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Ling Zhao - One of the best experts on this subject based on the ideXlab platform.
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chd7 deficiency delays leukemogenesis in mice induced by cbfb MYH11
Blood, 2017Co-Authors: Tao Zhen, Katherine R Hyde, Lemlem Alemu, Ling Zhao, Erika M Kwon, Ying Lu, Nancy A SpeckAbstract:Inversion of chromosome 16 is a consistent finding in patients with acute myeloid leukemia subtype M4 with eosinophilia, which generates a CBFB-MYH11 fusion gene. Previous studies showed that the interaction between CBFβ-smooth muscle myosin heavy chain (SMMHC; encoded by CBFB-MYH11) and RUNX1 plays a critical role in the pathogenesis of this leukemia. Recently, it was shown that chromodomain helicase DNA-binding protein-7 (CHD7) interacts with RUNX1 and suppresses RUNX1-induced expansion of hematopoietic stem and progenitor cells. These results suggest that CHD7 is also critical for CBFB-MYH11–induced leukemogenesis. To test this hypothesis, we generated Chd7f/fMx1-CreCbfb+/56M mice, which expressed the Cbfb-MYH11 fusion gene and deactivated Chd7 in hematopoietic cells upon inducing Cre with polyinosinic-polycytidylic acid. The Lin–Sca1–c-Kit+ (LK) population was significantly lower in Chd7f/fMx1-CreCbfb+/56M mice than in Mx1-CreCbfb+/56M mice. In addition, there were fewer 5-bromo-2′-deoxyuridine–positive cells in the LK population in Chd7f/fMx1-CreCbfb+/56M mice, and genes associated with cell cycle, cell growth, and proliferation were differentially expressed between Chd7f/fMx1-CreCbfb+/56M and Mx1-CreCbfb+/56M leukemic cells. In vitro studies showed that CHD7 interacted with CBFβ-SMMHC through RUNX1 and that CHD7 enhanced transcriptional activity of RUNX1 and CBFβ-SMMHC on Csf1r, a RUNX1 target gene. Moreover, RNA sequencing of c-Kit+ cells showed that CHD7 functions mostly through altering the expression of RUNX1 target genes. Most importantly, Chd7 deficiency delayed Cbfb-MYH11–induced leukemia in both primary and transplanted mice. These data indicate that Chd7 is important for Cbfb-MYH11–induced leukemogenesis by facilitating RUNX1 regulation of transcription and cellular proliferation.
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runx1 and cbfb MYH11 are required for the maintenance of inv 16 aml
Blood, 2016Co-Authors: Yiqian Wang, Lemlem Alemu, Ling Zhao, Kira Hannon, Lisa Richter, Michelle Becker, Lisa Garett, Cecilia Rivas, Katherine R HydeAbstract:The inversion of chromosome 16 (inv(16)) is found in 5-12% of human AML cases. Although considered a marker of favorable prognosis, approximately half of inv(16) AML patients eventually relapse. Inv(16) generates a fusion gene between the transcription factor gene CBFB and the MYH11 gene. Expression of the CBFB-MYH11 fusion gene, which encodes CBFβ-SMMHC, is the initiating event, but cooperating mutations are required for transformation to a frank leukemia. In previous work, we showed that CBFβ and CBFβ-SMMHC binding partner RUNX1 is required for efficient leukemia development. Small molecule inhibitors of the CBFβ-SMMHC: RUNX1 complex decrease leukemic burden and increase survival in mouse models, indicating that both proteins also play a role in leukemia maintenance. However, it is not currently known whether inhibition of this complex alone is sufficient to cure inv(16) AML. To test the requirement for CBFB-MYH11 after leukemic transformation, we generated knockin mice that have a Cbfb-MYH11 allele flanked by loxP sites ( Cbfb flMYH11 ), which allows for deletion of Cbfb-MYH11 by Cre recombinase (Cre). Chimeric founder mice were treated with N-ethyl-N-nitrosourea (ENU) to induce cooperating mutations and leukemia. Leukemia cells from three different founder mice had a similar histological appearance and immunophenotype as leukemia cells derived from previous Cbfb-MYH11 knockin models. Importantly, the leukemia was transplantable, with similar latency as the previous knockin models. These findings indicate that the Cbfb flMYH11 allele causes frank leukemia, similar to other Cbfb-MYH11 alleles. To induce excision of the fusion gene, Cbfb +/flMYH11 leukemia cells were transduced with a lentivirus expressing Cre and GFP. Excision of the Cbfb-MYH11 allele was verified by PCR and showed an average excision frequency of 72.3%, +/- 1.8. To test if deletion of Cbfb-MYH11 affected cell survival, leukemia cells were infected with Cre and control viruses, and stained for Annexin V. At 48 hours post-transduction, total Cre-infected Cbfb +/flMYH11 leukemia cells showed a statistically significant increase in Annexin V staining, as compared to cells infected with the control virus. Importantly, increased Annexin V staining was seen in Csf2rb - cells, a population we previously showed to be enriched for leukemia stem cells (LSCs). To test if loss of Cbfb-MYH11 induced differentiation of leukemia cells, we stained Cre and control transduced Cbfb flMYH11 leukemia cells for the myeloid differentiation markers Gr-1 and Mac-1. We found no difference in the expression of either marker with Cbfb-MYH11 excision. These findings indicate that Cbfb-MYH11 is required for the survival of leukemic cells, including the LSC population, and its loss does not cause differentiation. To test if Runx1 is required for Cbfb-MYH11 activities during leukemia maintenance, we utilized a lentiviral vector expressing an shRNA against Runx1 and infected Cbfb-MYH11 expressing mouse leukemia cells. Runx1 knockdown was verified by quantitative RT-PCR and by western blot. To test if Runx1 knockdown induced apoptosis, leukemia cells infected with Runx1 knockdown or a scrambled shRNA control virus were stained for Annexin V. With an average decrease in Runx1 of 60.0% +/- 0.17, we observed an overall increase in Annexin V staining, but not in the Csf2rb-, LSC enriched population. This implies that LSCs may be less sensitive to decreased RUNX1 activity, than non-LSCs. To examine the effect of Runx1 knockdown on LSC activity in vitro, we performed colony forming cell (CFC) assays. We found that cells with decreased expression of Runx1 produced significantly fewer colonies as compared to the scrambled shRNA-infected cells. While some of the observed colonies may have been due to rare cells that lost or silenced the shRNA vector, our preliminary data indicates some colonies retained Runx1 knockdown (70.0% decrease, as compared to control infected cells) after growth in culture. These findings indicate that Runx1 is required for leukemia maintenance, but that LSCs may be less sensitive to decreased RUNX1 activity than non-LSCs. Taken together, our results imply that both Cbfb-MYH11 and Runx1 are important for the maintenance of inv(16) AML, and that inhibition of CBFβ-SMMHCand RUNX1 have potential as a cure for inv(16) AML. Disclosures No relevant conflicts of interest to declare.
