study advances the understanding of the genetic characteristics of CML by identifying mutations in the NO- and H2S-producing genes and their complex connections with genes involved in apoptosis. The comprehensive genetic profile obtained by Sanger sequencing and NGS provides possibilities for identifying novel targets for therapy and personalized treatments for CML, therefore contributing to developments in hematological diseases.

E14a2 Transcript Favors Treatment-Free Remission in Chronic Myeloid Leukemia When Associated with Longer Treatment with Tyrosine Kinase Inhibitors and Sustained Deep Molecular Response

e13a2 and e14a2 are the most frequent transcript types of the BCR::ABL1 fusion gene in chronic myeloid leukemia (CML). The current goal with tyrosine kinase inhibitors (TKI) is to achieve sustained deep molecular response (DMR) in order to discontinue TKI treatment and remain in the so-called treatment-free remission (TFR) phase, but biological factors associated with these goals are not well established. This study aimed to determine the effect of transcript type on TFR in patients receiving frontline treatment with imatinib (IM) or second-generation TKI (2G-TKI). Patients treated at least 119 months with IM presented less post-discontinuation relapse than those that discontinued IM before 119 months (p = 0.005). In addition, cases with the e14a2 transcript type treated at least 119 months with IM presented a better TFR (p = 0.024). On the other hand, the type of transcript did not affect the cytogenetic or molecular response in 2G-TKI treated patients; however, the use of 2G-TKI may be associated with higher and earlier DMR in patients with the e14a2 transcript.

Clinical applications of abnormal DNA methylation in chronic myeloid leukemia.

DNA methylation, a crucial biochemical process within the human body, fundamentally alters gene expression without modifying the DNA sequence, resulting in stable changes. The changes in DNA methylation are closely related to numerous biological processes including cellular proliferation and differentiation, embryonic development, and the occurrence of immune diseases and tumor. Specifically, abnormal DNA methylation plays a crucial role in the formation, progression, and prognosis of chronic myeloid leukemia (CML). Moreover, DNA methylation offers substantial potential for diagnosing and treating CML. Accordingly, understanding the precise mechanism of DNA methylation, particularly abnormal changes in the methylation of specific genes in CML, can potentially promote the development of novel targeted therapeutic strategies. Such strategies could transform into clinical practice, effectively aiding diagnosis and treatment of CML patients.

The Role of BCR-ABL Gene on Chronic Myeloid Leukemia: An Overview

The Breakpoint Cluster Region is a protein encoded by the BCR gene with its partially discovered function to generate instructions for producing a protein and also to encode serine/threonine kinase protein. There are three Breakpoint cluster regions; major (M-bcr) which is high in CML patients, minor (m-bcr) with a 1-2% contribution in the CML, and micro (μ-bcr) which is very rare in the case of CML. The ABL1 encodes for the ABL1 protein which can function as a tyrosine kinase. The translocation of the ABL gene on chromosome 9 at the band 34 to the BCR gene on chromosome 22 at band 11 leads to the fusion of the BCR-ABL gene which is responsible for Chronic Myeloid Leukemia (CML), Acute Myeloid Leukemia (AML), and Acute Lymphoblastic Leukemia (ALL). Chromosome 22 with this hybrid gene called a Philadelphia chromosome codes for a BCR-ABL protein that enhances the activity of tyrosine kinase thereby activating numerous signaling pathways. If there is a translocation of BCR to chromosome 9, which will cause the emergence of the ABL-BCR gene (9q+). [1][4][5][6] The role of the BCR-ABL gene in CML is to increase the growth and proliferation of myeloid cells, avoid apoptosis, increase c-independent growth, induce aberrations in the cytoskeletons, etc.

Differences In Hematology And Bcr-Abl Molecular Profiles In Patients With Chronic Myeloid Leukemia Undergoing Tyrosine Kinase Inhibitor Therapy

