TRANSFORMING ACTIVITY OF THE NUP98-MLL FUSION IN A CONDITIONAL TRANSGENIC MOUSE MODEL
(Abstract release date: 05/19/16)
EHA Library. Thanasopoulou A. 06/10/16; 133167; P179
Ms. Angeliki Thanasopoulou
Contributions
Contributions
Abstract
Abstract: P179
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
Genes encoding for mixed-lineage leukaemia (MLL) and nucleoporin 98 (NUP98) are both involved in multiple recurrent chromosomal rearrangements associated with haematological malignancies. Inv(11)(p15q23), present as a sole cytogenetic abnormality in patients with myelodysplastic syndromes (MDS) or acute myeloid leukaemia (AML), was reported to lead to the expression of a fusion containing the FG-repeats of NUP98 and almost the entire open reading frame of MLL but lacking the N-terminal menin/LEDGF interaction domain, known to be critical for transformation of all currently known MLL fusions through expression of critical downstream targets like the HOXA gene cluster.
Aims
To address the transforming potential of the NUP98-MLL fusion we established conditional transgenic mice.
Methods
The human NUP98-MLL fusion ORF of 13kb was integrated into the Hprt gene locus under the control of a tetracycline (Tet) responsive element in mouse ES cells harbouring a reverse Tet transactivator (rtTA) in the Rosa26 locus and allowing conditional expression of the transgene by doxycycline (DOX) administration. The effects of conditional NUP98-MLL expression on the haematopoietic system were analysed.
Results
Ex vivo expression of NUP98-MLL in lineage-marker depleted bone marrow (BM) cells increased colony formation and replating in methylcellulose. In liquid cultures, NUP98-MLL increased proliferation and impaired differentiation of the cells shown by cell morphology and flow cytometric analysis for Gr-1, Mac-1, CD34 and c-Kit expression. In vivo induction of NUP98-MLL led to an MDS-like disease with progression to an AML phenotype after a median latency of 78 weeks. The pre-leukemic phenotype was characterized by the presence of dysplastic myeloid cells in the periphery, decreased Mac-1/Gr-1 expression in the BM, and extensive extramedullary haematopoiesis, especially erythropoiesis in the spleen. These mice also exhibited an increased number of haematopoietic stem and progenitor cells (HSPCs) that provided a significant competitive repopulation advantage when transplanted into irradiated syngeneic recipients. So far, 4 mice have progressed to AML characterized by increased white blood counts, presence of blasts in the periphery with infiltration of the BM, spleen, and other organs. The disease was transplantable into irradiated recipients and depended on NUP98-MLL expression since propagation of the disease was significantly impaired in the absence of DOX. As the fusion lacks the menin/LEDGF interaction domain we addressed the sensitivity to small molecules targeting this interface and expression of the HOX gene cluster. Interestingly, in contrast to cells immortalized by the MLL-AF9 or MLL-ENL fusions, NUP98-MLL expressing cells were resistant to the small molecule Mi2-2 menin inhibitor and did not express high levels of the HOX-A-B-C genes. In addition, treatment with the bromodomain inhibitor JQ-1 did not induce cell cycle arrest or significant cytotoxicity but increased the fraction of cells in >G2 phase suggesting alternative transforming mechanisms of NUP98-MLL implicating the cell cycle checkpoint control.
Conclusion
We show that transgenic NUP98-MLL expression in mice resulted in an “MDS-like” pre-leukemic state progressing to AML after a long latency. The transforming mechanisms of NUP98-MLL fusion differed from other MLL-fusions resulting in relative resistance to small molecules that are currently being explored for targeted AML therapy.
Session topic: Acute myeloid leukemia - Biology 1
Keyword(s): Acute myeloid leukemia, MDS/AML, NUP98, Transgenic mouse
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
Genes encoding for mixed-lineage leukaemia (MLL) and nucleoporin 98 (NUP98) are both involved in multiple recurrent chromosomal rearrangements associated with haematological malignancies. Inv(11)(p15q23), present as a sole cytogenetic abnormality in patients with myelodysplastic syndromes (MDS) or acute myeloid leukaemia (AML), was reported to lead to the expression of a fusion containing the FG-repeats of NUP98 and almost the entire open reading frame of MLL but lacking the N-terminal menin/LEDGF interaction domain, known to be critical for transformation of all currently known MLL fusions through expression of critical downstream targets like the HOXA gene cluster.
Aims
To address the transforming potential of the NUP98-MLL fusion we established conditional transgenic mice.