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runx1 is strictly required for cbfb MYH11 induced leukemia development
Blood, 2016Co-Authors: Tao Zhen, Katherine R Hyde, Ling Zhao, Guadalupe Lopez, Erika Mijin Kwon, Lemlem AlemuAbstract:Inversion of chromosome 16 is a consistent finding in patients with acute myeloid leukemia subtype M4 with eosinophilia (AML M4Eo), which generates a CBFB-MYH11 fusion gene. The prevailing hypothesis for the mechanism of leukemia development by CBFbeta-SMMHC, the fusion protein encoded by CBFB-MYH11, is that CBFbeta-SMMHC is a dominant negative repressor of RUNX1, a transcription factor that physically interacts with CBFbeta and CBFbeta-SMMHC. If this hypothesis is correct, reducing RUNX1 activity should facilitate leukemogenesis by CBFB-MYH11. In fact, loss-of-function mutations in RUNX1 are common in human AML, but not in inv(16) AML. However, we previously demonstrated that CBFB-MYH11 has RUNX1-repression independent functions (Hyde et al., Blood 115:1433, 2010). Moreover, we recently showed that a dominant negative allele of Runx1, Runx1-lz, delayed leukemogenesis by CBFB-MYH11 in a mouse model (Hyde et al., Leukemia 29:1771, 2015). These findings challenge the RUNX1-repression model for CBFbeta-SMMHC mediated leukemogenesis. However, our previous findings are not conclusive since the Runx1+/lz mice used in the previous study have one wild-type Runx1 allele, and still retain some Runx1 function. To definitively address this question, we crossed Cre-based conditional Runx1 knockout mice (Runx1f/f) with Cre-based conditional Cbfb-MYH11 knockin mice (Cbfb+/56M) and Mx1-Cre mice to generate Runx1f/f, Mx1-Cre, Cbfb+/56Mmice, which express CBFbeta-SMMHC but not Runx1 after pIpC (poly I:C) treatment to induce Cre expression. Runx1f/f, Mx1-Cre, Cbfb+/56Mmice had more severe platelet deficiencies and higher numbers of Lin-/Sca1-/C-kit+ progenitors and Lin-/Sca1+/C-kit+ hematopoietic stem cells in the bone marrow when comapred with Runx1f/f, Mx1-Cre mice. Unexpectedly Runx1f/f, Mx1-Cre, Cbfb+/56Mmice also developed severe macrocytic anemia within two weeks after pIpC induction, which was lethal in about 1/3 of the mice. However, none of the Runx1f/f, Mx1-Cre, Cbfb+/56M mice developed leukemia up to one year after pIpC treatment. In contrast, all Mx1-Cre, Cbfb+/56M mice developed leukemia with an average survival of 4 months, as reported previously. These results suggest that Runx1 is strictly required for Cbfb-MYH11 induced leukemogenesis. To further study the mechanism of leukemogenesis, we performed RNA-Seq on C-kit+ bone marrow cells isolated from mice two weeks after pIpC treatment, to explore the global gene expression changes caused by Runx1 knockout on Cbfb-MYH11 expressing mice. Our preliminary data analysis showed that 1688 genes were differential expressed (Padj ≤0.05, FC ≥ 2) between Runx1f/f, Mx1-Cre, Cbfb+/56M and Mx1-Cre, Cbfb+/56M mice. Interestingly, many of these genes (48%) are Runx1 target genes. The above results suggest that mis-regulating the expression of Runx1 target genes contributes to leukemogenesis by CBFbeta-SMMHC. Disclosures No relevant conflicts of interest to declare.
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runx1 is required for hematopoietic defects and leukemogenesis in cbfb MYH11 knock in mice
Leukemia, 2015Co-Authors: Ling Zhao, R K Hyde, L AlemuAbstract:Runx1 is required for hematopoietic defects and leukemogenesis in Cbfb-MYH11 knock-in mice
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runx1 is required by the inv 16 fusion gene cbfb MYH11
Blood, 2012Co-Authors: Katherine R Hyde, Ling Zhao, Lemlem AlemuAbstract:Abstract 2434 Acute myeloid leukemia (AML) is often associated with specific, recurrent chromosomal abnormalities, such as the inversion of chromosome 16 (Inv(16)) which is associated with subtype M4 with eosinophilia. This inversion creates a fusion between CBFB and MYH11, which encode Core Binding Factor beta and Smooth Muscle Myosin Heavy Chain, respectively. The resulting fusion gene, CBFB-MYH11, is known to be the initiating factor in Inv(16) AML, but its mechanism is not clear. Previous studies indicated that repression of RUNX1 is a potential mechanism. However, we found that Cbfb-MYH11 has activities independent of Runx1 repression. During primitive hematopoiesis, we showed that expression of Cbfb-MYH11 in knockin mouse embryos (Cbfb+/MYH11) caused defects in differentiation that were not seen in embryos nullizygous for Runx1 (Runx1−/−), indicating that Cbfb-MYH11 has activities in addition to the repression of Runx1. Moreover, we found that the defects in the primitive hematopoiesis were rescued in the Cbfb+/MYH11; Runx1−/− embryos, which suggests that Runx1 is required for Cbfb-MYH11 activity during primitive hematopoiesis. We next asked whether Cbfb-MYH11 was similarly dependent on Runx1 during definitive hematopoiesis. For this purpose we used mice expressing another allele of Runx1 in which a 39-truncated Runx1 is fused to the b-galactosidase gene, lacZ (Runx1lzd). This Runx1 allele has been reported to have dominant negative activities. Using an in vitro promoter assay, we found that co-expression of Cbfβ with Runx1 and Runx1-lzd resulted in decreased activation of the MCSFR promoter as compared to co-expressing Cbfβ and Runx1, indicating that Runx1-lzd has dominant negative activities. In addition, we found that expression of a single Runx1-lzd allele rescued the primitive blood defect in the Cbfb+/MYH11 embryos. Runx1+/lzd; Cbfb+/MYH11 embryos showed almost normal definitive hematopoiesis providing further evidence that Runx1-lzd has dominant negative activity. Previously we showed that induction of Cbfb-MYH11 results in a distinct population of pre-leukemic cells. By combining the Runx1-lzd allele with an inducible allele of Cbfb-MYH11, we examined the requirement for Runx1 activity in the production of pre-leukemic cells. We found that 7 days after induction of Cbfb-MYH11, Runx1+/lzd; Cbfb+/MYH11 mice showed a statistically significant decrease in the number of pre-leukemic cells as compared to Runx1+/+; Cbfb+/MYH11 mice. We also found a statistically significant decrease in BrdU incorporation in the bone marrow of Runx1+/lzd; Cbfb+/MYH11 mice as compared to Runx1+/+; Cbfb+/MYH11 mice. This indicates that Runx1 is important for Cbfb-MYH11 activity in adult hematopoietic cells. Consistent with this idea, we found that adult mice expressing Cbfb-MYH11 and the Runx1-lzd allele showed a significant delay in the development of leukemia as compared to their Cbfb+/MYH11; Runx1+/+ littermates. Collectively, this work implies that RUNX1 is important for CBFB-MYH11 activity and that inhibitors of RUNX1 have potential use for the treatment of Inv(16) leukemia. Disclosures: No relevant conflicts of interest to declare.
Lemlem Alemu - One of the best experts on this subject based on the ideXlab platform.
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chd7 deficiency delays leukemogenesis in mice induced by cbfb MYH11
Blood, 2017Co-Authors: Tao Zhen, Katherine R Hyde, Lemlem Alemu, Ling Zhao, Erika M Kwon, Ying Lu, Nancy A SpeckAbstract:Inversion of chromosome 16 is a consistent finding in patients with acute myeloid leukemia subtype M4 with eosinophilia, which generates a CBFB-MYH11 fusion gene. Previous studies showed that the interaction between CBFβ-smooth muscle myosin heavy chain (SMMHC; encoded by CBFB-MYH11) and RUNX1 plays a critical role in the pathogenesis of this leukemia. Recently, it was shown that chromodomain helicase DNA-binding protein-7 (CHD7) interacts with RUNX1 and suppresses RUNX1-induced expansion of hematopoietic stem and progenitor cells. These results suggest that CHD7 is also critical for CBFB-MYH11–induced leukemogenesis. To test this hypothesis, we generated Chd7f/fMx1-CreCbfb+/56M mice, which expressed the Cbfb-MYH11 fusion gene and deactivated Chd7 in hematopoietic cells upon inducing Cre with polyinosinic-polycytidylic acid. The Lin–Sca1–c-Kit+ (LK) population was significantly lower in Chd7f/fMx1-CreCbfb+/56M mice than in Mx1-CreCbfb+/56M mice. In addition, there were fewer 5-bromo-2′-deoxyuridine–positive cells in the LK population in Chd7f/fMx1-CreCbfb+/56M mice, and genes associated with cell cycle, cell growth, and proliferation were differentially expressed between Chd7f/fMx1-CreCbfb+/56M and Mx1-CreCbfb+/56M leukemic cells. In vitro studies showed that CHD7 interacted with CBFβ-SMMHC through RUNX1 and that CHD7 enhanced transcriptional activity of RUNX1 and CBFβ-SMMHC on Csf1r, a RUNX1 target gene. Moreover, RNA sequencing of c-Kit+ cells showed that CHD7 functions mostly through altering the expression of RUNX1 target genes. Most importantly, Chd7 deficiency delayed Cbfb-MYH11–induced leukemia in both primary and transplanted mice. These data indicate that Chd7 is important for Cbfb-MYH11–induced leukemogenesis by facilitating RUNX1 regulation of transcription and cellular proliferation.
