Gene In Chronic Myeloid Leukemia Research Articles

The Relationship Between the Regulation of TOB1 Gene with Cell Proliferation, Apoptosis and Cell Cycle in BCR-ABL Positive Cells

AbstractAbstract 5125The TOB1 gene is a transcription factor responsible for the transduction of the gene ERBB2. It is a member of a family of cell suppressor proliferation proteins called TOB/BTG1 family; also, this gene operates on the inhibition of neoplastic transformation. The TOB1 gene presents a decreased expression in several types of cancer such as lung, breast, thyroid and stomach cancer. However, the function of this gene in chronic myeloid leukemia (CML) remains unknown. Aiming to evaluate the inhibition of gene TOB1 into BCR-ABL positive cells and trying to elucidate the molecular mechanisms associated with the inhibition of this gene in the CML we proceed to a more detailed study of this gene. The inhibition of this gene in K562 cells was performed using specific lentivirus. The effect of silencing TOB1 in the proliferation of K562 cells was assessed by the MTT assay after 48 hours of culture; in shTOB1 the proliferation was increased in comparison with shControl cells. To evaluate the synergistic effect between the inhibition of kinase tyrosine activity of BCR-ABL and the inhibition of TOB1 we performed a treatment with different concentrations of imatinib (0. 1, 0. 5 and 1μM), but we observed the decrease in cell proliferation of shTOB1 cells to similar levels of shControl cells only at the 1μM concentration. Therefore, the TOB1 silencing increased the proliferation of K562 cells without an additional effect of a treatment with Imatinib. To analyze the clonogenicity, we performed a formation of colonies assay, in methylcellulose, to determine whether silencing TOB1 could cause a change in the clonal growth of positive BCR-ABL cells. There was no significant change in the number of colonies that grew in cell culture shTOB1 compared to shControl cells. These results suggest that silencing TOB1 in K562 cells may not change the clonogenicity. In the assessment of cell cycle, the flow cytometry analysis revealed a significant accumulation of K562 cells in S phase, with consequent reduction of cells in the G2 phase of the cell cycle in cells shTOB1 compared to cells shControl. The TOB1 gene silencing in K562 cells kept the cells in the S phase and prevented the entry of cells in the G2 phase showing that the inhibition of gene TOB1 induced an increase in proliferation of K562 BCR-ABL cells. The level of apoptosis was assessed by flow cytometry after labeling the cells with anexin-V/PI. The Imatinib treatment presented dose-response in the induction of apoptosis as expected. However, a cumulative effect with TOB1 silencing was not observed. Furthermore, the apoptosis was also assessed by assays of caspases 3, 8 and 9, which showed an increase of the caspase activity of shControl cells in relation of the shTOB1 cells, showing that inhibition of this gene also changes the level of apoptosis. These results corroborate the literature data that report the relationship of this tumour suppressor gene in signalling pathways related to angiogenesis, carcinogenesis, apoptosis and metastasis. When we relate the results obtained with the LMC, we can consider the possibility of TOB1 regulation changes be related to modification of important signalling pathways such as AKT, PI3K, STAT3 and STAT5, among others. Furthermore, the inhibition of TOB1 may be related with an increase on the number of BCR-ABL positive cells and subsequent disease progression. In conclusion, this study confirmed literature data showing that TOB1 gene works as a tumour suppressor protein in cells of many types of cancer. From this work we can infer that in CML the expression of this gene is transformed, resulting in changing of the capacity of induction of apoptosis, decrease tumour necrosis and increase cell proliferation. This work was supported by FAPESP and INCT. Disclosures:No relevant conflicts of interest to declare.

Metastasis Suppressor 1 Is Downregulated in CML Stem Cells and Overexpression Impairs Early Leukemic Cell Propagation.

