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Ling Zhao - One of the best experts on this subject based on the ideXlab platform.

  • title runx1 is indispensable for leukemia development induced by CBFB myh11
    Blood, 2018
    Co-Authors: Tao Zhen, Katherine R Hyde, Lemlem Alemu, Guadalupe Lopez, Erika Mijin Kwon, Ling Zhao, Paul Liu
    Abstract:

    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. It is generally considered that CBFβ-SMMHC, the fusion protein encoded by CBFB-MYH11, is a dominant negative repressor of RUNX1, a transcription factor that physically interacts with CBFβ and CBFβ-SMMHC. While loss-of-function mutations in RUNX1 are common in human AML, they have not been found in inv(16) AML. Moreover, we have demonstrated that CBFβ-SMMHC has RUNX1-repression independent functions (Hyde et al., Blood 115:1433, 2010), and Runx1 insufficiency (Runx1+/lz) delays CBFB-MYH11-induced leukemia in a mouse model (Hyde et al., Leukemia 29:1771, 2015). These findings challenge the RUNX1-repression model for CBFβ-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 generated mice with both Cre-based conditional Runx1 knockout and CBFB-MYH11 knockin (Runx1f/f, Mx1-Cre,CBFB+/56M), which express CBFB-MYH11 but no Runx1 after pIpC (poly I:C) treatment to induce Cre expression. Runx1f/f, Mx1-Cre,CBFB+/56Mmice had higher numbers of Lin-/Sca1-/c-Kit+ (LK) and Lin-/Sca1+/c-Kit+ cells in the bone marrow when comapred with Mx1-Cre, CBFB+/56M mice at 3 weeks after pIpC. However, none of the Runx1f/f, Mx1-Cre, CBFB+/56M mice developed leukemia up to one year after pIpC treatment. As reported previously, all Mx1-Cre,CBFB+/56M mice developed leukemia with an median survival time of 4 months. Moreoever, none of the recipients transplanted with bone marrow cells from Runx1f/f, Mx1-Cre,CBFB+/56M mice and then treated with pIpC developed leukemia, while all of the recipients of the Mx1-Cre,CBFB+/56M bone marrow cells did, suggesting that the loss of leukemogenesis is cell autonomous. All together, these results indicate that Runx1 is indispensable for CBFB-MYH11 induced leukemogenesis. Even though at 3 weeks after pIpC the Runx1f/f, Mx1-Cre,CBFB+/56M mice had more c-Kit+ cells, and more significantly the abnormal myeloid progenitors (AMPs) (LK/CD34-/Fc□RII/III+) from which the leukemia cells arise, these cells started to decrease and disappear 4 weeks after pIpC, suggesting that this is a critical stage for the failure of leukemogenesis in the Runx1f/f, Mx1-Cre,CBFB+/56M mice. We therefore performed RNA-seq on the AMP population isolated from Mx1-Cre,CBFB+/56M and Runx1f/f, Mx1-Cre,CBFB+/56M mice three weeks after pIpC treatment, to explore the global gene expression changes between them. Our preliminary data analysis showed that many genes (1635) were differential expressed (DEGs; Padj ≤0.05, absolute fold change ≥ 2) between Runx1f/f, Mx1-Cre, CBFB+/56M and Mx1-Cre, CBFB+/56M mice. Many of these DEGs (37.6%) are RUNX1 target genes (Mandoli et al., Leukemia. 28:770, 2014). Among the significantly enriched gene sets by gene set enrichment analysis of the DEGs, the "GAL_LEUKEMIC_STEM_CELL_DN" gene set, which contains genes down-regulated in leukemic stem cells, is positively correlated with DEGs upregulated in Runx1f/f, Mx1-Cre,CBFB+/56M cells, suggesting that the AMP population from Runx1f/f, Mx1-Cre,CBFB+/56M mice lost the leukemia initiating ability. We are preforming ChIP-seq on the AMP population to determine how loss of RUNX1 disrupts binding of CBFβ-SMMHC to target genes. The above results suggest that RUNX1 is required for the regulation of critical genes for leukemogenesis by CBFβ-SMMHC. Disclosures No relevant conflicts of interest to declare.

