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1
Shah, N. P., O. N. Witte, and C. T. Denny. "Characterization of the BCR promoter in Philadelphia chromosome-positive and -negative cell lines." Molecular and Cellular Biology 11, no. 4 (April 1991): 1854–60. http://dx.doi.org/10.1128/mcb.11.4.1854-1860.1991.
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The t(9;22) Philadelphia chromosome translocation fuses 5' regulatory and coding sequences of the BCR gene to the c-ABL proto-oncogene. This results in the formation of hybrid BCR-ABL mRNAs and proteins. The shift in ABL transcriptional control to the BCR promoter may play a role in cellular transformation mediated by this rearrangement. We have functionally localized the BCR promoter to a region 1 kb 5' of BCR exon 1 coding sequences by using a chloramphenicol acetyltransferase reporter gene assay. Nucleotide sequence analysis of this region revealed many consensus binding sequences for transcription factor SP1 as well as two potential CCAAT box binding factor sites and one putative helix-loop-helix transcription factor binding site. No TATA-like or "initiator" element sequences were found. Because of low steady-state levels of BCR mRNA and the high GC content (78%) of the promoter region, definitive mapping of transcription start sites required artificial amplification of BCR promoter-directed transcripts. Overexpression from the BCR promoter in a COS cell system was effective in demonstrating multiple transcription initiation sites. In order to assess the effects of chromosomal translocation on the transcriptional control of the BCR gene, we determined S1 nuclease protection patterns of poly(A)+ RNA from tumor cell lines. No differences were observed in the locations and levels of BCR transcription initiation sites between those lines that harbored the t(9;22) translocation and those that did not. This demonstrates that BCR promoter function remains intact in spite of genomic rearrangement. The BCR promoter is structurally similar to the ABL promoters. Together, this suggests that the structural fusion of BCR-ABL and not its transcriptional deregulation is primarily responsible for the transforming effect of the t(9;22) translocation.
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Shah, N. P., O. N. Witte, and C. T. Denny. "Characterization of the BCR promoter in Philadelphia chromosome-positive and -negative cell lines." Molecular and Cellular Biology 11, no. 4 (April 1991): 1854–60. http://dx.doi.org/10.1128/mcb.11.4.1854.
Full textAbstract:
The t(9;22) Philadelphia chromosome translocation fuses 5' regulatory and coding sequences of the BCR gene to the c-ABL proto-oncogene. This results in the formation of hybrid BCR-ABL mRNAs and proteins. The shift in ABL transcriptional control to the BCR promoter may play a role in cellular transformation mediated by this rearrangement. We have functionally localized the BCR promoter to a region 1 kb 5' of BCR exon 1 coding sequences by using a chloramphenicol acetyltransferase reporter gene assay. Nucleotide sequence analysis of this region revealed many consensus binding sequences for transcription factor SP1 as well as two potential CCAAT box binding factor sites and one putative helix-loop-helix transcription factor binding site. No TATA-like or "initiator" element sequences were found. Because of low steady-state levels of BCR mRNA and the high GC content (78%) of the promoter region, definitive mapping of transcription start sites required artificial amplification of BCR promoter-directed transcripts. Overexpression from the BCR promoter in a COS cell system was effective in demonstrating multiple transcription initiation sites. In order to assess the effects of chromosomal translocation on the transcriptional control of the BCR gene, we determined S1 nuclease protection patterns of poly(A)+ RNA from tumor cell lines. No differences were observed in the locations and levels of BCR transcription initiation sites between those lines that harbored the t(9;22) translocation and those that did not. This demonstrates that BCR promoter function remains intact in spite of genomic rearrangement. The BCR promoter is structurally similar to the ABL promoters. Together, this suggests that the structural fusion of BCR-ABL and not its transcriptional deregulation is primarily responsible for the transforming effect of the t(9;22) translocation.
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Makri, Maria, Chikashi Yoshida, Akihiko Muto, Kazuhiko Igarashi, and Junia V. Melo. "Transcriptional Regulation of the Bach2 B-Cell Differentiation and Apoptotic Factor by the Bcr-Abl Oncoprotein." Blood 106, no. 11 (November 16, 2005): 2991. http://dx.doi.org/10.1182/blood.v106.11.2991.2991.
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Abstract Transformation by the Bcr-Abl oncoprotein of CML is mediated by the activation of a variety of signalling pathways, leading to transcriptional regulation of genes conferring the malignant phenotype of increased proliferation, altered adhesion and inhibition of apoptosis. We previously reported that expression of the BACH2 gene is downregulated by Bcr-Abl. Bach2 is a B-lymphoid specific transcription factor, which regulates somatic hypermutation and class switch recombination of Ig genes. It is also a pro-apoptotic factor, coupling oxidative stress to transcription repression. It is possible that in an environment of increased genomic instability, Bcr-Abl transformed cells may repress pro-apoptotic signals by suppressing BACH2 transcription. To determine the direct association between Bcr-Abl and decreased BACH2 transcription, we infected human B-lymphoid cells with a retroviral vector expressing both p210Bcr-Abl and eGFP genes. Infected cells were treated with imatinib, an Abl tyrosine kinase inhibitor, prior to quantification of BACH2 transcripts by Real Time RT/PCR. Ectopic expression of BCR-ABL significantly decreased BACH2 mRNA levels, and this effect was completely abolished by imatinib. To investigate whether this regulation was exerted at the transcriptional level, we identified the BACH2 transcription initiation site (TIS), and then cloned and characterised a 3.9 Kb genomic DNA fragment including the BACH2 promoter region. By generating luciferase reporter constructs of various lengths of the BACH2 promoter we found that a region of 725 bp upstream the TIS conferred maximum promoter activity in human B-lymphoid cells. The effect of Bcr-Abl on promoter activity was demonstrated by co-transfection of the reporter and p210Bcr-Abl constructs. BACH2-promoter activity was reduced up to 60% in the presence of Bcr-Abl. Furthermore, when co-transfected cells were incubated with different concentrations of imatinib, the Bcr-Abl-mediated promoter repression was abrogated in a dose dependent manner, confirming the dependence of the effect on the tyrosine kinase activity of the oncoprotein. In support of these data, no effect on promoter activity was seen when the BACH2 promoter was co-transfected with a kinase-dead BCR-ABL construct. Moreover, treatment with imatinib of the BCR-ABL+ cell line BV173 transfected with the reporter induced a nearly 2-fold upregulation in its activity. Bioinformatics inspection of the promoter sequence revealed potential sites for the Pax5 B-cell differentiation factor and the Foxo3a transcription factor, a regulator of pro-apoptotic genes. In co-transfection experiments of either factor with the BACH2 promoter, both demonstrated a significant inducing effect on its activity. Gel shift and chromatin immunoprecipitation showed direct binding of Pax5 within the BACH2 promoter in vitro and in vivo. Moreover, Western analysis showed elevated Pax5 levels in BCR-ABL+ cell lines after imatinib treatment, indicating that inhibition of Bach2 expression by Bcr-Abl is mediated at least in part by Pax5. As to Foxo3a, it has been reported to be constitutively phosphorylated and inactivated in BCR-ABL+ cells, processes which prevent its translocation to the cell nucleus. Altogether, our data suggest that Bcr-Abl transcriptional repression of Bach2 via Pax5 could lead to a differentiation arrest in transformed B-cells, and that Foxo3a may induce imatinib-mediated apoptosis through up-regulation of the Bach2 apoptotic function.
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Burgess, Gem S., Elizabeth A. Williamson, Larry D. Cripe, Sara Litz-Jackson, Jay A. Bhatt, Kurt Stanley, Mark J. Stewart, Andrew S. Kraft, Harikrishna Nakshatri, and H. Scott Boswell. "Regulation of the c-jun Gene in p210 BCR-ABL Transformed Cells Corresponds With Activity of JNK, the c-jun N-Terminal Kinase." Blood 92, no. 7 (October 1, 1998): 2450–60. http://dx.doi.org/10.1182/blood.v92.7.2450.
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Abstract Activity of the c-jun N-terminal kinase (JNK) has been shown in hematopoietic cells transformed by p210 BCR-ABL. However, analysis has not been reported for hematopoietic cells on the consequences of this activity for c-jun promoter regulation within its distinctive proximal 8-base consensus CRE-like element, an element linked to JNK-mediated increase in c-jun transcription. In the present study, regulation of the proximal c-jun promoter was studied in murine myeloid cells transformed by p210 BCR-ABL. Promoter regulation in p210 BCR-ABL transformed cells was compared with regulation of the promoter in nontransformed interleukin-3 (IL-3)–dependent parental cells. The composition of nuclear AP-1 proteins contained within cells with p210 BCR-ABL, and their binding to the c-jun promoter proximal CRE-like element, was compared with the composition and binding of AP-1 proteins in IL-3–treated parental cells without p210 BCR-ABL. The present analysis found fivefold increased c-jun transcription occurring in p210 BCR-ABL transformed murine myeloid cells possessing a corresponding magnitude of increased kinase activity of JNK, compared with IL-3–stimulated parental cells. Augmented JNK activity was accompanied by increased nuclear abundance of c-jun and c-fos proteins that bound specifically to the proximal c-jun promoter CRE element. Also, representative human leukemic cell lines expressing p210 BCR-ABL and possessing abundant kinase activity of JNK, when compared with parental cells that were deficient in JNK activity, had increased c-jun and c-fosproteins. Finally, to show the relevance of these observations in model systems, we studied blast cells from patients with Philadelphia chromosome–positive acute leukemic transformation, and observed comparable activities of JNK catalysis and c-jun/AP-1 protein relative to the cell lines that possessed p210 BCR-ABL and JNK activity. These studies provide a basis for investigating the set of downstream genes which augmented c-jun/AP-1 activity enlists in the process of transformation by p210 BCR-ABL.
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Burgess, Gem S., Elizabeth A. Williamson, Larry D. Cripe, Sara Litz-Jackson, Jay A. Bhatt, Kurt Stanley, Mark J. Stewart, Andrew S. Kraft, Harikrishna Nakshatri, and H. Scott Boswell. "Regulation of the c-jun Gene in p210 BCR-ABL Transformed Cells Corresponds With Activity of JNK, the c-jun N-Terminal Kinase." Blood 92, no. 7 (October 1, 1998): 2450–60. http://dx.doi.org/10.1182/blood.v92.7.2450.2450_2450_2460.
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Activity of the c-jun N-terminal kinase (JNK) has been shown in hematopoietic cells transformed by p210 BCR-ABL. However, analysis has not been reported for hematopoietic cells on the consequences of this activity for c-jun promoter regulation within its distinctive proximal 8-base consensus CRE-like element, an element linked to JNK-mediated increase in c-jun transcription. In the present study, regulation of the proximal c-jun promoter was studied in murine myeloid cells transformed by p210 BCR-ABL. Promoter regulation in p210 BCR-ABL transformed cells was compared with regulation of the promoter in nontransformed interleukin-3 (IL-3)–dependent parental cells. The composition of nuclear AP-1 proteins contained within cells with p210 BCR-ABL, and their binding to the c-jun promoter proximal CRE-like element, was compared with the composition and binding of AP-1 proteins in IL-3–treated parental cells without p210 BCR-ABL. The present analysis found fivefold increased c-jun transcription occurring in p210 BCR-ABL transformed murine myeloid cells possessing a corresponding magnitude of increased kinase activity of JNK, compared with IL-3–stimulated parental cells. Augmented JNK activity was accompanied by increased nuclear abundance of c-jun and c-fos proteins that bound specifically to the proximal c-jun promoter CRE element. Also, representative human leukemic cell lines expressing p210 BCR-ABL and possessing abundant kinase activity of JNK, when compared with parental cells that were deficient in JNK activity, had increased c-jun and c-fosproteins. Finally, to show the relevance of these observations in model systems, we studied blast cells from patients with Philadelphia chromosome–positive acute leukemic transformation, and observed comparable activities of JNK catalysis and c-jun/AP-1 protein relative to the cell lines that possessed p210 BCR-ABL and JNK activity. These studies provide a basis for investigating the set of downstream genes which augmented c-jun/AP-1 activity enlists in the process of transformation by p210 BCR-ABL.
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Zhu, Qin-Shi, Nora Heisterkamp, and John Groffen. "Unique organization of the human BCR gene promoter." Nucleic Acids Research 18, no. 23 (1990): 7119–25. http://dx.doi.org/10.1093/nar/18.23.7119.
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Wong, Wei. "PTEN as a tumor promoter." Science Signaling 9, no. 423 (April 12, 2016): ec84-ec84. http://dx.doi.org/10.1126/scisignal.aaf8449.
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PTEN is generally considered to be a tumor suppressor because it limits the activity of the PI3K-Akt pathway, which usually promotes cell survival. However, in pre-B cells transformed with BCR-ABL1 or NRASG12D, oncogenes common to acute lymphoblastic leukemia (ALL), Shojaee et al. found that deletion of Pten resulted in cell death, and mice transplanted with the transformed pre-B cells in which Pten was also deleted did not develop leukemia. Pten deletion in transformed pre-B cells resulted in increased phosphorylation of Akt, which is activated downstream of the pre-B cell receptor through the tyrosine kinase Syk. Pharmacological inhibition of Akt or Syk reduced cell death caused by Pten deletion; it also prevented the cell death of autoreactive B cells, which are eliminated through negative selection because the pre-BCR binds to self-antigen. Pten deletion did not affect the abundance of the tumor suppressor p53 or the survival of BCR-ABL1–transformed chronic myeloid leukemia (CML) cells. In contrast, Pten deletion in BCR-ABL1–transformed pre-B ALL cells triggered the phosphorylation of p53 and its accumulation, effects that required Akt activity. Overexpression of the myeloid transcription factor C/EBP-α converts cells of the B cell lineage to the myeloid lineage, and Pten deletion increased glycolysis to a greater extent in pre-B ALL cells than in myeloid-reprogrammed cells, as indicated by increased glucose consumption and lactate production and depletion of ATP. Analysis of a genetic database of human cancers indicated that PTEN deletions or point mutations were not detected in pre-B ALL patient samples, and PTEN abundance was increased in pre-B ALL patient samples compared to that in patient samples of other types of lymphomas and leukemias. PTEN knockdown reduced cell viability in four different patient-derived pre-B ALL cell lines, and pharmacological inhibition of PTEN increased AKT signaling; the phosphorylation and accumulation of p53; and glycolytic metabolism in human pre-B ALL cells. Thus, PTEN may be a potential therapeutic target for the treatment of pre-B ALL (see also Fortin et al.). S. Shojaee, L. N. Chan, M. Buchner, V. Cazzaniga, K. N. Cosgun, H. Geng, Y. H. Qiu, M. Dühren-von Minden, T. Ernst, A. Hochhaus, G. Cazzaniga, A. Melnick, S. M. Kornblau, T. G. Graeber, H. Wu, H. Jumaa, M. Müschen, PTEN opposes negative selection and enables oncogenic transformation of pre-B cells. Nat. Med. 22,379–387 (2016). [PubMed] J. Fortin, C. Bassi, T. W. Mak, PTEN enables the development of pre-B acute lymphoblastic leukemia. Nat. Med. 22, 339–340 (2016). [PubMed]
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Gary-Gouy, Hélène, Julie Harriague, Georges Bismuth, Cornelia Platzer, Christian Schmitt, and Ali H. Dalloul. "Human CD5 promotes B-cell survival through stimulation of autocrine IL-10 production." Blood 100, no. 13 (December 15, 2002): 4537–43. http://dx.doi.org/10.1182/blood-2002-05-1525.