AbstractAbstract 2776The implementation of Bcr-Abl tyrosine kinase inhibitors (TKIs) has greatly improved the outcome of patients with chronic myeloid leukemia (CML). However, discontinuation of TKI therapy often results in relapse suggesting that leukemic stem cells (LSCs) survive despite treatment. More detailed investigations utilizing patient samples and murine CML models have confirmed that the leukemia-initiating cell population is usually not eradicated by inhibiting Bcr-Abl activity and that this is due to a lack of oncogene addiction of LSCs, showing that further research is required aiming to fully understand LSC biology.To identify new Bcr-Abl targets that are involved in LSC persistence, we performed a microarray analysis of the leukemia-initiating cell population in an inducible transgenic SCLtTAxBcr-Abl CML mouse model in which we had previously shown that these cells are not oncogene-addicted (Schemionek et al., BLOOD 2010; Hamilton et al., BLOOD 2012). One of the most downregulated genes in CML vs. normal stem cells was Metastasis Suppressor 1 (Mtss1/MIM). Although the multidomain protein Mtss1 may be involved in carcinogenesis of several solid tumors, its exact physiological role is still unknown. Current findings suggest that Mtss1 interacts with multiple partners and is involved in various signalling pathways that regulate actin dynamics and cell motility. Interestingly, Rac and Src are Mtss1 interacting partners, and both proteins are known to be involved in Bcr-Abl mediated leukemogenesis.We have previously shown that Mtss1 is downregulated in mouse LSCs and demonstrated that this is a Bcr-Abl kinase mediated effect in various human and murine CML cell lines. Moreover, we have shown that Mtss1 overexpression in 32D-Bcr-Abl cells induces a defect in Bcr-Abl mediated migration in vitro and reduces the potential to form solid tumors in vivo.Here we show that Bcr-Abl kinase-dependent regulation of Mtss1 expression was also evident in mononuclear cells and CD34+ progenitor cells from patients with CML upon IM or dasatinib treatment in vitro. Moreover, cells from IM-treated patients with chronic phase CML showed elevated Mtss1 expression levels within one to three weeks of treatment. Increasing Mtss1 expression upon 5-aza-2′-deoxycytidine-treatment of K562 and 32D-Bcr-Abl cells suggested that methylation might be involved in Mtss1 regulation. To determine a potential leukemia suppressing effect of Mtss1 overexpression, we performed colony assays using lineage negative SCLtTAxBcr-Abl (dtg) bone marrow (BM) cells that had been retrovirally infected to overexpress Mtss1 (dtg::Mtss1) or empty-vector (dtg::ev). Successfully transduced BM cells were FACS-sorted via GFP-expression, encoded by the retroviral vector. Mtss1 overexpression led to a 2.3-fold decrease in CFU numbers. In a second set of experiments we transplanted 1.3×105 GFP-FACS-sorted dtg::Mtss1 or dtg::ev cells into 9 Gy-irradiated recipients. While dtg::ev recipients contained 66% (+/−8%) of GFP-positive cells in the BM, these cells were decreased in dtg::Mtss1 transplanted mice to 23% (+/−21%), 12 days after transplantation. A similar effect was evident in the spleen [dtg::ev recipients: 90% (+/− 3%) versus dtg::Mtss1 recipients 59% (+/−20%)] suggesting that Mtss1 confers a disadvantage to Bcr-Abl positive BM cells in the early steps of leukemic cell propagation, compared to Bcr-Abl cells alone.Since the multidomain Mtss1 protein contains a putative Abl-SH2-binding site, we performed co-immunoprecipitations using 32D-Bcr-Abl-Flag-Mtss1 cells. These experiments showed that both proteins were direct binding partners and that Mtss1 was not phosphorylated by Bcr-Abl.Taken together, our data show that Mtss1 is downregulated via a Bcr-Abl kinase mediated mechanism and this might involve methylation. Moreover, additional inhibition of Mtss1 activity might be mediated through direct binding by Bcr-Abl. Forced expression of the potential tumor suppressor in CML stem and progenitor cells reduces leukemic cell propagation in vivo and may thus provide a rationale to contribute to LSC elimination in patients with CML. Disclosures:Mhairi:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria. Koschmieder:Novartis / Novartis Foundation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Chromosomal aberrations and fusion genes in myeloid malignancies

Since the discovery of the BCR–ABL1 fusion gene in chronic myeloid leukemia, many more fusion genes resulting from chromosomal rearrangements have been identified and characterized. The study of these fusion genes has been extremely important for our understanding of the role of chromosomal rearrangements in leukemogenesis and in oncology in general. In chronic myeloid leukemia, or related myeloproliferative malignancies caused by the expression of oncogenic fusion kinases, tyrosine kinase inhibitors are now successfully used to treat these diseases. In acute myeloid leukemias, the presence of chromosomal rearrangements, oncogenic fusion genes and point mutations in key oncogenic drivers has important prognostic value and determines the choice of therapy. In this review, the authors provide an overview of the important fusion genes present in various myeloid malignancies and their importance for clinical practice.

Intronic SNPs of TP53 gene in chronic myeloid leukemia: Impact on drug response

Background:TP53, located on chromosome 17p13, is one of the most mutated genes affecting many types of human cancers. Thus, we aimed at investigating the association of SNPs in TP53 gene with chronic myeloid leukemia (CML).Materials and Methods:A total of 236 CML and 157 control samples were analysed for mutations in TP53 gene using polymerase chain reaction followed by direct sequencing.Results:Sequencing analysis for mutations in exons 7–9 of the TP53 gene revealed four SNPs, three in intron 7 (C14181T, T14201G, and C14310T) and one SNP in intron 6 (A13463G) of TP53 gene. The mutation C14181T is located at position 72 base pairs downstream of the 3′-end of exon 7 of the P53 gene. This mutation is in complete linkage disequilibrium with a T14201G mutation, 20 base pairs further downstream occurring at position 14201. This mutation occurred only in the presence of C14181T mutation and these mutations showed association with advanced phase and cytogenetic poor response. Another two novel mutations, C14310T in intron 7 and A13463G in intron 6 were also found to be associated with cytogenetic poor response.Conclusion:Our study suggests that TP53 intronic SNPs might have a strong influence on disease progression and poor response in CML patients.

A Novel Dual AXL/Mertk and Aurora Kinase Inhibitor Active Against T Cell Acute Lymphoblastic Leukemia

