PMC0
10.1038%2Fonc.2014.108
introduction
Physiological and biological similarities of LSCs to those of HSCs are supposed to be the main causes of the difficulties in establishment of LSC-targeted therapy.>>1<<
Physiological and biological similarities of LSCs to those of HSCs are supposed to be the main causes of the difficulties in establishment of LSC-targeted therapy.1,>>2<<
(CML) is a myeloproliferative disorder by BCR–ABL, which can transform HSCs into LSCs with a limitless capacity for self-renewal, whereas LSCs of acute myeloid leukemia (AML) are mainly composed of more differentiated progenitor cells.>>3<<, 4, 5 The relentless march of CML from chronic phase (CP) to blast crisis (BC) phase can result in fatal survival outcomes.
(CML) is a myeloproliferative disorder by BCR–ABL, which can transform HSCs into LSCs with a limitless capacity for self-renewal, whereas LSCs of acute myeloid leukemia (AML) are mainly composed of more differentiated progenitor cells.3, >>4<<, 5 The relentless march of CML from chronic phase (CP) to blast crisis (BC) phase can result in fatal survival outcomes.
is a myeloproliferative disorder by BCR–ABL, which can transform HSCs into LSCs with a limitless capacity for self-renewal, whereas LSCs of acute myeloid leukemia (AML) are mainly composed of more differentiated progenitor cells.3, 4, >>5<< The relentless march of CML from chronic phase (CP) to blast crisis (BC) phase can result in fatal survival outcomes.
3, 4, 5 The relentless march of CML from chronic phase (CP) to blast crisis (BC) phase can result in fatal survival outcomes.>>6<< Despite substantial prognostic improvement of CML by a specific debulking of tumor burden with a tyrosine kinase inhibitor (TKI) targeting ABL kinase, imatinib-treated CP patients can relapse and progress to BC because of the remnant CML
prognostic improvement of CML by a specific debulking of tumor burden with a tyrosine kinase inhibitor (TKI) targeting ABL kinase, imatinib-treated CP patients can relapse and progress to BC because of the remnant CML stem cells.>>7<<, 8, 9, 10 Although recent findings have started to unveil the biological nature of CML stem cells,11, 12, 13, 14, 15, 16 further elucidation of the mechanisms controlling the self-renewal of these cells is still needed.
prognostic improvement of CML by a specific debulking of tumor burden with a tyrosine kinase inhibitor (TKI) targeting ABL kinase, imatinib-treated CP patients can relapse and progress to BC because of the remnant CML stem cells.7, >>8<<, 9, 10 Although recent findings have started to unveil the biological nature of CML stem cells,11, 12, 13, 14, 15, 16 further elucidation of the mechanisms controlling the self-renewal of these cells is still needed.
prognostic improvement of CML by a specific debulking of tumor burden with a tyrosine kinase inhibitor (TKI) targeting ABL kinase, imatinib-treated CP patients can relapse and progress to BC because of the remnant CML stem cells.7, 8, >>9<<, 10 Although recent findings have started to unveil the biological nature of CML stem cells,11, 12, 13, 14, 15, 16 further elucidation of the mechanisms controlling the self-renewal of these cells is still needed.
7, 8, 9, >>10<< Although recent findings have started to unveil the biological nature of CML stem cells,11, 12, 13, 14, 15, 16 further elucidation of the mechanisms controlling the self-renewal of these cells is still needed.
7, 8, 9, 10 Although recent findings have started to unveil the biological nature of CML stem cells,>>11<<, 12, 13, 14, 15, 16 further elucidation of the mechanisms controlling the self-renewal of these cells is still needed.
7, 8, 9, 10 Although recent findings have started to unveil the biological nature of CML stem cells,11, >>12<<, 13, 14, 15, 16 further elucidation of the mechanisms controlling the self-renewal of these cells is still needed.
7, 8, 9, 10 Although recent findings have started to unveil the biological nature of CML stem cells,11, 12, >>13<<, 14, 15, 16 further elucidation of the mechanisms controlling the self-renewal of these cells is still needed.
7, 8, 9, 10 Although recent findings have started to unveil the biological nature of CML stem cells,11, 12, 13, >>14<<, 15, 16 further elucidation of the mechanisms controlling the self-renewal of these cells is still needed.