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runx1 and cbfb MYH11 are required for the maintenance of inv 16 aml
Blood, 2016Co-Authors: Yiqian Wang, Lemlem Alemu, Ling Zhao, Kira Hannon, Lisa Richter, Michelle Becker, Lisa Garett, Cecilia Rivas, Katherine R HydeAbstract:The inversion of chromosome 16 (inv(16)) is found in 5-12% of human AML cases. Although considered a marker of favorable prognosis, approximately half of inv(16) AML patients eventually relapse. Inv(16) generates a fusion gene between the transcription factor gene CBFB and the MYH11 gene. Expression of the CBFB-MYH11 fusion gene, which encodes CBFβ-SMMHC, is the initiating event, but cooperating mutations are required for transformation to a frank leukemia. In previous work, we showed that CBFβ and CBFβ-SMMHC binding partner RUNX1 is required for efficient leukemia development. Small molecule inhibitors of the CBFβ-SMMHC: RUNX1 complex decrease leukemic burden and increase survival in mouse models, indicating that both proteins also play a role in leukemia maintenance. However, it is not currently known whether inhibition of this complex alone is sufficient to cure inv(16) AML. To test the requirement for CBFB-MYH11 after leukemic transformation, we generated knockin mice that have a Cbfb-MYH11 allele flanked by loxP sites ( Cbfb flMYH11 ), which allows for deletion of Cbfb-MYH11 by Cre recombinase (Cre). Chimeric founder mice were treated with N-ethyl-N-nitrosourea (ENU) to induce cooperating mutations and leukemia. Leukemia cells from three different founder mice had a similar histological appearance and immunophenotype as leukemia cells derived from previous Cbfb-MYH11 knockin models. Importantly, the leukemia was transplantable, with similar latency as the previous knockin models. These findings indicate that the Cbfb flMYH11 allele causes frank leukemia, similar to other Cbfb-MYH11 alleles. To induce excision of the fusion gene, Cbfb +/flMYH11 leukemia cells were transduced with a lentivirus expressing Cre and GFP. Excision of the Cbfb-MYH11 allele was verified by PCR and showed an average excision frequency of 72.3%, +/- 1.8. To test if deletion of Cbfb-MYH11 affected cell survival, leukemia cells were infected with Cre and control viruses, and stained for Annexin V. At 48 hours post-transduction, total Cre-infected Cbfb +/flMYH11 leukemia cells showed a statistically significant increase in Annexin V staining, as compared to cells infected with the control virus. Importantly, increased Annexin V staining was seen in Csf2rb - cells, a population we previously showed to be enriched for leukemia stem cells (LSCs). To test if loss of Cbfb-MYH11 induced differentiation of leukemia cells, we stained Cre and control transduced Cbfb flMYH11 leukemia cells for the myeloid differentiation markers Gr-1 and Mac-1. We found no difference in the expression of either marker with Cbfb-MYH11 excision. These findings indicate that Cbfb-MYH11 is required for the survival of leukemic cells, including the LSC population, and its loss does not cause differentiation. To test if Runx1 is required for Cbfb-MYH11 activities during leukemia maintenance, we utilized a lentiviral vector expressing an shRNA against Runx1 and infected Cbfb-MYH11 expressing mouse leukemia cells. Runx1 knockdown was verified by quantitative RT-PCR and by western blot. To test if Runx1 knockdown induced apoptosis, leukemia cells infected with Runx1 knockdown or a scrambled shRNA control virus were stained for Annexin V. With an average decrease in Runx1 of 60.0% +/- 0.17, we observed an overall increase in Annexin V staining, but not in the Csf2rb-, LSC enriched population. This implies that LSCs may be less sensitive to decreased RUNX1 activity, than non-LSCs. To examine the effect of Runx1 knockdown on LSC activity in vitro, we performed colony forming cell (CFC) assays. We found that cells with decreased expression of Runx1 produced significantly fewer colonies as compared to the scrambled shRNA-infected cells. While some of the observed colonies may have been due to rare cells that lost or silenced the shRNA vector, our preliminary data indicates some colonies retained Runx1 knockdown (70.0% decrease, as compared to control infected cells) after growth in culture. These findings indicate that Runx1 is required for leukemia maintenance, but that LSCs may be less sensitive to decreased RUNX1 activity than non-LSCs. Taken together, our results imply that both Cbfb-MYH11 and Runx1 are important for the maintenance of inv(16) AML, and that inhibition of CBFβ-SMMHCand RUNX1 have potential as a cure for inv(16) AML. Disclosures No relevant conflicts of interest to declare.
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runx1 is strictly required for cbfb MYH11 induced leukemia development
Blood, 2016Co-Authors: Tao Zhen, Katherine R Hyde, Ling Zhao, Guadalupe Lopez, Erika Mijin Kwon, Lemlem AlemuAbstract:Inversion of chromosome 16 is a consistent finding in patients with acute myeloid leukemia subtype M4 with eosinophilia (AML M4Eo), which generates a CBFB-MYH11 fusion gene. The prevailing hypothesis for the mechanism of leukemia development by CBFbeta-SMMHC, the fusion protein encoded by CBFB-MYH11, is that CBFbeta-SMMHC is a dominant negative repressor of RUNX1, a transcription factor that physically interacts with CBFbeta and CBFbeta-SMMHC. If this hypothesis is correct, reducing RUNX1 activity should facilitate leukemogenesis by CBFB-MYH11. In fact, loss-of-function mutations in RUNX1 are common in human AML, but not in inv(16) AML. However, we previously demonstrated that CBFB-MYH11 has RUNX1-repression independent functions (Hyde et al., Blood 115:1433, 2010). Moreover, we recently showed that a dominant negative allele of Runx1, Runx1-lz, delayed leukemogenesis by CBFB-MYH11 in a mouse model (Hyde et al., Leukemia 29:1771, 2015). These findings challenge the RUNX1-repression model for CBFbeta-SMMHC mediated leukemogenesis. However, our previous findings are not conclusive since the Runx1+/lz mice used in the previous study have one wild-type Runx1 allele, and still retain some Runx1 function. To definitively address this question, we crossed Cre-based conditional Runx1 knockout mice (Runx1f/f) with Cre-based conditional Cbfb-MYH11 knockin mice (Cbfb+/56M) and Mx1-Cre mice to generate Runx1f/f, Mx1-Cre, Cbfb+/56Mmice, which express CBFbeta-SMMHC but not Runx1 after pIpC (poly I:C) treatment to induce Cre expression. Runx1f/f, Mx1-Cre, Cbfb+/56Mmice had more severe platelet deficiencies and higher numbers of Lin-/Sca1-/C-kit+ progenitors and Lin-/Sca1+/C-kit+ hematopoietic stem cells in the bone marrow when comapred with Runx1f/f, Mx1-Cre mice. Unexpectedly Runx1f/f, Mx1-Cre, Cbfb+/56Mmice also developed severe macrocytic anemia within two weeks after pIpC induction, which was lethal in about 1/3 of the mice. However, none of the Runx1f/f, Mx1-Cre, Cbfb+/56M mice developed leukemia up to one year after pIpC treatment. In contrast, all Mx1-Cre, Cbfb+/56M mice developed leukemia with an average survival of 4 months, as reported previously. These results suggest that Runx1 is strictly required for Cbfb-MYH11 induced leukemogenesis. To further study the mechanism of leukemogenesis, we performed RNA-Seq on C-kit+ bone marrow cells isolated from mice two weeks after pIpC treatment, to explore the global gene expression changes caused by Runx1 knockout on Cbfb-MYH11 expressing mice. Our preliminary data analysis showed that 1688 genes were differential expressed (Padj ≤0.05, FC ≥ 2) between Runx1f/f, Mx1-Cre, Cbfb+/56M and Mx1-Cre, Cbfb+/56M mice. Interestingly, many of these genes (48%) are Runx1 target genes. The above results suggest that mis-regulating the expression of Runx1 target genes contributes to leukemogenesis by CBFbeta-SMMHC. Disclosures No relevant conflicts of interest to declare.