Chronic myeloid leukemia (CML) is a myeloproliferative malignancy due to the formation of the BCR-ABL fusion gene in chronic myeloid leukemia. This condition causes excessive cell proliferation, resulting in an increase in the number of leukocytes. Tyrosine Kinase Inhibitor (TKI) is a first-line therapy that helps reduce the percentage of the BCR-ABL fusion gene in patients with chronic myeloid leukemia. This study was conducted to determine differences in the hematological profiles (hemoglobin levels, leukocyte counts, platelet counts) and molecular BCR-ABL in patients with chronic myeloid leukemia before and after 12 months of tyrosine kinase inhibitors therapy. This analytic observational study was administered using a cross-sectional design to in analyzing the medical records of CML patients who underwent TKI therapy at the Sub Specialist Polyclinic of Internal Medicine Hematology Oncology Ulin Banjarmasin Regional Hospital from March 2021-April 2022. Dependent T-test and McNemar's test were performed in data analysis which results showed that 12 month tyrosine kinase inhibitor therapy could increase the hemoglobin levels, decrease leukocyte counts, platelet counts as well as decreasing the percentage of BCR-ABL gene fusion in patients with chronic myeloid leukemia. In conclusion, significant differences were found in the hematological profiles (p&lt;0.001) and the molecular BCR-ABL (p&lt;0.001) before and after 12 months of tyrosine kinase inhibitor therapy

Three-dimensional nuclear telomere architecture and differential expression of aurora kinase genes in chronic myeloid leukemia to measure cell transformation

BackgroundTelomere dysfunction results in aneuploidy, and ongoing chromosomal abnormalities. The three-dimensional (3D) nuclear organization of telomeres allows for a distinction between normal and tumor cells. On the other hand, aurora kinase genes (AURKA and AURKB) play an important role regulating the cell cycle. A correlation between overexpression of aurora kinase genes and clinical aggressiveness has been demonstrated in different types of neoplasias. To better understand cellular and molecular mechanisms of CML evolution, it was examined telomere dysfunction (alterations in the 3D nuclear telomere architecture), and the expression levels of AURKA and AURKB genes in two clinical distinct subgroups of CML samples, from the same patient.MethodsEighteen CML patients, in total, 36 bone marrow samples (18 patients, chronic vs. accelerated/blast phase) were eligible for 3D telomeric investigations. Quantitative 3D imaging, cytologic diagnosis and cytogenetic determination of additional chromosomal abnormalities were assessed according to standard protocols.ResultsUsing TeloView software, two CML subgroups were defined based on their 3D telomeric profiles, reflecting the different stages of the disease (chronic vs. accelerated/blast phase). Statistical analyses showed significant differences between the CML subgroups (p < 0.001). We also found that AURKA and AURKB mRNA were expressed at significantly higher levels in both CML subgroups, when compared with healthy donors. Our findings suggest that the evolution of CML progresses from a low to a high level of telomere dysfunction, that is, from an early stage to a more aggressive stage, followed by disease transformation, as demonstrated by telomere, additional chromosomal abnormalities, and gene expression profile dynamics.ConclusionsThus, we demonstrated that 3D telomere organization, in accordance with the genomic instability observed in CML samples were able to distinguish subgroup CML patients. Classifying CML patients based on these characteristics might represent an important strategy to define better therapeutic strategies.

Overview of Molecular Quantification of the BCR-ABL Oncogene in CML Patients

Chronic myeloid leukemia (CML) is considered a common blood cancers and accounts for approximately 15–20% of the total cases of leukemia. Recent studies indicated that above 95% of patients suffering of CML have been found with a distinctive Philadelphia chromosome that originates from a mutual translocation between both arms of chromosomes 9 and 22. During this mutation the translocation of the ABL gene located on chromosome 9 get transferred to the breakpoint cluster region (BCR) of chromosome 22 as an effect of a joined BCR-ABL gene. Furthermore, BCR-ABL oncogene is characteristically found in CML, causing cells to divide uncontrollably and inducing severe consequences among CML patients. In line with this, applying quantification technique of the BCR-ABL gene using molecular approaches is crucial for patient controlling, initiation of the proper treatment, measurement of response to therapy, and prediction of relapse. Of greater significance, molecular assay and monitoring of the BCR-ABL gene in CML using quantitative RT-PCR provides physicians with essential diagnostic and prognostic information.

