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Reprogramming of MLL-AF9 leukemia cells into pluripotent stem cells.

Liu Y, Cheng H, Gao S, Lu X, He F, Hu L, Hou D, Zou Z, Li Y, Zhang H, Xu J, Kang L, Wang Q, Yuan W, Gao S, Cheng T - Leukemia (2013)

Bottom Line: RNA-seq analysis showed reversible global gene expression patterns between these interchangeable leukemia and iPS cells on activation or reactivation of MLL-AF9, suggesting a sufficient epigenetic force in driving the leukemogenic process.This study represents an important step for further defining the potential interplay between oncogenic molecules and reprogramming factors during MLL leukemogenesis.More importantly, our reprogramming approach may be expanded to characterize a range of hematopoietic malignancies in order to develop new strategies for clinical diagnosis and treatment.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Center for Stem Cell Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China.

ABSTRACT
The 'Yamanaka factors' (Oct4, Sox2, Klf4 and c-Myc) are able to generate induced pluripotent stem (iPS) cells from different cell types. However, to what degree primary malignant cells can be reprogrammed into a pluripotent state has not been vigorously assessed. We established an acute myeloid leukemia (AML) model by overexpressing the human mixed-lineage leukemia-AF9 (MLL-AF9) fusion gene in mouse hematopoietic cells that carry Yamanaka factors under the control of doxycycline (Dox). On addition of Dox to the culture, the transplantable leukemia cells were efficiently converted into iPS cells that could form teratomas and produce chimeras. Interestingly, most chimeric mice spontaneously developed the same type of AML. Moreover, both iPS reprogramming and leukemia reinitiation paths could descend from the same leukemia-initiating cell. RNA-seq analysis showed reversible global gene expression patterns between these interchangeable leukemia and iPS cells on activation or reactivation of MLL-AF9, suggesting a sufficient epigenetic force in driving the leukemogenic process. This study represents an important step for further defining the potential interplay between oncogenic molecules and reprogramming factors during MLL leukemogenesis. More importantly, our reprogramming approach may be expanded to characterize a range of hematopoietic malignancies in order to develop new strategies for clinical diagnosis and treatment.

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Mechanism of MLL-AF9 gene silencing. (a) FACS analysis of GFP expression in 1° leukemia cells and L-iPS cells, revealing that GFP was not expressed in L-iPS cells. (b) qRT-PCR analysis of the MLL-AF9 fusion gene expression, showing that the fusion gene was silenced in L-iPS cells but not in 1° and 2° leukemia cells (n=3). Bars represent mean±s.d. (c) Bisulfite genomic sequencing of the 3′LTR of MLL-AF9 vector in 1° leukemia cells, L-iPS cells and 2° leukemia cells. Black and white circles indicate methylated and unmethylated cytosine-phosphate-guanine (CpGs), respectively. Differences in methylation between each type of cell that were statistically significant are indicated (n⩾2), *P<0.05. (d) qRT-PCR analysis of KAP1 gene expression in different types of cells (n=3). Bars represent mean±s.d. (e) qRT-PCR analysis of Dnmts expression in different types of cells (n=3). Bars represent mean±s.d.
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fig3: Mechanism of MLL-AF9 gene silencing. (a) FACS analysis of GFP expression in 1° leukemia cells and L-iPS cells, revealing that GFP was not expressed in L-iPS cells. (b) qRT-PCR analysis of the MLL-AF9 fusion gene expression, showing that the fusion gene was silenced in L-iPS cells but not in 1° and 2° leukemia cells (n=3). Bars represent mean±s.d. (c) Bisulfite genomic sequencing of the 3′LTR of MLL-AF9 vector in 1° leukemia cells, L-iPS cells and 2° leukemia cells. Black and white circles indicate methylated and unmethylated cytosine-phosphate-guanine (CpGs), respectively. Differences in methylation between each type of cell that were statistically significant are indicated (n⩾2), *P<0.05. (d) qRT-PCR analysis of KAP1 gene expression in different types of cells (n=3). Bars represent mean±s.d. (e) qRT-PCR analysis of Dnmts expression in different types of cells (n=3). Bars represent mean±s.d.