  • title runx1 is indispensable for leukemia development induced by CBFB myh11
    Blood, 2018
    Co-Authors: Tao Zhen, Katherine R Hyde, Guadalupe Lopez, Erika Mijin Kwon, Ling Zhao, Lemlem Alemu
    Abstract:

    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. It is generally considered that CBFβ-SMMHC, the fusion protein encoded by CBFB-MYH11, is a dominant negative repressor of RUNX1, a transcription factor that physically interacts with CBFβ and CBFβ-SMMHC. While loss-of-function mutations in RUNX1 are common in human AML, they have not been found in inv(16) AML. Moreover, we have demonstrated that CBFβ-SMMHC has RUNX1-repression independent functions (Hyde et al., Blood 115:1433, 2010), and Runx1 insufficiency (Runx1+/lz) delays CBFB-MYH11-induced leukemia in a mouse model (Hyde et al., Leukemia 29:1771, 2015). These findings challenge the RUNX1-repression model for CBFβ-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 generated mice with both Cre-based conditional Runx1 knockout and CBFB-MYH11 knockin (Runx1f/f, Mx1-Cre,CBFB+/56M), which express CBFB-MYH11 but no Runx1 after pIpC (poly I:C) treatment to induce Cre expression. Runx1f/f, Mx1-Cre,CBFB+/56Mmice had higher numbers of Lin-/Sca1-/c-Kit+ (LK) and Lin-/Sca1+/c-Kit+ cells in the bone marrow when comapred with Mx1-Cre, CBFB+/56M mice at 3 weeks after pIpC. However, none of the Runx1f/f, Mx1-Cre, CBFB+/56M mice developed leukemia up to one year after pIpC treatment. As reported previously, all Mx1-Cre,CBFB+/56M mice developed leukemia with an median survival time of 4 months. Moreoever, none of the recipients transplanted with bone marrow cells from Runx1f/f, Mx1-Cre,CBFB+/56M mice and then treated with pIpC developed leukemia, while all of the recipients of the Mx1-Cre,CBFB+/56M bone marrow cells did, suggesting that the loss of leukemogenesis is cell autonomous. All together, these results indicate that Runx1 is indispensable for CBFB-MYH11 induced leukemogenesis. Even though at 3 weeks after pIpC the Runx1f/f, Mx1-Cre,CBFB+/56M mice had more c-Kit+ cells, and more significantly the abnormal myeloid progenitors (AMPs) (LK/CD34-/Fc□RII/III+) from which the leukemia cells arise, these cells started to decrease and disappear 4 weeks after pIpC, suggesting that this is a critical stage for the failure of leukemogenesis in the Runx1f/f, Mx1-Cre,CBFB+/56M mice. We therefore performed RNA-seq on the AMP population isolated from Mx1-Cre,CBFB+/56M and Runx1f/f, Mx1-Cre,CBFB+/56M mice three weeks after pIpC treatment, to explore the global gene expression changes between them. Our preliminary data analysis showed that many genes (1635) were differential expressed (DEGs; Padj ≤0.05, absolute fold change ≥ 2) between Runx1f/f, Mx1-Cre, CBFB+/56M and Mx1-Cre, CBFB+/56M mice. Many of these DEGs (37.6%) are RUNX1 target genes (Mandoli et al., Leukemia. 28:770, 2014). Among the significantly enriched gene sets by gene set enrichment analysis of the DEGs, the "GAL_LEUKEMIC_STEM_CELL_DN" gene set, which contains genes down-regulated in leukemic stem cells, is positively correlated with DEGs upregulated in Runx1f/f, Mx1-Cre,CBFB+/56M cells, suggesting that the AMP population from Runx1f/f, Mx1-Cre,CBFB+/56M mice lost the leukemia initiating ability. We are preforming ChIP-seq on the AMP population to determine how loss of RUNX1 disrupts binding of CBFβ-SMMHC to target genes. The above results suggest that RUNX1 is required for the regulation of critical genes for leukemogenesis by CBFβ-SMMHC. Disclosures No relevant conflicts of interest to declare.

  • runx1 is strictly required for CBFB myh11 induced leukemia development
    Blood, 2016
    Co-Authors: Tao Zhen, Katherine R Hyde, Guadalupe Lopez, Erika Mijin Kwon, Ling Zhao, Lemlem Alemu
    Abstract:

    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.

  • runx1 and CBFB myh11 are required for the maintenance of inv 16 aml
    Blood, 2016
    Co-Authors: Yiqian Wang, Lemlem Alemu, Kira Hannon, Michelle Becker, Lisa Garett, Cecilia Rivas, Lisa Richter, Ling Zhao, Katherine R Hyde
    Abstract:

    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.

  • runx1 and CBFB myh11 are required for the maintenance of inv 16 aml
    Blood, 2016
    Co-Authors: Yiqian Wang, Lemlem Alemu, Kira Hannon, Michelle Becker, Lisa Garett, Cecilia Rivas, Lisa Richter, Ling Zhao, Katherine R Hyde
    Abstract:

    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.

Katherine R Hyde - One of the best experts on this subject based on the ideXlab platform.