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CD5 is a negative regulator of B-cell receptor (BCR) signaling that is up-regulated after BCR stimulation and likely contributes to B-cell tolerance in vivo. However, CD5 is constitutively expressed on the B-1 subset of B cells. Contrary to CD5− B-2 B cells, B-1 B cells are long-lived because of autocrine interleukin-10 (IL-10) production through unknown mechanisms. We demonstrate herein a direct relationship between CD5 expression and IL-10 production. Human peripheral blood CD5+ B cells produce more IL-10 than CD5− B cells after BCR activation. Introducing CD5 into CD5− B cells induces the production of IL-10 by activating its promoter and the synthesis of its mRNA. The cytoplasmic domain of CD5 is sufficient for this process. CD5 also protects normal human B cells from apoptosis after BCR stimulation while reducing the BCR-induced Ca2+ response. We conclude that CD5 supports the survival of B cells by stimulating IL-10 production and by concurrently exerting negative feedback on BCR-induced signaling events that can promote cell death.
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Binné, Ulrich K., Wolfgang Amon, and Paul J. Farrell. "Promoter Sequences Required for Reactivation of Epstein-Barr Virus from Latency." Journal of Virology 76, no. 20 (October 15, 2002): 10282–89. http://dx.doi.org/10.1128/jvi.76.20.10282-10289.2002.
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ABSTRACT A luciferase reporter system with stably transfected oriP plasmids in Akata Burkitt's lymphoma cells provides a quantitative assay for the BZLF1 Zp promoter in response to B-cell receptor (BCR) activation by cross-linking with anti-immunoglobulin. In this system, detailed kinetic studies of promoter activity are possible. Previously reported promoter elements upstream of −221 from the transcription start and the ZIIR sequence had little effect on the Zp promoter, but the ZI and ZIIIA elements were essential for early activation. The ZIIIB element mediates autoactivation. Mutation of the ZV repressor sequence greatly increased the induction of the promoter but did not make it constitutively active. Zp transcription in response to BCR cross-linking declined after a few hours; this decline was reduced and delayed by acyclovir or phosphonoacetic acid, indicating that viral DNA replication or a late viral gene can play a role in the switch off of the Zp promoter. Late expression of the LMP1 protein may account for this.
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Casolari, Debora A., Jun Ishiko, Michelle Perugini, Richard J. D'Andrea, and Junia V. Melo. "BCR-ABL-Induced Downregulation of GADD45G in Chronic Myeloid Leukemia." Blood 120, no. 21 (November 16, 2012): 3731. http://dx.doi.org/10.1182/blood.v120.21.3731.3731.
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Abstract Abstract 3731 Chronic myeloid leukemia (CML) is characterized by the presence of the BCR-ABL oncogene which encodes an activated tyrosine kinase. Despite the success of tyrosine kinase inhibitors (TKI), such as imatinib (IM), in CML treatment, a considerable percentage (12–50%) of patients develops resistance to TKIs. This can result in progression to blast crisis (BC), at which stage durable response to any TKI therapy is minimal. It is still unclear how additional genetic lesions, believed to play a major role in the transformation to BC, are generated, but it is likely that the BCR-ABL induction of DNA damage and low-fidelity DNA repair, added to inhibition of apoptosis contributes to this process. The Growth Arrest and DNA Damage 45 (GADD45A, B and G) proteins are tumor suppressors which coordinate cell cycle arrest, DNA repair and apoptosis in response to genotoxic and oncogenic stress. Failure to activate GADD45 expression, e.g. due to the presence of leukemic oncogenes or its promoter methylation, is associated with an attenuated DNA-damage response and impaired drug response. A microarray study showed that GADD45G is in the top 10% of genes downregulated in CML patients in BC compared to those in chronic phase (CP). Given the critical role of GADD45 proteins as key regulators of the cellular stress response, our hypothesis is that GADD45G reduced expression in CML-BC is mediated by BCR-ABL and contributes to the accumulation of mutations seen in BC. Therefore the aims of this work are to determine the mechanism of GADD45G downregulation by BCR-ABL and its functional effects on CML cells. We have confirmed the reduced GADD45G levels in CD34+ cells from BC patients versus CP and normal controls using quantitative RT-PCR. Furthermore, ectopic expression of BCR-ABL in a myeloid cell line resulted in decreased expression of GADD45G and, conversely, BCR-ABL inhibition with IM in several CML cell lines resulted in induction of GADD45G expression. These results confirm GADD45G as a down-modulated target of BCR-ABL. To determine the effect of GADD45G expression on CML cells, a CML cell line was transduced with GADD45G and showed significantly reduced proliferation, G1 cell cycle arrest and decrease in cell viability due to increased apoptosis. Once the GADD45G tumor suppressor effect in CML was confirmed, we investigated the mechanism by which BCR-ABL inhibits its expression. Silencing of GADD45G expression in solid tumors can occur by promoter methylation; therefore, we analyzed the GADD45G promoter methylation status in the presence or absence of BCR-ABL. Bisulfite genomic sequencing of BCR-ABL-positive and –negative hematopoietic cell lines and on peripheral blood mononuclear cells from CML-CP and BC patients, as well as of normal controls, showed no correlation between the presence of BCR-ABL and promoter methylation. Another mechanism possibly employed by BCR-ABL to regulate GADD45G expression is interference with transcription factor (TF) binding to the promoter. Bioinformatics analysis revealed, among several other TF binding sites, the presence of two highly conserved RUNX1/AML1 binding sites on the promoter. The TF RUNX1 is a key controller of hematopoiesis and heterodimerises with CBF-β to regulate gene expression. RUNX1 mutations in AML result in lower GADD45A expression and increase in DNA damage. In CML, RUNX1 was reported as mutated in >30% of patients in BC, and was suggested to cooperate with Bcr-Abl in the induction of AML in mice. It has also been shown to be a target of inhibitory phosphorylation by SRC kinases, which display increased activity in CML-BC. We therefore co-transfected a CML cell line with a GADD45G promoter luciferase reporter and both RUNX1 and CBF-β constructs. This resulted in a 10-fold increase in luciferase activity, indicating that RUNX1 is involved in the regulation of GADD45G expression in CML cells. Overall, our results suggest that GADD45G has a tumor suppressor role in CML and that its down-regulation by BCR-ABL could, therefore, contribute to the accumulation of DNA damage and progression to BC. In addition, our data identify RUNX1 as an inducer of GADD45G expression and, together with data from the literature, suggest that RUNX1 mutations or protein inactivation in CML could result in the observed reduced GADD45G expression. We are currently investigating this possibility. Disclosures: No relevant conflicts of interest to declare.
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Umemura, Tsukuru, Tatsuki Shibuta, Emi Honda, Hiromichi Shiotsu, Yuka Tanaka, Shalini Vellasamy, and Motoaki Shiratsuchi. "An Epigenetic Mechanism Of Imatinib Via Demethylation Of MiR-203." Blood 122, no. 21 (November 15, 2013): 3766. http://dx.doi.org/10.1182/blood.v122.21.3766.3766.
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Abstract Background MicroRNAs (miRNAs) are short noncoding RNAs regulating a variety of biological processes by post-transcriptionally silencing via targeting mRNA. Recently there are many reports demonstrating that epigenetic alterations correlate to the characteristics of tumor cells, and that miRNAs were also reported to be regulated by methylation of CpG islands within the promoter region. MiR-203 is epigenetically silenced in human BCR-ABL1-positive leukemic cell lines and primary chronic myelogenous leukemia (CML) cells by the methylation of promoter region. In this study, we analyzed the effect of imatinib, a tyrosine-kinase inhibitor specific to BCR-ABL1 protein, on the expression of miRNA in BCR-ABL1-positive cells. Materials & Methods Two CML cell lines (K562 and KU812) and one AML cell line (HL60) were treated with imatinib for 72 hours. Microarray analysis of miRNAs was conducted by 3D-Gene (TORAY) in K562 cells with/without imatinib. Methylation specific PCR and bisulfite direct sequencing was performed to evaluate methylation status of promoter region of miR-203. Validation of expressions of miRNAs, including miR-203, and mRNAs was analyzed by RT-qPCR. The expression of BCR-ABL protein was confirmed by Western blotting. The function of miR-203 for cell survival was evaluated by the transfection of anti-miRNA. Results The microarray analysis showed that 48 miRNAs of CpG-rich 212 miRNAs were upregulated over 2-fold after imatinib treatment, in K562 cells. The demethylated state of the promoter region of miR-203, one of 48 miRNAs, was confirmed by bisulfite direct sequencing. The expression of BCR-ABL mRNA, which is one of the target of miR-203, was inhibited with imatinib to 52% and 26% of the level in control cultures in K562 cells and KU812 cells, respectively. The expression of BCR-ABL protein was also inhibited. The addition of anti-miR-203 increased the expression level of BCR-ABL protein to 68.1% in the K562 cell culture with imatinib treatment. The expression of DNA methyltransferase (DNMT) mRNA was analyzed, and the expressions of DNMT1 and DNMT3B were significantly decreased after imatinib treatments in CML cell lines, whereas the expression of DNMT3A was not changed. Discussion & Conclusion We report, for the first time, that imatinib up-regulated miR-203 by inducing demethylation of the promoter region of miR-203 in CML cells. MiR-203 is the important miRNA to inhibit ABL1 and BCR-ABL1 mRNA, and imatinib-induced demethylation of miR-203 is the possible mechanism to suppress growth of BCR-ABL1-positive leukemic cells. It was suggested that the demethylation was partially caused by down regulation of DNMT1 and DNMT3B after imatinib treatments in CML cell lines. In conclusion, imatinib not only inhibits the activity of tyrosine kinase but also induces DNA demethylation of miR-203 in CML cells. Disclosures: No relevant conflicts of interest to declare.
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Mayerhofer, Matthias, Peter Valent, Wolfgang R. Sperr, James D. Griffin, and Christian Sillaber. "BCR/ABL induces expression of vascular endothelial growth factor and its transcriptional activator, hypoxia inducible factor-1α, through a pathway involving phosphoinositide 3-kinase and the mammalian target of rapamycin." Blood 100, no. 10 (November 15, 2002): 3767–75. http://dx.doi.org/10.1182/blood-2002-01-0109.
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Recent data suggest that vascular endothelial growth factor (VEGF), a cytokine involved in autocrine growth of tumor cells and tumor angiogenesis, is up-regulated and plays a potential role in myelogenous leukemias. In chronic myelogenous leukemia (CML), VEGF is expressed at high levels in the bone marrow and peripheral blood. We show here that the CML-associated oncogene BCR/ABL induces VEGF gene expression in growth factor–dependent Ba/F3 cells. Whereas starved cells were found to contain only baseline levels of VEGF mRNA, Ba/F3 cells induced to express BCR/ABL exhibited substantial amounts of VEGF mRNA. BCR/ABL also induced VEGF promoter activity and increased VEGF protein levels in Ba/F3 cells. Moreover, BCR/ABL was found to promote the expression of functionally active hypoxia-inducible factor-1 (HIF-1), a major transcriptional regulator of VEGF gene expression. BCR/ABL-induced VEGF gene expression was counteracted by the phosphoinositide 3-kinase (PI3-kinase) inhibitor LY294002 and rapamycin, an antagonist of mammalian target of rapamycin (mTOR), but not by inhibition of the mitogen-activated protein kinase pathway. Similarly, BCR/ABL-dependent HIF-1α expression was inhibited by the addition of LY294002 and rapamycin. Together, our data show that BCR/ABL induces VEGF- and HIF-1α gene expression through a pathway involving PI3-kinase and mTOR. BCR/ABL-induced VEGF expression may contribute to the pathogenesis and increased angiogenesis in CML.
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Maltzman, J. S., J. A. Carmen, and J. G. Monroe. "Transcriptional regulation of the Icam-1 gene in antigen receptor- and phorbol ester-stimulated B lymphocytes: role for transcription factor EGR1." Journal of Experimental Medicine 183, no. 4 (April 1, 1996): 1747–59. http://dx.doi.org/10.1084/jem.183.4.1747.
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Intercellular adhesion molecule (ICAM) 1/CD54 plays an important role in T cell dependent B cell activation and for function of B lymphocytes as antigen-presenting cells. ICAM-1 expression is upregulated as a consequence of B lymphocyte antigen receptor (BCR) signaling, thereby serving to render antigen-stimulated B cells more receptive to T cell-mediated costimulatory signals. We have investigated BCR-induced expression of the Icam-1 gene in primary B cells and B cell lines and have found it to be dependent on BCR-induced expression of the transcription factor EGR1. Icam-1 transcription, induced by BCR cross-linking or bypassing the BCR with phorbol ester, is absent in a B cell line in which the EGR1-encoding gene (egr-1) is methylated and not expressed. A potential EGR1-binding site was located at -701 bp upstream of the murine Icam-1 gene transcription start site and shown by electrophoretic mobility shift assay to bind to murine EGR1. Mutation of this site in the context of 1.1 kb of the Icam-1 promoter significantly abrogated transcriptional induction by phorbol ester and anti-mu stimulation in primary B cells. A direct effect of EGR1 on the Icam-1 promoter is suggested by the ability of EGR1 expressed from an SV40-driven expression vector transactivate the wild-type Icam-1 promoter, whereas mutation of the EGR1 mutation of the EGR1 binding motif at -701 bp markedly compromises this induction. These data identify EGR1 as a signaling intermediate in BCR-stimulated B cell functional responses, specifically linking BCR signal transduction to induction of the Icam-1 gene. Furthermore, similar findings for BCR-induced CD44 gene induction (Maltzman, J.S., J.A. Carman, and J.G. Monroe. 1996. Role of EGR1 in regulation of stimulus-dependent CD44 transcription in B lymphocytes. Mol. Cell. Biol. In press) suggest that EGR1 may be an important signaling molecule for regulating levels of migration and adhesion molecules during humoral immune responses.
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JuszczynsKi, Przemyslaw, Linfeng Chen, Jose M. Polo, Stella M. Ranuncolo, Riccardo Dalla-Favera, Ari Melnick, and Margaret A. Shipp. "BCL6 Regulates Tonic BCR Signaling in Diffuse Large B-Cell Lymphomas by Repressing the SYK Phosphatase, PTPROt." Blood 112, no. 11 (November 16, 2008): 802. http://dx.doi.org/10.1182/blood.v112.11.802.802.