7, 8, 9, 10 Although recent findings have started to unveil the biological nature of CML stem cells,11, 12, 13, 14, >>15<<, 16 further elucidation of the mechanisms controlling the self-renewal of these cells is still needed.
7, 8, 9, 10 Although recent findings have started to unveil the biological nature of CML stem cells,11, 12, 13, 14, 15, >>16<< further elucidation of the mechanisms controlling the self-renewal of these cells is still needed.
components that control hematopoiesis severely perturb normal development, one of which is ecotropic viral integration site 1 (Evi1), a predictor of poor outcomes in myeloid malignancies such as AML, myelodysplastic syndrome and CML-BC.>>17<<, 18, 19, 20, 21 In normal hematopoiesis, Evi1 is restricted to embryonic and adult HSCs22 and cumulative data have placed Evi1 as one of ‘stemness' genes.
that control hematopoiesis severely perturb normal development, one of which is ecotropic viral integration site 1 (Evi1), a predictor of poor outcomes in myeloid malignancies such as AML, myelodysplastic syndrome and CML-BC.17, >>18<<, 19, 20, 21 In normal hematopoiesis, Evi1 is restricted to embryonic and adult HSCs22 and cumulative data have placed Evi1 as one of ‘stemness' genes.
that control hematopoiesis severely perturb normal development, one of which is ecotropic viral integration site 1 (Evi1), a predictor of poor outcomes in myeloid malignancies such as AML, myelodysplastic syndrome and CML-BC.17, 18, >>19<<, 20, 21 In normal hematopoiesis, Evi1 is restricted to embryonic and adult HSCs22 and cumulative data have placed Evi1 as one of ‘stemness' genes.
17, 18, 19, >>20<<, 21 In normal hematopoiesis, Evi1 is restricted to embryonic and adult HSCs22 and cumulative data have placed Evi1 as one of ‘stemness' genes.
17, 18, 19, 20, >>21<< In normal hematopoiesis, Evi1 is restricted to embryonic and adult HSCs22 and cumulative data have placed Evi1 as one of ‘stemness' genes.
17, 18, 19, 20, 21 In normal hematopoiesis, Evi1 is restricted to embryonic and adult HSCs>>22<< and cumulative data have placed Evi1 as one of ‘stemness' genes.
17, 18, 19, 20, 21 In normal hematopoiesis, Evi1 is restricted to embryonic and adult HSCs22 and cumulative data have placed Evi1 as one of ‘stemness' genes.>>23<<, 24, 25 The recent gene expression profiling of bulk samples or selective populations have shown that EVI1 is one of LSC signature genes in AML and that stem cell-enriched CML CD34+ cells have high EVI1, underlining the relevance of EVI1
17, 18, 19, 20, 21 In normal hematopoiesis, Evi1 is restricted to embryonic and adult HSCs22 and cumulative data have placed Evi1 as one of ‘stemness' genes.23, >>24<<, 25 The recent gene expression profiling of bulk samples or selective populations have shown that EVI1 is one of LSC signature genes in AML and that stem cell-enriched CML CD34+ cells have high EVI1, underlining the relevance of EVI1 and
myeloid malignancies such as AML, myelodysplastic syndrome and CML-BC.17, 18, 19, 20, 21 In normal hematopoiesis, Evi1 is restricted to embryonic and adult HSCs22 and cumulative data have placed Evi1 as one of ‘stemness' genes.23, 24, >>25<< The recent gene expression profiling of bulk samples or selective populations have shown that EVI1 is one of LSC signature genes in AML and that stem cell-enriched CML CD34+ cells have high EVI1, underlining the relevance of EVI1 and LSCs.
recent gene expression profiling of bulk samples or selective populations have shown that EVI1 is one of LSC signature genes in AML and that stem cell-enriched CML CD34+ cells have high EVI1, underlining the relevance of EVI1 and LSCs.>>26<<,27 As EVI1 is an oncogenic transcription factor,28, 29, 30, 31, 32, 33, 34 prospective isolation of EVI1-high leukemic cells from clinical patients is unfeasible, so is the functional assessment of EVI1-high cells compared with EVI1-low
gene expression profiling of bulk samples or selective populations have shown that EVI1 is one of LSC signature genes in AML and that stem cell-enriched CML CD34+ cells have high EVI1, underlining the relevance of EVI1 and LSCs.26,>>27<< As EVI1 is an oncogenic transcription factor,28, 29, 30, 31, 32, 33, 34 prospective isolation of EVI1-high leukemic cells from clinical patients is unfeasible, so is the functional assessment of EVI1-high cells compared with EVI1-low
26,27 As EVI1 is an oncogenic transcription factor,>>28<<, 29, 30, 31, 32, 33, 34 prospective isolation of EVI1-high leukemic cells from clinical patients is unfeasible, so is the functional assessment of EVI1-high cells compared with EVI1-low cells intraindividually.