Potential functions of hsa-miR-155-5p and core genes in chronic myeloid leukemia and emerging role in human cancer: A joint bioinformatics analysis

Considering the critical roles of hsa-miR-155-5p participated in hematopoietic system, this study aims to clarify the possible pathogenesis of chronic myeloid leukemia (CML) induced by hsa-miR-155-5p.Three different strategies were employed, namely a network-based pipeline, a survival analysis and genetic screening method, and a simulation modeling approach, to assess the oncogenic role of hsa-miR-155-5p in CML. We identified new potential roles of hsa-miR-155-5p in CML, involving the BCR/ABL-mediated leukemogenesis through MAPK signaling. Several promising targets including E2F2, KRAS and FLI1 were screened as candidate diagnostic marker genes. The survival analysis revealed that mRNA expression of E2F2, KRAS and FLI1 was negatively correlated with hsa-miR-155-5p and these targets were significantly associated with poor overall survival. Furthermore, an overlap between CML-related genes and hsa-miR-155-5p target genes was revealed using competing endogenous RNA (ceRNA) networks analysis. Taken together, our results reveal the dynamic regulatory aspect of hsa-miR-155-5p as potential player in CML pathogenesis.

Expression of Hippo signaling pathway and Aurora kinase genes in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm resulting from clonal expansion of hematopoietic stem cells positive for the Philadelphia chromosome. The CML pathogenesis is associated with expression of the BCR-ABL1 oncogene, which encodes the Bcr-Abl protein with tyrosine kinase activity, promoting the leukemic cell exacerbated myeloproliferation and resistance to apoptosis. CML patients are usually treated with tyrosine kinase inhibitors (TKI), but some of them acquire resistance or are refractory to TKI. Thus, it is still relevant to elucidate the CML pathogenesis and seek new therapeutic targets, such as the Hippo signaling pathway and cell cycle regulatory genes fromtheAurora kinase family. The present study quantified the expression level of genes encoding components of the Hippo signaling pathway (LATS1, LATS2, YAP, and TAZ), AURKA and AURKB in CML patients at different stages of the disease, who were resistant or sensitive to imatinib mesylate therapy, and in healthy individuals. The expression levels of the target genes were correlated with the CML Sokal's prognostic score. The most striking results were the LATS2 and AURKA overexpression in CML patients, the overexpression of TAZ and AURKB in CML patients at advanced phasesand TAZ in CML IM-resistant. The development of drugs and/or identification of tumor markers for the Hippo signaling pathway and the Aurora kinase family, either alone or in combination, can optimize CML treatment by enhancing the susceptibility of leukemic cells to apoptosis and leading to a better disease prognosis.

Variation of LNK Gene in Chronic Myeloid Leukemia

To compare the mutation and single nucleotide polymorphism (SNP) of LNK gene between chronic myeloid leukemia(CML) and control groups, and to explore the relationship between LNK gene variation and the occurrence of CML. A total of 36 patients with CML were selected, 46 healthy persons were used as normal controls. DNA was extracted from bone marrow and peripheral blood, BCR/ABL1 fusion gene was detected by Q-PCR. The whole exon of LNK gene was amplified by PCR. The amplified sequences included the Rs3184504 (C/T) and Rs78894077 (A/C/G/T) affecting the expression of amino acids in LNK gene, and the Rs7973120 (A/T) unaffecting the expression of the amino acids. The mutations and SNP of LNK gene were analyzed by DNA sequencing. Thirty-six cases of CML had BCR/ABL1 mutation, while no mutation was found in the control group. One case of CML had LNK heterozygous mutation (A300V), and the mutation rate was 2.8%, no mutation was seen in normal control group. Rs3184504: C/T allele frequency was 50%/50% in the control group, 94.4%/5.6% in the CML group, and the C allele in CML group was significantly higher than that in the control group; CC genotype accounted for 94.4% (P<0.01). Rs78894077: C/T allele in the control group was 9.8%/90.2%, in CML group was 16.7%/83.3%, the difference was not statistically significant(P>0.05); but CC genotype in CML group was very statistically significant higher than that in control group(P<0.01). Rs7973120: A/T allele frequency was 10.9%/89.1% in the control group, 25%/75% in the CML group, the LNK A allele in CML group was very significantly higher than that in control group (P<0.01). CML patients have been confirmed to have LNK mutation; the SNPs of LNK are related with the development of CML, and the most CML patients carry the LNK Rs3184504 C allele and the Rs7973120 A allele.