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Abstract Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease in which subsets of tumors likely rely upon different survival pathways. We previously identified a subset of primary DLBCLs with increased abundance of multiple components of the BCR signaling cascade including the spleen tyrosine kinase, SYK (“BCR-type” tumors). In related studies, we found that BCR-type DLBCL cell lines and primary tumors exhibited tonic B-cell receptor signaling. Since BCR-associated SYK activation initiates downstream events and amplifies the original BCR signal, we previously evaluated the efficacy of targeted SYK inhibition. Inhibition of SYK-dependent tonic BCR signaling induced apoptosis in the majority of “BCR-type” cell lines and primary DLBCLs. Furthermore, an oral SYK inhibitor (FosD) had promising activity in a subset of relapsed/refractory DLBCLs in a recently completed phase I/II clinical trial. “BCR-type” tumors also have more abundant expression of the transcriptional repressor, BCL6, and more frequent BCL6 translocations, greater repression of BCL6 target genes and increased sensitivity to BCL6 inhibitors. Since the same subset of primary DLBCLs relies upon SYK-dependent BCR signaling and BCL6-mediated transcriptional repression, we evaluated potential connections between BCL6 and SYK. SYK is a major substrate of the tissue-specific and developmentally regulated protein tyrosine phosphatase, PTPROt. For this reason, we first assessed the relative expression of BCL6 and PTPROt in two large independent series of transcriptionally profiled primary DLBCLs and two independent groups of highly purified normal B-cell subpopulations (naïve, germinal center, memory). There was a highly significant reciprocal pattern of expression of PTPROt and BCL6 in both normal B cells and primary DLBCLs, prompting speculation that PTPROt might be a target of BCL6-mediated transcriptional repression. We next analyzed the PTPROt promoter region in silico and identified 3 candidate BCL6 binding sites. Using chromatin immunoprecipitation assays in BCR-type DLBCL cell lines, we demonstrated that BCL6 binding sites in the PTPROt promoter were occupied in vivo by the transcription factor. To assess the role of BCL6 in regulating PTPROt transcription, we cotransfected a luciferase reporter vector driven by the PTPROt promoter with constructs encoding either wild-type BCL6 or one of two inactive BCL6 mutants. Wild-type BCL6 suppressed PTPROt-driven luciferase activity in a dose-dependent manner whereas neither BCL6 mutant altered luciferase levels. Consistent with these observations, individual or combined mutations of the BCL6 binding sites in the PTPROt promoter reduced or abolished the response to BCL6. Next, we asked whether PTPROt was a physiologic target of BCL6 in normal B cells. Forced overexpression of BCL6 in normal naïve B cells was associated with a marked reduction in PTPROt transcript abundance. Since PTPROt functions as a SYK phosphatase, we then assessed the consequences of BCL6 depletion on SYK Y352 phosphorylation and BCR signaling in BCR-type DLBCL cell lines. BCL6 siRNA increased PTPROt transcript abundance 10-fold whereas other components of the BCR signaling pathway were unchanged. In cells transduced with BCL6 siRNA, tonic and anti-Ig associated phosphorylation of SYK Y352 and BLNK Y84 were significantly lower than in parental or mock-transduced cells. Taken together, these studies indicate that BCL6 represses the expression of the SYK phosphatase, PTPROt, and augments SYK-dependent tonic BCR signaling. Since BCL6 and SYK are both promising rational therapeutic targets in many DLBCLs, combined inhibition of these functionally related pathways warrants further study.
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Maltzman, J. S., J. A. Carman, and J. G. Monroe. "Role of EGR1 in regulation of stimulus-dependent CD44 transcription in B lymphocytes." Molecular and Cellular Biology 16, no. 5 (May 1996): 2283–94. http://dx.doi.org/10.1128/mcb.16.5.2283.
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The immediate-early gene egr-1 encodes a transcription factor (EGR1) that links B-cell antigen receptor (BCR) signals to downstream activation events through the regulation of previously unidentified target genes. Here we identify the gene encoding the lymphocyte homing and migration protein CD44 as a target of EGR1 regulation in B cells. BCR-induced increases in CD44 mRNA expression and transcription levels are shown to occur in EGR1-expressing but not in nonexpressing subclones of the B-cell line WEHI-231. Kinetics of egr-1 transcription and the appearance of nuclear EGR1 protein precede CD44 induction and occur within 30 min after stimulation in the EGR1-expressing subclone. A single EGR1 binding motif is demonstrated at bp -301 of the human CD44 promoter. Cotransfection of a CD44 promoter-chloramphenicol acetyltransferase reporter construct with an egr-1 expression vector resulted in a 6.5- to 8.5-fold induction of transcriptional activity relative to an empty expression vector. The EGR1 binding motif was shown to be necessary for stimulus-induced expression of a CD44 promoter-chloramphenicol acetyltransferase reporter construct in nontransformed B lymphocytes and was required for transactivation by an EGR1 expression vector in a B-cell line. These studies identify EGR1 as an intermediary linking BCR-derived signals to the induction of CD44. The relevance of these molecular events to BCR signal transduction and antigen-stimulated B-cell-mediated immune responses is discussed.
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Gesbert, Franck, and James D. Griffin. "Bcr/Abl activates transcription of theBcl-X gene through STAT5." Blood 96, no. 6 (September 15, 2000): 2269–76. http://dx.doi.org/10.1182/blood.v96.6.2269.
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Abstract Several tyrosine kinase oncogenes have been associated with myeloproliferative diseases, including Bcr/Abl, Tel/Abl, Tel/Jak2, and Tel/PDGFR. One target molecule shared by these oncogenes is known to be STAT5. We generated sublines of Ba/F3 cells in which either wild-type STAT5 or a constitutively active mutant of STAT5 (STAT5-1*6) were expressed under the control of a tetracycline-inducible promoter. These cell lines were compared with a Ba/F3 cell line in which the expression of p210Bcr/Abl was made inducible by a similar promoter. Before induction, all cells were dependent on interleukin 3 (IL-3) for growth and survival. Both STAT5-1*6 and Bcr/Abl enhanced viability and induced proliferation in the absence of IL-3. We found that the proviability protein Bcl-XL, but not Bcl-2, was induced by both p210Bcr/Abl and STAT5-1*6. Using a Bcl-X gene promoter construct fused to a luciferase complementary DNA (cDNA), both p210Bcr/Abl and STAT5-1*6 were shown to induce transcription of Bcl-X. The increase in transcription of the Bcl-X promoter and the increase in Bcl-X protein, due to p210Bcr/Abl, were blocked by expression of a dominant negative STAT5 mutant. Interestingly, however, STAT5-1*6 required the continued presence of IL-3 to cause a significant increase in Bcl-XL protein, whereas p210Bcr/Abl did not need IL-3. Studies with enzyme inhibitors suggest that the extra signal supplied by IL-3 may be supplied by the PI3K pathway. Overall, these data suggest that constitutively activated STAT5 can increase viability and proliferation of Ba/F3 cells. This may contribute to, but is not likely sufficient for, the enhanced viability associated with Bcr/Abl transformation.
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Gesbert, Franck, and James D. Griffin. "Bcr/Abl activates transcription of theBcl-X gene through STAT5." Blood 96, no. 6 (September 15, 2000): 2269–76. http://dx.doi.org/10.1182/blood.v96.6.2269.h8002269_2269_2276.
Full textAbstract:
Several tyrosine kinase oncogenes have been associated with myeloproliferative diseases, including Bcr/Abl, Tel/Abl, Tel/Jak2, and Tel/PDGFR. One target molecule shared by these oncogenes is known to be STAT5. We generated sublines of Ba/F3 cells in which either wild-type STAT5 or a constitutively active mutant of STAT5 (STAT5-1*6) were expressed under the control of a tetracycline-inducible promoter. These cell lines were compared with a Ba/F3 cell line in which the expression of p210Bcr/Abl was made inducible by a similar promoter. Before induction, all cells were dependent on interleukin 3 (IL-3) for growth and survival. Both STAT5-1*6 and Bcr/Abl enhanced viability and induced proliferation in the absence of IL-3. We found that the proviability protein Bcl-XL, but not Bcl-2, was induced by both p210Bcr/Abl and STAT5-1*6. Using a Bcl-X gene promoter construct fused to a luciferase complementary DNA (cDNA), both p210Bcr/Abl and STAT5-1*6 were shown to induce transcription of Bcl-X. The increase in transcription of the Bcl-X promoter and the increase in Bcl-X protein, due to p210Bcr/Abl, were blocked by expression of a dominant negative STAT5 mutant. Interestingly, however, STAT5-1*6 required the continued presence of IL-3 to cause a significant increase in Bcl-XL protein, whereas p210Bcr/Abl did not need IL-3. Studies with enzyme inhibitors suggest that the extra signal supplied by IL-3 may be supplied by the PI3K pathway. Overall, these data suggest that constitutively activated STAT5 can increase viability and proliferation of Ba/F3 cells. This may contribute to, but is not likely sufficient for, the enhanced viability associated with Bcr/Abl transformation.
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Maeda, Shintaro, Ken Morita, Kensho Suzuki, Hiroshi Sugiyama, Souichi Adachi, and Yasuhiko Kamikubo. "Targeting Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia with a Novel Transcriptional Inhibitor." Blood 128, no. 22 (December 2, 2016): 2737. http://dx.doi.org/10.1182/blood.v128.22.2737.2737.
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Abstract Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ALL) is caused by reciprocal translocation between chromosome 9 (region q34) and chromosome 22 (region q11). This translocation results in oncogenic BCR-ABL fusion gene that encodes a chimeric BCR-ABL protein, continuously activated tyrosine kinase which causes unregulated cell division and leukemia. Although tyrosine kinase inhibitors (TKIs) are the most useful treatment against Ph+ALL patients, a part of them steadily become refractory to current therapy mainly through acquired mutations in ATP binding sites of BCR-ABL, necessitating a novel strategy to treat TKI resistant Ph+ALL. Here we show that our novel small molecule which targets specific transcriptional factor induces Ph+ALL cell death through directly controlling the expression of BCR-ABL gene. We have previously reported the fundamental requirement of transcription factor A (TFA) in the development of acute myeloid leukemia (AML), another form of acute leukemia originating from myeloid progenitor cells. Since previous reports suggest the involvement of TFA family genes in the development of lymphoid cells also, we first addressed the role of TFA in Ph+ALL cells using tetracycline-responsive short hairpin RNA (shRNA)-mediated gene knockdown system. Upon doxycycline treatment, shRNAs targeting TFA (sh_TFA)-transduced SU-Ph2 (Ph+ALL) and SU/SR (SU-Ph2 with BCR-ABL T315I mutation, therefore resistant to Imatinib) cells showed decreased proliferation rate with cell cycle arrest at G0/G1 phase and profound apoptosis. Intriguingly, TFA knockdown suppressed the expression of BCR-ABL fusion gene both at mRNA and protein levels. Concomitant down-regulation of BCR-ABL cytoplasmic gene targets such as mTOR-AKT, STAT and MEK-ERK pathways were also observed in sh_TFA-transduced ALL cells. These results indicate the possible interaction between BCR-ABL and TFA. Since expression of BCR-ABL fusion gene is tightly regulated by gene promoter of BCR andthere exists a TFA-consensus binding site in the proximal promoter region of BCR, we next addressed the TFA-mediated transcriptional regulation of BCR-ABL. In silico data analysis of gene expression array sets extracted from ALL patients revealed the positive correlation of BCR and TFA expressions. Analysis of chromatin immunoprecipitation and sequencing (ChIP-Seq) in mouse ES cells disclosed the potential TFA binding in the proximal regulatory region of BCR, which we have confirmed by ChIP assay in SU/SR cells. These findings suggest that TFA positively and directly controls BCR-ABLexpression through BCR promoter binding in ALL cells. We next sought to explore the effect of TFA inhibition in ALL cells in vivo. Immunodeficient NOG mice transplanted with SU/SR cells that are transduced with sh_TFA or control vector were followed for overall survival and chimerism of ALL cells in the peripheral blood. As expected, TFA inhibition decelerated the dissemination of leukemia and prolonged overall survival period in these mice. We next challenged this ALL-xenografted mice with newly synthesized small compound which irreversibly attenuates the function of TFA as a transcription factor. Existence of T315I mutation in BCR-ABL gene in SU/SR cells naturally provides TKI-therapy resistance to these mice, and oral Imatinib treatment at 100 mg/kg body weight did not prolonged their survivals actually. In contrast, our novel compound was highly effective in these mice at 375 microgram/kg body weight and significantly prolonged the overall survival period without serious side effects, overcoming TKIs resistance. These findings collectively underscore the importance of TFA in the maintenance of ALL cells with resistance to TKIs and is a druggable target in the leukemia therapy. Disclosures No relevant conflicts of interest to declare.
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Kollmann, Karoline, Gerwin Heller, Rene Georg Ott, Ruth Scheicher, Eva Zebedin-Brandl, Christine Schneckenleithner, Olivia Simma, et al. "c-JUN promotes BCR-ABL–induced lymphoid leukemia by inhibiting methylation of the 5′ region of Cdk6." Blood 117, no. 15 (April 14, 2011): 4065–75. http://dx.doi.org/10.1182/blood-2010-07-299644.
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Abstract The transcription factor c-JUN and its upstream kinase JNK1 have been implicated in BCR-ABL–induced leukemogenesis. JNK1 has been shown to regulate BCL2 expression, thereby altering leukemogenesis, but the impact of c-JUN remained unclear. In this study, we show that JNK1 and c-JUN promote leukemogenesis via separate pathways, because lack of c-JUN impairs proliferation of p185BCR-ABL–transformed cells without affecting their viability. The decreased proliferation of c-JunΔ/Δ cells is associated with the loss of cyclin-dependent kinase 6 (CDK6) expression. In c-JunΔ/Δ cells, CDK6 expression becomes down-regulated upon BCR-ABL–induced transformation, which correlates with CpG island methylation within the 5′ region of Cdk6. We verified the impact of Cdk6 deficiency using Cdk6−/− mice that developed BCR-ABL–induced B-lymphoid leukemia with significantly increased latency and an attenuated disease phenotype. In addition, we show that reexpression of CDK6 in BCR-ABL–transformed c-JunΔ/Δ cells reconstitutes proliferation and tumor formation in Nu/Nu mice. In summary, our study reveals a novel function for the activating protein 1 (AP-1) transcription factor c-JUN in leukemogenesis by antagonizing promoter methylation. Moreover, we identify CDK6 as relevant and critical target of AP-1–regulated DNA methylation on BCR-ABL–induced transformation, thereby accelerating leukemogenesis.
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Chen, Huan, Jin Lou, Heng Wang, Na An, Yuming Pan, Weihong Chen, Xin Du, and Qiaoxia Zhang. "MXD1 Regulates the Imatinib Resistance of Chronic Myeloid Leukemiacells By Repressing BCR-ABL1 Expression." Blood 132, Supplement 1 (November 29, 2018): 5119. http://dx.doi.org/10.1182/blood-2018-99-113362.