26,27 As EVI1 is an oncogenic transcription factor,28, >>29<<, 30, 31, 32, 33, 34 prospective isolation of EVI1-high leukemic cells from clinical patients is unfeasible, so is the functional assessment of EVI1-high cells compared with EVI1-low cells intraindividually.
26,27 As EVI1 is an oncogenic transcription factor,28, 29, >>30<<, 31, 32, 33, 34 prospective isolation of EVI1-high leukemic cells from clinical patients is unfeasible, so is the functional assessment of EVI1-high cells compared with EVI1-low cells intraindividually.
26,27 As EVI1 is an oncogenic transcription factor,28, 29, 30, >>31<<, 32, 33, 34 prospective isolation of EVI1-high leukemic cells from clinical patients is unfeasible, so is the functional assessment of EVI1-high cells compared with EVI1-low cells intraindividually.
26,27 As EVI1 is an oncogenic transcription factor,28, 29, 30, 31, >>32<<, 33, 34 prospective isolation of EVI1-high leukemic cells from clinical patients is unfeasible, so is the functional assessment of EVI1-high cells compared with EVI1-low cells intraindividually.
26,27 As EVI1 is an oncogenic transcription factor,28, 29, 30, 31, 32, >>33<<, 34 prospective isolation of EVI1-high leukemic cells from clinical patients is unfeasible, so is the functional assessment of EVI1-high cells compared with EVI1-low cells intraindividually.
26,27 As EVI1 is an oncogenic transcription factor,28, 29, 30, 31, 32, 33, >>34<< prospective isolation of EVI1-high leukemic cells from clinical patients is unfeasible, so is the functional assessment of EVI1-high cells compared with EVI1-low cells intraindividually.
we have established multiple CML mice model with Evi1-Internal Ribosomal Entry Site (IRES)-green fluorescent protein (GFP) knock-in allele, in which Evi1-high CML cells can be separated directly and prospectively using a single GFP>>35<< to evaluate their capacity for leukemia development.
materials and methods
Target gene expression for bulk samples or sorted populations was evaluated by LightCycler 480 as described previously.>>35<< All assays were performed in triplicate and expression levels relative to 18s ribosomal RNA were determined.
Evi1-IRES-GFP knock-in (Evi1+/GFP) mice, Evi1 knock-out (Evi1+/−) mice and p210 BCR–ABL transgenic (BCR–ABLtg/−) mice on a C57BL/6 (Ly5.2) background were used and genotyped as previously described.>>25<<,35,57 CML development of BCR–ABLtg/− mice was confirmed by leukocyte elevation (>10 000 cells/μl) and >80% increase of Gr-1+ cells in PB.
Evi1-IRES-GFP knock-in (Evi1+/GFP) mice, Evi1 knock-out (Evi1+/−) mice and p210 BCR–ABL transgenic (BCR–ABLtg/−) mice on a C57BL/6 (Ly5.2) background were used and genotyped as previously described.25,>>35<<,57 CML development of BCR–ABLtg/− mice was confirmed by leukocyte elevation (>10 000 cells/μl) and >80% increase of Gr-1+ cells in PB.
Evi1-IRES-GFP knock-in (Evi1+/GFP) mice, Evi1 knock-out (Evi1+/−) mice and p210 BCR–ABL transgenic (BCR–ABLtg/−) mice on a C57BL/6 (Ly5.2) background were used and genotyped as previously described.25,35,>>57<< CML development of BCR–ABLtg/− mice was confirmed by leukocyte elevation (>10 000 cells/μl) and >80% increase of Gr-1+ cells in PB.