Abstract 1519 INTRODUCTION:Receptor tyrosine kinases AXL and MER belong to the TYRO3 kinase family, first identified as a transforming gene in chronic myeloid leukemia and are found at high levels in various cancers, including hematopoietic malignancies like T cell acute lymphoblastic leukemia (T-ALL). Aurora kinases play important roles in chromosome alignment and cytokinesis during mitosis and are also aberrantly expressed in ALL. We describe here characterization and pre-clinical testing of Huntsman Cancer Institute-2084 (HCI-2084), a small molecule inhibitor of AXL and MER kinases that also has activity against Aurora Kinases A and B, as a novel therapeutic for T-ALL treatment, a leukemia with poor prognosis. METHODS:Quantitative RT-PCR and Western Blot confirmed elevated levels of AXL/MER expression in Jurkat, a human T-ALL, cell line. Our compound, HCI-2084, was developed using a computational structure-based approach against AXL kinase. HCI-2084 activity against AXL/MER, in cell-based assays was evaluated utilizing ATPlite. To test activity against Aurora kinase, Jurkat cells were treated with various concentrations of HCI-2084 and VX 680, a known aurora kinase inhibitor, for 24 hours. Cell lysates were evaluated for phospho-AKT (Ser473), phospho-Aurora (Thr288) and phospho-Histone H3 (Ser10) using the Meso Scale Discovery platform (MSD). Cell cycle analysis was performed on Jurkat cells incubated in the absence (negative control) or presence of 100nM HCI-2084 or VX-680 for 24 hours, stained with propidium iodide and analyzed by flow cytometry on FACS Calibur. Immunostaining was performed to evaluate the levels of phospho-Histone H3 (Ser10) and phospho-Aurora (Thr288) during mitosis in Jurkat cells incubated in the absence (negative control) or presence of 100nM HCI-2084 and VX-680 for 24 hours. In addition to the anti-phospho-Histone H3 and anti-phospho-Aurora antibodies, anti-alpha-tubulin and DAPI were used to determine the presence of mitotic chromosomes.To examine HCI-2084 as a therapeutic agent for T-ALL treatment in vivo, we tested it using transgenic Zebrafish (Danio rerio) with T-ALL driven by human MYC (hMYC). Over-expression of endogenous D. rerio axl /mertk transcripts in T-ALL from hMYC fish were verified by qRT-PCR. Fish were treated with HCI-2084, Dexamethasone (a known T-ALL therapeutic; positive control), and DMSO vehicle (HCI-2084 is reconstituted in DMSO; negative control). Trials were conducted by housing fish with fluorescently-labeled T-ALL (GFP-tagged) in water containing the agent being tested, for 14 days with monitoring of disease response by fluorescent microscopy. RESULTS:In in vitro studies, HCI-2084 showed potent activity in cell viability assays with an IC50 of 12 nM against AXL, 60 nM against MERTK and 15 nM against Aurora kinase. MSD assays demonstrated efficacy at 1 uM for reduction of phospho-AKT, phospho-Histone H3 and phospho-Aurora, as seen with VX 680. Cell cycle analyses performed on HCI-2084-treated Jurkat cells showed a significantly increased G2/M population and an accumulation of cells with ≥4N DNA, indicative of Aurora B inhibition and endo-reduplication (Figure A & B). Immunofluorescence analyzed using fluorescent microscopy demonstrated mitotic arrest with loss of phospho-Histone H3 and phospho-Aurora staining, demonstrating inhibition of Aurora kinase activity. HCI-2084 was also a potent therapeutic against Zebrafish T-ALL. Fish treated at 1uM HCI-2084 for 14 days, showed complete responses (CR), with efficacy comparable to our dexamethasone positive control (Figure C). Following treatment, fish remained disease-free for several days, and overall survival was prolonged significantly relative to untreated controls. CONCLUSION:HCI-2084 shows dual action in vitro against Jurkat cells with potent cytotoxicity via AXL/MER kinase inhibition and anti-proliferative activity via Aurora kinase inhibition. In vivo, HCI-2084 demonstrates activity against a MYC-driven vertebrate model of T-ALL, prolongs survival, and is well tolerated. We conclude that HCI-2084 is a potent dual AXL/MER kinase and Aurora kinase inhibitor which should be explored further as a potential novel therapeutic in the treatment of human T-ALL. [Display omitted] Disclosures:Sharma:Millenium: Research Funding.

Abstract 3609: Homology structure-based design, synthesis and biological evaluation of a series of novel Axl and Mer kinase inhibitors

Abstract Axl and Mer kinases belong to the TAM family that was first identified as a transforming gene in chronic myeloid leukemia. Its intracellular region has the typical receptor tyrosine kinase (RTK) structure and its extracellular domain is similar to cadherin-type adhesion molecules in that it is composed of fibronectin type II and immunoglobulin (Ig) motifs. Axl binds various growth factors, with vitamin K-dependent protein growth-arrest-specific gene 6 (GAS6) being the best studied. Axl is involved in mesenchymal and neuronal development as well as in cell survival, adhesion and blood vessel function. High levels of Axl are found in various tumors, including malignant glioma and metastatic colon, ovarian and breast cancers, and it plays a role in cancer invasion. Recent development of Axl inhibitors suggested that inhibition of Axl activity may be impact both tumor angiogenesis and tumor growth. We describe here the design, synthesis, molecular modeling, and biological evaluation of a series of small molecule, inhibitors of Axl and Mer kinases. Our initial lead compound was identified via cross-docking experiments utilizing the homology model of Axl kinase and screening of a diverse in-house chemical library. We subsequently carried out structure-activity relationship studies and optimized the lead structure which has 2 to 6.1 μM inhibition activity to 2-fold improvement in the Axl and Mer kinase activities to 730 nM. As a starting point for further optimization, it was considered that the modest of these series could be improved by introducing a spacer at the aryl piperazine moiety and focused on maximizing the in vitro potency, addressing the SAR and molecular properties. We further explored Leu620, hydrophobic, Gly543, Phe547 and DFG motif sites through scaffold hoping lead to the identification of an electron-withdrawing functional groups lead to the compound HCI-2084 and HCI-2091 which exhibited potent Axl kinase inhibition activity of 7 and 12 nM in Axl kinase assay and 30-50 nM is panel of cancer cell lines. These novel series of compounds were synthesized in two-step procedure via a standard cross-coupling reaction or with various substituted aryl amine derivatives under amination conditions. In a subsequent step we employed Buchwald-Hartwig amination reaction to prepare target molecules. Due to the ready synthesis accessibility, we extensively investigated the SAR improving the RO5, solubility and permeability parameters. The details of these results will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3609. doi:10.1158/1538-7445.AM2011-3609