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Abstract Tyrosine kinase inhibitors have achieved unprecedented efficacy in the treatment of chronic myeloid leukemia (CML), however, imatinib resistance has emerged as a major problem in the clinic. Because the overexpression of BCR-ABL1 critically contributes to CML pathogenesis and drug resistance, targeting the regulation of BCR-ABL1 gene expression may be a novel therapeutic strategy. In this study, we found that the transcriptional repressor MXD1 showed low expression in CML patients and was negatively correlated with BCR-ABL1. Overexpression of MXD1 markedly inhibited the proliferation of K562 cells and sensitized the imatinib-resistant K562/G01 cell line to imatinib, with decreased BCR-ABL1 mRNA and protein. Further investigations using reporter gene analysis showed that MXD1 significantly inhibited the transcriptional activity of the BCR-ABL1 gene promoter. Taken together, these data show that MXD1 functions as a negative regulator of BCR-ABL1 expression and subsequently inhibits proliferation and sensitizes CML cells to imatinib treatment. Disclosures No relevant conflicts of interest to declare.
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Schulz, Alexandra, Yoon Jung Park, Rainer Claus, Hamid Kashkar, Jens Seeger, Julia Claasen, Christian P. Pallasch, et al. "TOSO Deficiency Exerts Impact On BAFF-R Expression, Thereby Causing Impaired B Cell Development in Vivo." Blood 120, no. 21 (November 16, 2012): 3898. http://dx.doi.org/10.1182/blood.v120.21.3898.3898.
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Abstract Abstract 3898 Apoptosis resistance concomitant with aberrant upregulation of pro-survival pathways is a main pathogenic mechanism in development and maintenance of chronic lymphocytic leukemia (CLL). Our group recently identified TOSO to be significantly overexpressed in CLL compared to other B cell lymphomas or healthy B cells. Interestingly, TOSO is thought to exert pro-survival signaling, although it remains still enigmatic, how TOSO is regulated and why TOSO is expressed extremely heterogeneous on different B cells entities. Moreover, we previously detected elevated TOSO expression to be associated with progressive disease, including unmutated IgVH status of the B cell receptor (BCR). Since the BCR is a driving force in CLL, TOSO expression was investigated after BCR crosslinking and resulted in an increase of TOSO. To date, the TOSO promoter has not been described yet. Here, we firstly identified the TOSO proximal region to exert promoter activity. Moreover, in silico analysis and phylogenetic footprinting exhibited existence of transcription factor binding sites for NF-κB and BCL6. In luciferase reporter assays, including targeted mutagenesis, NF-κB was confirmed as novel inducer of TOSO expression. Whereas BCL6 binding, confirmed by ChIP and luciferase assays, was shown to exert repressing activity on the TOSO promoter. Although it can explained now how TOSO is regulated by the BCR, the reason for its distinct basal expression levels in normal B cells and other B cell malignancies still remained unclear. Our data illustrate for the first time that DNA hypomethylation of the TOSO promoter is a conspicuous characteristic in CLL patients compared to healthy donors. Indeed, the methylation status seems to play a major role, since the methylation level correlates with TOSO expression also in other B cell lymphomas. Moreover, it is indispensible to clarify the biologic significance of TOSO, particularly in the CLL relevant B cells. Therefore, we generated a B cell-specific knockout mouse model and identified impaired B cell development characterized by diminished B cell count. Gene expression analysis and flow cytometry revealed a decrease of the B-cell activating factor receptor (BAFF-R). BAFF-R ligation is known to promote B cell survival in particular via degradation of IκBα and translocation of NF-κB to the nucleus, thus activating the NF-κB pathway. Thus, BAFF-R decrease caused by TOSO depletion might lead to the detected reduction of B lymphocytes, which corresponds to the previous observations. Taken together, this work reveals a counteractive TOSO regulation by transcription activator NF-κB and transcription repressor BCL6 in a BCR-dependent manner in B cells. Moreover, we detected CLL-specific hypomethylation of the TOSO promoter, which is supposed to be causative for elevated TOSO level in CLL. Moreover, our results might reveal a new function of TOSO in pro-survival signaling and B cell homeostasis, supporting the anti-apoptotic feature of TOSO in B cells. Identifying the regulating mechanisms and biological function of the anti-apoptotic TOSO, is an essential step towards elucidation of the underlying molecular causes for the development of CLL. Disclosures: No relevant conflicts of interest to declare.
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Horita, Machiko, Enrique Jose Andreu, Adalberto Benito, Cristina Arbona, Cristina Sanz, Isana Benet, Felipe Prosper, and Jose Luis Fernandez-Luna. "Blockade of the Bcr-Abl Kinase Activity Induces Apoptosis of Chronic Myelogenous Leukemia Cells by Suppressing Signal Transducer and Activator of Transcription 5–Dependent Expression of Bcl-XL." Journal of Experimental Medicine 191, no. 6 (March 13, 2000): 977–84. http://dx.doi.org/10.1084/jem.191.6.977.
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Bcr-Abl–expressing leukemic cells are highly resistant to apoptosis induced by chemotherapeutic drugs. Although a number of signaling molecules have been shown to be activated by the Bcr-Abl kinase, the antiapoptotic pathway triggered by this oncogene has not been elucidated. Here, we show that the interleukin 3-independent expression of the antiapoptotic protein, Bcl-xL, is induced by Bcr-Abl through activation of signal transducer and activator of transcription (Stat)5. Inhibition of the Bcr-Abl kinase activity in Bcr-Abl–expressing cell lines and CD34+ cells from chronic myelogenous leukemia (CML) patients induces apoptosis by suppressing the capacity of Stat5 to interact with the bcl-x promoter. Interestingly, after inhibition of the Bcr-Abl kinase, the expression of Bcl-xL is downregulated more rapidly in chronic phase than in blast crisis CML cells, suggesting an involvement of this protein in disease progression. Overall, we describe a novel antiapoptotic pathway triggered by Bcr-Abl that may contribute to the resistance of CML cells to undergo apoptosis.
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Lavens, Sandra, Emmanuel A. Faust, Fang Lu, Michele Jacob, Messele Leta, Paul M. Lieberman, and Ellen Puré. "Identification of Protein Tyrosine Kinases Required for B-Cell- Receptor-Mediated Activation of an Epstein-Barr Virus Immediate-Early Gene Promoter." Journal of Virology 78, no. 16 (August 15, 2004): 8543–51. http://dx.doi.org/10.1128/jvi.78.16.8543-8551.2004.
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ABSTRACT Epstein-Barr Virus (EBV) is a potentially oncogenic herpesvirus that infects >90% of the world's population. EBV exists predominantly as a latent infection in B lymphocytes, with periodic lytic-cycle reactivation essential for cellular and host transmission. Viral reactivation can be stimulated by ligand-induced activation of B-cell-receptor (BCR)-coupled signaling pathways. The critical first step in the transition from latency to the lytic cycle is the expression of the viral immediate-early gene BZLF1 through the transcription activation of its promoter, Zp. However, the BCR-coupled signal transduction cascade(s) leading to the induction of Zp and the expression of the BZLF1 gene product, Zta, is currently unclear. A major obstacle to delineating the relevant signal transduction events has been the lack of a model of EBV infection that is amenable to genetic manipulation. The use of the avian B-cell line DT40 has proven to be a powerful tool for delineating BCR-mediated signal transduction pathways that appear to be highly conserved between avian and mammalian systems. We demonstrate that the DT40 cell line is a robust and genetically tractable system for the study of BCR-mediated signaling pathways leading to transcriptional activation of BZLF1. Using this system, we demonstrate that activation of Zp requires the BCR-coupled protein tyrosine kinases Syk and Btk and that it is positively regulated by Lyn. Thus, the use of DT40 cells has allowed us to delineate the early signaling components required for BCR-dependent reactivation of latent EBV, and this system is likely to prove useful for further dissection of the downstream signaling cascades involved.
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Marega, Manuela, Rocco Piazza, Iris Meneghetti, Sara Redaelli, Angela Mogavero, and Carlo Gambacorti. "BCR and BCR/ABL Regulation during Myeloid Differentiation in Healthy Donors and in Chronic Phase/Blast Crisis CML Patients." Blood 112, no. 11 (November 16, 2008): 3204. http://dx.doi.org/10.1182/blood.v112.11.3204.3204.
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Abstract The fusion protein BCR/ABL leads to chronic myeloid leukaemia (CML). The corresponding fusion gene is under the transcriptional control of BCR promoter. It is known that in CML progenitors the ability to block myeloid differentiation is directly related to BCR/ABL levels. These observations open new questions about BCR/ABL and BCR expression control. However, up to date only few studies have been focused on the characterization of the BCR promoter, so little is known about the transcriptional regulation of this gene. We studied BCR expression in sorted myeloid precursors in healthy donors (HD) and in CML patients in Chronic Phase (CP) and Blast Crisis (BC). CML samples were also analyzed for BCR/ABL. The expression level was analyzed by Real Time PCR normalized against GUS as housekeeping gene, in haematopoietic stem cells (HSCs defined as CD34+/CD38−/Thy+/−), common myeloid and granulocyte-monocyte progenitors (CMPs and GMPs, respectively defined as CD34+/CD38+/IL−3Ralo/CD45RA− and CD34+/CD38+/IL−3Ralo/CD45RA+,) sorted by Fluorescence Activated Cell Sorting (FACS). Preliminarly, we analyzed the BCR and BCR/ABL mRNA halflife. We performed a RNA stability assay using Actinomycin D in the K562 cell line and in CP and BC samples: the halflife of both genes were comparable thus excluding major differences in RNA stability. Then, we analyzed BCR levels in healthy donors. A statistically significant BCR downregulation was noted during myeloid maturation in HD (HSCs [0.815±0.27SD] vs CMPs [0.176±0.1] and vs GMPs [0.167±0.17], p=0.0079 in both cases). The same analysis performed in CP patients showed that both BCR and BCR/ABL were downregulated upon committement to differentiation (BCR: HSCs [0.326±0.24] vs CMPs [0.0899±0.054], p=0.0078, and vs GMPs [0.0277±0.07], p=0.0008; BCR/ABL: HSCs [2.99±1.76] vs CMPs [0.781±0.41], p=0.0005 and vs GMPs [0.304±0.46], p=0.028). However, while BCR levels were lower in CML samples compared to HD (HSC samples [0.815±0.27 vs 0.326±0.24]: p=0.0120), probably due to haploinsufficiency, BCR/ABL values were higher than BCR ones, when compared to CML (HSCs: p=0.0020) and even to HD samples (HSCs: p=0.0074). These data indicate that in CML a potent upregulation of BCR/ABL compared to BCR is present, probably caused by a selective mechanism acting on BCR/ABL. In a limited set of BC samples (4 patients), the decrease of BCR and BCR/ABL was less evident and did not reach statistical significance (BCR: HSCs 0.339±0.235; CMPs: 0.135±0.12; GMPs: 0.126±0.06; BCR/ABL: HSCs: 8.58±5.78; CMPs: 5.55±7.39; GMPs: 3.36±2.61); thus as already indicated by Jamieson and colleagues (NEJM, 351:657, 2004), in BC the downregulation of BCR/ABL during maturation is impaired. Moreover, we detected a tendency for higher expression of BCR/ABL in BC than CP in all the subpopulations under study, which reached statistical significance only in GMPs (HSCs [8.58±5.78 BC vs 2.99±1.76 CP]: p=0.1483; CMPs [5.55±7.39 BC vs 0.781±0.41 CP]: p=0.1483; GMPs [3.36±2.61 BC vs 0.304±0.46 CP]: p=0.028); BCR was upregulated only in GMPs (HSCs [BC 0.339±0.235 vs 0.326±0.24 CP]: p=0.9399; CMPs [0.135±0.12 vs 0.0899±0.054]: p=0.4818; GMPs [0.126±0.06 vs 0.0277±0.07]: p=0.0755). In conclusion, BCR is physiologically downregulated during myeloid maturation, and this regulation is conserved in CP-CML, not only for BCR but also for BCR/ABL. In BC-CML, this mechanism of regulation seems to be impaired for BCR/ABL and possibly for BCR too. In addition, a potent upregulation of BCR/ABL compared to BCR is present in all stages of CML, probably caused by a selective mechanism acting on BCR/ABL.
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Tomasello, Luisa, Marzia Vezzalini, Christian Boni, Massimiliano Bonifacio, Luigi Scaffidi, Mohamed Yassin, Nader Al-Dewik, Paul Takam Kamga, Mauro Krampera, and Claudio Sorio. "Regulative Loop between β-catenin and Protein Tyrosine Receptor Type γ in Chronic Myeloid Leukemia." International Journal of Molecular Sciences 21, no. 7 (March 26, 2020): 2298. http://dx.doi.org/10.3390/ijms21072298.
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Protein tyrosine phosphatase receptor type γ (PTPRG) is a tumor suppressor gene, down-regulated in Chronic Myeloid Leukemia (CML) cells by the hypermethylation of its promoter region. β-catenin (CTNNB1) is a critical regulator of Leukemic Stem Cells (LSC) maintenance and CML proliferation. This study aims to demonstrate the antagonistic regulation between β-catenin and PTPRG in CML cells. The specific inhibition of PTPRG increases the activation state of BCR-ABL1 and modulates the expression of the BCR-ABL1- downstream gene β-Catenin. PTPRG was found to be capable of dephosphorylating β-catenin, eventually causing its cytosolic destabilization and degradation in cells expressing PTPRG. Furthermore, we demonstrated that the increased expression of β-catenin in PTPRG-negative CML cell lines correlates with DNA (cytosine-5)-methyl transferase 1 (DNMT1) over-expression, which is responsible for PTPRG promoter hypermethylation, while its inhibition or down-regulation correlates with PTPRG re-expression. We finally confirmed the role of PTPRG in regulating BCR-ABL1 and β-catenin phosphorylation in primary human CML samples. We describe here, for the first time, the existence of a regulative loop occurring between PTPRG and β-catenin, whose reciprocal imbalance affects the proliferation kinetics of CML cells.
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Kumarylakshmikuttyamma, Asha, Elodie Pastural, John F. DeCoteau, and C. Ronald Geyer. "BCR-ABL Induces Autocrine IGF-1 Signaling." Blood 110, no. 11 (November 16, 2007): 4545. http://dx.doi.org/10.1182/blood.v110.11.4545.4545.
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Abstract BCR-ABL is responsible for the initial phase of chronic myelogenous leukemia (CML), which is effectively treated by the BCR-ABL tyrosine kinase inhibitor imatinib. Over time, patients become resistant to treatment and progress to blast crisis, an event that is driven by additional genetic and epigenetic aberrations. Recently, we showed that RIZ1 expression decreases in blast crisis and that re-expression of RIZ1 inhibits IGF-1 expression. IGF-1 signaling is required in many stages of hematopoiesis and inappropriate activation of autocrine IGF-1 signaling may facilitate transformation to blast crisis. In this study, we examined mechanisms used by CML blast crisis cells to activate IGF-1 expression. We monitored the effects of small molecule inhibitors and shRNAs of BCR-ABL, HCK, and STAT5b on IGF-1 expression, proliferation, and apoptosis in CML blast crisis cell lines. We used chromatin immunoprecipitation assays to confirm that STAT5b associates with the IGF-1 promoter in a BCR-ABL-dependent manner. We found that BCR-ABL activates autocrine IGF-1 signaling using HCK and STAT5b. Imatinib blocks HCK phosphorylation, STAT5b phosphorylation, and the association of STAT5b with the IGF-1 promoter. Inhibition of these signaling components using small molecule drugs or shRNA inhibits IGF-1 expression, decreases proliferation, and enhances apoptosis. The effects of imatinib are partially reversed by the addition of exogenous IGF-1. Furthermore, treatment of CML blast crisis cell lines with the IGF-1 receptor inhibitor AG1024 decreases proliferation and enhances apoptosis and this activity correlates with IGF-1 expression levels. Our study highlights autocrine IGF-1 signaling as an important event in the transformation to blast crisis and provides a novel mechanism to explain the activity of IGF-1R and HCK inhibitors in blocking blast crisis phenotypes. Together, our results provide further support for the therapeutic potential of IGF-1 signaling components in treating CML, especially in cases associated with elevated autocrine IGF-1 expression.