  • title runx1 is indispensable for leukemia development induced by CBFB myh11
    Blood, 2018
    Co-Authors: Tao Zhen, Katherine R Hyde, Lemlem Alemu, Guadalupe Lopez, Erika Mijin Kwon, Ling Zhao, Paul Liu
    Abstract:

    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. It is generally considered that CBFβ-SMMHC, the fusion protein encoded by CBFB-MYH11, is a dominant negative repressor of RUNX1, a transcription factor that physically interacts with CBFβ and CBFβ-SMMHC. While loss-of-function mutations in RUNX1 are common in human AML, they have not been found in inv(16) AML. Moreover, we have demonstrated that CBFβ-SMMHC has RUNX1-repression independent functions (Hyde et al., Blood 115:1433, 2010), and Runx1 insufficiency (Runx1+/lz) delays CBFB-MYH11-induced leukemia in a mouse model (Hyde et al., Leukemia 29:1771, 2015). These findings challenge the RUNX1-repression model for CBFβ-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 generated mice with both Cre-based conditional Runx1 knockout and CBFB-MYH11 knockin (Runx1f/f, Mx1-Cre,CBFB+/56M), which express CBFB-MYH11 but no Runx1 after pIpC (poly I:C) treatment to induce Cre expression. Runx1f/f, Mx1-Cre,CBFB+/56Mmice had higher numbers of Lin-/Sca1-/c-Kit+ (LK) and Lin-/Sca1+/c-Kit+ cells in the bone marrow when comapred with Mx1-Cre, CBFB+/56M mice at 3 weeks after pIpC. However, none of the Runx1f/f, Mx1-Cre, CBFB+/56M mice developed leukemia up to one year after pIpC treatment. As reported previously, all Mx1-Cre,CBFB+/56M mice developed leukemia with an median survival time of 4 months. Moreoever, none of the recipients transplanted with bone marrow cells from Runx1f/f, Mx1-Cre,CBFB+/56M mice and then treated with pIpC developed leukemia, while all of the recipients of the Mx1-Cre,CBFB+/56M bone marrow cells did, suggesting that the loss of leukemogenesis is cell autonomous. All together, these results indicate that Runx1 is indispensable for CBFB-MYH11 induced leukemogenesis. Even though at 3 weeks after pIpC the Runx1f/f, Mx1-Cre,CBFB+/56M mice had more c-Kit+ cells, and more significantly the abnormal myeloid progenitors (AMPs) (LK/CD34-/Fc□RII/III+) from which the leukemia cells arise, these cells started to decrease and disappear 4 weeks after pIpC, suggesting that this is a critical stage for the failure of leukemogenesis in the Runx1f/f, Mx1-Cre,CBFB+/56M mice. We therefore performed RNA-seq on the AMP population isolated from Mx1-Cre,CBFB+/56M and Runx1f/f, Mx1-Cre,CBFB+/56M mice three weeks after pIpC treatment, to explore the global gene expression changes between them. Our preliminary data analysis showed that many genes (1635) were differential expressed (DEGs; Padj ≤0.05, absolute fold change ≥ 2) between Runx1f/f, Mx1-Cre, CBFB+/56M and Mx1-Cre, CBFB+/56M mice. Many of these DEGs (37.6%) are RUNX1 target genes (Mandoli et al., Leukemia. 28:770, 2014). Among the significantly enriched gene sets by gene set enrichment analysis of the DEGs, the "GAL_LEUKEMIC_STEM_CELL_DN" gene set, which contains genes down-regulated in leukemic stem cells, is positively correlated with DEGs upregulated in Runx1f/f, Mx1-Cre,CBFB+/56M cells, suggesting that the AMP population from Runx1f/f, Mx1-Cre,CBFB+/56M mice lost the leukemia initiating ability. We are preforming ChIP-seq on the AMP population to determine how loss of RUNX1 disrupts binding of CBFβ-SMMHC to target genes. The above results suggest that RUNX1 is required for the regulation of critical genes for leukemogenesis by CBFβ-SMMHC. Disclosures No relevant conflicts of interest to declare.

  • title runx1 is indispensable for leukemia development induced by CBFB myh11
    Blood, 2018
    Co-Authors: Tao Zhen, Katherine R Hyde, Guadalupe Lopez, Erika Mijin Kwon, Ling Zhao, Lemlem Alemu
    Abstract:

    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. It is generally considered that CBFβ-SMMHC, the fusion protein encoded by CBFB-MYH11, is a dominant negative repressor of RUNX1, a transcription factor that physically interacts with CBFβ and CBFβ-SMMHC. While loss-of-function mutations in RUNX1 are common in human AML, they have not been found in inv(16) AML. Moreover, we have demonstrated that CBFβ-SMMHC has RUNX1-repression independent functions (Hyde et al., Blood 115:1433, 2010), and Runx1 insufficiency (Runx1+/lz) delays CBFB-MYH11-induced leukemia in a mouse model (Hyde et al., Leukemia 29:1771, 2015). These findings challenge the RUNX1-repression model for CBFβ-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 generated mice with both Cre-based conditional Runx1 knockout and CBFB-MYH11 knockin (Runx1f/f, Mx1-Cre,CBFB+/56M), which express CBFB-MYH11 but no Runx1 after pIpC (poly I:C) treatment to induce Cre expression. Runx1f/f, Mx1-Cre,CBFB+/56Mmice had higher numbers of Lin-/Sca1-/c-Kit+ (LK) and Lin-/Sca1+/c-Kit+ cells in the bone marrow when comapred with Mx1-Cre, CBFB+/56M mice at 3 weeks after pIpC. However, none of the Runx1f/f, Mx1-Cre, CBFB+/56M mice developed leukemia up to one year after pIpC treatment. As reported previously, all Mx1-Cre,CBFB+/56M mice developed leukemia with an median survival time of 4 months. Moreoever, none of the recipients transplanted with bone marrow cells from Runx1f/f, Mx1-Cre,CBFB+/56M mice and then treated with pIpC developed leukemia, while all of the recipients of the Mx1-Cre,CBFB+/56M bone marrow cells did, suggesting that the loss of leukemogenesis is cell autonomous. All together, these results indicate that Runx1 is indispensable for CBFB-MYH11 induced leukemogenesis. Even though at 3 weeks after pIpC the Runx1f/f, Mx1-Cre,CBFB+/56M mice had more c-Kit+ cells, and more significantly the abnormal myeloid progenitors (AMPs) (LK/CD34-/Fc□RII/III+) from which the leukemia cells arise, these cells started to decrease and disappear 4 weeks after pIpC, suggesting that this is a critical stage for the failure of leukemogenesis in the Runx1f/f, Mx1-Cre,CBFB+/56M mice. We therefore performed RNA-seq on the AMP population isolated from Mx1-Cre,CBFB+/56M and Runx1f/f, Mx1-Cre,CBFB+/56M mice three weeks after pIpC treatment, to explore the global gene expression changes between them. Our preliminary data analysis showed that many genes (1635) were differential expressed (DEGs; Padj ≤0.05, absolute fold change ≥ 2) between Runx1f/f, Mx1-Cre, CBFB+/56M and Mx1-Cre, CBFB+/56M mice. Many of these DEGs (37.6%) are RUNX1 target genes (Mandoli et al., Leukemia. 28:770, 2014). Among the significantly enriched gene sets by gene set enrichment analysis of the DEGs, the "GAL_LEUKEMIC_STEM_CELL_DN" gene set, which contains genes down-regulated in leukemic stem cells, is positively correlated with DEGs upregulated in Runx1f/f, Mx1-Cre,CBFB+/56M cells, suggesting that the AMP population from Runx1f/f, Mx1-Cre,CBFB+/56M mice lost the leukemia initiating ability. We are preforming ChIP-seq on the AMP population to determine how loss of RUNX1 disrupts binding of CBFβ-SMMHC to target genes. The above results suggest that RUNX1 is required for the regulation of critical genes for leukemogenesis by CBFβ-SMMHC. Disclosures No relevant conflicts of interest to declare.

  • runx1 is strictly required for CBFB myh11 induced leukemia development
    Blood, 2016
    Co-Authors: Tao Zhen, Katherine R Hyde, Guadalupe Lopez, Erika Mijin Kwon, Ling Zhao, Lemlem Alemu
    Abstract:

    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.

  • runx1 and CBFB myh11 are required for the maintenance of inv 16 aml
    Blood, 2016
    Co-Authors: Yiqian Wang, Lemlem Alemu, Kira Hannon, Michelle Becker, Lisa Garett, Cecilia Rivas, Lisa Richter, Ling Zhao, Katherine R Hyde
    Abstract:

    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.

  • runx1 is strictly required for CBFB myh11 induced leukemia development
    Blood, 2016
    Co-Authors: Tao Zhen, Katherine R Hyde, Guadalupe Lopez, Erika Mijin Kwon, Ling Zhao, Lemlem Alemu
    Abstract:

    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.

Lemlem Alemu - One of the best experts on this subject based on the ideXlab platform.

  • title runx1 is indispensable for leukemia development induced by CBFB myh11
    Blood, 2018
    Co-Authors: Tao Zhen, Katherine R Hyde, Lemlem Alemu, Guadalupe Lopez, Erika Mijin Kwon, Ling Zhao, Paul Liu
    Abstract:

    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. It is generally considered that CBFβ-SMMHC, the fusion protein encoded by CBFB-MYH11, is a dominant negative repressor of RUNX1, a transcription factor that physically interacts with CBFβ and CBFβ-SMMHC. While loss-of-function mutations in RUNX1 are common in human AML, they have not been found in inv(16) AML. Moreover, we have demonstrated that CBFβ-SMMHC has RUNX1-repression independent functions (Hyde et al., Blood 115:1433, 2010), and Runx1 insufficiency (Runx1+/lz) delays CBFB-MYH11-induced leukemia in a mouse model (Hyde et al., Leukemia 29:1771, 2015). These findings challenge the RUNX1-repression model for CBFβ-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 generated mice with both Cre-based conditional Runx1 knockout and CBFB-MYH11 knockin (Runx1f/f, Mx1-Cre,CBFB+/56M), which express CBFB-MYH11 but no Runx1 after pIpC (poly I:C) treatment to induce Cre expression. Runx1f/f, Mx1-Cre,CBFB+/56Mmice had higher numbers of Lin-/Sca1-/c-Kit+ (LK) and Lin-/Sca1+/c-Kit+ cells in the bone marrow when comapred with Mx1-Cre, CBFB+/56M mice at 3 weeks after pIpC. However, none of the Runx1f/f, Mx1-Cre, CBFB+/56M mice developed leukemia up to one year after pIpC treatment. As reported previously, all Mx1-Cre,CBFB+/56M mice developed leukemia with an median survival time of 4 months. Moreoever, none of the recipients transplanted with bone marrow cells from Runx1f/f, Mx1-Cre,CBFB+/56M mice and then treated with pIpC developed leukemia, while all of the recipients of the Mx1-Cre,CBFB+/56M bone marrow cells did, suggesting that the loss of leukemogenesis is cell autonomous. All together, these results indicate that Runx1 is indispensable for CBFB-MYH11 induced leukemogenesis. Even though at 3 weeks after pIpC the Runx1f/f, Mx1-Cre,CBFB+/56M mice had more c-Kit+ cells, and more significantly the abnormal myeloid progenitors (AMPs) (LK/CD34-/Fc□RII/III+) from which the leukemia cells arise, these cells started to decrease and disappear 4 weeks after pIpC, suggesting that this is a critical stage for the failure of leukemogenesis in the Runx1f/f, Mx1-Cre,CBFB+/56M mice. We therefore performed RNA-seq on the AMP population isolated from Mx1-Cre,CBFB+/56M and Runx1f/f, Mx1-Cre,CBFB+/56M mice three weeks after pIpC treatment, to explore the global gene expression changes between them. Our preliminary data analysis showed that many genes (1635) were differential expressed (DEGs; Padj ≤0.05, absolute fold change ≥ 2) between Runx1f/f, Mx1-Cre, CBFB+/56M and Mx1-Cre, CBFB+/56M mice. Many of these DEGs (37.6%) are RUNX1 target genes (Mandoli et al., Leukemia. 28:770, 2014). Among the significantly enriched gene sets by gene set enrichment analysis of the DEGs, the "GAL_LEUKEMIC_STEM_CELL_DN" gene set, which contains genes down-regulated in leukemic stem cells, is positively correlated with DEGs upregulated in Runx1f/f, Mx1-Cre,CBFB+/56M cells, suggesting that the AMP population from Runx1f/f, Mx1-Cre,CBFB+/56M mice lost the leukemia initiating ability. We are preforming ChIP-seq on the AMP population to determine how loss of RUNX1 disrupts binding of CBFβ-SMMHC to target genes. The above results suggest that RUNX1 is required for the regulation of critical genes for leukemogenesis by CBFβ-SMMHC. Disclosures No relevant conflicts of interest to declare.

  • title runx1 is indispensable for leukemia development induced by CBFB myh11
    Blood, 2018
    Co-Authors: Tao Zhen, Katherine R Hyde, Guadalupe Lopez, Erika Mijin Kwon, Ling Zhao, Lemlem Alemu
    Abstract:

    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. It is generally considered that CBFβ-SMMHC, the fusion protein encoded by CBFB-MYH11, is a dominant negative repressor of RUNX1, a transcription factor that physically interacts with CBFβ and CBFβ-SMMHC. While loss-of-function mutations in RUNX1 are common in human AML, they have not been found in inv(16) AML. Moreover, we have demonstrated that CBFβ-SMMHC has RUNX1-repression independent functions (Hyde et al., Blood 115:1433, 2010), and Runx1 insufficiency (Runx1+/lz) delays CBFB-MYH11-induced leukemia in a mouse model (Hyde et al., Leukemia 29:1771, 2015). These findings challenge the RUNX1-repression model for CBFβ-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 generated mice with both Cre-based conditional Runx1 knockout and CBFB-MYH11 knockin (Runx1f/f, Mx1-Cre,CBFB+/56M), which express CBFB-MYH11 but no Runx1 after pIpC (poly I:C) treatment to induce Cre expression. Runx1f/f, Mx1-Cre,CBFB+/56Mmice had higher numbers of Lin-/Sca1-/c-Kit+ (LK) and Lin-/Sca1+/c-Kit+ cells in the bone marrow when comapred with Mx1-Cre, CBFB+/56M mice at 3 weeks after pIpC. However, none of the Runx1f/f, Mx1-Cre, CBFB+/56M mice developed leukemia up to one year after pIpC treatment. As reported previously, all Mx1-Cre,CBFB+/56M mice developed leukemia with an median survival time of 4 months. Moreoever, none of the recipients transplanted with bone marrow cells from Runx1f/f, Mx1-Cre,CBFB+/56M mice and then treated with pIpC developed leukemia, while all of the recipients of the Mx1-Cre,CBFB+/56M bone marrow cells did, suggesting that the loss of leukemogenesis is cell autonomous. All together, these results indicate that Runx1 is indispensable for CBFB-MYH11 induced leukemogenesis. Even though at 3 weeks after pIpC the Runx1f/f, Mx1-Cre,CBFB+/56M mice had more c-Kit+ cells, and more significantly the abnormal myeloid progenitors (AMPs) (LK/CD34-/Fc□RII/III+) from which the leukemia cells arise, these cells started to decrease and disappear 4 weeks after pIpC, suggesting that this is a critical stage for the failure of leukemogenesis in the Runx1f/f, Mx1-Cre,CBFB+/56M mice. We therefore performed RNA-seq on the AMP population isolated from Mx1-Cre,CBFB+/56M and Runx1f/f, Mx1-Cre,CBFB+/56M mice three weeks after pIpC treatment, to explore the global gene expression changes between them. Our preliminary data analysis showed that many genes (1635) were differential expressed (DEGs; Padj ≤0.05, absolute fold change ≥ 2) between Runx1f/f, Mx1-Cre, CBFB+/56M and Mx1-Cre, CBFB+/56M mice. Many of these DEGs (37.6%) are RUNX1 target genes (Mandoli et al., Leukemia. 28:770, 2014). Among the significantly enriched gene sets by gene set enrichment analysis of the DEGs, the "GAL_LEUKEMIC_STEM_CELL_DN" gene set, which contains genes down-regulated in leukemic stem cells, is positively correlated with DEGs upregulated in Runx1f/f, Mx1-Cre,CBFB+/56M cells, suggesting that the AMP population from Runx1f/f, Mx1-Cre,CBFB+/56M mice lost the leukemia initiating ability. We are preforming ChIP-seq on the AMP population to determine how loss of RUNX1 disrupts binding of CBFβ-SMMHC to target genes. The above results suggest that RUNX1 is required for the regulation of critical genes for leukemogenesis by CBFβ-SMMHC. Disclosures No relevant conflicts of interest to declare.

  • runx1 and CBFB myh11 are required for the maintenance of inv 16 aml
    Blood, 2016
    Co-Authors: Yiqian Wang, Lemlem Alemu, Kira Hannon, Michelle Becker, Lisa Garett, Cecilia Rivas, Lisa Richter, Ling Zhao, Katherine R Hyde
    Abstract:

    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.

  • runx1 is strictly required for CBFB myh11 induced leukemia development
    Blood, 2016
    Co-Authors: Tao Zhen, Katherine R Hyde, Guadalupe Lopez, Erika Mijin Kwon, Ling Zhao, Lemlem Alemu
    Abstract:

    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.

  • runx1 is strictly required for CBFB myh11 induced leukemia development
    Blood, 2016
    Co-Authors: Tao Zhen, Katherine R Hyde, Guadalupe Lopez, Erika Mijin Kwon, Ling Zhao, Lemlem Alemu
    Abstract:

    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.

Martha Kirby - One of the best experts on this subject based on the ideXlab platform.

  • CBFB runx1 repression independent blockage of differentiation and accumulation of csf2rb expressing cells by CBFB myh11
    Blood, 2010
    Co-Authors: Katherine R Hyde, Yasuhiko Kamikubo, Martha Kirby, Lemlem Alemu, Stacie M. Anderson, Ling Zhao
    Abstract:

    It is known that CBFB-MYH11, the fusion gene generated by inversion of chromosome 16 in human acute myeloid leukemia, is causative for oncogenic transformation. However, the mechanism by which CBFB-MYH11 initiates leukemogenesis is not clear. Previously published reports showed that CBFB-MYH11 dominantly inhibits RUNX1 and CBFB, and such inhibition has been suggested as the mechanism for leukemogenesis. Here we show that CBFB-MYH11 caused CBFB/Runx1 repression–independent defects in both primitive and definitive hematopoiesis. During primitive hematopoiesis, CBFB-MYH11 delayed differentiation characterized by sustained expression of Gata2, Il1rl1, and Csf2rb, a phenotype not found in CBFB and Runx1 knockout mice. Expression of CBFB-MYH11 in the bone marrow induced the accumulation of abnormal progenitor-like cells expressing Csf2rb in preleukemic mice. The expression of all 3 genes was detected in most human and murine CBFB-MYH11+ leukemia samples. Interestingly, CBFB-MYH11+ preleukemic progenitors and leukemia-initiating cells did not express Csf2rb, although the majority of leukemia cells in our CBFB-MYH11 knockin mice were Csf2rb+. Therefore Csf2rb can be used as a negative selection marker to enrich preleukemic progenitor cells and leukemia-initiating cells from CBFB-MYH11 mice. These results suggest that CBFB/Runx1 repression–independent activities contribute to leukemogenesis by CBFB-MYH11.

