Expression of the ETS transcription factor GABPα is positively correlated to the BCR-ABL1/ABL1 ratio in CML patients and affects imatinib sensitivity in vitro
2015; Elsevier BV; Volume: 43; Issue: 10 Linguagem: Inglês
10.1016/j.exphem.2015.05.011
ISSN1873-2399
AutoresGeorgi Manukjan, Tim Ripperger, Laura Santer, Nils von Neuhoff, Arnold Ganser, Axel Schambach, Brigitte Schlegelberger, Doris Steinemann,
Tópico(s)Chronic Lymphocytic Leukemia Research
Resumo•GA-binding protein α subunit (GABPα) expression is positively correlated to the BCR-ABL1/ABL1 ratio in patients with chronic myeloid leukemia (CML).•GABPα levels directly mediate imatinib sensitivity in K-562 cells.•Imatinib-resistant NALM-1 cells are sensitized to imatinib after functional impairment of GABP.•PRKD2 and RAC2 are discussed as putative GABP targets with an impact on CML signaling. In Philadelphia-positive chronic myeloid leukemia (CML), imatinib resistance frequently emerges because of point mutations in the ABL1 kinase domain, but may also be the consequence of uncontrolled upstream signaling. Recently, the heteromeric transcription factor GA-binding protein (GABP) was found to promote CML-like myeloproliferative disease in mice. In a cohort of 70 CML patients, we found that expression of the GABP α subunit (GABPα) is positively correlated to the BCR–ABL1/ABL1 ratio. Moreover, significantly higher GABPα expression was detected in blast crisis than in chronic phase CML after performing data mining on 91 CML patients. In functional studies, imatinib sensitivity is enhanced after GABPα knockdown in tyrosine kinase inhibitors (TKI)–sensitive K-562, as well as by overexpression of a deletion mutant in TKI-resistant NALM-1 cells. Moreover, in K-562 cells, GABP-dependent expression variations of PRKD2 and RAC2, relevant signaling mediators in CML, were observed. Notably, protein kinase D2 (Prkd2) was reported to be a GABP target gene in mice. In line with this, we detected a positive correlation between GABPA and PRKD2 expression in primary human CML, indicating that the effects of GABP are mediated by PRKD2. These findings illustrate an important role for GABP in disease development and imatinib sensitivity in human CML. In Philadelphia-positive chronic myeloid leukemia (CML), imatinib resistance frequently emerges because of point mutations in the ABL1 kinase domain, but may also be the consequence of uncontrolled upstream signaling. Recently, the heteromeric transcription factor GA-binding protein (GABP) was found to promote CML-like myeloproliferative disease in mice. In a cohort of 70 CML patients, we found that expression of the GABP α subunit (GABPα) is positively correlated to the BCR–ABL1/ABL1 ratio. Moreover, significantly higher GABPα expression was detected in blast crisis than in chronic phase CML after performing data mining on 91 CML patients. In functional studies, imatinib sensitivity is enhanced after GABPα knockdown in tyrosine kinase inhibitors (TKI)–sensitive K-562, as well as by overexpression of a deletion mutant in TKI-resistant NALM-1 cells. Moreover, in K-562 cells, GABP-dependent expression variations of PRKD2 and RAC2, relevant signaling mediators in CML, were observed. Notably, protein kinase D2 (Prkd2) was reported to be a GABP target gene in mice. In line with this, we detected a positive correlation between GABPA and PRKD2 expression in primary human CML, indicating that the effects of GABP are mediated by PRKD2. These findings illustrate an important role for GABP in disease development and imatinib sensitivity in human CML. Chronic myeloid leukemia (CML) is caused by the Philadelphia translocation t(9;22)(q34;q11) forming the BCR-ABL1 fusion in more than 95% of cases [1Rowley J.D. Letter: A new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and Giemsa staining.Nature. 