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MECOM oncogene expression correlates with chronic myeloid leukaemia (CML) progression. Here we show that the knockdown of MECOM (E) and MECOM (ME) isoforms reduces cell division at low cell density, inhibits colony-forming cells by 34% and moderately reduces BCR-ABL1 mRNA and protein expression but not tyrosine kinase catalytic activity in K562 cells. We also show that both E and ME are expressed in CD34+ selected cells of both CML chronic phase (CML-CP), and non-CML (normal) origin. Furthermore, MECOM mRNA and protein expression were repressed by imatinib mesylate treatment of CML-CP CD34+ cells, K562 and KY01 cell lines whereas imatinib had no effect in non-CML BCR-ABL1 −ve CD34+ cells. Together these results suggest that BCR-ABL1 tyrosine kinase catalytic activity regulates MECOM gene expression in CML-CP progenitor cells and that the BCR-ABL1 oncoprotein partially mediates its biological activity through MECOM. MECOM gene expression in CML-CP progenitor cells would provide an in vivo selective advantage, contributing to CML pathogenesis.
Chronic Myeloid Leukaemia (CML) is a disorder of haemopoietic stem cells (HSC) (Hamilton et al, 2010), characterized by the Philadelphia (Ph) chromosome (Kurzrock et al, 1988). The balanced translocation t(9;22) creates a novel fusion gene BCR-ABL1 (Ben-Neriah et al, 1986) which encodes a spatially (Wetzler et al, 1993) and functionally (Konopka et al, 1984) de-regulated tyrosine kinase, BCR-ABL1. BCR-ABL1 inappropriately activates the MAPK, PI3K and JAK-STAT signal transduction pathways (Pendergast et al, 1993; Skorski et al, 1997; Carlesso et al, 1996) contributing to abnormal myeloid cell proliferation, differentiation, transformation and survival (Smith et al, 2003).
CML usually progresses through three stages, which are designated as chronic (CP), accelerated (AP) and terminal blast crisis (BC) phases (Irvine et al, 2010). CML-CP and CML-BC resemble myeloproliferative disorder and acute leukaemia respectively; the transition between stages is unpredictable, non-time limited and inevitable unless treated (Elrick et al, 2005). Imatinib mesylate (IM; Glivec®; Novartis Pharma, Frimley, UK), a rationally designed tyrosine kinase inhibitor (TKI) that selectively inhibits BCR-ABL1 tyrosine kinase catalytic activity, is the currently favoured therapeutic agent that successfully manages the majority of patients with CML-CP (Valent, 2010), although it is much less effective when administered at advanced stages (CML-AP/BC) (O'Hare et al, 2006).
The t(9:22) translocation is a CML disease-initiating progenitor cell genetic change, the mutation being present in cells at all stages of the disease (Melo & Barnes, 2007). Disease progression requires the acquisition of new genetic abnormalities and various genes have been implicated in the majority of cases (reviewed in Melo & Barnes, 2007). Indeed, enhanced expression of MECOM (MDS1 and EVI1 complex locus, also known as EVI1, MDS1), a proto-oncogene located on chromosome 3q26, is frequently observed in CML-BC (Russell et al, 1993; Carapeti et al, 1996; Ogawa et al, 1996; De Weer et al, 2008). The MECOM gene encodes a zinc finger transcription factor with important roles both in normal development and leukemogenesis (Wieser, 2007). MECOM belongs to the positive regulatory (PR) domain family and is expressed as multiple naturally occurring alternatively spliced variants (Huang, 1999; Alzuherri et al, 2006). One form, designated MECOM (E), encodes the originally described protein (Morishita et al, 1990a) whereas another results from splicing of the coding region of the MDS1 gene with exon 2 of the EVI1 gene (encoding the PR domain) translating MECOM (ME) protein (Fears et al, 1996). Both proteins contain two domains of 7 (ZF1) and 3 (ZF2) repeats of the zinc finger motif (Morishita et al, 1988), function as DNA binding transcription factors (Wieser, 2007) and contribute to the progression of acute leukaemia (Morishita et al, 1992). Enhanced expression of MECOM in CML-BC implicates this transcription factor in disease progression (Ogawa et al, 1996).
Previous studies have shown that normal human CD34+ haemopoietic cells express MECOM (Gerhardt et al, 1997). Furthermore, this gene has been shown to have a role in self-renewal, proliferation and the repopulating capacity of murine HSC in Mecom null mice (Yuasa et al, 2005). However, MECOM's role in CML has not been fully determined. MECOM over-expression in CML-BC has been found both in the presence and absence of chromosome 3q26 abnormalities (Morishita et al, 1990b). Some chromosome 3q26 translocations generate enhanced expression of intact MECOM (E) including t(3:9;17;22), t(3;7), t(2;3), inv(3) and t(3;8) (De Weer et al, 2008; Henzan et al, 2004; Stevens-Kroef et al, 2004; Suzukawa et al, 1997; Lin et al, 2009) whereas others create novel fusion proteins involving ETV6 t(3;12) (Nakamura et al, 2002) or RUNX1 t(3;21) (Mitani et al, 1994). Many of the same genetic changes are also present in poor prognosis acute myeloid leukaemia.