  • CBFB runx1 repression independent blockage of differentiation and accumulation of csf2rb expressing cells by CBFB myh11
    Blood, 2010
    Co-Authors: Katherine R Hyde, Yasuhiko Kamikubo, Martha Kirby, Lemlem Alemu, Stacie M. Anderson, Ling Zhao
    Abstract:

    It is known that CBFB-MYH11, the fusion gene generated by inversion of chromosome 16 in human acute myeloid leukemia, is causative for oncogenic transformation. However, the mechanism by which CBFB-MYH11 initiates leukemogenesis is not clear. Previously published reports showed that CBFB-MYH11 dominantly inhibits RUNX1 and CBFB, and such inhibition has been suggested as the mechanism for leukemogenesis. Here we show that CBFB-MYH11 caused CBFB/Runx1 repression–independent defects in both primitive and definitive hematopoiesis. During primitive hematopoiesis, CBFB-MYH11 delayed differentiation characterized by sustained expression of Gata2, Il1rl1, and Csf2rb, a phenotype not found in CBFB and Runx1 knockout mice. Expression of CBFB-MYH11 in the bone marrow induced the accumulation of abnormal progenitor-like cells expressing Csf2rb in preleukemic mice. The expression of all 3 genes was detected in most human and murine CBFB-MYH11+ leukemia samples. Interestingly, CBFB-MYH11+ preleukemic progenitors and leukemia-initiating cells did not express Csf2rb, although the majority of leukemia cells in our CBFB-MYH11 knockin mice were Csf2rb+. Therefore Csf2rb can be used as a negative selection marker to enrich preleukemic progenitor cells and leukemia-initiating cells from CBFB-MYH11 mice. These results suggest that CBFB/Runx1 repression–independent activities contribute to leukemogenesis by CBFB-MYH11.

  • c kit with d816y v mutations cooperate with CBFB myh11 to accelerate leukemogenesis in mice
    Blood, 2009
    Co-Authors: Ling Zhao, Jan Joseph Melenhorst, Yasuhiko Kamikubo, Martha Kirby, Lemlem Alemu, Shelley Hoogstratenmiller, Stacie M. Anderson, Grant D Gilliland
    Abstract:

    Abstract 274 CBF leukemia is a group of acute myeloid leukemia (AML) caused by the fusion genes involving core binding factors (CBFs), which include RUNX1 and CBFB. C-KIT mutations are common in CBF leukemias. For example, 10–45% of AML cases with inv(16), which generates a fusion gene between CBFB and MYH11, carry activating mutations in c-KIT. In general, the presence of c-KIT mutations in inv(16) AML suggests poor clinical prognosis. Therefore, it is important to verify that c-KIT mutations cooperate with CBFB-MYH11, and to investigate the underlying mechanism during leukemogenesis. Here, we transduced bone marrow (BM) cells from conditional CBFB-MYH11 knockin mice with retroviral vectors carrying c-KIT mutations,D816V and D816Y (located in the kinase domain of c-KIT and can cause constitutive activation of c-KIT), which are common in inv(16) AML. Wild type c-KIT and empty retroviral vectors were used as controls. The transduced BM cells were then transplanted to sub-lethally irradiated recipient mice. In 2–5 months, 60% of mice carrying D816Y and 50% of mice carrying D816V c-KIT mutations developed leukemia, while none of the mice transduced with the control vectors developed leukemia up to one year after transplantation. In addition, BM cells from wildtype mice transduced with the c-KIT mutant vectors did not induce leukemia >5 months after transplantation. Analysis of signaling pathways revealed that Stat3-serine phosphorylation and P44/42 MAPK pathways, but not AKT and Stat5 pathways, were activated (phosphorylated) in the leukemia cells from the CBFB-MYH11 knockin mice expressing mutated c-KIT. In vitro colony forming assays indicated that CBFB-MYH11 BM cells expressing c-KIT D816V/Y mutants produced similar number of colonies as CBFB-MYH11 BM cells transduced with wild type c-KIT control vector. However, they had increased CFU-G and CFU-GEMM and decreased CFU-E colonies, suggesting that the c-KIT mutants drove CBFB-MYH11 BM cells towards an earlier progenitor state. Secondary transplantation with 1×106 leukemia cells from donors with c-KIT mutations showed an average life span of three weeks compared to six weeks from donors without c-KIT mutation. Further limiting dilution transplantation with 100 leukemia cells from one donor with c-KIT mutation led to lethal leukemia in 4 out of 5 mice 2.5 months after transplantation, indicating a very high frequency of leukemia initiating cells in this sample. These data suggest a strong cooperation between the mutant c-KIT and CBFB-MYH11 for leukemogenesis in mice. This mouse model can also serve as a clinically relevant model for small chemical screening and the development of novel therapeutic approaches. Disclosures: No relevant conflicts of interest to declare.