1973; 243: 290-293Crossref PubMed Scopus (3306) Google Scholar, 2Maru Y. Witte O.N. The BCR gene encodes a novel serine/threonine kinase activity within a single exon.Cell. 1991; 67: 459-468Abstract Full Text PDF PubMed Scopus (210) Google Scholar, 3Kantarjian H. Sawyers C. Hochhaus A. et al.Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia.N Engl J Med. 2002; 346: 645-652Crossref PubMed Scopus (1810) Google Scholar, 4Druker B.J. Translation of the Philadelphia chromosome into therapy for CML.Blood. 2008; 112: 4808-4817Crossref PubMed Scopus (567) Google Scholar]. CML evolves from a single transformed leukemic stem cell (LSC) [5Reya T. Morrison S.J. Clarke M.F. Weissman I.L. Stem cells, cancer, and cancer stem cells.Nature. 2001; 414: 105-111Crossref PubMed Scopus (7693) Google Scholar], in which the constitutively active ABL1 kinase gives rise to a survival advantage. Phenotypically, this results in hyperplasia of the myeloid compartment and elevated premature and mature myeloid cell counts in bone marrow and peripheral blood. As standard CML therapy, competitive first-line or second-line tyrosine kinase inhibitors (TKIs), for example, imatinib, nilotinib, and dasatinib, were developed [6Jabbour E. Kantarjian H. Chronic myeloid leukemia: 2012 update on diagnosis, monitoring, and management.Am J Hematol. 2012; 87: 1037-1045Crossref PubMed Scopus (107) Google Scholar, 7Baccarani M. Deininger M.W. Rosti G. et al.European LeukemiaNet recommendations for the management of chronic myeloid leukemia: 2013.Blood. 2013; 122: 872-884Crossref PubMed Scopus (1468) Google Scholar]. However, drug resistance is frequently observed and, in about 50% of cases, is caused by point mutations in the ATP-binding kinase domain of BCR-ABL1 [4Druker B.J. Translation of the Philadelphia chromosome into therapy for CML.Blood. 2008; 112: 4808-4817Crossref PubMed Scopus (567) Google Scholar]. However, BCR-ABL1-independent mechanisms driving CML pathogenesis, as well as TKI resistance, are under debate [8Kirschner K.M. Baltensperger K. Erythropoietin promotes resistance against the Abl tyrosine kinase inhibitor imatinib (STI571) in K562 human leukemia cells.Mol Cancer Res. 2003; 1: 970-980PubMed Google Scholar, 9Quentmeier H. Eberth S. Romani J. Zaborski M. Drexler H.G. BCR-ABL1-independent PI3Kinase activation causing imatinib-resistance.J Hematol Oncol. 2011; 4: 6Crossref PubMed Scopus (87) Google Scholar, 10Hamad A. Sahli Z. El Sabban M. Mouteirik M. Nasr R. Emerging therapeutic strategies for targeting chronic myeloid leukemia stem cells.Stem Cells Int. 2013; 2013: 724360Crossref PubMed Scopus (36) Google Scholar]. The heteromeric transcription factor GA-binding protein (GABP) consists of two distinct subunits, GABPα and GABPβ [11Rosmarin A.G. Resendes K.K. Yang Z. McMillan J.N. Fleming S.L. GA-binding protein transcription factor: a review of GABP as an integrator of intracellular signaling and protein-protein interactions.Blood Cells Mol Dis. 2004; 32: 143-154Crossref PubMed Scopus (151) Google Scholar]. GABPα belongs to the E26 transformation-specific (ETS) family of transcription factors and bears the DNA-binding domain (DBD), which targets GGAA/T consensus motifs, but lacks transactivation capability [12Brown T.A. McKnight S.L. Specificities of protein–protein and protein–DNA interaction of GABP alpha and two newly defined ets-related proteins.Genes Dev. 1992; 6: 2502-2512Crossref PubMed Scopus (233) Google Scholar]. In humans, GABPβ is expressed in four isoforms as a result of alternative splicing. It contains the transactivation (TAD) and nuclear localization signal (NLS) domains, whereas it does not physically interact with DNA [12Brown T.A. McKnight S.L. Specificities