Identification of Imatinib-Sensitizing Genes in Chronic Myeloid Leukemia with a Genome-Scale Crispr Knock-out Screen

Background: Resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) can either originate from mutations in the BCR-ABL1 gene, which are mostly well characterized, or emerge from unknown alternative mutations elsewhere in the genome. Small hairpin (sh)RNA screens have been used to discover such genes but are becoming limited due to sup-optimal protein depletion and non-reliable off-target effects. More efficient screening techniques in human cells are now available as a result of the increasing understanding of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) /Cas9 system.Aims: Our goal is to uncover imatinib (IM)-sensitizing genes that cause IM resistance when knocked-out. Characterizing these genes may help understand the mechanisms of IM uptake, metabolism, degradation and/or activity in CML cells. Additionally, we also expect to unveil alternative, BCR-ABL1 independent, oncogenic pathways in CML cells.Methods: In order to find other genes involved in IM resistance, we performed a genome scale CRISPR knock-out (GeCKO) screen, which contains 121,413 sgRNAs that target 20,914 protein coding genes and miRNAs. We transduced one sgRNA per cell and challenged the K562-GeCKO cell pool to IM selection. We compared the abundance of sgRNAs between pre/post-IM treatment by next generation sequencing (NGS).Results: After IM selection, the sgRNAs from surviving cells were identified by NGS and unveiled potential IM-sensitizing genes. The most enriched sgRNAs (FDR < 0.01) targeted genes involved in transcriptional (KLF1, MED24) and translational (EIF2AK1, UBE2M) regulation, apoptosis (BAX, BCL2L11) and cell cycle regulation (BAP1, SPRED2). Subsequent screens on LAMA84 cells are currently underway in order to validate our findings. Additionally, the establishment of individual gene knock-out cell lines are in progress in order to fully understand the role of each gene in IM resistance.Summary/Conclusion: Using a CRISPR knock-out screen, we produced a list of 19 genes (FDR < 0.05) that may play a role in IM resistance. Encouragingly, a subset of these genes (BAX, BAP1, BCL2L11 and SPRED2) have already been correlated to CML progression and/or TKI resistance in the past. We aim to bolster our findings by establishing individual gene KO cell lines and study resistance in LAMA84 cells. The utilization of CRISPR libraries may not only help understand TKI resistance in CML, but also help identify numerous novel genes involved in drug resistances for a myriad of different diseases. DisclosuresMahon:ARIAD: Honoraria; PFIZER: Honoraria; BMS: Consultancy, Honoraria; NOVARTIS PHARMA: Consultancy, Honoraria, Research Funding.

3210 Mutation analysis of TGF-beta-Smad pathway genes in chronic myeloid leukemia by next generation sequencing

3210 Mutation analysis of TGF-beta-Smad pathway genes in chronic myeloid leukemia by next generation sequencing

MicroRNA-320a acts as a tumor suppressor by targeting BCR/ABL oncogene in chronic myeloid leukemia.

Accumulating evidences demonstrated that the induction of epithelial-mesenchymal transition (EMT) and aberrant expression of microRNAs (miRNAs) are associated with tumorigenesis, tumor progression, metastasis and relapse in cancers, including chronic myeloid leukemia (CML). We found that miR-320a expression was reduced in K562 and in CML cancer stem cells. Moreover, we found that miR-320a inhibited K562 cell migration, invasion, proliferation and promoted apoptosis by targeting BCR/ABL oncogene. As an upstream regulator of BCR/ABL, miR-320a directly targets BCR/ABL. The enhanced expression of miR-320a inhibited the phosphorylation of PI3K, AKT and NF-κB; however, the expression of phosphorylated PI3K, AKT and NF-κB were restored by the overexpression of BCR/ABL. In K562, infected with miR-320a or transfected with SiBCR/ABL, the protein levels of fibronectin, vimentin, and N-cadherin were decreased, but the expression of E-cadherin was increased. The expression of mesenchymal markers in miR-320a-expressing cells was restored to normal levels by the restoration of BCR/ABL expression. Generally speaking, miR-320a acts as a novel tumor suppressor gene in CML and miR-320a can decrease migratory, invasive, proliferative and apoptotic behaviors, as well as CML EMT, by attenuating the expression of BCR/ABL oncogene.