Methylation status of DDIT3 gene in Chronic Myeloid Leukemia

BackgroundDNA-damage-inducible transcript 3 (DDIT3), a candidate tumor suppressor gene (TSG), has been found involved in the regulation of cellular growth and differentiation. The epigenetic changes of TSGs are recently recognized as an abnormal mechanism contributing to the development of chronic myeloid leukemia (CML). The aim of this study was to investigate the methylation status of DDIT3 gene in CML patients.MethodsThe methylation status of DDIT3 promoter was detected in the bone marrow mononuclear cells from 53 patients with CML using methylation-specific PCR (MSP). The expression levels of DDIT3 and bcr/abl transcript were determined by real-time quantitative PCR (RQ-PCR). Clinical data of these patients were collected and analyzed.ResultsThe aberrant methylation of DDIT3 gene promoter was found in 35 of 53 (66%) CML cases. Correlation was not found between DDIT3 promoter hypermethylation and the age, sex, hemoglobin concentration, platelet counts, chromosomal abnormalities, bcr/abl transcript, and staging of CML patients (P > 0.05), but found between DDIT3 promoter hypermethylation and WBC counts of CML cases (R = 0.781, P < 0.001). The level of DDIT3 transcript in CML patients was significantly lower than that in controls (median 3.28 vs 19.69, P < 0.001), however, there was no difference in the level of DDIT3 transcript between methylation-positive CML cases (0.05-65.32, median 2.13) and methylation- negative CML cases (0.12-126.04, median 3.92) (P > 0.05).ConclusionOur results demonstrate that aberrant methylation of DDIT3 occurs in CML frequently.

Myeloid zinc finger 1 induces migration, invasion, and in vivo metastasis through Axl gene expression in solid cancer.

Myeloid zinc finger 1 (MZF1) is a member of the SCAN domain family transcription factors that form dimers through their highly conserved SCAN motifs. Silencing of MZF1 inhibits cell proliferation, and abnormal expression of MZF1 results in cancer development. However, a potential role of MZF1 in metastasis remains unclear. Axl is a receptor tyrosine kinase and was first identified as a transforming gene in chronic myeloid leukemia. Axl overexpression induces proliferation, migration, and invasion and is highly expressed in different human cancers. In this study, we show that overexpression of MZF1 induces migration and invasion in colorectal (Rko, SW480) and cervical (HeLa) cancer cells. In addition, we show that MZF1 binds to the Axl promoter, transactivates promoter activity, and enhances Axl-mRNA and protein expression in a dose-dependent manner. In vitro, sh-RNA knockdown of Axl reduced MZF1-induced migration and invasion in HeLa and Rko cells (P = 0.05). Additionally, Rko cells overexpressing MZF1 showed increased tumor formation and liver metastasis in the chicken-embryo-metastasis assay in vivo. Furthermore, the expression of MZF1 and Axl was significantly higher in resected colorectal tumors compared with corresponding normal tissues (P = 0.02; P = 0.05), and MZF1 expression was positively correlated with Axl gene expression in tumor tissues (P < 0.01). Taken together, this is the first study to show that MZF1 induces invasion and in vivo metastasis in colorectal and cervical cancer, at least in part by regulating Axl gene expression.

Dramatic and durable efficacy of imatinib in an advanced angiosarcoma without detectable KIT and PDGFRA mutations

The tyrosine kinase (TK) inhibitor, imatinib, has revolutionized therapy of malignancies that are addicted to one of its target kinases, c-abl, c-kit, and PDGF-R. This addiction is generally dependent on the acquisition of an activating kinase mutation, e.g. the bcr-abl fusion gene in chronic myeloid leukemia, or point mutations of KIT or PDGFRA in gastrointestinal stroma tumors. Other types of sarcomas are generally considered to be insensitive to imatinib. We have observed a striking and durable remission of an advanced angiosarcoma to imatinib that can only be explained by TK addiction. Unexpectedly, GIST-type KIT and PDGFRA were absent in this case. This case illustrates the diagnostic challenges in identifying individual candidate patients for TK inhibitor therapy.

Expression of the CDH13 gene and BCR/ABL fusion gene in chronic myeloid leukemia patients and their relationship

To investigate the expression of the CDH13 gene and BCR/ABL fusion gene in chronic myeloid leukemia(CML) patients at different stages and explore their relationship. RNA was isolated from peripheral blood in 30 healthy adults, 22 primary CML patients and 25 blastic crisis of CML patients. We examined the expression of the CDH13 gene and BCR/ABL fusion gene using EVA Green real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The expression of the BCR/ABL fusion gene in the patients with blastic crisis CML was 4.72 folds as that of the patients with primary CML. The expression of the CDH13 gene in the primary and blastic crisis CML patients was significantly reduced to 36% and 25% of that in healthy controls, respectively. Moreover, negative correlation was found between the expressions of these two genes. The study showed that the reduction of the CDH13 expression at different clinical stage of CML may account for the defective cell adhesion in CML, and the expression of the CDH13 gene was probably down-regulated by the BCR/ABL fusion gene.