of protein–protein and protein–DNA interaction of GABP alpha and two newly defined ets-related proteins.Genes Dev. 1992; 6: 2502-2512Crossref PubMed Scopus (233) Google Scholar, 13Sawa C. Goto M. Suzuki F. Watanabe H. Sawada J. Handa H. Functional domains of transcription factor hGABP beta1/E4TF1-53 required for nuclear localization and transcription activation.Nucleic Acids Res. 1996; 24: 4954-4961Crossref PubMed Scopus (47) Google Scholar, 14Batchelor A.H. Piper D.E. de la Brousse F.C. McKnight S.L. Wolberger C. The structure of GABPalpha/beta: an ETS domain-ankyrin repeat heterodimer bound to DNA.Science. 1998; 279: 1037-1041Crossref PubMed Scopus (266) Google Scholar]. Thus, both heteromer-forming subunits share an obligate codependence, a unique phenomenon among the ETS transcription factor family [11Rosmarin A.G. Resendes K.K. Yang Z. McMillan J.N. Fleming S.L. GA-binding protein transcription factor: a review of GABP as an integrator of intracellular signaling and protein-protein interactions.Blood Cells Mol Dis. 2004; 32: 143-154Crossref PubMed Scopus (151) Google Scholar, 14Batchelor A.H. Piper D.E. de la Brousse F.C. McKnight S.L. Wolberger C. The structure of GABPalpha/beta: an ETS domain-ankyrin repeat heterodimer bound to DNA.Science. 1998; 279: 1037-1041Crossref PubMed Scopus (266) Google Scholar]. GABP was described to influence the development and lineage commitment in lymphoid as well as myeloid compartments [15Xue H.H. Bollenbacher J. Rovella V. et al.GA binding protein regulates interleukin 7 receptor alpha-chain gene expression in T cells.Nat Immunol. 2004; 5: 1036-1044Crossref PubMed Scopus (119) Google Scholar, 16Xue H.H. Bollenbacher-Reilley J. Wu Z. et al.The transcription factor GABP is a critical regulator of B lymphocyte development.Immunity. 2007; 26: 421-431Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 17Yang Z.F. Drumea K. Cormier J. Wang J. Zhu X. Rosmarin A.G. GABP transcription factor is required for myeloid differentiation, in part, through its control of Gfi-1 expression.Blood. 2011; 118: 2243-2253Crossref PubMed Scopus (28) Google Scholar, 18Yu S. Cui K. Jothi R. et al.GABP controls a critical transcription regulatory module that is essential for maintenance and differentiation of hematopoietic stem/progenitor cells.Blood. 2011; 117: 2166-2178Crossref PubMed Scopus (67) Google Scholar]. The possible role of GABP in CML was recently addressed in two independent murine studies [19Yu S. Jing X. Colgan J.D. Zhao D.M. Xue H.H. Targeting tetramer-forming GABPbeta isoforms impairs self-renewal of hematopoietic and leukemic stem cells.Cell Stem Cell. 2012; 11: 207-219Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 20Yang Z.F. Zhang H. Ma L. et al.GABP transcription factor is required for development of chronic myelogenous leukemia via its control of PRKD2.Proc Natl Acad Sci U S A. 2013; 110: 2312-2317Crossref PubMed Scopus (19) Google Scholar]. Homozygous loss of Gabpa induces cell cycle arrest of BCR-ABL1-transformed hematopoietic stem cells (HSCs), leading to prolonged survival of recipient mice [20Yang Z.F. Zhang H. Ma L. et al.GABP transcription factor is required for development of chronic myelogenous leukemia via its control of PRKD2.Proc Natl Acad Sci U S A. 2013; 110: 2312-2317Crossref PubMed Scopus (19) Google Scholar]. Moreover, protein kinase D2 (Prkd2) was established as a GABP target and mediator of CML pathogenesis in mice [20Yang Z.F. Zhang H. Ma L. et al.GABP transcription factor is required for development of chronic myelogenous leukemia via its control of PRKD2.Proc Natl Acad Sci U S A. 2013; 110: 2312-2317Crossref PubMed Scopus (19) Google Scholar]. Studies on Gabpb knockout mice reported that heterotetramer-forming GABPβ isoforms are critical for