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runx1 is required by the inv 16 fusion gene cbfb MYH11
Blood, 2012Co-Authors: Katherine R Hyde, Ling Zhao, Lemlem AlemuAbstract:Abstract 2434 Acute myeloid leukemia (AML) is often associated with specific, recurrent chromosomal abnormalities, such as the inversion of chromosome 16 (Inv(16)) which is associated with subtype M4 with eosinophilia. This inversion creates a fusion between CBFB and MYH11, which encode Core Binding Factor beta and Smooth Muscle Myosin Heavy Chain, respectively. The resulting fusion gene, CBFB-MYH11, is known to be the initiating factor in Inv(16) AML, but its mechanism is not clear. Previous studies indicated that repression of RUNX1 is a potential mechanism. However, we found that Cbfb-MYH11 has activities independent of Runx1 repression. During primitive hematopoiesis, we showed that expression of Cbfb-MYH11 in knockin mouse embryos (Cbfb+/MYH11) caused defects in differentiation that were not seen in embryos nullizygous for Runx1 (Runx1−/−), indicating that Cbfb-MYH11 has activities in addition to the repression of Runx1. Moreover, we found that the defects in the primitive hematopoiesis were rescued in the Cbfb+/MYH11; Runx1−/− embryos, which suggests that Runx1 is required for Cbfb-MYH11 activity during primitive hematopoiesis. We next asked whether Cbfb-MYH11 was similarly dependent on Runx1 during definitive hematopoiesis. For this purpose we used mice expressing another allele of Runx1 in which a 39-truncated Runx1 is fused to the b-galactosidase gene, lacZ (Runx1lzd). This Runx1 allele has been reported to have dominant negative activities. Using an in vitro promoter assay, we found that co-expression of Cbfβ with Runx1 and Runx1-lzd resulted in decreased activation of the MCSFR promoter as compared to co-expressing Cbfβ and Runx1, indicating that Runx1-lzd has dominant negative activities. In addition, we found that expression of a single Runx1-lzd allele rescued the primitive blood defect in the Cbfb+/MYH11 embryos. Runx1+/lzd; Cbfb+/MYH11 embryos showed almost normal definitive hematopoiesis providing further evidence that Runx1-lzd has dominant negative activity. Previously we showed that induction of Cbfb-MYH11 results in a distinct population of pre-leukemic cells. By combining the Runx1-lzd allele with an inducible allele of Cbfb-MYH11, we examined the requirement for Runx1 activity in the production of pre-leukemic cells. We found that 7 days after induction of Cbfb-MYH11, Runx1+/lzd; Cbfb+/MYH11 mice showed a statistically significant decrease in the number of pre-leukemic cells as compared to Runx1+/+; Cbfb+/MYH11 mice. We also found a statistically significant decrease in BrdU incorporation in the bone marrow of Runx1+/lzd; Cbfb+/MYH11 mice as compared to Runx1+/+; Cbfb+/MYH11 mice. This indicates that Runx1 is important for Cbfb-MYH11 activity in adult hematopoietic cells. Consistent with this idea, we found that adult mice expressing Cbfb-MYH11 and the Runx1-lzd allele showed a significant delay in the development of leukemia as compared to their Cbfb+/MYH11; Runx1+/+ littermates. Collectively, this work implies that RUNX1 is important for CBFB-MYH11 activity and that inhibitors of RUNX1 have potential use for the treatment of Inv(16) leukemia. Disclosures: No relevant conflicts of interest to declare.
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runx1 is required for cbfb MYH11 activity during primitive hematopoiesis
Blood, 2011Co-Authors: Katherine R Hyde, Ling Zhao, Lemlem AlemuAbstract:Abstract 1357 Inversion of chromosome 16 (Inv(16)) is found in nearly all patients with acute myeloid leukemia (AML) subtype M4 with eosinophilia. Inv(16) results in the fusion of the transcription factor gene CBFB , and the MYH11 gene, which encodes Smooth Muscle Myosin Heavy Chain (SMMHC). It has been proposed that the Inv(16) fusion protein, CBFβ-SMMHC, initiates leukemogenesis through repression of the transcription factor RUNX1. However, we recently found that CBFβ-SMMHC also has activities that are independent of Runx1 repression. Mice expressing a knockin allele of Cbfb-MYH11 ( Cbfb +/MYH11 ) show a severe differentiation defect during primitive hematopoiesis. Interestingly, this severe defect is not seen in mice homozygous for a null allele of Runx1 ( Runx1 −/− ). We also found genes deregulated uniquely in Cbfb +/MYH11 embryos were also expressed in leukemic cells from mice and Inv(16) patients. These findings imply that CBFβ-SMMHC9s Runx1 repression independent activities play a role during leukemogenesis. There are two potential models to describe these observations: a Runx1-independent model and a Runx1-dependent model. In the first model, Cbfb-MYH11 initiates leukemia through a novel mechanism that does not involve Runx1 . In the second model, Cbfb-MYH11 cooperates with Runx1 , but rather than repressing Runx1 activity, Cbfb-MYH11 alters it. To test these two models, we generated mice that express a conditional allele of Cbfb-MYH11 on a Runx1 −/− background. Surprisingly, we found a complete rescue of the Cbfb-MYH11 induced primitive hematopoietic defect: the primitive blood from Runx1 −/− ; Cbfb +/MYH11 embryos was indistinguishable from Runx1 −/− ; Cbfb +/+ embryos. Preliminary data showed that Cbfb-MYH11 expression in the embryos was not affected by the loss of Runx1 , indicating that Runx1 is not regulating Cbfb-MYH11 expression, but is required for its activity. Interestingly, another allele of Runx1 in which Runx1 is fused to the β-galactosidase gene, lacZ ( Runx1 lzd ) and that has been shown to have dominant negative activities (Nancy Speck, personal communication) also rescued the primitive blood defect in the Cbfb +/MYH11 embryos. This demonstrates the Cbfb-MYH11 requires Runx1 activity for its primitive hematopoietic differentiation defect. Based on this finding, we hypothesize that Cbfb-MYH11 may have a similar requirement for Runx1 activity during leukemogenesis. Consistent with this idea, we found that adult mice expressing Cbfb-MYH11 and the Runx1-lzd allele showed a significant delay in the development of leukemia as compared to their Cbfb +/MYH11 ; Runx1 +/+ littermates. Disclosures: No relevant conflicts of interest to declare.