Methods
The human NUP98-MLL fusion ORF of 13kb was integrated into the Hprt gene locus under the control of a tetracycline (Tet) responsive element in mouse ES cells harbouring a reverse Tet transactivator (rtTA) in the Rosa26 locus and allowing conditional expression of the transgene by doxycycline (DOX) administration. The effects of conditional NUP98-MLL expression on the haematopoietic system were analysed.
Results
Ex vivo expression of NUP98-MLL in lineage-marker depleted bone marrow (BM) cells increased colony formation and replating in methylcellulose. In liquid cultures, NUP98-MLL increased proliferation and impaired differentiation of the cells shown by cell morphology and flow cytometric analysis for Gr-1, Mac-1, CD34 and c-Kit expression. In vivo induction of NUP98-MLL led to an MDS-like disease with progression to an AML phenotype after a median latency of 78 weeks. The pre-leukemic phenotype was characterized by the presence of dysplastic myeloid cells in the periphery, decreased Mac-1/Gr-1 expression in the BM, and extensive extramedullary haematopoiesis, especially erythropoiesis in the spleen. These mice also exhibited an increased number of haematopoietic stem and progenitor cells (HSPCs) that provided a significant competitive repopulation advantage when transplanted into irradiated syngeneic recipients. So far, 4 mice have progressed to AML characterized by increased white blood counts, presence of blasts in the periphery with infiltration of the BM, spleen, and other organs. The disease was transplantable into irradiated recipients and depended on NUP98-MLL expression since propagation of the disease was significantly impaired in the absence of DOX. As the fusion lacks the menin/LEDGF interaction domain we addressed the sensitivity to small molecules targeting this interface and expression of the HOX gene cluster. Interestingly, in contrast to cells immortalized by the MLL-AF9 or MLL-ENL fusions, NUP98-MLL expressing cells were resistant to the small molecule Mi2-2 menin inhibitor and did not express high levels of the HOX-A-B-C genes. In addition, treatment with the bromodomain inhibitor JQ-1 did not induce cell cycle arrest or significant cytotoxicity but increased the fraction of cells in >G2 phase suggesting alternative transforming mechanisms of NUP98-MLL implicating the cell cycle checkpoint control.
Conclusion
We show that transgenic NUP98-MLL expression in mice resulted in an “MDS-like” pre-leukemic state progressing to AML after a long latency. The transforming mechanisms of NUP98-MLL fusion differed from other MLL-fusions resulting in relative resistance to small molecules that are currently being explored for targeted AML therapy.
Session topic: Acute myeloid leukemia - Biology 1
Keyword(s): Acute myeloid leukemia, MDS/AML, NUP98, Transgenic mouse
Abstract: P179
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
Genes encoding for mixed-lineage leukaemia (MLL) and nucleoporin 98 (NUP98) are both involved in multiple recurrent chromosomal rearrangements associated with haematological malignancies. Inv(11)(p15q23), present as a sole cytogenetic abnormality in patients with myelodysplastic syndromes (MDS) or acute myeloid leukaemia (AML), was reported to lead to the expression of a fusion containing the FG-repeats of NUP98 and almost the entire open reading frame of MLL but lacking the N-terminal menin/LEDGF interaction domain, known to be critical for transformation of all currently known MLL fusions through expression of critical downstream targets like the HOXA gene cluster.
Aims
To address the transforming potential of the NUP98-MLL fusion we established conditional transgenic mice.
Methods
The human NUP98-MLL fusion ORF of 13kb was integrated into the Hprt gene locus under the control of a tetracycline (Tet) responsive element in mouse ES cells harbouring a reverse Tet transactivator (rtTA) in the Rosa26 locus and allowing conditional expression of the transgene by doxycycline (DOX) administration. The effects of conditional NUP98-MLL expression on the haematopoietic system were analysed.
Results
Ex vivo expression of NUP98-MLL in lineage-marker depleted bone marrow (BM) cells increased colony formation and replating in methylcellulose. In liquid cultures, NUP98-MLL increased proliferation and impaired differentiation of the cells shown by cell morphology and flow cytometric analysis for Gr-1, Mac-1, CD34 and c-Kit expression. In vivo induction of NUP98-MLL led to an MDS-like disease with progression to an AML phenotype after a median latency of 78 weeks. The pre-leukemic phenotype was characterized by the presence of dysplastic myeloid cells in the periphery, decreased Mac-1/Gr-1 expression in the BM, and extensive extramedullary haematopoiesis, especially erythropoiesis in the spleen. These mice also exhibited an increased number of haematopoietic stem and progenitor cells (HSPCs) that provided a significant competitive repopulation advantage when transplanted into irradiated syngeneic recipients. So far, 4 mice have progressed to AML characterized by increased white blood counts, presence of blasts in the periphery with infiltration of the BM, spleen, and other organs. The disease was transplantable into irradiated recipients and depended on NUP98-MLL expression since propagation of the disease was significantly impaired in the absence of DOX. As the fusion lacks the menin/LEDGF interaction domain we addressed the sensitivity to small molecules targeting this interface and expression of the HOX gene cluster. Interestingly, in contrast to cells immortalized by the MLL-AF9 or MLL-ENL fusions, NUP98-MLL expressing cells were resistant to the small molecule Mi2-2 menin inhibitor and did not express high levels of the HOX-A-B-C genes. In addition, treatment with the bromodomain inhibitor JQ-1 did not induce cell cycle arrest or significant cytotoxicity but increased the fraction of cells in >G2 phase suggesting alternative transforming mechanisms of NUP98-MLL implicating the cell cycle checkpoint control.