CML initiation and propagation [19Yu S. Jing X. Colgan J.D. Zhao D.M. Xue H.H. Targeting tetramer-forming GABPbeta isoforms impairs self-renewal of hematopoietic and leukemic stem cells.Cell Stem Cell. 2012; 11: 207-219Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar]. In this context, a synergistic effect of GABP impairment and imatinib treatment was observed. The functional reason discussed was that GABP deficiency causes diminished cycling and maintenance of HSCs and, hence, BCR-ABL1+ LSCs [19Yu S. Jing X. Colgan J.D. Zhao D.M. Xue H.H. Targeting tetramer-forming GABPbeta isoforms impairs self-renewal of hematopoietic and leukemic stem cells.Cell Stem Cell. 2012; 11: 207-219Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 20Yang Z.F. Zhang H. Ma L. et al.GABP transcription factor is required for development of chronic myelogenous leukemia via its control of PRKD2.Proc Natl Acad Sci U S A. 2013; 110: 2312-2317Crossref PubMed Scopus (19) Google Scholar]. In the present study, we asked whether GABP also plays a role in human CML. Therefore, we analyzed expression of GABP subunits and found a positive correlation between the GABPα subunit (GABPA) and the BCR-ABL1/ABL1 ratio, a molecular indicator of tumor cell burden. Together with elevated GABPA expression in blast crisis CML, illustrated by in silico analysis of a publicly available data set [21Radich J.P. Dai H. Mao M. et al.Gene expression changes associated with progression and response in chronic myeloid leukemia.Proc Natl Acad Sci U S A. 2006; 103: 2794-2799Crossref PubMed Scopus (448) Google Scholar], a leukemogenic effect of GABP on development and progression of human CML can be supposed. In addition, a significant correlation between GABPA and PRKD2 expression was detected in primary human CML, indicating that GABP may drive a CML-promoting kinase pathway with the opportunity for targeted therapeutic intervention. In functional in vitro studies using BCR-ABL1+ human cell lines, GABPα dosage was found to inversely affect imatinib sensitivity, confirming GABP's leukemogenic potential. In line with this, the TKI-resistant CML cell line NALM-1 could be sensitized to imatinib after functional GABP impairment. All together, our data indicate that GABP is also a relevant transcription factor in human CML. Herewith, GABP-implicated pathways may be considered as alternative targets in therapy of blast crisis or TKI-resistant CML. The CML patient cohort consisted of 44 male and 26 female patients with a median age of 61 years. All patients were in chronic phase and positive for the BCR-ABL1 b2a2 fusion. Patients did not receive TKI therapy before sampling. RNA was extracted from peripheral blood samples according to standard protocols. BCR-ABL1/ABL1 ratios were determined with the LightCycler FastStart DNA MasterPLUS HybProbe Kit (Roche Diagnostics, Mannheim, Germany) [22Emig M. Saussele S. Wittor H. et al.Accurate and rapid analysis of residual disease in patients with CML using specific fluorescent hybridization probes for real time quantitative RT-PCR.Leukemia. 1999; 13: 1825-1832Crossref PubMed Scopus (247) Google Scholar]. Values were corrected by a lab-specific conversion factor (0.674), which was defined in an interlaboratory comparison trial [23Hughes T. Deininger M. Hochhaus A. et al.Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for harmonizing current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results.Blood. 