Mentions: The convertible phenotypes between leukemia and iPS normalcy prompted us to further obtain a molecular basis underlying leukemogenesis versus reprogramming. MLL-AF9 is driven by the LTR promoter in the retroviral vector.29, 30, 31 LTR promoter activity can be silenced in ES cells mainly by KAP1 (KRAB-associated protein 1)- or Dnmts (DNA methyltransferases)-mediated methylation.32, 33, 34 As expected, GFP was not expressed in L-iPS cells (Figure 3a), indicating that the retroviral vector was silenced in L-iPS cells. qRT-PCR and RNA-Seq analyses demonstrated that MLL-AF9 was not expressed in all tested L-iPS cells (Figure 3b and Supplementary Figure S5A). In contrast, it was expressed in most (five/eight) chimeras on day 30 (Supplementary Figure S5B) and in all mice when leukemia was fully developed (Figure 3b and Supplementary Figure S5A). Bisulfite genomic sequencing of the 3′LTR of the MLL-AF9 vector demonstrated a higher level of methylation of the vector in L-iPS cells than in primary leukemia cells or recurrent leukemia cells (Figure 3c), which confirmed that MLL-AF9 silencing was due to methylation of the retroviral vector. Furthermore, an analysis of KAP1 expression by qRT-PCR revealed that ES, N-iPS and L-iPS all highly expressed the KAP1 gene compared with leukemia cells (Figure 3d), consistent with its role in silencing the retroviral vector in ES cells. Examination of Dnmts (including Dnmt1, Dnmt3a, Dnmt3b and Dnmt3l) using qRT-PCR revealed a higher expression of Dnmt3b in L-iPS cells than in ES, N-iPS and leukemia cells (Figure 3e), suggesting a more dominant role of Dnmt3b compared with other Dnmts in silencing the MLL-AF9 transgene.


Reprogramming of MLL-AF9 leukemia cells into pluripotent stem cells.

Liu Y, Cheng H, Gao S, Lu X, He F, Hu L, Hou D, Zou Z, Li Y, Zhang H, Xu J, Kang L, Wang Q, Yuan W, Gao S, Cheng T - Leukemia (2013)