Lucio H Castilla - One of the best experts on this subject based on the ideXlab platform.
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cbfb MYH11 hinders early t cell development and induces massive cell death in the thymus
Blood, 2007Co-Authors: Ling Zhao, Stacie M Anderson, Martha Kirby, Lucio H Castilla, Jennifer L Cannons, Liping Xu, Pamela L Schwartzberg, Remy BosselutAbstract:Recent studies suggest that the chromosome 16 inversion, associated with acute myeloid leukemia M4Eo, takes place in hematopoietic stem cells. If this is the case, it is of interest to know the effects of the resulting fusion gene, CBFB-MYH11, on other lineages. Here we studied T-cell development in mice expressing Cbfb-MYH11 and compared them with mice compound-heterozygous for a Cbfb null and a hypomorphic GFP knock-in allele (Cbfb−/GFP), which had severe Cbfb deficiency. We found a differentiation block at the DN1 stage of thymocyte development in Cbfb-MYH11 knock-in chimeras. In a conditional knock-in model in which Cbfb-MYH11 expression was activated by Lck-Cre, there was a 10-fold reduction in thymocyte numbers in adult thymus, resulting mainly from impaired survival of CD4+CD8+ thymocytes. Although Cbfb-MYH11 derepressed CD4 expression efficiently in reporter assays, such derepression was less pronounced in vivo. On the other hand, CD4 expression was derepressed and thymocyte development was blocked at DN1 and DN2 stages in E17.5 Cbfb−/GFP thymus, with a 20-fold reduction of total thymocyte numbers. Our data suggest that Cbfb-MYH11 suppressed Cbfb in several stages of T-cell development and provide a mechanism for CBFB-MYH11 association with myeloid but not lymphoid leukemia.
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cbfb MYH11 induces expansion of a lin kit sca1 abnormal progenitor compartment that predisposes acute myeloid leukemia in mice
Blood, 2004Co-Authors: Susan Ann Heilman, Amy Chen, Rachel Gernstein, Scott C Kogan, Lucio H CastillaAbstract:Acute myeloid leukemia (AML) samples with chromosome 16 inversion express the CBFb-MYH11 fusion gene. One of three RUNX genes (RUNX1, RUNX2, and RUNX3) encode the α-subunit and CBFb encodes the β subunit of the heterodimeric transcription factor CBF. This transcription factor is a key regulator of multiple steps on hematopoietic differentiation. Studies in the mouse have determined that Cbfb-MYH11 expression impairs hematopoiesis, and that it induces AML in collaboration with other mutations. To further study the effects of Cbfb-MYH11 expression in hematopoiesis and leukemogenesis, we created a Cbfb-MYH11 conditional knock-in model (Cbfb56M), using the Cre-loxP recombination system. In this model, the floxed Cbfb allele expresses wildtype Cbfb, and the downstream Cbfb-MYH11 is induced upon Cre-mediated deletion. Floxed heterozygous and homozygous Cbfbfb56M mice are disease-free, indicating that Cbfb expressed from the floxed allele is functional. In addition, Cbfb-MYH11 was efficiently induced in over 80% of bone marrow cells from Cbfb56M/+/Mx1-Cre mice after pIpC injection. The preleukemic effects of Cbfb-MYH11 in hematopoiesis were analyzed using induced mice and non-competitive repopulation assays. First, circulating B-cells were reduced soon after Cbfb-MYH11 induction, and a significant differentiation block at the pre-pro B-cell stage was detected in the bone marrow. Second, thymic T-cell differentiation of induced mice showed impairment of DN2 to DN3 stage and reduction of thymic size in 3/10 induced mice analyzed. Interestingly, the number of circulating T cells was unaffected in repopulation assays. Third, platelets were reduced 50% in peripheral blood and megakaryocyte number was reduced in bone marrow. Fourth, we found an expanded abnormal progenitor compartment (Lin-kit+Sca1-) that accumulated in the bone marrow and spleen. In vitro differentiation assays showed a 2-to-3 fold increase of Cbfb-MYH11 colonies when compared to controls. The colony size was smaller, and showing partial differentiation deficiency. Interestingly, the colony numbers declined upon serial plating below controls. Taken together these results indicate that Cbfb-MYH11 induce accumulation of late progenitors (primarily myeloid progenitors) with limited self-renewal potential. Acute myeloid leukemia arised spontaneously 4 to 6 months after Cbfb-MYH11 induction, with expansion of blast- and monoblastic-like leukemic cells defined as Lin-kit+Sca1-. Leukemic mice showed infiltration in several tissues, including spleen, liver, brain, and lungs. To test whether a second “hit” is necessary in this model, we used bone marrow transduction assays to co-express Cbfb-MYH11 and the candidate cooperating gene Runx2. Recipient mice developed AML with similar phenotype 6 to 12 weeks post transplantation, while control mice remained healthy for 6 months. This study demonstrates that Cbfb-MYH11 expression (i) defines a preleukemic stage with hematopoietic differentiation block at stages associated with Runx function, (ii) the accumulation of an abnormal progenitor cell population (Lin-/kit+/Sca1-), (iii) induces additional mutations to efficiently develop AML, and (iv) synergizes with Runx2 in AML development.
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Cbfb-MYH11 Induces Expansion of a Lin-Kit+Sca1- Abnormal Progenitor Compartment that Predisposes Acute Myeloid Leukemia in Mice.
Blood, 2004Co-Authors: Susan Ann Heilman, Amy Chen, Rachel Gernstein, Scott C Kogan, Lucio H CastillaAbstract:Acute myeloid leukemia (AML) samples with chromosome 16 inversion express the CBFb-MYH11 fusion gene. One of three RUNX genes (RUNX1, RUNX2, and RUNX3) encode the α-subunit and CBFb encodes the β subunit of the heterodimeric transcription factor CBF. This transcription factor is a key regulator of multiple steps on hematopoietic differentiation. Studies in the mouse have determined that Cbfb-MYH11 expression impairs hematopoiesis, and that it induces AML in collaboration with other mutations. To further study the effects of Cbfb-MYH11 expression in hematopoiesis and leukemogenesis, we created a Cbfb-MYH11 conditional knock-in model (Cbfb56M), using the Cre-loxP recombination system. In this model, the floxed Cbfb allele expresses wildtype Cbfb, and the downstream Cbfb-MYH11 is induced upon Cre-mediated deletion. Floxed heterozygous and homozygous Cbfbfb56M mice are disease-free, indicating that Cbfb expressed from the floxed allele is functional. In addition, Cbfb-MYH11 was efficiently induced in over 80% of bone marrow cells from Cbfb56M/+/Mx1-Cre mice after pIpC injection. The preleukemic effects of Cbfb-MYH11 in hematopoiesis were analyzed using induced mice and non-competitive repopulation assays. First, circulating B-cells were reduced soon after Cbfb-MYH11 induction, and a significant differentiation block at the pre-pro B-cell stage was detected in the bone marrow. Second, thymic T-cell differentiation of induced mice showed impairment of DN2 to DN3 stage and reduction of thymic size in 3/10 induced mice analyzed. Interestingly, the number of circulating T cells was unaffected in repopulation assays. Third, platelets were reduced 50% in peripheral blood and megakaryocyte number was reduced in bone marrow. Fourth, we found an expanded abnormal progenitor compartment (Lin-kit+Sca1-) that accumulated in the bone marrow and spleen. In vitro differentiation assays showed a 2-to-3 fold increase of Cbfb-MYH11 colonies when compared to controls. The colony size was smaller, and showing partial differentiation deficiency. Interestingly, the colony numbers declined upon serial plating below controls. Taken together these results indicate that Cbfb-MYH11 induce accumulation of late progenitors (primarily myeloid progenitors) with limited self-renewal potential. Acute myeloid leukemia arised spontaneously 4 to 6 months after Cbfb-MYH11 induction, with expansion of blast- and monoblastic-like leukemic cells defined as Lin-kit+Sca1-. Leukemic mice showed infiltration in several tissues, including spleen, liver, brain, and lungs. To test whether a second “hit” is necessary in this model, we used bone marrow transduction assays to co-express Cbfb-MYH11 and the candidate cooperating gene Runx2. Recipient mice developed AML with similar phenotype 6 to 12 weeks post transplantation, while control mice remained healthy for 6 months. This study demonstrates that Cbfb-MYH11 expression (i) defines a preleukemic stage with hematopoietic differentiation block at stages associated with Runx function, (ii) the accumulation of an abnormal progenitor cell population (Lin-/kit+/Sca1-), (iii) induces additional mutations to efficiently develop AML, and (iv) synergizes with Runx2 in AML development.