2006; 108: 28-37Crossref PubMed Scopus (991) Google Scholar]. Primer and probe sequences are as follows: ABL1 (forward: CTTCAGCGGCCAGTAGCATC; reverse: CATCAGAAGCAGTGTTGATCCT); BCR/ABL1-b2a2 (forward: CAGATGCTGACCAACTCGTGT; reverse: CAGGAGTGTTTCTCCAGACTG); ABL1_probe_1 (GCATAACTAAAGGTGAAAAGCTCCGGGLC-fluorescein); ABL1_probe_2 (LC Red-640CTTAGGCTATAATCACAATGGGGAATGG). The investigation was approved by the Hannover Medical School Ethics Committee (No. 2899), and written consent was obtained from each patient in accordance with the Declaration of Helsinki. BCR-ABL1+ TKI-sensitive K-562 cells (ACC-10, DSMZ, Braunschweig, Germany) were cultured under standard conditions in RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum and 1% penicillin/streptomycin/L-glutamine (Biochrom, Berlin, Germany). BCR-ABL1+ TKI-resistant NALM-1 cells (ACC-131, DSMZ) were cultivated similarly using 20% heat-inactivated fetal calf serum. ABL1 mutations were excluded as the cause of TKI resistance [9Quentmeier H. Eberth S. Romani J. Zaborski M. Drexler H.G. BCR-ABL1-independent PI3Kinase activation causing imatinib-resistance.J Hematol Oncol. 2011; 4: 6Crossref PubMed Scopus (87) Google Scholar]. Short hairpin RNAs (shRNAs) targeting GABPA and a scrambled control were designed using the siRNA Wizard algorithm (http://www.invivogen.com/sirna-wizard) and cloned into the lentiviral vector pLVTHM (Addgene, Cambridge, MA). VSV-G-pseudotyped lentiviral particles were produced using standard calcium phosphate transformation of 293T cells (ACC-635, DSMZ, Braunschweig, Germany). For ectopic overexpression, human GABPA and GABPB1 transcript variant beta-2 cDNAs, as well as the GABPB1.ΔTAD construct, were cloned into the γ-retroviral expression vector pRSF91.IRES.eGFP.pre* or pRSF91.IRES.dTomato.pre* [24Schambach A. Mueller D. Galla M. et al.Overcoming promoter competition in packaging cells improves production of self-inactivating retroviral vectors.Gene Ther. 2006; 13: 1524-1533Crossref PubMed Scopus (114) Google Scholar]. Retroviral particles were produced as described above using the RD114/TR envelope [25Sandrin V. Boson B. Salmon P. et al.Lentiviral vectors pseudotyped with a modified RD114 envelope glycoprotein show increased stability in sera and augmented transduction of primary lymphocytes and CD34+ cells derived from human and nonhuman primates.Blood. 2002; 100: 823-832Crossref PubMed Scopus (243) Google Scholar]. Transductions of K-562 and NALM-1 cells were performed in RetroNectin-coated plates following the manufacturer's instructions (Takara BIO Europe/Clonetech, Saint-Germain-en-Laye, France). For subsequent expression and proliferation analyses, enhanced green fluorescent protein (eGFP) or dTomato-positive cells were isolated by fluorescence-activated cell sorting (FACS). K-562 cells seeded at a density of 1 × 105 cells/mL in 96-well plates were treated with imatinib mesylate (Santa Cruz Biotechnology, Heidelberg, Germany) resolved in dimethyl sulfoxide (DMSO) at the indicated concentrations. As a solvent control, 0.1% DMSO (Sigma-Aldrich, Steinheim, Germany) was used. Every 24 hours, cell proliferation was determined using the WST-1 assay as recommended by the manufacturer (Roche Diagnostics). Clonogenic capacity of K-562 cells was investigated in Methocult H4100 methylcellulose (StemCell Technologies, Grenoble, France). Transduced and FACS-purified K-562 cells were seeded at a density of 1 × 104 cells/mL in 2 mL methylcellulose/IMDM supplemented with 10% heat-inactivated fetal bovine serum and the respective compounds. Colonies, defined as cell populations in close contact with n ≥ 8 cells, were counted 3 days after culture induction. Colony area was determined by measuring colony diameter after 6 days using ImageJ software, Version 1.37 (National Institutes of Health, Bethesda, MD). For viability quantification by means of competition assays, unsorted NALM-1 cells were seeded at a density of 2 × 105 cells/mL and treated with the solvent control or with imatinib mesylate at the indicated concentrations. After 2 and 6 days, the amount of