Mechanism of MLL-AF9 gene silencing. (a) FACS analysis of GFP expression in 1° leukemia cells and L-iPS cells, revealing that GFP was not expressed in L-iPS cells. (b) qRT-PCR analysis of the MLL-AF9 fusion gene expression, showing that the fusion gene was silenced in L-iPS cells but not in 1° and 2° leukemia cells (n=3). Bars represent mean±s.d. (c) Bisulfite genomic sequencing of the 3′LTR of MLL-AF9 vector in 1° leukemia cells, L-iPS cells and 2° leukemia cells. Black and white circles indicate methylated and unmethylated cytosine-phosphate-guanine (CpGs), respectively. Differences in methylation between each type of cell that were statistically significant are indicated (n⩾2), *P<0.05. (d) qRT-PCR analysis of KAP1 gene expression in different types of cells (n=3). Bars represent mean±s.d. (e) qRT-PCR analysis of Dnmts expression in different types of cells (n=3). Bars represent mean±s.d.
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fig3: Mechanism of MLL-AF9 gene silencing. (a) FACS analysis of GFP expression in 1° leukemia cells and L-iPS cells, revealing that GFP was not expressed in L-iPS cells. (b) qRT-PCR analysis of the MLL-AF9 fusion gene expression, showing that the fusion gene was silenced in L-iPS cells but not in 1° and 2° leukemia cells (n=3). Bars represent mean±s.d. (c) Bisulfite genomic sequencing of the 3′LTR of MLL-AF9 vector in 1° leukemia cells, L-iPS cells and 2° leukemia cells. Black and white circles indicate methylated and unmethylated cytosine-phosphate-guanine (CpGs), respectively. Differences in methylation between each type of cell that were statistically significant are indicated (n⩾2), *P<0.05. (d) qRT-PCR analysis of KAP1 gene expression in different types of cells (n=3). Bars represent mean±s.d. (e) qRT-PCR analysis of Dnmts expression in different types of cells (n=3). Bars represent mean±s.d.
Mentions: The convertible phenotypes between leukemia and iPS normalcy prompted us to further obtain a molecular basis underlying leukemogenesis versus reprogramming. MLL-AF9 is driven by the LTR promoter in the retroviral vector.29, 30, 31 LTR promoter activity can be silenced in ES cells mainly by KAP1 (KRAB-associated protein 1)- or Dnmts (DNA methyltransferases)-mediated methylation.32, 33, 34 As expected, GFP was not expressed in L-iPS cells (Figure 3a), indicating that the retroviral vector was silenced in L-iPS cells. qRT-PCR and RNA-Seq analyses demonstrated that MLL-AF9 was not expressed in all tested L-iPS cells (Figure 3b and Supplementary Figure S5A). In contrast, it was expressed in most (five/eight) chimeras on day 30 (Supplementary Figure S5B) and in all mice when leukemia was fully developed (Figure 3b and Supplementary Figure S5A). Bisulfite genomic sequencing of the 3′LTR of the MLL-AF9 vector demonstrated a higher level of methylation of the vector in L-iPS cells than in primary leukemia cells or recurrent leukemia cells (Figure 3c), which confirmed that MLL-AF9 silencing was due to methylation of the retroviral vector. Furthermore, an analysis of KAP1 expression by qRT-PCR revealed that ES, N-iPS and L-iPS all highly expressed the KAP1 gene compared with leukemia cells (Figure 3d), consistent with its role in silencing the retroviral vector in ES cells. Examination of Dnmts (including Dnmt1, Dnmt3a, Dnmt3b and Dnmt3l) using qRT-PCR revealed a higher expression of Dnmt3b in L-iPS cells than in ES, N-iPS and leukemia cells (Figure 3e), suggesting a more dominant role of Dnmt3b compared with other Dnmts in silencing the MLL-AF9 transgene.

Bottom Line: RNA-seq analysis showed reversible global gene expression patterns between these interchangeable leukemia and iPS cells on activation or reactivation of MLL-AF9, suggesting a sufficient epigenetic force in driving the leukemogenic process.This study represents an important step for further defining the potential interplay between oncogenic molecules and reprogramming factors during MLL leukemogenesis.More importantly, our reprogramming approach may be expanded to characterize a range of hematopoietic malignancies in order to develop new strategies for clinical diagnosis and treatment.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Experimental Hematology, Institute of Hematology & Blood Diseases Hospital, Center for Stem Cell Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China.

ABSTRACT
The 'Yamanaka factors' (Oct4, Sox2, Klf4 and c-Myc) are able to generate induced pluripotent stem (iPS) cells from different cell types. However, to what degree primary malignant cells can be reprogrammed into a pluripotent state has not been vigorously assessed. We established an acute myeloid leukemia (AML) model by overexpressing the human mixed-lineage leukemia-AF9 (MLL-AF9) fusion gene in mouse hematopoietic cells that carry Yamanaka factors under the control of doxycycline (Dox). On addition of Dox to the culture, the transplantable leukemia cells were efficiently converted into iPS cells that could form teratomas and produce chimeras. Interestingly, most chimeric mice spontaneously developed the same type of AML. Moreover, both iPS reprogramming and leukemia reinitiation paths could descend from the same leukemia-initiating cell. RNA-seq analysis showed reversible global gene expression patterns between these interchangeable leukemia and iPS cells on activation or reactivation of MLL-AF9, suggesting a sufficient epigenetic force in driving the leukemogenic process. This study represents an important step for further defining the potential interplay between oncogenic molecules and reprogramming factors during MLL leukemogenesis. More importantly, our reprogramming approach may be expanded to characterize a range of hematopoietic malignancies in order to develop new strategies for clinical diagnosis and treatment.

Show MeSH
Related in: MedlinePlus