Differential Methylation Pattern of ID4, SFRP1, and SHP1 between Acute Myeloid Leukemia and Chronic Myeloid Leukemia

To gain insight into the differential mechanism of gene promoter hypermethylation in acute and chronic leukemia, we identified the methylation status on one part of 5'CpG rich region of 8 genes, DAB2IP, DLC-1, H-cadherin, ID4, Integrin α4, RUNX3, SFRP1, and SHP1 in bone marrows from acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) patients. Also, we compared the methylation status of genes in AML and CML using methylation-specific PCR (MSP). The frequencies of DNA methylation of ID4, SFRP1, and SHP1 were higher in AML patients compared to those in CML patients. In contrast, no statistical difference between AML and CML was detected for other genes such as DLC-1, DAB2IP, H-cadherin, Integrin α4, and RUNX3. Taken together, these results suggest that these methylation-controlled genes may have different roles in AML and CML, and thus, may act as a biological marker of AML.

Expression Profiling of ABC Transporter Genes in Chronic Myeloid Leukemia (CML) and Responsiveness to Imatinib

The deregulation of ATP-binding cassette (ABC) transporters responsible for the efflux of anticancer agents may involve mutations or single nucleotide polymorphisms (SNPs) or an increase in their expression level. Consequently, chemoresistance may develop. We have previously shown that the expression level and transcription of ABC drug transporters in CML cells is affected by c-Myc. Our results demonstrated that c-Myc is highly expressed in CD34+ cells from newly diagnosed chronic phase (CP)-CML patients, and that it can significantly upregulate the expression of several ABC genes, particularly, the ABCC1, ABCC4 and ABCG2, while it downregulates the expression of ABCC3. We have also demonstrated that c-Myc was a direct regulator and physically associated with the promoter of tested ABC genes, as assessed by Chromatin Immunoprecipitation in a cell line derived from a Ph+ CML patient. Taken together, our findings supported the model of a direct and coordinate regulation of a large set of ABC genes by the c-Myc transcription factor. Our study also supported prior findings that deregulation of specific set of ABC genes could be an important molecular mechanism altering imatinib transport. Based on these observations we have started to investigate the role of ABC transport genes expression in newly diagnosed CP-CML patients treated with imatinib. RNA extracted from white blood cells of 5 patients who achieved a stable major molecular response (MMR) by 12 months (responders) and 15 patients who didn't show a partial cytogenetic response (CgR) by 6 months nor a complete CgR by 12 months (suboptimal responders according to European LeukemiaNet recommendations). All pts were enrolled on GIMEMA CML Working Party-sponsored clinical trials of imatinib. A panel of ABC genes including ABCB1, ABCB9, ABCC1, ABCC3, ABCC4, ABCE1 and ABCG2 was interrogated by Q-PCR for the level of expression. Results were normalized to the expression of three reference genes, i.e., GUSB, b-actin and GAPDH. Our results show that suboptimal responders display high expression levels of ABCG2 (p<0.01) and very low levels of ABCC3 (p<0.0001) as compared to patients with responders. Interestingly, when ABC expression profile of the same patient was evaluated before starting imatinib treatment and compared with a measurement obtained at the time of suboptimal response, we could not observe any significant difference between the two conditions. That suggest that this specific ABC transporter expression profile could be present at diagnosis. Although preliminary, our findings suggest that profiling of ABC drug transporter genes in CML patients could provide a novel modality of investigating their responsiveness to imatinib. Analysis of a larger series of patients is ongoing to further explore the potential predictive value of this expression profile. We speculate that ABCC3, which is strongly repressed possibly through a typical epigenetic mechanisms such as promoter hypermethylation and/or chromatin condensation, could be re-activated by means of demethylating agents or inhibitors of chromatin condensation. Thus, our study proposes that an important novel approach to optimize imatinib response in such patients merits further investigations.

The chemokine interleukin-8 and the surface activation protein CD69 are markers for Bcr–Abl activity in chronic myeloid leukemia

We have identified differentially regulated genes in chronic myeloid leukemia (CML) cells upon short treatment with the broad-spectrum Bcr–Abl inhibitor dasatinib. The highly specific Bcr–Abl inhibitor nilotinib caused a very similar gene expression signature, validating the identified differentially regulated genes as a read-out of Bcr–Abl activity and implying that Bcr–Abl is the functionally central target of dasatinib in CML cells. Among the strongest downregulated genes, we have further validated the activation marker CD69 and the chemokine interleukin (IL)-8. Expression of both proteins is upregulated upon Bcr–Abl expression and inhibited by dasatinib and nilotinib. IL-8 may thus be a useful marker for the monitoring of CML inhibitor efficacy and play a potential pathophysiological role in CML.

Identification and structural comparison of deleterious mutations in nsSNPs of ABL1 gene in chronic myeloid leukemia: A bio-informatics study

Single nucleotide polymorphism (SNP) serve as frequent genetic markers along the chromosome. They can, however, have important consequences for individual susceptibility to disease and reactions to medical treatment. Also, genetics of the human phenotype variation could be understood by knowing the functions of these SNPs. Currently, a vast literature exists reporting possible associations between SNPs and diseases. It is still a major challenge to identify the functional SNPs in a disease related gene. In this work, we have analyzed the genetic variation that can alter the expression and the function in chronic myeloid leukemia (CML) by ABL1 gene through computational methods. Out of the total 827 SNPs, 18 were found to be non-synonymous (nsSNPs). Among the 30 SNPs in the untranslated region, 3 SNPs were found in 5′ and 27 SNPs were found in 3′ untranslated regions (UTR). It was found that 16.7% nsSNPs were found to be damaging by both SIFT and PolyPhen server. UTR resource tool suggested that 6 out of 27 SNPs in the 3′ UTR region were functionally significant. The two major mutations that occurred in the native protein (1OPL) coded by ABL1 gene were at positions 159 (L → P) and 178 (G → S). Val (6), Ala (7) and Trp (344) were found to be stabilizing residues in the native protein (1OPL) coded by ABL1 gene. Even though all the three residues were found in the mutant protein 178 (G → S), only two of them Val (6) and Ala (7) were acting as stabilizing residue in another mutant 159 (L → P). We propose from the overall results obtained in this work that, both the mutations 159 (L → P) and 178 (G → S) should be considered important in the chronic myeloid leukemia caused by ABL1 gene. Our results on this computational study will find good application with the cancer biologist working on experimental protocols.