dTomato-positive cells was measured by flow cytometry. Normalization was performed against DMSO-treated cells on day 2. For apoptosis quantification, annexin V/7-aminoactinomycin (7-AAD) co-staining was used according to the manufacturer's recommendations (BD Pharmingen, Heidelberg, Germany). For analyses of data acquired, FlowJo software, Version 7.6.5. (Tree Star, Ashland, OR), was used. For quantitative reverse transcription polymerase chain reaction (RT-PCR), we used the QuantiTect SYBR Green RT-PCR Master Mix (Qiagen, Hilden, Germany) on a StepOnePlus real-time cycler (Life Technologies, Darmstadt, Germany) with StepOnePlus software, Version 2.2 (Life Technologies). Relative expression was calculated with the ΔΔCt method in correlation to the housekeeping genes SDHA and/or TBP. Primer sequences are as follows: GABPA (forward: TTTTTCAGCGGGTTCCTCG; reverse: GTACTTTGGCTGCTTTCGC); GABPB1 transcript variants beta-1, beta-2, gamma-1, gamma-2 (forward: GGTCAAGATGATGAAGTTCG; reverse: CTGGCATCTCTGCTCACAC); PRKD2 (forward: GCGTGATCATGTACGTCAG; reverse: AGCAGGTTGTTGATGAGGTC); RAC2 (forward: GTCTTCCTCATCTGCTTCTC, reverse: TATTTCACCGAGTCAATCTCC); SDHA (forward: GCCATCCACTACATGACG; reverse: TCCATATAAGGTGTGCAATAGC); TBP (forward: CCTAAAGACCATTGCACTTCG; reverse: CTGGACTGTTCTTCACTCTTG). For co-immunoprecipitation (Co-IP), we used approximately 6 × 107 cells grown under standard conditions. Cells were lysed using a non-denaturing lysis buffer (20 mM Tris–HCl, pH 8, 137 mmol/L NaCl, 2 mmol/L EDTA, pH 8, 10% glycerol, 1% Igepal CA-630). After pre-clearance using protein G Sepharose beads (GE Healthcare, Braunschweig, Germany), 2 μg of murine IgG (sc-2025, Santa Cruz Biotechnology) or a GABPα antibody mixture (sc-28311 and sc-28312, 1 μg each, Santa Cruz Biotechnology) was used together with protein G Sepharose beads for IP. Finally, IP eluates and supernatants were analyzed by SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) and Western blotting. Western blotting of RIPA-lysed whole-cell protein extracts was performed in accordance with standard procedures. The following primary antibodies were used: anti-GABPα (sc-28312), anti-GABPβ1 (sc-28684), anti-RAC2 (sc-96), anti-GAPDH (sc-32233) (all Santa Cruz), and anti-PRKD2 (8188S, New England Biolabs, Frankfurt/Main, Germany). Investigating the expression of GABPα (GABPA) and GABPβ (GABPB1) in primary human CML (n = 70) at the time of diagnosis, a significant positive correlation between GABPA expression and the BCR-ABL1/ABL1 ratio was detected (R2 = 0.16) (Fig. 1A). Moreover, comparison of the lower and upper tertiles of the patient cohort, that is, the third of patients with lowest and the third with the highest BCR-ABL1/ABL1 ratio, respectively, revealed that mean relative GABPA expression was significantly higher in the upper tertile (p < 0.001) (Fig. 1B). In contrast to GABPA, no significant correlation was observed between GABPB1 expression and BCR-ABL1/ABL1 ratio (Fig. 1C and D). In addition, we analyzed global gene expression profiles of 91 CML patients within chronic phase (n = 42), accelerated phase (n = 17), or blast crisis (n = 32) for GABPA expression (GEO data set 4170) [21Radich J.P. Dai H. Mao M. et al.Gene expression changes associated with progression and response in chronic myeloid leukemia.Proc Natl Acad Sci U S A. 2006; 103: 2794-2799Crossref PubMed Scopus (448) Google Scholar]. Comparison of the clinical phases of CML revealed that overall GABPA median expression is significantly higher in the blast phase than in the accelerated and chronic phases (Fig. 1E and F). For GABPB1, no annotation was given in the data set. To study the influence of GABP on TKI sensitivity in the BCR-ABL1+ human CML model cell line K-562, we modified GABPα protein levels by means