In experiments with the Evi1-IRES-GFP knock-in mouse, a ‘fluorescence minus one' littermate control was analyzed in parallel to set GFP gates.>>35<< Antibodies are listed in Supplementary Table S2.
Plat-E packaging cells>>58<< were transiently transfected with retroviral constructs by FuGENE6 (Roche).
5FU-primed BM cells were incubated in RPMI1640 medium (Wako, Osaka, Japan) with cytokines (50 ng/ml stem cell factor, 50 ng/ml thrombopoietin, 10 ng/ml interleukin-6) for 24 h as previously described,>>25<<,35 and cultured cells were infected with retrovirus on RetroNectin (TAKARA)-coated plate.
5FU-primed BM cells were incubated in RPMI1640 medium (Wako, Osaka, Japan) with cytokines (50 ng/ml stem cell factor, 50 ng/ml thrombopoietin, 10 ng/ml interleukin-6) for 24 h as previously described,25,>>35<< and cultured cells were infected with retrovirus on RetroNectin (TAKARA)-coated plate.
Cells (1 × 103) were plated into MethoCult GF M3434 (StemCell Technologies, Vancouver, BC, Canada) as described previously.>>59<< The number of colonies was counted at day 7. Images were taken with a Nikon Eclipse TE2000-U (Nikon, Tokyo, Japan).
Cells were incubated with 5 ng/ml Hoechst 33342 (Invitrogen/Life Technologies) and 25 μg/ml verapamil at 37 °C for 45 min.>>35<<
OP-9 cells were pre-seeded on 24-well plates at day –1.>>59<<,60 One hundred LSK cells from Evi1-reporter CML-CP mice were cultured from day 0 in alpha-minimum essential medium with 20% fetal calf serum, 1% penicillin+streptomycin, 2 μmol/l L-glutamine (Gibco/Invitrogen/Life Technologies), 1 μmol/l
OP-9 cells were pre-seeded on 24-well plates at day –1.59,>>60<< One hundred LSK cells from Evi1-reporter CML-CP mice were cultured from day 0 in alpha-minimum essential medium with 20% fetal calf serum, 1% penicillin+streptomycin, 2 μmol/l L-glutamine (Gibco/Invitrogen/Life Technologies), 1 μmol/l
results
Microarray data from Radich et al.>>1<< revealed that EVI1 in the whole BM is upregulated in advanced phase (accelerated phase (AP) and BC) of CML compared with CML-CP, possibly implying the limited EVI1 in CML-CP stem cells and the extended EVI1 in BM of CML-AP and CML-BC
To clarify the precise behavior of Evi1-high CML LSK cells in vivo, we crossed Evi1+/GFP mice with p210 BCR–ABL transgenic mice (BCR–ABLtg/−),>>36<< which develop a CML-like disease (Supplementary Figure S3a), to analyze undamaged BM of CML-CP (Figure 2e).
The median survival of Evi1+/GFP BCR–ABLtg/− mice in our experiments was 287 days, consistent with the previous report,>>36<< which revealed that Evi1-IRES-GFP allele had no unforeseen effect on CML development.
Given that Evi1+/− mice are fertile with no sign of BM failure over a year,>>25<< these data suggest that Evi1 has a distinctive role in CML development.
To evaluate a leukemogenic function of Evi1-high cells in CML-BC, BCR–ABL and NUP98–HOXA9 were co-transduced>>37<< into 5FU-primed BM of Evi1+/GFP mice to establish Evi1-reporter CML-BC mice (Figure 4a).
Based on the criteria of Bethesda proposals,>>38<< the disease was finally diagnosed as ‘Myeloid leukemia with maturation'.
Given that the duration of CML-CP development by BCR–ABL retrovirus is about 3–4 weeks, and Evi1 alone could induce myelodysplastic syndrome/AML in mice after 6 months or more,>>39<<,40 these results indicate that Evi1 can have a causative role in blastic transformation of CML.
Given that the duration of CML-CP development by BCR–ABL retrovirus is about 3–4 weeks, and Evi1 alone could induce myelodysplastic syndrome/AML in mice after 6 months or more,39,>>40<< these results indicate that Evi1 can have a causative role in blastic transformation of CML.
discussion
Even in the era of TKI treatment in CML, blastic transformation can occur with the translocation involving EVI1 locus,>>41<<,42 which is crucial because the clinical outcomes of CML plummet from CP (80%) to BC (20%).