Effect of Inhibition of Grb2 Expression on BCR-ABL Mediated Transformation of Primary Human Hematopoietic Cells.

The critical mechanisms underlying human hematopoietic stem cell (HSC) transformation by the BCR-ABL gene in chronic myeloid leukemia (CML) are still not well understood. Since treatment with imatinib fails to eliminate primitive CML hematopoietic cells, there is a pressing need to identify additional mechanisms that can be targeted to enhance elimination of CML stem cells. A tyrosine residue at position 177 (Y177) in the BCR-ABL protein binds the adapter protein Grb2 and appears to play an important role in BCR-ABL induced myeloid leukemogenesis in murine CML models. We have recently shown that a tyrosine to phenylalanine mutation of Y177 (Y177F) also results in significant reduction of abnormalities in proliferation and differentiation in BCR-ABL expressing human CD34+ cells and in BCR-ABL induced activation of Ras, Akt and STAT5 signaling (Cancer Res 2007 67:7045). Since Grb2 signaling is implicated in several other human malignancies and likely plays an important role in signaling downstream of BCR-ABL, we were interested in investigating the role of Grb2 in BCR-ABL-mediated transformation of primary human hematopoietic cells. Cord blood (CB) CD34+ cells were transduced with bicistronic retrovirus vectors coexpressing wild type or Y177F mutated BCR-ABL genes with the GFP gene. Coimmunoprecipitation studies confirmed the association of Grb2 with BCR-ABL and was abrogated by the Y177F mutation. We next investigated the effect of inhibition of Grb2 expression in BCR-ABL transduced human CD34+ cells. CB CD34+ cells were co-transduced with retroviral vectors coexpressing the BCR-ABL and GFP genes or control vectors expressing the GFP gene alone together with lentivirus vectors coexpressing Grb2 shRNA constructs and the dsRed gene or the dsRed gene alone. Cells expressing CD34+, GFP and dsRed were selected using flow cytometry. Western blot analysis indicated that Grb2 levels were reduced by 80.8±14.5% (n=3, P < 0.014); in CD34+ cells transduced with BCR-ABL and Grb2 shRNA compared with cells expressing BCR-ABL alone. Expression of Grb2 shRNA resulted in significant reduction in expansion of BCR-ABL expressing CD34+ cells compared with cells expressing BCR-ABL alone after 7 days of culture in serum free medium (SFM) with low concentrations of growth factor similar to those present in bone marrow stroma conditioned medium (BA alone 48.5±7.7; BA+Grb2 shRNA, 16.7±4.7, n=3). Co-expression of Grb2 shRNA also resulted in significant reduction in the total number of colonies generated by BCR-ABL expressing CD34+ cells in methylcellulose progenitor culture (BA, 123±31; BA+Grb2, 40±9; n=3, p<0.03), with reduction being seen mainly for erythroid colonies. Expression of Grb2 shRNA also reduced cell expansion from control CD34+ expressing GFP alone but the difference was not statistically significant. Similarly Grb2 inhibition did not result in a significant difference in the number of colonies generated from control CD34+ cells. Grb2 inhibition was associated with reduced levels of P-MAPK, but not P-AKT in BCR-ABL expressing CD34+ cells. Interestingly Grb2 inhibition also results in decreased levels of P-STAT5 consistent with the observed reduction in erythroid colonies. In conclusion RNAi mediated inhibition of Grb2 expression results in significant inhibition of BCR-ABL induced proliferation of human CD34+ cells proliferation, indicating an important role for Grb2 in BCR-ABL mediated transformation of CML cells. These observations support further evaluation of inhibition of Grb2 signaling in targeting of CML stem cells.

DNA Methylation Analysis of 807 Genes in Chronic Myeloid Leukemia and Acute Promyelocytic Leukemia.