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Vidovic, Karina, Emelie Svensson, Ake Borg, Johan Vallon-Christersson, Carin Lassen, Petra Hakansson, Johan Richter, Tor Olofsson, Thoas Fioretos, and Urban Gullberg. "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." Blood 108, no. 11 (November 16, 2006): 4322. http://dx.doi.org/10.1182/blood.v108.11.4322.4322.
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Abstract 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.
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Riether, Carsten, Christian M. Schürch, Ramin Radpour, Magdalena Hinterbrandner, Anne-Laure Huguenin, and Adrian F. Ochsenbein. "CD70/CD27 Signaling Mediates Resistance of Chronic Myeloid Leukemia Stem Cells to Tyrosine Kinase Inhibitors By Compensatory Activation of the Wnt Pathway." Blood 124, no. 21 (December 6, 2014): 400. http://dx.doi.org/10.1182/blood.v124.21.400.400.
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Abstract The introduction of BCR/ABL-specific tyrosine kinase inhibitors (TKIs) a decade ago revolutionized chronic myelogenous leukemia (CML) therapy. However, disease-initiating leukemia stem cells (LSCs) in CML are resistant to TKIs despite BCR/ABL inhibition. Therefore, CML will ultimately relapse upon drug discontinuation. We have previously shown that blocking CD70/CD27 signaling targets LSCs by inhibiting the activation of the Wnt pathway. Here, we investigated a combination therapy of TKIs and CD70/CD27 blocking monoclonal antibodies in human and murine CML. We demonstrate that TKI-mediated BCR/ABL inhibition down-regulates miR-29, leading to increased expression of specificity protein 1 (SP1), a transcription factor with binding site in the CD70 promoter. In addition, TKI treatment reduced the expression of DNA methyltransferases resulting in de-methylation of the CD70 promoter. These combined effects resulted in CD70 up-regulation on LSCs, enhanced CD70/CD27 signaling and compensatory Wnt pathway activation. Combined BCR/ABL and CD70/CD27 inhibition synergistically reduced Wnt signaling and eradicated leukemia cells in vitro. More importantly, combination therapy effectively eliminated CD34+ CML stem/progenitor cells in murine xenografts and LSCs in a murine CML model. Therefore, TKI-induced CD70 up-regulation triggers CD70/CD27 signaling leading to compensatory Wnt activation. These findings identify an important targetable TKI resistance mechanism of CML LSCs and may lead to new therapeutic strategies to directly target LSCs to overcome treatment resistance. Disclosures No relevant conflicts of interest to declare.
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Thompson, AA, WJ Jr Wood, MJ Gilly, MA Damore, SA Omori, and R. Wall. "The promoter and 5' flanking sequences controlling human B29 gene expression." Blood 87, no. 2 (January 15, 1996): 666–73. http://dx.doi.org/10.1182/blood.v87.2.666.bloodjournal872666.
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The product of the B-cell-specific B29 gene (B29, Ig beta, CD79b) is essential for Ig-mediated B-cell activation via the B-cell antigen receptor complex (BCR) on human and murine B lymphocytes. To better understand the regulation of this pivotal gene, we have analyzed the human genomic DNA sequence upstream of the B29 ATG start codon for transcriptional control activity. The human B29 gene lacks either a TATA or a CAAT box and transcription is initiated at multiple sites. The minimal promoter of the human B29 gene is contained within a 193-bp region 5′ of these multiple start sites. This minimal promoter exhibits B-cell-specific activity and contains SP1, ETS, OCT, and IKAROS/LYF-1 transcription factor motifs. All these motifs are strikingly conserved in sequence and placement relative to the previously characterized murine B29 promoter. Additional upstream gene segments dramatically affected B29 minimal promoter activity. A newly identified motif called the B29 conserved sequence (BCS), found upstream of both human and murine B29 promoters, appears to stimulate B29 transcription through a novel mechanism. A single BCS had little effect either on the minimal B29 promoter or on a heterologous promoter. Instead, the BCS stimulated transcription by counteracting 5′ negative regulatory DNA sequences that block the activity of the B29 minimal promoter in its absence. These findings indicate that B29 gene expression is controlled by the complex interplay of positive and negative regulatory elements.
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Burke, B. A., and M. Carroll. "BCR–ABL: a multi-faceted promoter of DNA mutation in chronic myelogeneous leukemia." Leukemia 24, no. 6 (May 6, 2010): 1105–12. http://dx.doi.org/10.1038/leu.2010.67.
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Sengupta, Amitava, Ashley M. Ficker, Susan K. Dunn, Malav Madhu, and Jose A. Cancelas. "Bmi1 reprograms CML B-lymphoid progenitors to become B-ALL–initiating cells." Blood 119, no. 2 (January 12, 2012): 494–502. http://dx.doi.org/10.1182/blood-2011-06-359232.
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The characterization and targeting of Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL)–initiating cells remains unresolved. Expression of the polycomb protein Bmi1 is up-regulated in patients with advanced stages of chronic myelogenous leukemia (CML). We report that Bmi1 transforms and reprograms CML B-lymphoid progenitors into stem cell leukemia (Scl) promoter-driven, self-renewing, leukemia-initiating cells to result in B-lymphoid leukemia (B-ALL) in vivo. In vitro, highly proliferating and serially replatable myeloid and lymphoid colony-forming cultures could be established from BCR-ABL and Bmi1 coexpressing progenitors. However, unlike in vivo expanded CML B-lymphoid progenitors, hematopoietic stem cells, or multipotent progenitors, coexpressing BCR-ABL and Bmi1 did not initiate or propagate leukemia in a limiting dilution assay. Inducible genetic attenuation of BCR-ABL reversed Bmi1-driven B-ALL development, which was accompanied by induction of apoptosis of leukemic B-lymphoid progenitors and by long-term animal survival, suggesting that BCR-ABL is required to maintain B-ALL and that BCR-ABL and Bmi1 cooperate toward blast transformation in vivo. Our data indicate that BCR-ABL targeting itself is required to eradicate Ph+/Bmi1+ B-ALL–initiating cells and confirm their addiction to BCR-ABL signaling.
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Juszczynski, Przemyslaw, Linfeng Chen, Evan O'Donnell, Jose M. Polo, Stella M. Ranuncolo, Riccardo Dalla-Favera, Ari Melnick, and Margaret A. Shipp. "BCL6 modulates tonic BCR signaling in diffuse large B-cell lymphomas by repressing the SYK phosphatase, PTPROt." Blood 114, no. 26 (December 17, 2009): 5315–21. http://dx.doi.org/10.1182/blood-2009-02-204362.
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Abstract Tonic B-cell receptor (BCR) signaling is a key survival pathway during normal B-cell ontogenesis and in a subset of diffuse large B-cell lymphomas (DLBCLs). We previously demonstrated that BCR-dependent DLBCL cell lines and primary tumors underwent apoptosis after treatment with an ATP-competitive inhibitor of the BCR-associated spleen tyrosine kinase (SYK). These “BCR-type” tumors also have more abundant expression of the transcriptional repressor, BCL6, and increased sensitivity to BCL6 inhibition. Herein, we evaluated potential connections between BCL6-mediated transcriptional repression and SYK-dependent BCR signaling. In transcriptionally profiled normal B-cell subsets (naive, germinal center, and memory B cells) and in primary DLBCLs, there were reciprocal patterns of expression of BCL6 and the SYK tyrosine phosphatase PTPROt. BCL6 repressed PTPROt transcription via a direct interaction with functional BCL6 binding sites in the PTPROt promoter. Enforced expression of BCL6 in normal naive B cells and RNAi-mediated depletion of BCL6 in germinal center B cells directly modulated PTPROt expression. In “BCR-type” DLBCLs, BCL6 depletion increased PTPROt expression and decreased phosphorylation of SYK and the downstream adaptor protein BLNK. Because BCL6 augments BCR signaling and BCL6 and SYK are both promising therapeutic targets in many DLBCLs, combined inhibition of these functionally related pathways warrants further study.
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Perazzona, Bastianella, Yan Wang, and Ralph B. Arlinghaus. "Role of BCR in Down-Modulation of BCR-ABL Oncogenicity in CML." Blood 112, no. 11 (November 16, 2008): 3210. http://dx.doi.org/10.1182/blood.v112.11.3210.3210.
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Abstract Bcr-Abl acquires its transforming ability through its up-regulated Abl tyrosine kinase activity. Bcr is a phosphoprotein with a novel serine/threonine kinase activity encoded by its first exon. Over-expression of BCR in K562 cells produces a phosphoserine form of Bcr and interferes with the oncogenic effects of BCR-ABL in mice (Lin et al., Oncogene 2001). We have recently shown the inhibitory effects of Bcr on Bcr-Abl, in a nude mouse solid tumor model. Expression of BCR/GFP in TonB210 cells used for injection delayed tumor formation and tumors were 50% smaller compared to the TonB210/GFP control. In contrast two point mutants in the BCR kinase domain (Y360F and S354A), not only blocked Bcr’s inhibitory effects but enhanced the oncogenic effects of BCRABL (Perazzona et.al. Oncogene, 2008). Similar Bcr effects were observed in a mouse leukemia model. We are investigating the mechanism of the interaction between Bcr and Bcr-Abl proteins. Using TonB210 cells, in which BCR-ABL expression is controlled by a tetracycline-inducible promoter and Bcr is stably transduced by lentivirus infection, we observed that increasing levels of Bcr-Abl expression increased the levels of the Bcr protein. Treatment of TonB210 cells with imatinib mesylate decreased the levels of Bcr- Abl and surprisingly the Bcr protein as well, indicating that the tyrosine kinase function of Bcr-Abl is required to up-regulate Bcr protein expression. In addition, withdrawal of doxycycline also reduced Bcr-Abl and Bcr protein levels, confirming that Bcr-Abl is required for increased expression of the Bcr protein. In order to examine the levels of Bcr in cells lacking Bcr-Abl, we transduced BCR/GFP with lentivirus infection into BaF3 and 32D cells. Surprisingly, these cell lines expressed extremely low levels of Bcr, despite 90% expression of the GFP marker. Expression of Bcr was restored by overnight treatment with the proteasome inhibitor calpain inhibitor I. Forced expression of Bcr-Abl in BCR- transduced cells restored high expression of Bcr protein, confirming that Bcr- Abl is required for preventing degradation of the Bcr protein. Together these findings indicate that Bcr-Abl up-regulated Bcr expression by interfering with proteasomemediated degradation of the Bcr protein. Additional studies indicated that Bcr increases expression of the myeloid membrane surface marker Mac-1 in Bcr-Abl TonB210 cells, which originated from the mouse pro-B cell BaF3. We propose that Bcr may play a role in generating the myeloid phenotype caused by Bcr-Abl in CML patients and may be an important player in the chronic phase of CML by down-modulating Bcr-Abl.
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Katsuta, Hitoshi, Sachiyo Tsuji, Yoshiyuki Niho, Tomohiro Kurosaki, and Daisuke Kitamura. "Cutting Edge: Lyn-Mediated Down-Regulation of B Cell Antigen Receptor Signaling: Inhibition of Protein Kinase C Activation by Lyn in a Kinase-Independent Fashion." Journal of Immunology 160, no. 4 (February 15, 1998): 1547–51. http://dx.doi.org/10.4049/jimmunol.160.4.1547.
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Abstract Stimulation of the B cell Ag receptor (BCR) induces activation of tyrosine kinases such as Lyn and Syk, phosphorylation and activation of multiple signaling components, and eventually, the expression of several genes including c-myc. Syk is required for activation of phospholipase C-γ2 and the subsequent phosphatidylinositol hydrolysis, leading to protein kinase C (PKC) activation and intracellular Ca2+ increase. In contrast, the function of Lyn remains obscure. Here, we report that BCR-mediated induction of c-myc promoter activity and of PKC activity, but not the expression level of functional PKC, was markedly augmented in Lyn-deficient chicken B cells. This enhancement was reversed to the level of wild-type cells by the expression of exogenous Lyn of kinase-inactive form. These results indicate that Lyn inhibits BCR-mediated activation of a large portion of PKC isozymes in a kinase-independent fashion. This finding reveals a novel role of Lyn in negative regulation of BCR signaling.
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Hjort, Elizabeth, Weiqi Huang, and Elizabeth A. Eklund. "Bcr-Abl Represses IRF8 Gene Transcription in a Stat5-Dependent Manner." Blood 120, no. 21 (November 16, 2012): 3497. http://dx.doi.org/10.1182/blood.v120.21.3497.3497.
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Abstract Abstract 3497 The interferon consensus sequence binding protein is a member of the interferon regulatory factor family of transcription factors (referred to as Icsbp or Irf8). The first described functions for Icsbp involved regulation of phagocyte and B-cell effector genes, including genes encoding components of the phagocyte NADPH-oxidase, Toll-like receptors and interleukin receptors. However, subsequent studies in murine models and human disease indicated that Icsbp also functions as a myeloid leukemia suppressor. For example, decreased Icsbp expression is found in chronic myeloid leukemia (CML) in association with uncontrolled disease, drug resistance and progression to blast crisis (BC). Decreased Icsbp expression is also found in the bone marrow of subjects with some subtypes of acute myeloid leukemia (AML). Consistent with this clinical correlative data, IRF8−/− mice exhibit a myeloproliferative neoplasm that is similar to CML and progresses to BC over time. However, the mechanism for decreased Icsbp expression in leukemia is not known, although preliminary studies indicate that DNA-methylation of the IRF8 locus is not altered. Therefore, in these studies, we investigate the effects of Bcr-abl on IRF8 transcription. This is clinically relevant, because previous studies in our laboratory identified a set of Icsbp-target-genes that contribute to the pathogenesis of CML. We find that Bcr-abl decreases expression of Icsbp mRNA and protein in a kinase dependent manner. Since it is unlikely that Bcr-abl directly binds to the promoter to regulate gene transcription, we hypothesized that Bcr-abl regulates IRF8 through an intermediary transcription factor. In this study, we determine that Stat5 negatively regulates IRF8 transcription through a proximal promoter cis-element. We also find that Stat5 repression activity is necessary for Bcr-abl dependent regulation of IRF8. Bcr-abl is known to phosphorylate and activate Stat5 in CML. In our studies, we find that Stat5 protein (but not mRNA) is also increased in Bcr-abl+ cells. Stat5 is a known substrate for calpain; a serine protease. We previously demonstrated that Icsbp regulates calpain protease activity through repression of the gene encoding Gas2; an endogenous calpain inhibitor. Consistent with this, our current studies demonstrate that Stat5 protein stability is increased in Bcr-abl+ cells in an Icsbp/Gas2/calpain-dependent manner. These results identify novel mechanisms by which Bcr-abl-kinase activity controls a positive feedback loop that leads to decreased Icsbp expression and stabilization of Stat5 protein. These studies suggest that targeting Gas2/calpain might be a novel therapeutic approach to CML. Disclosures: No relevant conflicts of interest to declare.