A combination of STI571 and BCR-ABL1 siRNA with overexpressed p15INK4B induced enhanced proliferation inhibition and apoptosis in chronic myeloid leukemia

p15INK4B, a cyclin-dependent kinase inhibitor, has been recognized as a tumor suppressor. Loss of or methylation of the p15INK4B gene in chronic myeloid leukemia (CML) cells enhances myeloid progenitor formation from common myeloid progenitors. Therefore, we examined the effects of overexpressed p15INK4B on proliferation and apoptosis of CML cells. Overexpression of p15INK4B inhibited the growth of K562 cells by downregulation of cyclin-dependent kinase 4 (CDK4) and cyclin D1 expression. Overexpression of p15INK4B also induced apoptosis of K562 cells by upregulating Bax expression and downregulating Bcl-2 expression. Overexpression of p15INK4B together with STI571 (imatinib) or BCR-ABL1 small interfering RNA (siRNA) also enhanced growth inhibition and apoptosis induction of K562 cells. The enhanced effect was also mediated by reduction of cyclin D1 and CDK4 and regulation of Bax and Bcl-2. In conclusion, our study may provide new insights into the role of p15INK4B in CML and a potential therapeutic target for overcoming tyrosine kinase inhibitor resistance in CML.

Construction and in vitro study of eukaryotic expression vector carrying GITRL and IL-21 gene

The aim of this study was to construct the eukaryotic expression vector carrying glucocorticoid-induced tumor necrosis factor receptor ligand (GITRL) and interleukin-21 (IL-21) gene for transfection into chronic myeloid leukemia (CML) dervied dendritic cell (DC), so as to provide an effective platform for exploring the function of target gene in CML. The recombinant eukaryotic expression vector was transfected to the purified DC by Liposome-mediated method, the interleukin-2 (IL-2) and Interferon-γ (IFN-γ) expression of transfected DC were analyzed by ELISA. Further, the transfected DC with purified NK were mixed and cultured to be DC-CIK, the lactate dehydrogenase release assay was performed to measure the killing activity of DC-CIK. The results indicated that the sequence of cloned target gene was same as that in GenBank. The size of endonuclease products by restriction enzyme were same as the predict one. The concentration of Interleukin-2 (IL-2) and interferon-γ (IFN-γ) in transfected DC all increased. The NK kill activity became stronger while induced by transfected DC. It is concluded that DC transfected by IL-21 and GITRL gene has the ability of self-activation, up-regulate cytokine secretion. Further, the results would be help to provide the theoretical evidence of advanced immunotherapy for treatment of CML patients who showed no reaction to tyrosine kinase inhibitor.

Methylation status of CEBPA gene promoter in chronic myeloid leukemia

CCAAT/enhancer binding protein alpha is one of the crucial transcription factors for myeloid cell development that has been found to be involved in hematopoietic differentiation and leukemiogenesis. Recently, epigenetic regulation of CEBPA expression through DNA methylation has been demonstrated in leukemia. The aim of this study was to investigate the methylation status of CEBPA gene in chronic myeloid leukemia (CML) patients. The methylation status of CEBPA promoter was studied in 100 patients with CML and 98 normal healthy individuals from Hyderabad, India, using methylation-specific polymerase chain reaction. The aberrant methylation of CEBPA gene promoter was found in 32 of the 100 CML cases. A highly significant association was found between the frequency of CEBPA gene promoter hypermethylation and the CML stages (P = 0.017), but association with respect to age and gender of the patient was not found. The results suggest that aberrant methylation in the CpG island of the promoter region of this gene might be a common event in CML, and systemic expression studies will be needed to unfold the role of CEBPA promoter methylation in the development, progression, and prognosis of CML.

Differential Expression of BIRC Family Genes In The Course Of Chronic Myeloid Leukemia – BIRC3 and BIRC8 As Potential New Candidates To Identify Disease Progression