Between 60–70% of CML-BC cells express MECOM in the absence of detectable gross cytogenetic abnormalities but in these cases it is unclear if expression is a marker or a driver of disease (Ogawa et al, 1996). These and other studies suggest that MECOM is not expressed in CML-CP mononuclear cells from bone marrow or peripheral blood, but CML-CP CD34+ cells have not been previously examined. This study investigated MECOM gene expression, the effect of IM treatment and the biological activity of this gene in primary CML-CP CD34+ progenitor cells as well as CML-derived cell lines.
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This study showed that MECOM has a role in cell proliferation in Ph+ cells. KD of MECOM in K562 cells reduced their proliferative capacity in CFC as well as single cell proliferation assays. These observations were the same for KD either of all MECOM isoforms (with HB14-treated cells) or when MECOM (Δ324) expression only was retained (HB11-treated cells), showing this latter truncated protein, which lacks transforming activity (Kilbey & Bartholomew, 1998) cannot compensate for reduced MECOM (E) and MECOM (ME) isoforms. It is possible that our studies underestimate the impact of MECOM repression due to partial KD only because other studies including Mecom KO mice showed a more severe effect on HSC numbers (Yuasa et al, 2005) and an almost complete cell cycle arrest was reported in MECOM KD K562 cells (Lugthart et al, 2011).
The MECOM (ME) 185 kDa protein was not detected in K562 or KY01 cells using α-MECOM antibody, despite qRT-PCR data demonstrating RNA expression of this isoform. It is currently unclear if the MECOM (ME) protein is not produced in K562 cells or is at levels below the detection limits of our assays. This study might have underestimated the abundance of MECOM (E) encoding transcripts, as there are multiple transcription initiation sites (1a, 1b, 1c and 3L) that are not detected by the specific primers and probe set used here (Aytekin et al, 2005; Lugthart et al, 2008). Expression of MECOM (E) alone is associated with poor prognosis acute leukaemia (Barjesteh van Waalwijk van Doorn-Khosrovani et al, 2003). Therefore higher levels of MECOM (E) relative to MECOM (ME) would be likely to contribute to disease progression. A similar observation has been made for another PR family gene, PRDM2, where the shorter PR domain-deleted forms are always found in chromosome 1p36-linked malignancies, consistent with the view that the long PR domain-containing forms have tumour suppressor activity (Huang, 1999).
We also showed here that MECOM expression is not only seen in K562 and KY01 cell lines but also in primary CML-CP cells, whereas previous reports have suggested it is only observed in CML-BC (Ogawa et al, 1996). It is likely that the discrepancy between our results and previous data are due to increased sensitivity of detection resulting from the analysis of a CD34+ subpopulation of peripheral blood mononuclear cells, which express the highest levels of MECOM. Both CD34+ non-CML cells and CD34+ CML cells expressed similar levels of both MECOM (E) and MECOM (ME) gene transcripts. Previous studies show both isoforms are present in normal tissues (Fears et al, 1996; Wimmer et al, 1998). Only one of the samples in this study, donor 010, was normal but the relative abundance of both transcripts was similar here to that of the other non-CML and CML samples examined. Therefore, these data show: (i) that CD34+ cells express high levels of MECOM and (ii) that both transcripts are present in CML cells at similar levels to normal and non-CML cells.
In this study we observed that MECOM KD had no effect on BCR-ABL1 kinase activity, so the oncogenic catalytic kinase was then switched-off by treatment with the TKI, IM. IM mediated inhibition of BCR-ABL1 resulted in a rapid repression of both MECOM (ME) and MECOM (E) gene expression in CML CD34+, K562 and KY01 cells. This effect is likely to be specific, as IM treatment had no effect on MECOM expression in BCR-ABL1-ve non-CML CD34+ cells (Fig 6C, D). Western blot analysis showed a dramatic reduction of the 145 kDa MECOM (E) protein in IM-treated K562 and KY01 cells. This repression was not a consequence of general inhibition of cell proliferation but was BCR-ABL1 specific, as treatment with the anti-metabolite, HC did not alter MECOM (E) protein levels. Furthermore, this was not the result of general cell toxicity, as trypan blue exclusion studies showed the cells retained viability for at least 24 h of IM treatment and there was no impact on cellular levels of numerous other proteins examined, including CrKL, GAPDH, STAT5, ERK1/2, AKT and BCR-ABL1 (data not shown).