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identification of genes that synergize with cbfb MYH11 in the pathogenesis of acute myeloid leukemia
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Lucio H Castilla, Susan Ann Heilman, Lisa Garrett, Paola N Perrat, Natalia Martinez, Sean Landrette, R Keys, Sarah R Oikemus, J Flanegan, Amalia DutraAbstract:Acute myeloid leukemia subtype M4 with eosinophilia is associated with a chromosome 16 inversion that creates a fusion gene CBFB-MYH11. We have previously shown that CBFB-MYH11 is necessary but not sufficient for leukemogenesis. Here, we report the identification of genes that specifically cooperate with CBFB-MYH11 in leukemogenesis. Neonatal injection of Cbfb-MYH11 knock-in chimeric mice with retrovirus 4070A led to the development of acute myeloid leukemia in 2-5 months. Each leukemia sample contained one or a few viral insertions, suggesting that alteration of one gene could be sufficient to synergize with Cbfb-MYH11. The chromosomal position of 67 independent retroviral insertion sites (RISs) was determined, and 90% of the RISs mapped within 10 kb of a flanking gene. In total, 54 candidate genes were identified; six of them were common insertion sites (CISs). CIS genes included members of a zinc finger transcription factors family, Plag1 and Plagl2, with eight and two independent insertions, respectively. CIS genes also included Runx2, Myb, H2T24, and D6Mm5e. Comparison of the remaining 48 genes with single insertion sites with known leukemia-associated RISs indicated that 18 coincide with known RISs. To our knowledge, this retroviral genetic screen is the first to identify genes that cooperate with a fusion gene important for human myeloid leukemia.
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role of cbfb in hematopoiesis and perturbations resulting from expression of the leukemogenic fusion gene cbfb MYH11
Blood, 2002Co-Authors: Mondira Kundu, Martha Kirby, Lucio H Castilla, Liping Xu, Amy Chen, Stacie Anderson, David BodineAbstract:Core-binding factor β (CBFβ) and CBFα2 form a heterodimeric transcription factor that plays an important role in hematopoiesis. The genes encoding either CBFβ or CBFα2 are involved in chromosomal rearrangements in more than 30% of cases of acute myeloid leukemia (AML), suggesting that CBFβ and CBFα2 play important roles in leukemogenesis. Inv(16)(p13;q22) is found in almost all cases of AML M4Eo and results in the fusion of CBFB with MYH11 , the gene encoding smooth muscle myosin heavy chain. Mouse embryos heterozygous for a Cbfb-MYH11 knock-in gene lack definitive hematopoiesis, a phenotype shared by Cbfb−/− embryos. In this study we generated a Cbfb-GFP knock-in mouse model to characterize the normal expression pattern of Cbfβ in hematopoietic cells. In midgestation embryos, Cbfβ was expressed in populations enriched for hematopoietic stem cells and progenitors. This population of stem cells and progenitors was not present in mouse embryos heterozygous for the Cbfb-MYH11 knock-in gene. Together, these data suggest that Cbfb-MYH11 blocks embryonic hematopoiesis at the stem-progenitor cell level and that Cbfb is essential for the generation of hematopoietic stem and progenitor cells. In adult mice, Cbfβ was expressed in stem and progenitor cells, as well as mature myeloid and lymphoid cells. Although it was expressed in erythroid progenitors, Cbfβ was not expressed during the terminal stages of erythropoiesis. Our data indicate that Cbfb is required for myeloid and lymphoid differentiation; but does not play a critical role in erythroid differentiation.
Katherine R Hyde - One of the best experts on this subject based on the ideXlab platform.
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il1rl1 is dynamically expressed on cbfb MYH11 leukemia stem cells and promotes cell survival
Scientific Reports, 2019Co-Authors: Yiqian Wang, Lisa Richter, Michelle Becker, Catalina Amador, Katherine R HydeAbstract:Acute myeloid leukemia (AML) is often characterized by the presence of specific, recurrent chromosomal abnormalities. One of the most common aberrations, inversion of chromosome 16 [inv(16)], generates the fusion oncogene CBFB-MYH11. Previously, we used a mouse knock-in model to show that Cbfb-MYH11 induces changes in gene expression and results in the accumulation of abnormal myeloid cells, a subset of which are enriched for leukemia stem cell (LSC) activity. One gene upregulated by Cbfb-MYH11 encodes the cytokine receptor IL1RL1 (ST2). IL1RL1 and its ligand IL-33 are known regulators of mature myeloid cells, but their roles in AML are not known. Here, we use Cbfb-MYH11 knock-in mice to show that IL1RL1 is expressed by cell populations with high LSC activity, and that the cell surface expression of IL1RL1 is dynamic, implying that the expression of IL1RL1 is not restricted to a specific stage of differentiation. We also show that treatment with IL-33 increased serial replating ability and expression of pro-survival proteins in vitro. Finally, we show that IL1RL1+ cells can survive chemotherapy better than IL1RL1− cells in vivo. Collectively, our results indicate that IL1RL1 is dynamically expressed in Cbfb-MYH11+ leukemia cells and promotes their survival.
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chd7 deficiency delays leukemogenesis in mice induced by cbfb MYH11
Blood, 2017Co-Authors: Tao Zhen, Katherine R Hyde, Lemlem Alemu, Ling Zhao, Erika M Kwon, Ying Lu, Nancy A SpeckAbstract:Inversion of chromosome 16 is a consistent finding in patients with acute myeloid leukemia subtype M4 with eosinophilia, which generates a CBFB-MYH11 fusion gene. Previous studies showed that the interaction between CBFβ-smooth muscle myosin heavy chain (SMMHC; encoded by CBFB-MYH11) and RUNX1 plays a critical role in the pathogenesis of this leukemia. Recently, it was shown that chromodomain helicase DNA-binding protein-7 (CHD7) interacts with RUNX1 and suppresses RUNX1-induced expansion of hematopoietic stem and progenitor cells. These results suggest that CHD7 is also critical for CBFB-MYH11–induced leukemogenesis. To test this hypothesis, we generated Chd7f/fMx1-CreCbfb+/56M mice, which expressed the Cbfb-MYH11 fusion gene and deactivated Chd7 in hematopoietic cells upon inducing Cre with polyinosinic-polycytidylic acid. The Lin–Sca1–c-Kit+ (LK) population was significantly lower in Chd7f/fMx1-CreCbfb+/56M mice than in Mx1-CreCbfb+/56M mice. In addition, there were fewer 5-bromo-2′-deoxyuridine–positive cells in the LK population in Chd7f/fMx1-CreCbfb+/56M mice, and genes associated with cell cycle, cell growth, and proliferation were differentially expressed between Chd7f/fMx1-CreCbfb+/56M and Mx1-CreCbfb+/56M leukemic cells. In vitro studies showed that CHD7 interacted with CBFβ-SMMHC through RUNX1 and that CHD7 enhanced transcriptional activity of RUNX1 and CBFβ-SMMHC on Csf1r, a RUNX1 target gene. Moreover, RNA sequencing of c-Kit+ cells showed that CHD7 functions mostly through altering the expression of RUNX1 target genes. Most importantly, Chd7 deficiency delayed Cbfb-MYH11–induced leukemia in both primary and transplanted mice. These data indicate that Chd7 is important for Cbfb-MYH11–induced leukemogenesis by facilitating RUNX1 regulation of transcription and cellular proliferation.