Conclusion
We show that transgenic NUP98-MLL expression in mice resulted in an “MDS-like” pre-leukemic state progressing to AML after a long latency. The transforming mechanisms of NUP98-MLL fusion differed from other MLL-fusions resulting in relative resistance to small molecules that are currently being explored for targeted AML therapy.
Session topic: Acute myeloid leukemia - Biology 1
Keyword(s): Acute myeloid leukemia, MDS/AML, NUP98, Transgenic mouse
Type: Poster Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 17:15 - 18:45
Location: Poster area (Hall H)
Background
Genes encoding for mixed-lineage leukaemia (MLL) and nucleoporin 98 (NUP98) are both involved in multiple recurrent chromosomal rearrangements associated with haematological malignancies. Inv(11)(p15q23), present as a sole cytogenetic abnormality in patients with myelodysplastic syndromes (MDS) or acute myeloid leukaemia (AML), was reported to lead to the expression of a fusion containing the FG-repeats of NUP98 and almost the entire open reading frame of MLL but lacking the N-terminal menin/LEDGF interaction domain, known to be critical for transformation of all currently known MLL fusions through expression of critical downstream targets like the HOXA gene cluster.
Aims
To address the transforming potential of the NUP98-MLL fusion we established conditional transgenic mice.
Methods
The human NUP98-MLL fusion ORF of 13kb was integrated into the Hprt gene locus under the control of a tetracycline (Tet) responsive element in mouse ES cells harbouring a reverse Tet transactivator (rtTA) in the Rosa26 locus and allowing conditional expression of the transgene by doxycycline (DOX) administration. The effects of conditional NUP98-MLL expression on the haematopoietic system were analysed.
Results
Ex vivo expression of NUP98-MLL in lineage-marker depleted bone marrow (BM) cells increased colony formation and replating in methylcellulose. In liquid cultures, NUP98-MLL increased proliferation and impaired differentiation of the cells shown by cell morphology and flow cytometric analysis for Gr-1, Mac-1, CD34 and c-Kit expression. In vivo induction of NUP98-MLL led to an MDS-like disease with progression to an AML phenotype after a median latency of 78 weeks. The pre-leukemic phenotype was characterized by the presence of dysplastic myeloid cells in the periphery, decreased Mac-1/Gr-1 expression in the BM, and extensive extramedullary haematopoiesis, especially erythropoiesis in the spleen. These mice also exhibited an increased number of haematopoietic stem and progenitor cells (HSPCs) that provided a significant competitive repopulation advantage when transplanted into irradiated syngeneic recipients. So far, 4 mice have progressed to AML characterized by increased white blood counts, presence of blasts in the periphery with infiltration of the BM, spleen, and other organs. The disease was transplantable into irradiated recipients and depended on NUP98-MLL expression since propagation of the disease was significantly impaired in the absence of DOX. As the fusion lacks the menin/LEDGF interaction domain we addressed the sensitivity to small molecules targeting this interface and expression of the HOX gene cluster. Interestingly, in contrast to cells immortalized by the MLL-AF9 or MLL-ENL fusions, NUP98-MLL expressing cells were resistant to the small molecule Mi2-2 menin inhibitor and did not express high levels of the HOX-A-B-C genes. In addition, treatment with the bromodomain inhibitor JQ-1 did not induce cell cycle arrest or significant cytotoxicity but increased the fraction of cells in >G2 phase suggesting alternative transforming mechanisms of NUP98-MLL implicating the cell cycle checkpoint control.
Conclusion
We show that transgenic NUP98-MLL expression in mice resulted in an “MDS-like” pre-leukemic state progressing to AML after a long latency. The transforming mechanisms of NUP98-MLL fusion differed from other MLL-fusions resulting in relative resistance to small molecules that are currently being explored for targeted AML therapy.
Session topic: Acute myeloid leukemia - Biology 1
Keyword(s): Acute myeloid leukemia, MDS/AML, NUP98, Transgenic mouse
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