of GABPA knockdown or ectopic overexpression. For this, we performed retroviral transductions resulting in stable and sufficient GABPα protein level alterations (Fig. 2A). In subsequent proliferation assays (Fig. 2B), treatment with 100 nM imatinib resulted in significantly reduced proliferation capacity in GABPA knockdown cells. Cells overexpressing GABPA exhibited a drastic proliferation benefit compared with the empty vector control. Even in the presence of 200 nM imatinib, higher proliferation capacity was observed in cells with GABPA overexpression. No alteration of basal proliferation rates was seen in any of the transduced cell populations treated with the solvent control DMSO. To elucidate the imatinib-sensitizing effect of the GABPA knockdown in more detail, apoptosis rates were studied by means of annexin V/7-AAD costaining following treatment with 100 nM imatinib for 2 days (Fig. 2C). Relative apoptotic cell fractions (annexin V+/7-AAD−) were significantly increased in GABPA knockdown cells compared with the scrambled control (40 ± 1.5% versus 21 ± 2.9%). Next, the clonogenic capacity of K-562 cells after GABPA knockdown or overexpression was examined to investigate proliferation of single cells. For this, cells were plated in methylcellulose containing the respective substances (Fig. 3). To address initial proliferation dynamics, colonies were counted on day 3. A colony was defined as cell population with n ≥ 8 cells in close contact. In addition, areas of definite colonies were measured on day 6 after imatinib treatment induction. K-562 cells with GABPA knockdown treated with 100 nM imatinib displayed significant decreases in colony number and colony area compared with the scrambled control (Fig. 3A and B). GABPA-overexpressing cells treated with 100 nM imatinib exhibited no significant difference. However, twice as many colonies were counted compared with the empty vector control when 200 nM imatinib was used (Fig. 3C and D). Colony areas were elevated for both GABPA-overexpressing imatinib treatment groups. No differences in clonogenic capacities were seen in untreated controls. To assess downstream consequences of different GABPα expression levels, we investigated transcript and protein levels of PRKD2 and RAC2, two putative GABP target genes involved in CML pathogenesis. In K-562 cells, GABPA knockdown led to significant reductions in amounts of PRKD2 transcript and protein (Fig. 4A and B). In contrast, ectopic overexpression of GABPA or GABPA/GABPB1 led to increased levels of PRKD2 transcript as well as protein (Fig. 4C and D). PRKD2 protein abundances in Western blots after overexpression of GABPA alone or combined overexpression of GABPA and GABPB1 were comparable (Fig. 4D). Because it was previously reported that RAC2 is not expressed in K-562 cells on the protein level [26Muthukrishnan R. Skalnik D.G. Identification of a minimal cis-element and cognate trans-factor(s) required for induction of Rac2 gene expression during K562 cell differentiation.Gene. 2009; 440: 63-72Crossref PubMed Scopus (6) Google Scholar], we focused on analyses of RAC2 expression after GABP overexpression. Comparable to PRKD2, GABPA overexpression induced an increase in RAC2 expression (Fig. 4C). However, in contrast to PRKD2, RAC2 protein was detectable only when both GABP subunits were simultaneously overexpressed (Fig. 4D). Comparison of CML patients with the lowest GABPA expression to those with the highest expression (n = 8 per group) revealed a significant positive correlation with PRKD2 expression (p < 0.01) (Fig. 4E), whereas no significant difference was seen studying RAC2. To examine GABP's influence on imatinib resistance independent of ABL1 kinase domain mutations, the BCR-ABL1+ and TKI-resistant cell line NALM-1 was studied. In comparison to K-562 cells, expression of both GABP subunits as well as PRKD2 is quite prominent in NALM-1 cells (Fig. 5A). Unexpectedly, transduction of NALM-1 cells using the lentiviral shRNA vector system was not efficient. However, to achieve impaired GABP functionality, a GABPβ1 deletion mutant (GABPB1.