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runx1 and cbfb MYH11 are required for the maintenance of inv 16 aml
Blood, 2016Co-Authors: Yiqian Wang, Lemlem Alemu, Ling Zhao, Kira Hannon, Lisa Richter, Michelle Becker, Lisa Garett, Cecilia Rivas, Katherine R HydeAbstract:The inversion of chromosome 16 (inv(16)) is found in 5-12% of human AML cases. Although considered a marker of favorable prognosis, approximately half of inv(16) AML patients eventually relapse. Inv(16) generates a fusion gene between the transcription factor gene CBFB and the MYH11 gene. Expression of the CBFB-MYH11 fusion gene, which encodes CBFβ-SMMHC, is the initiating event, but cooperating mutations are required for transformation to a frank leukemia. In previous work, we showed that CBFβ and CBFβ-SMMHC binding partner RUNX1 is required for efficient leukemia development. Small molecule inhibitors of the CBFβ-SMMHC: RUNX1 complex decrease leukemic burden and increase survival in mouse models, indicating that both proteins also play a role in leukemia maintenance. However, it is not currently known whether inhibition of this complex alone is sufficient to cure inv(16) AML. To test the requirement for CBFB-MYH11 after leukemic transformation, we generated knockin mice that have a Cbfb-MYH11 allele flanked by loxP sites ( Cbfb flMYH11 ), which allows for deletion of Cbfb-MYH11 by Cre recombinase (Cre). Chimeric founder mice were treated with N-ethyl-N-nitrosourea (ENU) to induce cooperating mutations and leukemia. Leukemia cells from three different founder mice had a similar histological appearance and immunophenotype as leukemia cells derived from previous Cbfb-MYH11 knockin models. Importantly, the leukemia was transplantable, with similar latency as the previous knockin models. These findings indicate that the Cbfb flMYH11 allele causes frank leukemia, similar to other Cbfb-MYH11 alleles. To induce excision of the fusion gene, Cbfb +/flMYH11 leukemia cells were transduced with a lentivirus expressing Cre and GFP. Excision of the Cbfb-MYH11 allele was verified by PCR and showed an average excision frequency of 72.3%, +/- 1.8. To test if deletion of Cbfb-MYH11 affected cell survival, leukemia cells were infected with Cre and control viruses, and stained for Annexin V. At 48 hours post-transduction, total Cre-infected Cbfb +/flMYH11 leukemia cells showed a statistically significant increase in Annexin V staining, as compared to cells infected with the control virus. Importantly, increased Annexin V staining was seen in Csf2rb - cells, a population we previously showed to be enriched for leukemia stem cells (LSCs). To test if loss of Cbfb-MYH11 induced differentiation of leukemia cells, we stained Cre and control transduced Cbfb flMYH11 leukemia cells for the myeloid differentiation markers Gr-1 and Mac-1. We found no difference in the expression of either marker with Cbfb-MYH11 excision. These findings indicate that Cbfb-MYH11 is required for the survival of leukemic cells, including the LSC population, and its loss does not cause differentiation. To test if Runx1 is required for Cbfb-MYH11 activities during leukemia maintenance, we utilized a lentiviral vector expressing an shRNA against Runx1 and infected Cbfb-MYH11 expressing mouse leukemia cells. Runx1 knockdown was verified by quantitative RT-PCR and by western blot. To test if Runx1 knockdown induced apoptosis, leukemia cells infected with Runx1 knockdown or a scrambled shRNA control virus were stained for Annexin V. With an average decrease in Runx1 of 60.0% +/- 0.17, we observed an overall increase in Annexin V staining, but not in the Csf2rb-, LSC enriched population. This implies that LSCs may be less sensitive to decreased RUNX1 activity, than non-LSCs. To examine the effect of Runx1 knockdown on LSC activity in vitro, we performed colony forming cell (CFC) assays. We found that cells with decreased expression of Runx1 produced significantly fewer colonies as compared to the scrambled shRNA-infected cells. While some of the observed colonies may have been due to rare cells that lost or silenced the shRNA vector, our preliminary data indicates some colonies retained Runx1 knockdown (70.0% decrease, as compared to control infected cells) after growth in culture. These findings indicate that Runx1 is required for leukemia maintenance, but that LSCs may be less sensitive to decreased RUNX1 activity than non-LSCs. Taken together, our results imply that both Cbfb-MYH11 and Runx1 are important for the maintenance of inv(16) AML, and that inhibition of CBFβ-SMMHCand RUNX1 have potential as a cure for inv(16) AML. Disclosures No relevant conflicts of interest to declare.
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runx1 is strictly required for cbfb MYH11 induced leukemia development
Blood, 2016Co-Authors: Tao Zhen, Katherine R Hyde, Ling Zhao, Guadalupe Lopez, Erika Mijin Kwon, Lemlem AlemuAbstract:Inversion of chromosome 16 is a consistent finding in patients with acute myeloid leukemia subtype M4 with eosinophilia (AML M4Eo), which generates a CBFB-MYH11 fusion gene. The prevailing hypothesis for the mechanism of leukemia development by CBFbeta-SMMHC, the fusion protein encoded by CBFB-MYH11, is that CBFbeta-SMMHC is a dominant negative repressor of RUNX1, a transcription factor that physically interacts with CBFbeta and CBFbeta-SMMHC. If this hypothesis is correct, reducing RUNX1 activity should facilitate leukemogenesis by CBFB-MYH11. In fact, loss-of-function mutations in RUNX1 are common in human AML, but not in inv(16) AML. However, we previously demonstrated that CBFB-MYH11 has RUNX1-repression independent functions (Hyde et al., Blood 115:1433, 2010). Moreover, we recently showed that a dominant negative allele of Runx1, Runx1-lz, delayed leukemogenesis by CBFB-MYH11 in a mouse model (Hyde et al., Leukemia 29:1771, 2015). These findings challenge the RUNX1-repression model for CBFbeta-SMMHC mediated leukemogenesis. However, our previous findings are not conclusive since the Runx1+/lz mice used in the previous study have one wild-type Runx1 allele, and still retain some Runx1 function. To definitively address this question, we crossed Cre-based conditional Runx1 knockout mice (Runx1f/f) with Cre-based conditional Cbfb-MYH11 knockin mice (Cbfb+/56M) and Mx1-Cre mice to generate Runx1f/f, Mx1-Cre, Cbfb+/56Mmice, which express CBFbeta-SMMHC but not Runx1 after pIpC (poly I:C) treatment to induce Cre expression. Runx1f/f, Mx1-Cre, Cbfb+/56Mmice had more severe platelet deficiencies and higher numbers of Lin-/Sca1-/C-kit+ progenitors and Lin-/Sca1+/C-kit+ hematopoietic stem cells in the bone marrow when comapred with Runx1f/f, Mx1-Cre mice. Unexpectedly Runx1f/f, Mx1-Cre, Cbfb+/56Mmice also developed severe macrocytic anemia within two weeks after pIpC induction, which was lethal in about 1/3 of the mice. However, none of the Runx1f/f, Mx1-Cre, Cbfb+/56M mice developed leukemia up to one year after pIpC treatment. In contrast, all Mx1-Cre, Cbfb+/56M mice developed leukemia with an average survival of 4 months, as reported previously. These results suggest that Runx1 is strictly required for Cbfb-MYH11 induced leukemogenesis. To further study the mechanism of leukemogenesis, we performed RNA-Seq on C-kit+ bone marrow cells isolated from mice two weeks after pIpC treatment, to explore the global gene expression changes caused by Runx1 knockout on Cbfb-MYH11 expressing mice. Our preliminary data analysis showed that 1688 genes were differential expressed (Padj ≤0.05, FC ≥ 2) between Runx1f/f, Mx1-Cre, Cbfb+/56M and Mx1-Cre, Cbfb+/56M mice. Interestingly, many of these genes (48%) are Runx1 target genes. The above results suggest that mis-regulating the expression of Runx1 target genes contributes to leukemogenesis by CBFbeta-SMMHC. Disclosures No relevant conflicts of interest to declare.