DNA methylation changes are a common finding in leukemia, and hypermethylation of CpG island promoters is associated with aberrant gene silencing. Some abnormal cancer related methylation changes have been associated with clinical phenotype including pathologic features, prognosis, and treatment response. However, other DNA methylation changes do not appear to have phenotypic consequences and may reflect a stochastic event or a downstream event of tumorogenesis, such as the CpG island methylator phenotype (CIMP). In order to obtain a better understanding of the DNA methylation changes found in leukemia we analyzed 18 acute promyelocytic leukemia (APL) and 36 chronic myeloid leukemia (CML) patients. We specifically chose to study APL and CML as these leukemia are initiated by specific genetic translocation events, t(15:17) and t(9:22) respectively. To measure the DNA methylation status, we used the GoldenGate Assay for Methylation and BeadArray technology from Illumina, Inc. The Standard Methylation Cancer Panel I from Illumina interrogates 1505 CpG sites, selected from 807 genes (231 genes contain one CpG site per gene, 463 genes contain two CpG sites and 114 genes have three or more CpG sites). In our study we found 142 and 269 genes that were hypermethylated in CML and APL. 31 genes were uniquely hypermethylated in CML, 158 genes were hypermethylated only in APL, and 111 genes were hypermethylated in both leukemias. There was a unique pattern of hypermethylated genes in each cancer; such there was a high concordance of hypermethylated genes within each leukemia type. These data suggest that the epigenetic events were a result of the genetic translocation BCR/ABL or PML/RARα (associated with chromosomal aberrations t(9:22) or t(15:17)) that initiates these leukemias. Analysis of the number of hypermethylated genes in these two leukemias showed a bimodal distribution suggestive of CIMP, however, closer examination showed that this bimodal distribution could be attributed to the two different types of leukemia. APL patients had mean of 280 genes hypermethylated while CML patients only had a mean of 193 genes hypermethylated. APL had a stronger methylator phenotype than CML for the loci studied, which underscores the possible relationship of CIMP to a genetic phenotype. Subset analysis of our CML samples by chronic phase (23 patients), accelerated phase (5 patients), and blast crisis (8 patients) revealed 42 genes that became hypermethylated with progression of CML. It is possible that hypermethylation of these genes are clinically important in the leukemia phenotype, and maybe targets for epigenetic therapy. We examined the DNA methylation changes induced by the DNA methylation inhibitor, azacitidine, in a patient with blast crisis CML and refractory to imatinib mesylate therapy. Azacitidine could reverse the aberrant hypermethylation associated with progression of CML to blast crisis and supports the use of this drug as an epigenetic therapy. Our data show that the majority of DNA hypermethylation events in leukemia are dependent on genetic events, but there is a subset of DNA hypermethylation events that are involved in the progression of leukemia and may be therapeutically reversed by DNA methylation inhibitors.

Molecular Signature of Distinct CD34+ Hematopoietic Stem and Progenitor Cell Subsets of Patients with CML in Chronic Phase.

During the last decade, chronic myeloid leukemia (CML) has been mainly characterized by the reciprocal translocation between chromosomes 9 and 22, resulting in the formation of the protooncogene BCR-ABL. This constitutively active tyrosine kinase is widely considered as the cause of the disease. Even though BCR-ABL transcripts are found in every dividing hematopoietic cell and thus, the disease is likely to originate from a primitive stem cell, the “cell of origin” is still a matter of debate. Despite the active “leukemia stem cell” discussion, very few characteristics of the “cancer stem cell” are established to date. In order to get further molecular insights into CML stem and progenitor cells, we examined CD34+ cell subsets obtained from bone marrow of 7 patients with CML in chronic phase in comparison with 5 healthy volunteers. CD34+ cells were immunomagnetically selected and high-speed cell sorting of lineage-negative, CD34+, CD38−, hematopoietic stem cells and myeloid progenitors was performed. Progenitors were further subdivided by anti-IL-3Ralpha and anti-CD45RA staining. Following RNA extraction, a two-cycle amplification procedure was used to generate cDNA for the hybridization with Affymetrix U133A2.0 arrays. After performing smoothening spline normalization, we applied the perfect match-mismatch difference model algorithm to calculate expression values (dChip). Hierarchical cluster analysis was performed using a correlation based centroid linkage algorithm. Hereby we could discriminate the HSCs, CMPs, and MEP subsets. Corroboration of RNA expression was performed by real-time RT-PCR for selected genes. Comparing the HSC subsets of CML patients with healthy controls we found 98 differentially expressed genes. 87 genes had a lower expression level in CML HSCs whereas 11 genes had a higher one. Among the downregulated genes in CML were transcriptions factors involved in myelogenesis and proliferation and several adhesion molecules associated with homing and migration of the HSCs. On the other hand, the Leptin receptor and BCR-ABL downstream targets were found to be upregulated. Within the common myeloid progenitor (CMP) compartment 37 genes were significantly differentially regulated. Twenty genes had a higher expression level in CML CMPs, 17 genes were downlegulated. Hematopoietic cell-specific cell cycle inhibitor MS4A3 was among the significantly downregulated genes whereas genes of the retinoblastoma and E2F families as well as inhibitors of the Wnt-signaling pathway were upregulated. Looking at megakaryocte-erythrocyte progenitors (MEP) in CML, key mediators of G2-M cell cycle transition were downregulated indicating a lower proliferative capacity of this subset. No transcriptional differences have been observed between granulocyte-macrophage progenitors from CML patients and healthy volunteers. Interestingly, among all other subsets myeloperoxidase (MPO) was downregulated in the CML samples and the Leptin receptor was upregulated. Our results provide novel insights into the biology of CML and potentially provide the basis for the characterization of a candidate CML stem cell.

Epigenetic regulation of PRAME gene in chronic myeloid leukemia

Tumor associated antigens (TAA) provide attractive targets for cancer-specific immunotherapy. PRAME is a TAA gene up-regulated in advanced phases of chronic myeloid leukemia (CML). To date, molecular mechanisms for the expression of PRAME have never been studied. We found that some Ph’-positive cell lines did not express PRAME. The expression of PRAME was restored in these cell lines by treatment with 5′-aza-2′-deoxycytidine, suggesting that the expression of PRAME is mainly suppressed by hypermethylation. Bisulfite sequencing analysis of the CpG sites of the PRAME exon 2 in these cancer cell lines revealed a close relationship between the methylation status of the PRAME gene and its expression. A methylation-specific PCR analysis demonstrated that hypomethylation of PRAME was significantly more frequent in CML blast crisis (70%) than in chronic phase (36%) ( P = 0.01) and was correlated with high expression levels of PRAME transcripts ( P < 0.0001). These results suggest that hypomethylation of PRAME up-regulates its expression in CML and might play a significant role in the progression of the disease.