Although majority of chronic myeloid leukemia (CML) patients benefit from targeted therapy, there is an unmet need to identify as early as possible patients who develop resistance to tyrosine kinase inhibitors (TKI) and/or patients who progress to blastic phase (CML-BP). Searching for potential candidates of disease progression we have focused on BIRC (baculoviral IAP repeat-containing) genes expression in various stages of CML. This family includes eight functionally- and structurally-related proteins, most of them are believed to serve as endogenous inhibitors of apoptosis. Overexpression of various BIRC genes has been associated with cancer progression, multidrug resistance, poor prognosis and short survival in several types of neoplasms including hematological malignancies. In CML so far expression of BIRC5 (encoding survivin) and BIRC4 (encoding XIAP) has been associated with progression of the disease. However, there is no data on the role of other BIRC members in myeloproliferative diseases.To study the expression of BIRC genes in CML we employed RT-qPCR following MIQE guidelines. We analyzed sequential samples of peripheral blood leukocytes obtained from CML patients at various stages of the disease. Initially we looked at the samples from patients in chronic phase (CML-CP): at the diagnosis and after development of TKI resistance (confirmed as a loss of cytogenetic response, n=5). Then we analyzed samples from patients who progressed either to accelerated phase (CML-AccPh) or to blastic phase (CML-BP) (n=6). Among eight BIRC genes we observed significant decrease in BIRC3 (encoding cIAP2) and BIRC8 (encoding ILP2) expression. This was associated both with TKI resistance and with progression of CML to accelerated or blastic phase. Simultaneously, we observed marked increase in BIRC5 expression in samples from CML-AccPh and BP as compared to chronic phase (as was previously shown by others) but we observed no significant difference in BIRC5 expression between CML-CP diagnostic and TKI-resistant samples. Expression of BIRC1 (NAIP), BIRC2 (cIAP1), BIRC4 (XIAP), BIRC6 (BRUCE) and BIRC7 (LIVIN) was comparable in sequential samples from CML-CP and CML-BC and was not related to TKI-resistance.We verified these results by comparing larger group of patients in either CML-CP at the diagnosis prior to any treatment (n=15) or CML-BP (n=11). To compare the expression of BIRC family in CML to normal hematopoietic cells, we included also cDNA from healthy blood donors (n=10). In accordance with paired samples analysis, we observed downregulation of BIRC3 and BIRC8 expression in CML-BP (as compared to CML-CP and healthy blood donors), while BIRC5 was upregulated in CML-BP patients (as compared to CML-CP and healthy blood donors). There was no difference in expression of other BIRC family members.In conclusion, this is the first comprehensive analysis of the expression of all eight BIRC genes in the course of CML. In addition to the previously described upregulation of BIRC5, we observed significant downregulation of BIRC3 and BIRC8 associated with TKI-resistance and also with progression to accelerated or blastic phase. Recently Rossi D. et al. (Blood 2012;119: 2854-62) described potential role of disruption of BIRC3 (through mutation or gene deletion) in resistance to fludarabine in chronic lymphocytic leukemia. Together with our results this shows that current view linking BIRC genes upregulation with tumor progression and drug resistance may not be true for all of BIRC genes. Our results suggest novel and unexpected role of BIRC3 and BIRC8 in the clonal evolution of CML and open a new area for further exploration of the role of BIRC in CML progression. Disclosures:No relevant conflicts of interest to declare.

Expression of LYN and PTEN genes in chronic myeloid leukemia and their importance in therapeutic strategy

Tyrosine kinase inhibitors (TKIs), imatinib, nilotinib and dasatinib, are the current treatment of chronic myeloid leukemia (CML). BCR-ABL1 point mutations are the principal cause of resistance to treatment; however other mechanisms could be involved in failure to TKI therapy. LYN is a src kinase protein that regulates survival and responsiveness of tumor cells by a BCR-ABL1 independent mechanism. PTEN tumor suppressor gene is downregulated by BCR-ABL1 in CML stem cells and its deletion is associated with acceleration of disease. In this study we evaluated the expression of LYN, PTEN and the ratio of both genes in 40 healthy donors (HD) and in 139 CML patients; 88 of them resistant to TKI in different phases of disease and 51 in chronic phase classified as optimal responders (OR) to TKI [40 treated with imatinib or nilotinib (OR-IN) and 11 treated with dasatinib (OR-D) therapy]. When we analyzed the gene expression values of LYN, an increase was observed only in advanced stages of the disease, however, when we analyzed the ratio between LYN and PTEN genes, the group of resistant patients in chronic phase in imatinib or nilotinib treatment (CP-IN) also showed a significant increase. Resistant patients treated with dasatinib, a src kinase inhibitor, presented a similar ratio to the observed in HD. In addition, the LYN/PTEN ratio and the LYN expression showed a direct significant correlation with BCR-ABL1 transcript levels in unmutated resistant patients treated with non-src kinase inhibitors. We were able to identify 8/35 (23%) of cases in CP-IN and 4/12 (33%) in accelerated phase and blast phase (AP/BC-IN), in which resistance could be associated with an increase in the ratio of the LYN/PTEN. Our data suggest that the LYN/PTEN expression ratio may be a sensitive monitor of disease progression in unmutated CML patients under imatinib or nilotinib treatment. This ratio could detect cases when resistance is related to altered LYN expression, suggesting that the treatment change to a src kinase inhibitor would be most suitable to overcome resistance.