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Tian, Yamin, Yan Dong, Seiichiro Kobayashi, Manabu Ozawa, Kiyoko Izawa, Yuansong Bai, Yasushi Soda, et al. "Leukemogenic Fusion Gene (p190 BCR-ABL) Transduction Into Hematopoietic Stem/Progenitor Cells In the Common Marmoset." Blood 116, no. 21 (November 19, 2010): 4323. http://dx.doi.org/10.1182/blood.v116.21.4323.4323.
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Abstract Abstract 4323 [Introduction] Patients with Philadelphia chromosome (p190 BCR-ABL fusion gene)-positive acute lymphoblastic leukemia have a poor prognosis despite intensive therapeutic intervention. Although a rodent model of this leukemia was previously established, the genetic and physiological differences between humans and rodents make it difficult to extrapolate the results from these models and apply these findings to human cases. Primates are more genetically related to humans than rodents. In this study, we attempted to develop a leukemia non-human primate model that mimics various human systems. [Methods and results] (1) A third-generation VSV.G pseudotyped lentiviral vector expressing the p190 BCR-ABL fusion gene driven by CMV or PGK promoter was produced (HIV-CMV/PGK-BCR-ABL). Ba/F3 cells, a mIL-3-dependent murine hematopoietic cell line, were transfected with this vector and cultured without mIL-3. These cells rapidly expanded after 12 days, indicating that p190 BCR-ABL gene expression allowed the Ba/F3 cells to grow autonomously. Next, using a biotin-labeled anti-marmoset CD34 monoclonal antibody (clone MA24) which was produced in our laboratory, MACS-sorted bone marrow CD34+ cells were transduced with the lentiviral vector (HIV-CMV/PGK-BCR-ABL) and subjected to the colony formation assay. In the majority of examined colonies, p190 BCR-ABL gene was detected regardless of the promoter. Taken together, the above findings indicate that p190 BCR-ABL gene was efficiently transduced into marmoset hematopoietic stem/progenitor cells. (2) Peripheral blood mononuclear cells (PBMNCs) were collected from individual marmosets after mobilizing the hematopoietic stem/progenitor cells with G-CSF. These cells were stimulated with cytokines (hIL3, hSCF and hTPO), followed by the transduction with the lentiviral vector. These cells were transplanted into marmosets preconditioned with busulfan. In this ex vivo transduction method, p190 BCR-ABL gene expression which was detected in PBMNCs by nested RT-PCR disappeared after day 56 and 100 in two marmosets. (3) Concentrated lentiviral vector was directly injected into the bone marrow cavity of individual marmosets pretreated with 5-fluorouracil and prednisolone. In this in vivo direct injection method, p190 BCR-ABL gene expression was maintained for more than one year and a half. Transduction of p190 BCR-ABL gene into hematopoietic stem/progenitor cells was confirmed by colony forming assay. In this model, one marmoset unexpectedly developed myelofibrosis-like disease. However, none of the marmosets have developed leukemia to date. [Conclusion] We successfully achieved sustained p190 BCR-ABL gene expression in vivo. This novel in vivo approach will help to develop a marmoset leukemia model in the future. Because a multiple-hit model of oncogenesis has been proposed for various human cancers, a genetic mutation in addition to p190 BCR-ABL may be required for the malignant transformation of hematopoietic stem/progenitor cells in the common marmoset. Disclosures: No relevant conflicts of interest to declare.
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Muvarak, Nidal E., Shannon M. Kelley, Mario Tarasco, Maria R. Baer, Kara A. Scheibner, and Feyruz Rassool. "C-MYC and C-MYC-Regulated Micrornas Increase The Activity Of The Error-Prone ALT NHEJ Pathway Through Upregulation Of LIG3 and PARP1 In Tyrosine Kinase-Activated Leukemias." Blood 122, no. 21 (November 15, 2013): 809. http://dx.doi.org/10.1182/blood.v122.21.809.809.
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Abstract Constitutively activated tyrosine kinases (TK) BCR-ABL1 and FLT3/ITD not only increase cell survival and proliferation, but also increase levels of endogenous DNA damage and activity of an error-prone DNA double-strand break (DSB) repair pathway. This genomic instability leads to acquisition of genomic alterations that can result in disease progression and/or resistance to therapy. We have previously demonstrated that, in TK-activated leukemias, activity of the classic non homologous end-joining (C-NHEJ) pathway that repairs DSBs is decreased, and, as a consequence, an alternative, highly error-prone form of NHEJ (ALT NHEJ) predominates, evidenced by increased expression of DNA ligase IIIα (LIG3) and PARP1 (components of ALT NHEJ), increased frequency of large DNA deletions, and repair using DNA microhomologies. In this study, we sought to elucidate the role of a key downstream target of TKs, c-MYC, in upregulating LIG3 and PARP1 expression and consequently increasing ALT NHEJ and genomic instability. We demonstrated that MYC increases the expression of LIG3 and PARP1 through two mechanisms: 1) Increased binding to the promoters of LIG3 and PARP1, leading to increased transcription, and 2) Repression of microRNAs (miRs) that putatively regulate LIG3 and PARP1. Chemical and siRNA-mediated knockdown of MYC in MO7e-BCR/ABL and FLT3/ITD(+) MOLM14 cells results in significant reduction (p<0.05) in LIG3 and PARP1 mRNA and protein compared to controls. Chromatin immunoprecipitation assays revealed MYC binding to the promoters of LIG3 and PARP1 in AML (MOLM14) and CML (K562 and MO7e-BCR/ABL) cell lines. Additionally, transfection of PARP1 and LIG3 promoter-luciferase constructs into TK-activated (32D-FLT3/ITD, MO7e-BCR/ABL) cells showed significantly (p<0.01) increased LIG3 and PARP1 promoter activity compared to parental controls (32D, MO7e). Moreover, knockdown of MYC in 32D-FLT3/ITD and MO7e-BCR/ABL cells resulted in a significant reduction of promoter activity in luciferase assays (p<0.05). Conversely, overexpression of c-MYC in 293T cells caused an increase (p<0.05) in LIG3 and PARP1 promoter activity. We next determined whether MYC-repressed miRs that have predicted binding sites in the 3’-UTR and coding regions of LIG3 and PARP1 are involved in regulating expression of LIG3 and PARP1. We found that there was a significant inverse correlation between LIG3 expression and miR-22, miR-23a, and miR-150 (Pearson’s r ≤ -0.3, p<0.05). Similarly, there was a significant inverse correlation between PARP1 expression and miR-22, miR-23a, miR-27a, and miR-150 (Pearson r ≤ -0.3, p<0.05). Over-expression of miR-22 in the CML cell line K562 decreased both LIG3 and PARP1 protein levels by 52% and 63% respectively. Similar results were seen upon over-expression of miR-34a (59% and 45%) and miR-150 (46% and 62%) for LIG3 and PARP1. This indicates that MYC-regulated miRs may function coordinately to regulate NHEJ repair. Importantly, our functional NHEJ assays demonstrate an overall significant (p<0.05) reduction in the average size of deletions at the sites of DSB repair when MYC is knocked down, indicating a reduction in ALT NHEJ activity. To determine whether increased expression of LIG3 and PARP1 correlated with MYC expression in primary leukemia samples, we examined mRNA levels from bone marrow of 21 CML patients (12 chronic phase, 1 accelerated, 7 blast crisis, and 1 unknown). Twelve patients were resistant to Imatinib, 7 were responsive, and 2 undetermined. There was a strong positive correlation between levels of MYC and PARP1 (Pearson’s r= 0.75, p=0.001), as well as MYC and LIG3 (Pearson’s r =0.45, p=0.03). While there was no correlation between levels of gene expression and disease phase, we found that the majority of samples with elevated levels of MYC, LIG3 and PARP1 were from Imatinib-resistant patients (64%), compared to samples from Imatinib-sensitive patients (36%) (p=0.03). Additionally, 2 patient samples with TKI-resistant T315I mutation in BCR-ABL1 exhibited elevated levels of MYC, LIG3 and PARP1. Thus, increased MYC expression, and repression of miRs 22, 150 and 34a augment expression of LIG3 and PARP1, generating DSB repair errors that may lead to resistance to TKI therapy. Altered expression of MYC, LIG3, PARP1 and miRs 22, 150 and 34a may be biomarkers for those patients likely to become resistant to TKI therapy. Disclosures: No relevant conflicts of interest to declare.
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Sanders, Mathijs A., Anikó Szabó, Carla Exalto, Remco Hoogenboezem, Annelieke Zeilemaker, Jasper E. Koenders, Peter van Geel, et al. "Extensive RAG-Mediated Rearrangements and Mutations in BCR-ABL1 and BCR-ABL1-like Adult Acute Lymphoblastic Leukemia." Blood 128, no. 22 (December 2, 2016): 4067. http://dx.doi.org/10.1182/blood.v128.22.4067.4067.
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Abstract BCR-ABL1 and BCR-ABL1-like acute lymphoblastic leukemia (ALL) are two major pre-B cell acute leukemia subtypes characterized by genetic alterations affecting lymphoid-specific transcription factors. Studies examining the chain of genetic events necessary to develop leukemia established that the BCR-ABL1 fusion gene and kinase-activating BCR-ABL1-like lesions are initiating events, however, insufficient for leukemia development. Secondary genetic events targeting B cell development genes are therefore an essential requirement for overt ALL. A recent study (Papaemmanuil et al, Nat. Genet., 2014) revealed that illegitimate RAG-mediated recombination is the predominant mutational mechanism establishing these secondary genetic events in ETV6-RUNX1 ALL. Of note, ETV6-RUNX1ALL is mainly restricted to pediatric cases and it remains unanswered whether this mutational process also plays a prominent role in adult ALL pathogenesis. We carried out a detailed genomic characterization to determine whether aberrant RAG activity is also a prominent mutational driver in certain adult B cell ALL (B-ALL) subtypes. Diagnostic material of 53 unselected B-ALL cases and matched remission specimens were characterized using DNA mapping arrays to discern copy number alterations (CNAs). We observed multiple BCR-ABL1/BCR-ABL1-like patients with abundant genetic lesions and selected 5 cases for targeted sequencing of CNA boundaries to determine whether these lesions were driven by RAG-mediated recombination. Whole genome sequencing (WGS) for a single BCR-ABL1-like patient was used to asses this mutational mechanism genome-wide. In total 64 structural variants (SVs) could be analyzed at base-pair level. De novo motif detection on breakpoint sequences revealed the prominence of the heptamer CACAGTG (E-value=5.68x10-91), a constituent of the recombination signal sequence (RSS), present in 121 out of 128 breakpoints (94.5%). RSS detection revealed that 58 out of 64 SVs (90.6%) had a cryptic RSS (cRSS) on one or both sides of the lesion. Incorporation of non-templated sequences was observed for 54 out of the 64 (84.4%) SVs. Superimposition of breakpoints on chromatin marks revealed a strong enrichment for active promoters and enhancers (p < 2.2x10-16). WGS data revealed cRSS motifs and incorporation of non-templated sequences for 23 out of 26 SVs (88.5%). Integrative analysis of all 6 cases confirmed 125 unique SV breakpoints strongly enriched for the active chromatin marks H3K4me3 and H3K27ac. STAT5 binding, a postulated regulator of V(D)J recombination, is similarly enriched at the breakpoints. Promiscuous binding of RAG1 and RAG2 was previously noted in human thymocytes and murine pre-B cells (Teng et al, Cell, 2015). Strikingly, the breakpoints are frequently bound by RAG2 in human thymocytes. In total 66 out of 125 breakpoints could be translated to the murine genome and revealed a strong enrichment of RAG1 and RAG2 binding at homologous positions in murine pre-B cells. Exhaustive mutation detection revealed complex somatic mutations within cRSS motifs, which are rare V(D)J recombination products introduced by erroneous cleavage and error-prone repair (open-and-shut joints). Strikingly, 4 out of 6 BCR-ABL1/BCR-ABL1-like cases had mutations in the BTLA promoter-situated cRSS, frequently in combination with a RAG-mediated deletion of the other allele (Figure 1). Genomic screening in 142 B-ALL patients confirmed 8 additional cases with BTLA promoter mutations, predominantly (6 out of 8) belonging to the BCR-ABL1/BCR-ABL1-like subgroups. We provide strong evidence that aberrant RAG activity plays a pivotal role in the development of BCR-ABL1/BCR-ABL1-like adult ALL. We demonstrate that breakpoints are strongly enriched for RAG binding implying a predisposition for illegitimate V(D)J recombination. Importantly, we report on a novel mutational mechanism introducing mutations in cRSS motifs through open-and-shut joints, frequently resulting in the biallelic inactivation of BTLA. Proliferation and V(D)J recombination during pre-B cell development is orchestrated by the interplay of IL7R and pre-BCR signalling. Strikingly, most kinase-activating lesions constitutively activate these signalling cascades and could enact, in concert with BTLA inactivation, constant proliferation, pro-survival and V(D)J recombination-initiating signals with disastrous consequences. Disclosures No relevant conflicts of interest to declare.
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Wang, Zhanxiang, Seiji Fukuda, and Louis M. Pelus. "Disruption of the Inhibitor-Of-Apoptosis Protein, Survivin, Sensitizes Bcr-abl Positive Cells to STI571 (Gleevec®)-Induced Apoptosis." Blood 104, no. 11 (November 16, 2004): 2578. http://dx.doi.org/10.1182/blood.v104.11.2578.2578.