Even in the era of TKI treatment in CML, blastic transformation can occur with the translocation involving EVI1 locus,41,>>42<< which is crucial because the clinical outcomes of CML plummet from CP (80%) to BC (20%).
Even in the era of TKI treatment in CML, blastic transformation can occur with the translocation involving EVI1 locus,41,42 which is crucial because the clinical outcomes of CML plummet from CP (80%) to BC (20%).>>43<<,44 Allogeneic HSC transplantation is so far the only potential remedy for high EVI1 cases of CML-BC as well as AML, emphasizing the need for new therapy targeting
Even in the era of TKI treatment in CML, blastic transformation can occur with the translocation involving EVI1 locus,41,42 which is crucial because the clinical outcomes of CML plummet from CP (80%) to BC (20%).43,>>44<< Allogeneic HSC transplantation is so far the only potential remedy for high EVI1 cases of CML-BC as well as AML, emphasizing the need for new therapy targeting
43,44 Allogeneic HSC transplantation is so far the only potential remedy for high EVI1 cases of CML-BC as well as AML, emphasizing the need for new therapy targeting EVI1.>>18<<
43,44 Allogeneic HSC transplantation is so far the only potential remedy for high EVI1 cases of CML-BC as well as AML, emphasizing the need for new therapy targeting EVI1.18,>>21<<
43,44 Allogeneic HSC transplantation is so far the only potential remedy for high EVI1 cases of CML-BC as well as AML, emphasizing the need for new therapy targeting EVI1.18,21,>>45<<
43,44 Allogeneic HSC transplantation is so far the only potential remedy for high EVI1 cases of CML-BC as well as AML, emphasizing the need for new therapy targeting EVI1.18,21,45,>>46<<
When seen against previous studies,>>47<<, 48, 49 the novelty of this study lies in that high Evi1 could distinguish CML-CP stem cells even from SLAM-LSK cells or non-SLAM-LSK cells.
When seen against previous studies,47, >>48<<, 49 the novelty of this study lies in that high Evi1 could distinguish CML-CP stem cells even from SLAM-LSK cells or non-SLAM-LSK cells.
When seen against previous studies,47, 48, >>49<< the novelty of this study lies in that high Evi1 could distinguish CML-CP stem cells even from SLAM-LSK cells or non-SLAM-LSK cells.
proliferative potential in vitro, a superior leukemia-initiating capacity in vivo and nilotinib resistance. The resistant aspect of Evi1-high cells to nilotinib would fit into clinical data that high EVI1 is related to TKI resistance.>>17<< The in vivo quiescent status of Evi1-high CML-CP cells, which could proliferate aggressively in vitro, may be controlled by hypoxic BM niche microenvironment.
would fit into clinical data that high EVI1 is related to TKI resistance.17 The in vivo quiescent status of Evi1-high CML-CP cells, which could proliferate aggressively in vitro, may be controlled by hypoxic BM niche microenvironment.>>50<<,51 In line with less dependence of CML stem cells on BCR–ABL,2,52,53 Evi1-high CML-CP LSK cells showed a comparable BCR–ABL to their Evi1-low counterpart, reflecting little addiction to BCR–ABL (Figure 2d).
would fit into clinical data that high EVI1 is related to TKI resistance.17 The in vivo quiescent status of Evi1-high CML-CP cells, which could proliferate aggressively in vitro, may be controlled by hypoxic BM niche microenvironment.50,>>51<< In line with less dependence of CML stem cells on BCR–ABL,2,52,53 Evi1-high CML-CP LSK cells showed a comparable BCR–ABL to their Evi1-low counterpart, reflecting little addiction to BCR–ABL (Figure 2d).
50,51 In line with less dependence of CML stem cells on BCR–ABL,>>2<<,52,53 Evi1-high CML-CP LSK cells showed a comparable BCR–ABL to their Evi1-low counterpart, reflecting little addiction to BCR–ABL (Figure 2d).
50,51 In line with less dependence of CML stem cells on BCR–ABL,2,>>52<<,53 Evi1-high CML-CP LSK cells showed a comparable BCR–ABL to their Evi1-low counterpart, reflecting little addiction to BCR–ABL (Figure 2d).