Global DNA Methylation Analysis Identifies Key Pathway Differences Between Poor (Low OCT-1 Activity) and Standard Risk CP-CML Patients At Diagnosis

AbstractAbstract 3730 Background:DNA methylation, specifically CpG methylation, is an essential mediator of epigenetic gene expression which is of vital importance to many biological processes and human malignancies. DNA hypermethylation has been previously described in a small number of genes in chronic myeloid leukemia (CML); however, current published studies have only examined the methylation status of selected genes, often based on the results of studies in other malignancies. Therefore, the global DNA methylation profile of chronic phase-CML (CP-CML) remains poorly understood, as does the impact of the epigenome on patient response to tyrosine kinase inhibitors (TKIs) including imatinib. The organic cation transport-1 (OCT-1) protein is the major active protein involved in imatinib transport, and we have previously demonstrated that measuring its function in leukemic mononuclear cells, expressed as OCT-1 activity (OA), in patient cells prior to imatinib therapy, provides a strong prognostic indicator. Notably, very low OA (poor risk cohort) is associated with patients at significant risk for poor molecular response, mutation development and leukemic transformation on imatinib therapy. Therefore, it is of particular interest to ascertain whether epigenetic changes are distinct and potentially biologically relevant in these poor risk patients. Aim:To investigate the global DNA methylation profile in CP-CML patients with a particular focus on poor risk patients (very low OCT-1 activity), and to ascertain whether aberrant epigenetic programming may underlie their poor response. Method:Cells were isolated from the blood of 55 CP-CML patients at diagnosis and 5 normal individuals. CP-CML patients were classified according to their OA values, with 29 classified as poor (very low OA) and 26 standard risk (high OA). Whole genome DNA methylation analysis was performed using the Illumina Infinium® HumanMethylation450 BeadChip. Analysis of array data was performed with R v2.15.0, using the minfiBioconductor package. Results:The methylation profile of CP-CML was significantly different to that of normal individuals, as shown in Table 1. GeneGo enrichment analysis revealed a significant enrichment in CML for genes known to be involved in other leukemias (p=4.92e−26) particularly AML and CLL, suggesting similar pathways may be under epigenetic control in CML. A significant number of polycomb group (BMI1 and EZH2) target genes were also identified, suggesting the likely involvement of this pathway in CML.Analysis CriteriaBenjamini-Hochburg Adjusted P<0.05Adjusted P<0.05 & Fold Change > 4SamplesSignificant CpGsCorresponding GenesSignificant CpGsCorresponding GenesCP-CML vs. Normal25,8298,4593,467 (2,870 hyper & 597 hypo)1,827Poor vs. Standard Risk9,8616,29610 (5 hyper & 5 hypo)10Table 1: Summary of significant CpGs and corresponding genes when comparisons of CP-CML to normal individuals, and poor to standard risk patients, are made using methylation profiles.A significant difference was also observed when the methylation profiles of poor and standard risk CP-CML patients were analysed (Table 1). GeneGo analysis again identified polycomb group (SUZ12 and EZH2) target enrichment and significant enrichment of NOTCH, Hedgehog and WNT signalling (p=7.93e−9, p=2.42e−5 and p=3.66e−5 respectively) in poor risk patients, indicating these pathways may play a significant role in the unfavourable responses observed in many of these patients. Of particular interest were the ten CpGs where a fold change >4 was observed between the methylation profiles of poor and standard risk patients. Using the Prediction Analysis of Microarrays supervised learning algorithm, a classifier for patient OA prediction based on this 10 CpG signature was evaluated. This classifier had an overall accuracy of 94% (sensitivity: 95%, specificity: 93%). Conclusion:We present a comprehensive global DNA methylation analysis of CP-CML that indicates significant and widespread changes to the CML epigenome, compared with that of normal individuals. Importantly, we have generated a classifier, which identifies the poor risk patient subgroup (very low OA) with 94% accuracy. Validation of this classifier is currently in progress. The epigenetic changes identified here may contribute to CML pathogenesis, and also to the response heterogeneity observed between CP-CML patients treated with TKI therapy. Disclosures:Hughes:Novartis Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. White:Novartis Oncology: Honoraria, Research Funding; BMS: Research Funding; CSL: Research Funding.