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Abstract The Bcr-abl oncogene is found in cells of >95% of patients with CML and encodes a cytoplasmic protein with constitutive tyrosine kinase activity. Bcr-abl induces hematopoietic cell transformation and protects cells from apoptosis induced by numerous stimuli. Bcr-abl activates Myc, Ras, raf, Pl3K, and c-jun kinases that are critical for transforming activity, however, the signaling pathways between Bcr-abl and the apoptosis machinery are just beginning to be determined. It is known that Bcr-abl can exert an antiapoptotic effect by blocking mitochondrial release of cytochrome C. Although increased knowledge of Bcr-abl pathways resulted in the design of selective tyrosine kinase inhibitors such as STI571 (Gleevec), the development of drug resistance limits efficacy. Survivin is a member of the highly conserved inhibitor-of-apoptosis (IAP) family of endogenous caspase inhibitors. Similar to other IAPs, Survivin blocks apoptosis by inhibiting caspases 3, 7 and 9. However in contrast to other IAPs, Survivin is not expressed in most adult tissues but aberrantly overexpressed in all cancers and hematopoietic malignancies. Targeting of Survivin by antisense (AS) or dominant-negative (DN) strategies in transformed cell models induces apoptosis. Survivin is expressed in Bcr-abl+ CML cells in blast crisis, but not in cells from patients with Bcr-ablneg CML. Furthermore, high expression of Survivin is found in Adriamycin resistant Bcr-abl+ K562 cells. These findings led us to investigate whether Survivin is involved in the antiapoptotic effects of Bcr-abl and if Survivin disruption can facilitate apoptosis in Bcr-abl+ cells. Transient transfection of human Mo7e and mouse BaF3 hematopoietic cells with the Bcr-abl oncongene results in significantly elevated expression of Survivin mRNA and protein. The mRNA of 2 other Survivin splice variants, Survivin-2B and Survivin-ΔEx3 were also upregulated. In transfected Mo7e cells, Survivin promoter activity was upregulated 2–4 fold compared to parental Mo7e cells, determined using a luciferase reporter construct. In addition to Survivin, the IAP family member ILP was also upregulated by Bcr-abl. Disruption of Survivin expression/function in K562 cells that contain endogenous Bcr-abl and in Bcr-abl transfected Mo7e cells by ectopic expression of AS or DN T34A or C84A mutant Survivin constructs, significantly promoted apoptosis induced by the Bcr-abl tyrosine kinase inhibitor, Gleevec by ≥2-fold, in a time and dose dependent manner. Enhanced apoptosis induced by AS or DN Survivin was accompanied by caspase dependent cleavage of Bcr-abl oncoprotein (>50% decrease in Bcr-abl protein) that was blocked by the caspase inhibitor Z-VAD-fmk; disruption of mitochondria membrane potential (>35% increase in TMRMnegexpression); and enhanced cytochrome C release, quantified by westerns. Similarly, disruption of Survivin mRNA in K562 cells by a 20-mer antisense oligonucleotide resulted in >40% increase in mitochondrial disruption. In contrast, forced expression of wild-type Survivin in K562 cells protected cells from Gleevec-induced apopotosis. In summary, our results demonstrate that the Bcr-abl oncogene regulates Survivin transcription and production, which represents a new signaling pathway downstream of Bcr-abl that may be helpful in understanding the pathophysiology of CML. Targeted Survivin disruption may sensitize Bcr-abl+ CML cells to Gleevec-induced apoptosis and have therapeutic potential, particularly in the development of drug resistance.
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Sattler, Martin, Shalini Verma, Christopher H. Byrne, Gautam Shrikhande, Thomas Winkler, Paul A. Algate, Larry R. Rohrschneider, and James D. Griffin. "BCR/ABL Directly Inhibits Expression of SHIP, an SH2-Containing Polyinositol-5-Phosphatase Involved in the Regulation of Hematopoiesis." Molecular and Cellular Biology 19, no. 11 (November 1, 1999): 7473–80. http://dx.doi.org/10.1128/mcb.19.11.7473.
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ABSTRACT The BCR/ABL oncogene causes chronic myelogenous leukemia (CML), a myeloproliferative disorder characterized by clonal expansion of hematopoietic progenitor cells and granulocyte lineage cells. The SH2-containing inositol-5-phosphatase SHIP is a 145-kDa protein which has been shown to regulate hematopoiesis in mice. Targeted disruption of the murine SHIP gene results in a myeloproliferative syndrome characterized by a dramatic increase in numbers of granulocyte-macrophage progenitor cells in the marrow and spleen. Also, hematopoietic progenitor cells from SHIP−/−mice are hyperresponsive to certain hematopoietic growth factors, a phenotype very similar to the effects of BCR/ABL in murine cells. In a series of BCR/ABL-transformed hematopoietic cell lines, Philadelphia chromosome (Ph)-positive cell lines, and primary cells from patients with CML, the expression of SHIP was found to be absent or substantially reduced compared to untransformed cell lines or leukemia cells lacking BCR/ABL. Ba/F3 cells in which expression of BCR/ABL was under the control of a tetracycline-inducible promoter showed rapid loss of p145 SHIP, coincident with induction of BCR/ABL expression. Also, an ABL-specific tyrosine kinase inhibitor, CGP57148B (STI571), rapidly caused reexpression of SHIP, indicating that BCR/ABL directly, but reversibly, regulates the expression of SHIP protein. The estimated half-life of SHIP protein was reduced from 18 h to less than 3 h. However, SHIP mRNA also decreased in response to BCR/ABL, suggesting that SHIP protein levels could be affected by more than one mechanism. Reexpression of SHIP in BCR/ABL-transformed Ba/F3 cells altered the biological behavior of cells in culture. The reduction of SHIP due to BCR/ABL is likely to directly contribute to the pathogenesis of CML.
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Singh, Abhishek, Megha Dubey, Deepika Awasthi, Sheela Nagaroti, Manoj Barthwal, Rabindra Jena, Anil K. Tripathi, and Madhu Dikshit. "Glutathionylation of NF-Kb and Procaspase-3 Regulates Inducible Nitric Oxide Synthase Expression and Apoptosis of Chronic Myeloid Leukemia Cells." Blood 124, no. 21 (December 6, 2014): 3131. http://dx.doi.org/10.1182/blood.v124.21.3131.3131.
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Abstract 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. Disclosures No relevant conflicts of interest to declare.
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Sillaber, Christian, Franck Gesbert, David A. Frank, Martin Sattler, and James D. Griffin. "STAT5 activation contributes to growth and viability in Bcr/Abl-transformed cells." Blood 95, no. 6 (March 15, 2000): 2118–25. http://dx.doi.org/10.1182/blood.v95.6.2118.
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Abstract The transcription factor STAT5 is constitutively tyrosine phosphorylated and activated after transformation of hematopoietic cells by p210Bcr/Abl. A truncated form of STAT5B (▵STAT5; aa, 1-683) that lacks tyrosine 699 and the transcriptional activation domain was introduced into Ba/F3p210 cells under the control of a tetracycline-inducible promoter. Treatment of these cells with doxycycline, a tetracycline analogue, induced expression of ▵STAT5 and inhibited STAT5-dependent transcription. ▵STAT5 coprecipitated with STAT5 and decreased Bcr/Abl-dependent tyrosine phosphorylation of endogenous STAT5. Induction of ▵STAT5 inhibited growth of Ba/F3p210 cells (26%-52% of control levels at 4 days) but did not cause cell-cycle arrest. ▵STAT5 reduced viability of Ba/F3p210 cells and increased sensitivity of the cells to the cytotoxic drugs hydroxyurea and cytarabine. These results indicate that high-level expression of ▵STAT5, as achieved here by using a tetracycline-inducible promoter, inhibits STAT5 activity, reduces the growth rate of Ba/F3p210 cells by inhibiting viability, and results in increased sensitivity to chemotherapeutic drugs. It is therefore likely that STAT5 activation plays a role in the transformation of hematopoietic cell lines by p210Bcr/Abl.
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Dasgupta, Yashodhara, Mateusz Koptyra, Margaret Nieborowska-Skorska, Elisabeth Bolton Gillespie, Tomasz Stoklosa, Grazyna Hoser, Mariusz Wasik, Markus Muschen, Christine Richardson, and Tomasz Skorski. "Normal ABL1 Is a Tumor Suppressor and Therapeutic Target In BCR-ABL1–positive Leukemias." Blood 122, no. 21 (November 15, 2013): 1466. http://dx.doi.org/10.1182/blood.v122.21.1466.1466.
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Abstract BCR-ABL1 results from t(9;22)(q34;q11) reciprocal translocation resulting in BCR-ABL1 kinase expression, initiating chronic myeloid leukemia in chronic phase (CML-CP). At the initial stages of CML-CP both oncogenic BCR-ABL1 kinase and normal ABL1 kinase are expressed, however, loss of ABL1 kinase expression in CML-CP can result from an interstitial deletion in the normal chromosome 9 [del(9q34)] which may be combined with the transcriptional silencing of the alternative ABL1 promoter within the translocation eventually leading to disease progression and drug resistance. We found that BCR-ABL1 Abl1-/- cells generated a CML-blast phase (BP)-like disease phenotype in SCID mice compared to CML-CP-like disease from BCR-ABL1 Abl1+/+ cells. To determine the mechanisms responsible for blastic transformation of BCR-ABL1 Abl1-/- cells, we examined the role of ABL1 in proliferation, differentiation, apoptosis, genomic instability, and stemness. The presence of ABL1 inhibited proliferation in BCR-ABL1 cells as BCR-ABL1 Abl1-/- cells had higher clonogenic activity and proliferative rate compared to their wild-type counterparts. ABL1 is essential for myeloid differentiation since BCR-ABL1 Abl1-/- cells showed an immature blast phenotype when stained with Wright-Giemsa and myeloid differentiation markers Gr-1 and CD11b. ABL1 promoted apoptosis in response to genotoxic stress as revealed by reduced clonogenicity and elevated expression of p53, phosphoserine-15 p53 and activated caspase 3 in BCR-ABL1 Abl1 +/+ compared to knock-out cells. Although the absence of ABL1 did not enhance ROS and oxidative DNA damage, it appears that an impaired DNA damage response may be responsible for higher chromosome numbers and an accumulation of high numbers of chromosomal aberrations in BCR-ABL1 Abl1-/- cells. We detected an expansion of Lin-c-Kit+Sca-1+ leukemia stem cells (LSCs) in BCR-ABL1 Abl1-/- cells compared to BCR-ABL1 Abl1+/+ or non-transformed counterparts; among the LSCs, there was a higher percentage of CD34-Flt3- long-term and CD34+Flt3-short-term stem cells. These results showed that ABL1 is involved in regulating the LSC compartment in BCR-ABL1 cells. DNA microarray analysis revealed changes in mRNA levels of several genes involved in proliferation, myeloid differentiation, apoptosis, DNA damage response and stemness in BCR-ABL1 Abl1-/- cells in comparison to BCR-ABL1 Abl1+/+ cells. Together, these results demonstrated a critical role of ABL1 in BCR-ABL1-induced leukemia, prolonging survival in mice by suppressing proliferation and expansion of LSC, inducing myeloid differentiation, apoptosis and DNA damage response in BCR-ABL1 cells. Thus, it appears that ABL1 acts as a tumor suppressor in BCR-ABL1 –positive CML cells. Moreover, we hypothesized that the enhancement of the tumor suppressor function of ABL1 may have a significant impact on CML treatment. A small molecule activator of ABL1 kinase, 5-(1,3-diaryl-1H-pyrazol-4-yl)hydantoin (DPH), had been reported to interact with the myristoyl-binding site of ABL1 and destabilize the bent conformation of the α-1 helix, thereby preventing the auto-inhibitory conformation. DPH partially restored ABL1 activity in imatinib-treated cells. DPH-mediated stimulation of ABL1 tumor suppressor activity enhanced the effect of imatinib and ponatinib against CML CD34+ cells, Philadelphia chromosome-positive B-ALL (Ph+B-ALL) cells and relapsed Ph+B-ALL cells harboring T315I mutation without affecting normal counterparts. In summary, ABL1 is a potential tumor suppressor in BCR-ABL1-induced leukemia and stimulation of its function may play a significant role in the development of novel therapeutic strategies for CML and Ph+ALL. Disclosures: No relevant conflicts of interest to declare.
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Yasuda, Takahiko, Fumihiko Hayakawa, Shingo Kurahashi, and Tomoki Naoe. "PAX5 Phosphorylation by MAP Kinase Signal Potentially Suppresses PAX5 Function and Might Be a Trigger of the Plasma Cell Differentiation." Blood 116, no. 21 (November 19, 2010): 3898. http://dx.doi.org/10.1182/blood.v116.21.3898.3898.
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Abstract Abstract 3898 Pax5 is an essential transcription factor to maintain B cell identity. Pax5 is expressed in stages from pro-B to mature B cells and promotes the B cell differentiation program by transcriptional activation of many B cell receptor (BCR)-related genes such as CD19, CD79a, and BLNK. On the contrary, it inhibits plasma cell differentiation by suppressing the expression of Blimp1 and XBP-1, transcription factors essential for plasma cell differentiation. After BCR stimulation by antigen, upregulation of Blimp1 and XBP-1 and subsequent suppression of Pax5 by Blimp1 were observed and thought to be the trigger of plasma cell differentiation. However, the first trigger that represses PAX5 function is yet to be revealed. Here, we investigated the relevance between Pax5 and MAP kinase signal, a main component of BCR signal. We demonstrated PAX5 phosphorylation by ERK2 in vitro and in vivo (Figure. A). The sites of PAX5 phosphorylation were identified by PCR mutagenesis assay. In the luciferase reporter assays, PAX5 phosphorylation by MAP kinase signal reduced the transcriptional activity of PAX5 when CD19 promoter sequence was used for reporter gene. On the other hand, PAX5 caused transcriptional repression of Blimp1 promoter, which was released by PAX5 phosphorylation by MAP kinase signal. These data supported the reported findings that PAX5 had dual function, and suggested that the PAX5 function was inhibited by its phosphorylation. We demonstrated that B cell receptor stimulation with anti-IgM antibody induced strong MAP kinase activation, phosphorylation of endogenous Pax5 (Figure. B), and upregulation of Blimp1 mRNA (Figure. C). All of these phenomena were inhibited by U0126, MEK inhibitor. These results suggested that transcriptional repression of Blimp1 by PAX5 was released by its phosphorylation by MAP kinase signal. This might be a trigger of plasma cell differentiation. Our findings give a new insight into the regulation of the terminal differentiation of B cells. Disclosures: Hayakawa: Otsuka Pharmaceutical Co., Ltd.: Research Funding. Naoe:Chugai pharmaceutical, Zenyaku pharmaceutical, Kyowa-Kirin pharmaceutical, Dainippon-Sumitomo pharmaceutical, Novartis pharmaceutical, Janssen pharmaceutical, Otsuka pharmaceutical: Research Funding.
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Fernández de Mattos, Silvia, Abdelkader Essafi, Inês Soeiro, Alexandra M. Pietersen, Kim U. Birkenkamp, Corinne S. Edwards, Anthony Martino, et al. "FoxO3a and BCR-ABL Regulate cyclin D2 Transcription through a STAT5/BCL6-Dependent Mechanism." Molecular and Cellular Biology 24, no. 22 (November 15, 2004): 10058–71. http://dx.doi.org/10.1128/mcb.24.22.10058-10071.2004.
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ABSTRACT Cell cycle arrest by FoxO transcription factors involves transcriptional repression of cyclin D, although the exact mechanism remains unclear. In this study, we used the BCR-ABL-expressing cell line BV173 as a model system to investigate the mechanisms whereby FoxO3a regulates cyclin D2 expression. Inhibition of BCR-ABL by STI571 results in down-regulation of cyclin D2 expression, activation of FoxO3a activity, and up-regulation of BCL6 expression. Using reporter gene assays, we demonstrate that STI571, FoxO3a, and BCL6 can repress cyclin D2 transcription through a STAT5/BCL6 site located within the cyclin D2 promoter. We propose that BCR-ABL inhibition leads to FoxO3a activation, which in turn induces the expression of BCL6, culminating in the repression of cyclin D2 transcription through this STAT5/BCL6 site. This process was verified by mobility shift and chromatin immunoprecipitation analyses. We find that conditional activation of FoxO3a leads to accumulation of BCL6 and down-regulation of cyclin D2 at protein and mRNA levels. Furthermore, silencing of FoxO3a and BCL6 in BCR-ABL-expressing cells abolishes STI571-mediated effects on cyclin D2. This report establishes the signaling events whereby BCR-ABL signals are relayed to cyclin D2 to mediate cell cycle progression and defines a potential mechanism by which FoxO proteins regulate cyclin D2 expression.