As was seen for K562 cell line, IM-mediated inhibition of BCR-ABL1 results in a rapid decline in MECOM gene expression at both the mRNA and protein level in primary CML-CP cells. MECOM expression also declined, by 50%, following culture of primary CML-CP cells for 12–24 h. This reduction is not as great as in the presence of IM and might reflect maturation of cells or ex-vivo culture not adequately replicating in vivo conditions. A similar partial decline in MECOM expression was also observed in non-CML CD34+ cells cultured for 12 h or more.
Inhibition of MECOM expression by IM demonstrates for the first time that its expression is regulated in BCR-ABL1+ cells by the catalytic activity of this aberrant kinase. Regulation of mRNA expression is rapid, suggesting it is a direct response to inhibition of tyrosine kinase-mediated signalling from the BCR-ABL1 protein. Several studies have previously identified many potential BCR-ABL1 target genes but MECOM was not described (Håkansson et al, 2008; Nunoda et al, 2007; Bianchini et al, 2007). This study represents the first report of a signal transduction pathway that regulates MECOM gene expression. Although BCR-ABL1 transmits an aberrant signal, these results suggest MECOM expression is modulated by one or more of the three major pathways, JAK-STAT, MAPK or PI3K, activated by this promiscuous kinase (Pendergast et al, 1993; Skorski et al, 1997; Carlesso et al, 1996). The particular pathway involved is currently under investigation. The molecular basis of BCR-ABL1-mediated MECOM regulation should facilitate how expression of this developmentally important gene is controlled in HSC and other tissues where it is normally expressed (Yuasa et al, 2005; Goyama et al, 2008; Hoyt et al, 1997).
These data establish a link between BCR-ABL1 kinase catalytic activity and MECOM gene expression. We propose that BCR-ABL1 positively regulates MECOM gene expression. The level of MECOM gene expression is not elevated by BCR-ABL1 kinase relative to expression levels observed in non-CML and normal primitive haemopoetic cells. However, it is deregulated, because the mechanism regulating MECOM gene expression in normal CD34+ cells is distinct from that in CML-CP CD34+ cells, given that, in the latter, expression is dependent upon BCR-ABL1 catalytic activity. The BCR-ABL1 kinase might activate a pathway that normally regulates MECOM production in primitive haemopoietic cells. Since BCR-ABL1 kinase is constitutively active, it will continuously stimulate the pathway leading to sustained de-regulated MECOM gene expression. It is unlikely that MECOM expression is repressed by inhibition of other receptors (PDGFRΑ, PDGFRΒ or KIT) that are known to be inactivated by IM (Fabbro et al, 1999) because IM has no effect on BCR-ABL1 −ve CD34+ cells (Fig 6C, D).
Maintenance of MECOM expression in primitive HSC is probably a selective advantage. Retroviral tagging studies showed that proviral insertions are frequently seen in the MECOM locus in dominant non-malignant HSC clones retrieved from transplant recipients (Kustikova et al, 2005) and primary myeloid CD34+ex-vivo cultures are enriched for viral insertions in this gene (Sellers et al, 2010). Furthermore, MECOM is required for the survival and proliferation of HSC (Yuasa et al, 2005; Goyama et al, 2008). The MECOM KD studies in K562 cells described above support this notion, as these cells show a reduced proliferative capacity in CFC and single cell proliferation assays. Our results suggest that BCR-ABL1 tyrosine kinase catalytic activity sustains MECOM gene expression in CML-CP CD34+ cells and that this gives cells a selective advantage in a manner analogous to retroviral insertion. It is possible the impact of MECOM KD in primary CML-CP cells would be even greater than seen with K562, which is an immortal cell line with a number of additional genetic abnormalities. We are currently investigating this.
Sustained expression of MECOM could be one of the mechanisms by which BCR-ABL1 contributes a selective advantage to primitive haemopoietic cells in CML, resulting in an increased production of mature cells in peripheral blood. In this case, gross chromosome abnormalities are not required as the BCR-ABL1 mutation causes de-regulation of MECOM gene expression. Interestingly, MECOM translocations are frequently observed in CML patients treated with TKI inhibitors that progress to blast crisis (Paquette et al, 2011) and enhanced MECOM expression is a predictor of poor prognosis in TKI-resistant CML-CP (Daghistani et al, 2010). This suggests that inhibition of BCR-ABL1 kinase may select for cells that de-regulate MECOM expression by alternative mechanisms. Mutations causing elevated levels of MECOM (E) relative to MECOM (ME), or its fusion proteins that can occur in CML-BC might be necessary for other MECOM-mediated biological activities including inhibition of terminal cell differentiation. Indeed, previous studies show that MECOM mediated inhibition of granulocyte differentiation is dependent on the level of expression (Khanna-Gupta et al, 1996). Our results suggest BCR-ABL1-mediated MECOM gene expression represents a novel mechanism of de-regulating this gene in leukaemia.