50,51 In line with less dependence of CML stem cells on BCR–ABL,2,52,>>53<< Evi1-high CML-CP LSK cells showed a comparable BCR–ABL to their Evi1-low counterpart, reflecting little addiction to BCR–ABL (Figure 2d).
4h). Taken together, this is the first report that visualizes Evi1 upregulation in in vivo leukemia models. The finding is consistent with the previous report that showed the transcriptional regulation of Evi1 by NUP98–HOXA9 in vitro.>>54<< In our CML-BC model, HOXA9 showed no difference between Evi1-high and Evi1-low LK cells, eliminating the possible dependence of Evi1-high cells on NUP98–HOXA9 (Supplementary Figure S4b).
Although complete loss of Evi1 causes embryonic lethality in mice,>>25<<,55 Evi1+/− mice have no sign of BM failure over a year and are fertile with a decreased size and function of HSCs.
Although complete loss of Evi1 causes embryonic lethality in mice,25,>>55<< Evi1+/− mice have no sign of BM failure over a year and are fertile with a decreased size and function of HSCs.
a functional role of Evi1 in CML pathogenesis. Although complete loss of Evi1 causes embryonic lethality in mice,25,55 Evi1+/− mice have no sign of BM failure over a year and are fertile with a decreased size and function of HSCs.>>25<<,35 EVI1 has been reported to be relevant to BCR–ABL tyrosine kinase activity.27 Collectively, it is supposed that Evi1 reduction may permit the reversal of CML at the partial expense of HSCs.
a functional role of Evi1 in CML pathogenesis. Although complete loss of Evi1 causes embryonic lethality in mice,25,55 Evi1+/− mice have no sign of BM failure over a year and are fertile with a decreased size and function of HSCs.25,>>35<< EVI1 has been reported to be relevant to BCR–ABL tyrosine kinase activity.27 Collectively, it is supposed that Evi1 reduction may permit the reversal of CML at the partial expense of HSCs.
Evi1 causes embryonic lethality in mice,25,55 Evi1+/− mice have no sign of BM failure over a year and are fertile with a decreased size and function of HSCs.25,35 EVI1 has been reported to be relevant to BCR–ABL tyrosine kinase activity.>>27<< Collectively, it is supposed that Evi1 reduction may permit the reversal of CML at the partial expense of HSCs.
We extended this study to establish a new model of AML by BCR–ABL and Evi1. It is meaningful that overexpression of Evi1 itself is the driver in blastic transformation of CML not only by Evi1-related fusions. Cuenco et al.>>56<< have previously reported that EVI1 with BCR–ABL induces not leukemias but a fatal myeloproliferative disorder in mice.
DNA (in MSCV retroviral vector) in murine BMT model of Cuenco's study, we utilized murine Evi1 for Evi1 overexpression. Murine Evi1 especially in pMYs vector is suitable for establishing myeloid leukemia in mouse BMT model (Jones et al.>>42<< and unpublished), which resulted in a new AML model by Evi1 plus BCR–ABL. These findings can remind us of the importance of controlling Evi1 to impair CML-BC development by BCR–ABL and NUP98–HOXA9.
As authentic Evi1 targets,>>22<<,33,34 such as Gata2, Pten and Pbx1, showed no difference in expression levels between Evi1-high and Evi1-low CML cells (data not shown), the further exploration of CML-specific Evi1 targets would be warranted.
As authentic Evi1 targets,22,>>33<<,34 such as Gata2, Pten and Pbx1, showed no difference in expression levels between Evi1-high and Evi1-low CML cells (data not shown), the further exploration of CML-specific Evi1 targets would be warranted.
As authentic Evi1 targets,22,33,>>34<< such as Gata2, Pten and Pbx1, showed no difference in expression levels between Evi1-high and Evi1-low CML cells (data not shown), the further exploration of CML-specific Evi1 targets would be warranted.
stem cell disease such as CML. Although Evi1 could not enrich MLL-ENL AML LSCs in our model, it is possible that Evi1 reduction in the bulk leukemia cells would be a key in amelioration of MLL-related leukemia as previously investigated.>>25<< Exact introduction of these oncogenes to HSCs by different approaches such as via a transgene or a knocking-in technology could unravel the relation between AML stem cells and Evi1.
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