Direct and Coordinate Regulation of Multidrug Resistance Genes by the c-Myc Oncoprotein.

The deregulation of ATP-binding cassette (ABC) transporters responsible for the efflux of anticancer agents may be achieved either by mutations or single nucleotide polymorphisms (SNPs) affecting the biophysical and biochemical properties of the transporters or by an increase in their expression level. Consequently, chemoresistance will develop. In this study we have investigated regulatory mechanisms involved in the activation of ABC transporters. We have first examined how ABC genes are regulated at the transcriptional level and which transcription factors concur to such a control. The expression level of all 48 human ABC drug transporters was determined as a function of c-Myc expression in a p493 lymphoblastoid cell line harboring c-Myc driven by a tetracycline-responsive promoter. Our results demonstrated that c-Myc affected the transcription of several ABC genes, such as ABCA2, ABCB1, ABCB9, ABCC1, ABCC4, ABCE1, ABCF1, ABCF2, ABCF3, a majority of which, has been found implicated in chemoresistance. To evaluate whether c-Myc affects the transcription of those ABC genes directly, chromatin immunoprecipitation (ChIP) was performed in p493 cells. The results have shown a direct binding of c-Myc to the promoters of the ABC genes tested, with the only exception of ABCB1. To further confirm a direct role of c-Myc on ABC gene transcription, ABC gene promoters were cloned into a luciferase reporter assay and their activity was tested in various lymphoblastoid cell line variants, expressing different levels of c-Myc. Results proved a dose-dependent transcription activation of ABC reporters induced by c-Myc, which suggested that the c-Myc oncoprotein could regulate the level of expression of a large number of ABC genes in lymphocytes. Furthermore, we have investigated the expression of c-Myc and ABC genes in chronic myeloid leukemia (CML) in order to verify whether high expression levels of c-Myc may affect transcription of ABC drug transporters also in CML cells. In fact, recent studies reported that Bcr-Abl may positively regulate c-Myc expression. Our results showed that c-Myc is highly expressed in CD34+ cells from newly diagnosed chronic phase (CP)-CML patients, and that it can significantly upregulate the expression of several ABC genes, particularly that of the ABCC1 and ABCC4. We have demonstrated that c-Myc was physically associated with the promoter of tested ABC genes and their direct regulator as assessed by ChIP in the Kasumi-4 cell line, which has been derived from a Ph+ CML patient and expresses the CD34 antigen. Taken together, our findings support the model of a direct and coordinate regulation of a large set of ABC genes by the c-Myc transcription factor. Our study suggests that c-myc deregulation of ABC genes could be an important molecular mechanism altering imatinib transport. Thus, we concluded that c-Myc could be involved in the development of chemoresistance in CML, as well as resistance to targeted drugs, such as imatinib. Currently, we are further assessing transcriptional deregulation of specific ABC genes, and SNPs affecting the functional properties of ABC transporters, by comparing CP-CML patients responsive to imatinib, which have been shown to represent over 90% of treated individuals, and patients who developed resistance.

Signaling from the Oncogenic Fusion Protein BCR/ABL1 Leads to Expression of Wilms' Tumor Gene 1 Protein (WT1), Which Induces Transcriptional Repression of Interferon Consensus Sequence Binding Protein (ICSBP) in Human Cells.

The Wilms' tumor gene 1 (WT1) encodes a transcription factor highly expressed in most leukemias. Overexpression of WT1 and the fusion protein AML1-ETO in transgenic mice rapidly induces acute myeloid leukemia (AML), indicating an oncogenic effect by WT1. However, mechanisms behind expression of WT1 in leukemic cells, as well as mechanisms downstream of WT1, are largely unknown. Here, we report that the fusion protein BCR/ABL1, associated with chronic myeloid leukemia (CML), increases expression of WT1 mRNA and protein, which induces transcriptional represssion of ICSBP. Inhibition of BCR/ABL1 signaling by imatinib mesylate reduced WT1 mRNA levels in five different human CML cell lines, confirming previous reports (Cilloni D et al. Cancer 101:979, 2004). In extended investigations, we show that signaling via the phosphatidylinositol-3 kinase (PI3K)-Akt pathway is critical for BCR/ABL1 induced WT1 expression, while independent of JAK-STAT signaling. BCR/ABL1 signaling did not affect half-life of WT1 mRNA, but inhibition of BCR/ABL1 or PI3K strongly suppressed the transcriptional activity of WT1 promoter/enhancer reporters, indicating that BCR/ABL1 signaling increases transcription of the WT1 gene. Downstream of WT1, we report the interferon consensus sequence binding protein (ICSBP) gene as a new target gene of WT1. Increased levels of WT1 reduced the amount of ICSBP mRNA in both normal progenitors and in U937 leukemic cells. Moreover, WT1 repressed transcription from the ICSBP-promoter. Finally, we report that forced expression of BCR/ABL1 in human CD34+ bone marrow progenitor cells resulted in increased expression of WT1 mRNA and protein and repressed levels of ICSBP mRNA and protein, indicating that WT1 can repress expression of ICSBP. In contrast to the almost ubiquitously high expression of WT1 in leukemia, expression of ICSBP is very low or absent in most leukemias, consistent with the notion of WT1 as a repressor of ICSBP in vivo. Moreover, several reports point out ICSBP as an important suppressor gene in CML. Our results provide a mechanistic explanation for BCR/ABL1 induced repression of ICSBP via induction of WT1, and a general mechanism by which high expression of WT1 could contribute to repression of ICSBP also in other leukemias.