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Issa, Jean-Pierre J., Hagop Kantarjian, Avinash Mohan, Susan O’Brien, Jorge Cortes, Sherry Pierce, and Moshe Talpaz. "Methylation of the ABL1 Promoter in Chronic Myelogenous Leukemia: Lack of Prognostic Significance." Blood 93, no. 6 (March 15, 1999): 2075–80. http://dx.doi.org/10.1182/blood.v93.6.2075.406k29_2075_2080.
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The BCR-ABL chromosomal translocation is a central event in the pathogenesis of chronic myelogenous leukemia (CML). One of theABL1 promoters (Pa) and the coding region of the gene are usually translocated intact to the BCR locus, but the translocated promoter appears to be silent in most cases. Recently, hypermethylation of Pa was demonstrated in CML and was proposed to mark advanced stages of the disease. To study this issue, we measured Pa methylation in CML using Southern blot analysis. Of 110 evaluable samples, 23 (21%) had no methylation, 17 (15%) had minimal (<15%) methylation, 12 (11%) had moderate methylation (15% to 25%), and 58 (53%) had high levels of methylation (>25%) at the ABL1locus. High methylation was more frequent in advanced cases of CML. Among the 76 evaluable patients in early chronic phase (ECP), a major cytogenetic response with interferon-based therapy was observed in 14 of 34 patients with high methylation compared with 19 of 42 among the others (41% v 45%; P value not significant). At a median follow-up of 7 years, there was no significant difference in survival by ABL1 methylation category. Among patients who achieved a major cytogenetic response, low levels of methylation were associated with a trend towards improved survival, but this trend did not reach statistical significance. Thus, Pa methylation in CML is associated with disease progression but does not appear to predict for survival or response to interferon-based therapy.
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Asimakopoulos, Fotis A., Pesach J. Shteper, Svetlana Krichevsky, Eitan Fibach, Aaron Polliack, Eliezer Rachmilewitz, Yinon Ben-Neriah, and Dina Ben-Yehuda. "ABL1 Methylation Is a Distinct Molecular Event Associated With Clonal Evolution of Chronic Myeloid Leukemia." Blood 94, no. 7 (October 1, 1999): 2452–60. http://dx.doi.org/10.1182/blood.v94.7.2452.419k23_2452_2460.
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Methylation of the proximal promoter of the ABL1 oncogene is a common epigenetic alteration associated with clinical progression of chronic myeloid leukemia (CML). In this study we queried whether both the Ph′-associated and normal ABL1 alleles undergo methylation; what may be the proportion of hematopoietic progenitors bearing methylated ABL1 promoters in chronic versus acute phase disease; whether methylation affects the promoter uniformly or in patches with discrete clinical relevance; and, finally, whether methylation of ABL1 reflects a generalized process or is gene-specific. To address these issues, we adapted the techniques of methylation-specific PCR and bisulfite-sequencing to study the regulatory regions of ABL1 and other genes with a role in DNA repair or genotoxic stress response. In cell lines established from CML blast crisis, which only carry a single ABL1 allele nested within the BCR-ABL fusion gene, ABL1 promoters were universally methylated. By contrast, in clinical samples from patients at advanced stages of disease, both methylated and unmethylated promoter alleles were detectable. To distinguish between allele-specific methylation and a mixed cell population pattern, we studied the methylation status of ABL1 in colonies derived from single hematopoietic progenitors. Our results showed that both methylated and unmethylated promoter alleles coexisted in the same colony. Furthermore, ABL1 methylation was noted in the vast majority of colonies from blast crisis, but not chronic-phase CML. Both cell lines and clinical samples from acute-phase CML showed nearly uniform hypermethylation along the promoter region. Finally, we showed that ABL1 methylation does not reflect a generalized process and may be unique among DNA repair/genotoxic stress response genes. Our data suggest that specific methylation of the Ph′-associatedABL1 allele accompanies clonal evolution in CML.
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Yasuda, Takahiko, Fumihiko Hayakawa, Shingo Kurahashi, and Tomoki Naoe. "B Cell Receptor-ERK1/2 Signal Cancels PAX5-Dependent Repression of BLIMP1 Probably Through PAX5 Phosphorylation: A Mechanism of Antigen-Triggering Plasma Cell Differentiation." Blood 118, no. 21 (November 18, 2011): 1109. http://dx.doi.org/10.1182/blood.v118.21.1109.1109.
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Abstract Abstract 1109 Plasma cell differentiation is initiated by antigen stimulation of B cell receptor (BCR). Until BCR stimulation, BLIMP1, a master regulator of plasma cell differentiation, is suppressed by PAX5, a key transcriptional repressor to maintain B cell identity. After BCR stimulation, upregulation of BLIMP1 and subsequent suppression of PAX5 by BLIMP1 are observed and thought to be the trigger of plasma cell differentiation; however, the trigger that derepresses BLIMP1 expression is yet to be revealed. Here, we demonstrated PAX5 phosphorylation by ERK1/2, the main component of BCR signal, in vitro and in vivo. The sites of PAX5 phosphorylation were identified by PCR mutagenesis assay. In luciferase reporter assays, transcriptional repression on BLIMP1 promoter by PAX5 was canceled by PAX5 phosphorylation. Furthermore, transcriptional repression by phosphorylation-defective mutant of PAX5 was attenuated by CA-MEK1 co-expression to a significantly lesser extent than that by wild-type PAX5, indicating its resistance to ERK1/2 signal-dependent cancelation of the transcriptional repression (Figure A). Finally, BCR stimulation induced strong ERK1/2 activation, phosphorylation of endogenous PAX5 (Figure B), and upregulation of BLIMP1 mRNA expression in B cells. These phenomena were inhibited by U0126, MEK1 inhibitor. These data imply that PAX5 phosphorylation by BCR signal is the initial event in plasma cell differentiation (Figure C). Disclosures: Naoe: Kyowa-Hakko Kirin.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Chugai Pharma.: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Zenyaku-Kogyo: Research Funding; Otsuka Pharma.: Research Funding.
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Wang, Y. Lynn, Ji Ma, Vinay Mallikarjuna, Pin Lu, Shengchun Wang, Sahar Poureghbali, Mariusz A. Wasik, Reza Nejati, and Wenjun Wu. "SHP1 Loss Augments DLBCL Cellular Response to Ibrutinib: A Candidate Predictive Biomarker." Blood 138, Supplement 1 (November 5, 2021): 3490. http://dx.doi.org/10.1182/blood-2021-146086.
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Abstract Background B-cell receptor (BCR) signaling plays a key role in B-cell non-Hodgkin lymphoma (NHL). Protein tyrosine kinases such as LYN, SYK and BTK are essential in the activation of BCR leading to B-cell survival, proliferation and differentiation. SHP1 is a cytosolic protein tyrosine phosphatase functioning as a negative regulator of the BCR signaling cascade. It dephosphorylates and counteracts the activities of the tyrosine kinases, including CD79A/CD79B, SYK and BLNK (Curr. Opin. Immunol.12, 307). Diffuse large B-cell lymphoma (DLBCL) is the most common type of aggressive NHL. DLBCL demonstrates heterogeneity in clinical presentation, genomic alterations and cell signaling. BCR signature has been identified in a subset of DLBCL (Monti Blood 105, 1851). Chronic active BCR plays a particular role in the subtype of activated B-cell like (ABC) DLBCL by cell-of-origin subclassification. Based on the success of ibrutinib in other B-cell NHL, clinical trials of ibrutinib in DLBCL was conducted and the initial results showed a 37% response rate in ABC-lymphomas vs 5% in GCB-DLBCL (Wilson Nat Med 21, 922). However, a large phase 3 clinical trial of ibrutinib-containing regimen in >800 non-GCB patients did not find any substantial difference in response between those received ibrutinib vs placebo. Thus, biomarker based on cell-of-origin did not seem to help stratify patients as expected (Younes JCO 27, 1285). New predictive biomarkers are needed to identify patients who likely benefit from ibrutinib or other BCR-directed therapeutic agents. We hypothesized that SHP1 may play a role in defining the level of the activity of the BCR pathway. Suppressed SHP1 expression in various types of NHL has been reported. We hypothesized that lack of SHP1 may contribute to the hyperactivity of BCR in DLBCL; and tumors with low SHP1 may respond better to ibrutinib. In this work, we set out to explore the relationship between SHP1 & ibrutinib sensitivity in DLBCL. Methods and Results We first reviewed the published literature on SHP1 in NHL using a meta-analysis to provide a quantitative and thus unbiased analysis. Seventeen articles were selected based on criteria including inclusion of primary tumors with normal benign controls. The analysis of the SHP1 protein expression by immunohistochemistry (IHC) revealed that lack of SHP1 expression is found in 178 of 242 (73.6%) NHL tissues vs. 32 of 264 (12%) normal/benign controls. The odds ratio derived by the meta-analysis was 119.58 with a 95% confidence interval of 37.60-380.15, suggesting loss of SHP1 is strongly associated with NHL including DLBCL. Likewise, the analysis of the methylation studies revealed a strong association between SHP1 promoter hypermethylation and NHL (OR 69.46, 95% CI 12.32-391.74). To further understand the range of variability of SHP1 expression in subtypes of DLBCL tumors, we constructed a tissue microarray with 48 DLBCL tumors and 12 cell lines. By immunohistochemistry, we found a wide range of variability with regards to the expression levels and no substantial difference between the GCB and non-GCB subtypes. Using 12 cell lines together with resting B-cells from normal donors, we identified a strong reverse linear correlation between SHP1 protein expression and promoter methylation (r = 0.85, P=0.0002) suggesting that promoter hypermethylation may be responsible for reduced SHP1 expression in DLBCL cells. Using CRISPR, we knocked out SHP1 in both GCB and ABC cell lines. The phosphorylation of early BCR components, BTK, SYK and LYN, were increased in the SHP1 knockout clones compared to the wild type ones. The results demonstrate that SHP1 depletion results in increased proximal BCR signaling activity. Moreover, the knock out clones demonstrated increased sensitivity of ibrutinib. We further showed that re-introduction of SHP1 vector to the knockout clones via stable lentiviral transduction restored normal sensitivity of cells to ibrutinib. Conclusions Together, our results show that SHP1 expression is suppressed in a substantial fraction of DLBCL tumors regardless of cell-of-origin subclassifications. Using genetic approaches, we demonstrated that loss of SHP1 increases BCR signaling activity and sensitizes tumor cells to the inhibition by ibrutinib. Our results suggest that loss of SHP1 may be used as an alternative biomarker to cell-of-origin to identify patients who potentially benefit from ibrutinib treatment. Disclosures Wang: Incyte Research Institute: Ended employment in the past 24 months.
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Modur, Vi j. ay, Dean C. Bottino, Susan Branford, Tillmann Krahnke, Fabrizio Pane, Marc Rudoltz, Yu Jin, et al. "Mathematical Simulation of BCR-ABL Real Time Quantitative Polymerase Chain Reaction (RQ-PCR) for Chronic Myeloid Leukemia (CML) Response Monitoring Provides Insight on the Basis of International Standardization." Blood 112, no. 11 (November 16, 2008): 2124. http://dx.doi.org/10.1182/blood.v112.11.2124.2124.
Full textAbstract:
Abstract BACKGROUND: Cytogenetic assessment to measure CML response to Glivec or Tasigna is expressed as a ratio of Ph+ CML to total cells. Molecular response determination for CML by RQ-PCR achieves a similar goal by obtaining a ratio of transcript levels of BCR-ABL expressed exclusively in Ph+ CML cells to the BCR or ABL control gene which is expressed in both Ph+ and normal cells. However when a RQ-PCR assay result is obtained, a different RQ-PCR ratio will be obtained for the same sample using BCR vs. ABL as control gene due to the intrinsic expression level differences in the two control genes. Experiments have demonstrated empirically that international standardization of RQ-PCR data obtained by assays using these different control genes is possible, thus allowing direct comparison of data. We have developed a mathematical simulation that explains the underlying basis for international standardization and this was experimentally confirmed to show how standardization is feasible in practice as a part of the Tyrosine Kinase Optimization and Selectivity (TOPS) trial. METHODS: We have mathematically simulated a correlation between theoretical cytogenetic response (Ph+ to total cell ratio) with the theoretical molecular response (BCR-ABL to control gene transcript ratio) by using the following assumptions; 1) BCR and BCR-ABL expression occur from similar promoters and likely have similar expression levels. 2) Ph+ cells have one normal BCR resulting in 1x level of BCR transcription and normal cells have two normal copies of BCR resulting in 2x level of BCR transcription (x=BCR transcription rate from one promoter). This simulation was extended to the ABL control gene as data of the relative expression level of ABL vs. BCR was available from samples in the TOPS trial, and the ABL transcript level is similar in both Ph+ and normal cells as the ABL PCR primers amplify both ABL and BCR-ABL transcripts. To experimentally test the simulation, RQ-PCR samples from patients in TOPS were exchanged between the study’s regional RQ-PCR laboratories. Each sample was assayed at 2 labs, one using BCR and the other ABL as the control gene. RESULTS: Mathematical simulation suggests that RQ-PCR ratios with BCR but not ABL as control gene has a nonlinear relationship with the Ph+ to the total cell ratio across the entire range from 0 to 100%. However at clinically relevant RQ-PCR ratio of ≤10%, irrespective of whether BCR or ABL is used as a control gene, RQ-PCR ratios are linear but with different slopes due to higher ratios obtained with ABL as control gene. Despite the different slopes, RQ-PCR ratios obtained with either control gene can be easily inter-converted via a multiplicative factor to neutralize the bias between methods as is currently recommended in international standardization. Experimental testing in the TOPS trial showed that; 1) ABL transcript copy number is significantly lower than BCR transcript copy number leading to higher RQ-PCR ratios with ABL as control gene. 2) Mathematical correction of bias between BCR and ABL as control gene allows for direct comparison of RQ-PCR ratios with an 88.2% concordance when ratios are below 10%. CONCLUSIONS: Mathematical simulation shows that for BCR and ABL as the control gene, the basis for international standardization is a consistent linear relationship that can be easily inter-converted when PCR ratios are ≤ 10%. This can be experimentally demonstrated by the high level of concordance obtained when the same sample is analyzed using different control genes. For RQ-PCR ratios > 10%, standardization is not possible using the currently applied neutralization of bias method of conversion, but may be possible by using a formula that accounts for the non-linear relationship of BCR as a control gene. Simulation studies also highlight the importance of not only obtaining precise but also accurate RQ-PCR ratios thus enabling consistent standardization.