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runx1 is required by the inv 16 fusion gene cbfb MYH11
Blood, 2012Co-Authors: Katherine R Hyde, Ling Zhao, Lemlem AlemuAbstract:Abstract 2434 Acute myeloid leukemia (AML) is often associated with specific, recurrent chromosomal abnormalities, such as the inversion of chromosome 16 (Inv(16)) which is associated with subtype M4 with eosinophilia. This inversion creates a fusion between CBFB and MYH11, which encode Core Binding Factor beta and Smooth Muscle Myosin Heavy Chain, respectively. The resulting fusion gene, CBFB-MYH11, is known to be the initiating factor in Inv(16) AML, but its mechanism is not clear. Previous studies indicated that repression of RUNX1 is a potential mechanism. However, we found that Cbfb-MYH11 has activities independent of Runx1 repression. During primitive hematopoiesis, we showed that expression of Cbfb-MYH11 in knockin mouse embryos (Cbfb+/MYH11) caused defects in differentiation that were not seen in embryos nullizygous for Runx1 (Runx1−/−), indicating that Cbfb-MYH11 has activities in addition to the repression of Runx1. Moreover, we found that the defects in the primitive hematopoiesis were rescued in the Cbfb+/MYH11; Runx1−/− embryos, which suggests that Runx1 is required for Cbfb-MYH11 activity during primitive hematopoiesis. We next asked whether Cbfb-MYH11 was similarly dependent on Runx1 during definitive hematopoiesis. For this purpose we used mice expressing another allele of Runx1 in which a 39-truncated Runx1 is fused to the b-galactosidase gene, lacZ (Runx1lzd). This Runx1 allele has been reported to have dominant negative activities. Using an in vitro promoter assay, we found that co-expression of Cbfβ with Runx1 and Runx1-lzd resulted in decreased activation of the MCSFR promoter as compared to co-expressing Cbfβ and Runx1, indicating that Runx1-lzd has dominant negative activities. In addition, we found that expression of a single Runx1-lzd allele rescued the primitive blood defect in the Cbfb+/MYH11 embryos. Runx1+/lzd; Cbfb+/MYH11 embryos showed almost normal definitive hematopoiesis providing further evidence that Runx1-lzd has dominant negative activity. Previously we showed that induction of Cbfb-MYH11 results in a distinct population of pre-leukemic cells. By combining the Runx1-lzd allele with an inducible allele of Cbfb-MYH11, we examined the requirement for Runx1 activity in the production of pre-leukemic cells. We found that 7 days after induction of Cbfb-MYH11, Runx1+/lzd; Cbfb+/MYH11 mice showed a statistically significant decrease in the number of pre-leukemic cells as compared to Runx1+/+; Cbfb+/MYH11 mice. We also found a statistically significant decrease in BrdU incorporation in the bone marrow of Runx1+/lzd; Cbfb+/MYH11 mice as compared to Runx1+/+; Cbfb+/MYH11 mice. This indicates that Runx1 is important for Cbfb-MYH11 activity in adult hematopoietic cells. Consistent with this idea, we found that adult mice expressing Cbfb-MYH11 and the Runx1-lzd allele showed a significant delay in the development of leukemia as compared to their Cbfb+/MYH11; Runx1+/+ littermates. Collectively, this work implies that RUNX1 is important for CBFB-MYH11 activity and that inhibitors of RUNX1 have potential use for the treatment of Inv(16) leukemia. Disclosures: No relevant conflicts of interest to declare.
G Heil - One of the best experts on this subject based on the ideXlab platform.
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detection and quantification of cbfb MYH11 fusion transcripts in patients with inv 16 positive acute myeloblastic leukemia by real time rt pcr
Genes Chromosomes and Cancer, 2001Co-Authors: Jurgen Krauter, Wolf Hoellge, Mike P Wattjes, Stefan Nagel, Olaf Heidenreich, Donald Bunjes, Arnold Ganser, G HeilAbstract:We used a newly established real-time RT-PCR assay for the quantification of the leukemia-specific CBFB/MYH11 transcripts in inv(16)-positive acute myeloblastic leukemia. CBFB/MYH11 could be quantified over a five log range, with a detection limit of 10 molecules of a CBFB/MYH11 plasmid and a 1:105 dilution of RNA of the inv(16)-positive ME-1 cell line, respectively. The fusion transcripts were also quantified in 19 patients with acute myeloblastic leukemia and an inv(16) at initial diagnosis. The expression of CBFB/MYH11 varied over a two log range without correlation to clinical response or relapse rate. In nine patients, CBFB/MYH11 was also quantified during/after chemotherapy and autologous or allogeneic stem cell transplantation. All of these patients showed a similar decline of CBFB/MYH11 after intensive therapy. Six of these patients are in complete remission with a stable low-level or absent CBFB/MYH11 expression. Three patients relapsed, and their CBFB/MYH11 transcripts rose again to pretreatment levels. In two patients, this increase in CBFB/MYH11 could be detected by real-time PCR before hematological relapse. These data indicate that real-time RT-PCR can be used for the sensitive detection and quantification of CBFB/MYH11 transcripts in the follow-up of patients with inv(16)-positive AML. © 2001 Wiley-Liss, Inc.
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Detection and quantification of CBFB/MYH11 fusion transcripts in patients with inv(16)-positive acute myeloblastic leukemia by real-time RT-PCR
Genes Chromosomes and Cancer, 2001Co-Authors: Jurgen Krauter, Wolf Hoellge, Mike P Wattjes, Stefan Nagel, Olaf Heidenreich, Donald Bunjes, Arnold Ganser, G HeilAbstract:We used a newly established real-time RT-PCR assay for the quantification of the leukemia-specific CBFB/MYH11 transcripts in inv(16)-positive acute myeloblastic leukemia. CBFB/MYH11 could be quantified over a five log range, with a detection limit of 10 molecules of a CBFB/MYH11 plasmid and a 1:105 dilution of RNA of the inv(16)-positive ME-1 cell line, respectively. The fusion transcripts were also quantified in 19 patients with acute myeloblastic leukemia and an inv(16) at initial diagnosis. The expression of CBFB/MYH11 varied over a two log range without correlation to clinical response or relapse rate. In nine patients, CBFB/MYH11 was also quantified during/after chemotherapy and autologous or allogeneic stem cell transplantation. All of these patients showed a similar decline of CBFB/MYH11 after intensive therapy. Six of these patients are in complete remission with a stable low-level or absent CBFB/MYH11 expression. Three patients relapsed, and their CBFB/MYH11 transcripts rose again to pretreatment levels. In two patients, this increase in CBFB/MYH11 could be detected by real-time PCR before hematological relapse. These data indicate that real-time RT-PCR can be used for the sensitive detection and quantification of CBFB/MYH11 transcripts in the follow-up of patients with inv(16)-positive AML. © 2001 Wiley-Liss, Inc.