ΔTAD) was ectopically overexpressed. Even though the deletion mutant lacks the TAD, it is still capable of forming complexes with GABPα (Fig. 5B) and most likely acts as a dominant-negative mutant. To study the effects of GABPB1.ΔTAD overexpression in NALM-1 cells treated with imatinib, competition assays were performed. After transduction, maintenance of dTomato-positive cells was monitored in mass cultures with untransduced dTomato-negative cells by flow cytometry (Fig. 5C). NALM-1 cells overexpressing GABPB1.ΔTAD displayed a proliferation disadvantage in comparison to untransduced cells. This was not observed in the empty vector control. However, treatment with 5 μM imatinib enhanced the proliferation disadvantage of GABPB1.ΔTAD-overexpressing cells. Annexin V/7-AAD costaining assays were used to elucidate the imatinib-sensitizing effect of the GABPβ1 deletion mutant (Fig. 5D) in more detail. After treatment with the topoisomerase I inhibitor camptothecin, NALM-1 cells undergo apoptosis, as indicated by elevated fractions of annexin V+/7-AAD− cells (data not shown). In contrast to imatinib-sensitive K-562 cells, no apoptosis was observed after imatinib treatment of NALM-1 cells. However, prominent annexin V+/7-AAD+ cell fractions were observed, even under standard culture conditions, indicating non-apoptotic cell death. Remarkably, treatment with 5 μM imatinib or ectopic overexpression of GABPB1.ΔTAD led to an increase in double-positive cell fractions, and the combination had an additive effect, inducing cytotoxicity in more than 50 % of the cells. In recent murine studies, GABP was found to be a potential leukemogenic driver in CML-like myeloproliferative disease (MPD). A conditional Gabpa knockout in BCR-ABL1+ HSCs caused prolonged survival of recipient mice, accompanied by decreased cycling of Gabpa−/− HSCs and LSCs [20Yang Z.F. Zhang H. Ma L. et al.GABP transcription factor is required for development of chronic myelogenous leukemia via its control of PRKD2.Proc Natl Acad Sci U S A. 2013; 110: 2312-2317Crossref PubMed Scopus (19) Google Scholar]. Remarkably, BCR-ABL1-expressing cells were still detectable in small amounts in peripheral blood for several months, contributing to each hematopoietic lineage in Gabpa null mice [20Yang Z.F. Zhang H. Ma L. et al.GABP transcription factor is required for development of chronic myelogenous leukemia via its control of PRKD2.Proc Natl Acad Sci U S A. 2013; 110: 2312-2317Crossref PubMed Scopus (19) Google Scholar]. This supposes that GABP is involved in proliferation of LSCs and leukemic blasts, whereas its loss still allows terminal differentiation of BCR-ABL1+ cells. In GABPβ mouse studies, the two isoforms, GABPβ1L and GABPβ2, contributing to the assembly of GABP heterotetramer complexes were targeted simultaneously [19Yu S. Jing X. Colgan J.D. Zhao D.M. Xue H.H. Targeting tetramer-forming GABPbeta isoforms impairs self-renewal of hematopoietic and leukemic stem cells.Cell Stem Cell. 2012; 11: 207-219Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar]. Mice transplanted with BCR-ABL1-expressing GABPβ1L−/− and GABPβ2−/− double knockout HSCs exhibited prolonged survival, most likely as a result of reduced LSC self-renewal. This was in line with the synergistic effect of elevated imatinib sensitivity on survival rates [19Yu S. Jing X. Colgan J.D. Zhao D.M. Xue H.H. Targeting tetrame
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