  • CBFB myh11 hinders early t cell development and induces massive cell death in the thymus
    Blood, 2007
    Co-Authors: Ling Zhao, Martha Kirby, Lucio H. Castilla, Liping Xu, Jennifer L Cannons, Pamela L Schwartzberg, Stacie M. Anderson, Remy Bosselut
    Abstract:

    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.

  • CBFB myh11 hinders early t cell development and induces massive cell death in the thymus
    Blood, 2007
    Co-Authors: Ling Zhao, Martha Kirby, Lucio H. Castilla, Liping Xu, Jennifer L Cannons, Pamela L Schwartzberg, Stacie M. Anderson, Remy Bosselut
    Abstract:

    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.

R Mittra - One of the best experts on this subject based on the ideXlab platform.

  • a new de embedding technique for the analysis of printed circuits and antennas based on the characteristic basis function method
    European Conference on Antennas and Propagation, 2012
    Co-Authors: R Mittra, Chiara Pelletti, G Bianconi, Agostino Monorchio
    Abstract:

    In this paper we present a new de-embedding algorithm for an efficient evaluation of the S-parameter for printed microwave circuits or antennas. The proposed technique is based on the Characteristic Basis Function Method (CBFM) with exponential type of Characteristic Basis Functions (CBFs) which can be exploited in order to mitigate the low frequency problems associated with the conventional de-embedding techniques. A numerical example that demonstrates the numerical accuracy and efficiency of the proposed method is included.

  • solution of wide band scattering problems using the characteristic basis function method
    Iet Microwaves Antennas & Propagation, 2012
    Co-Authors: M De Gregorio, Agostino Monorchio, Gianluigi Tiberi, R Mittra
    Abstract:

    A numerically efficient method for the evaluation of wide band scattering characteristics is presented. This approach, which is based on the characteristic basis function method (CBFM), utilises the characteristic basis functions (CBFs) that are derived for the highest frequency in the range of interest. These CBFs, once generated, also capture the electromagnetic behaviour of the lower frequencies as well. The use of these bases, which will be referred to as ultra-wide band characteristic basis functions (UCBFs), enables one to solve the scattering for any frequency sample in the band without going through the time-consuming process to generate the CBFs anew. Numerical results shown validate the accuracy and the time efficiency of this method.

  • solution of large scattering problems using a multilevel scheme in the context of characteristic basis finite element method
    IEEE Antennas and Propagation Society International Symposium, 2010
    Co-Authors: Ozlem Ozgun, R Mittra, Mustafa Kuzuoglu
    Abstract:

    The Characteristic Basis Finite Element Method (CBFEM) [1–3] is a new descendant of the Characteristic Basis Function Method (CBFM), which is a non-iterative domain decomposition approach for solving large-scale electromagnetic problems by using characteristic basis functions (CBFs). The CBFM was originally introduced in the context of Method of Moments [4], but has rapidly evolved, with appropriate modifications, into a general-purpose approach that is also applicable to Finite Element Method (FEM). Some of the features that are common to all of the CBFM-based approaches are: (i) utilization of high-level physics-based basis functions — called CBFs — to represent the fields inside each sub-domain; (ii) reduced-matrix that can be handled by using direct solvers; (iii) parallelization to reduce the overall CPU time and memory. The basic steps of the CBFEM algorithm are as follows: (i) computational domain is divided into a number of sub-domains; (ii) CBFs are generated for each sub-domain; (iii) unknowns are expressed as a weighted sum of CBFs; (iv) original matrix is transformed into a reduced-matrix by using the Galerkin procedure, which uses the CBFs as both basis and testing functions; (v) reduced matrix is solved for the weight coefficients, which are substituted into the series expressions to solve for the unknowns in the entire computational domain.

  • electromagnetic scattering by finite periodic arrays using the characteristic basis function and adaptive integral methods
    IEEE Transactions on Antennas and Propagation, 2010
    Co-Authors: R Mittra
    Abstract:

    We introduce a novel technique that combines the AIM algorithm with the characteristic basis function method (CBFM) to solve the problem of electromagnetic scattering by large but finite periodic arrays. An important advantage of using the CBFM for this problem is that we only need to analyze a single unit cell to construct the characteristic basis functions (CBFs) for the entire array. The CBFs are generated by illuminating a single unit cell with a plane wave incident from different angles, for both the θ- and φ-polarizations. The initial set of CBFs, generated in the manner described above, are then downselected by applying a singular value decomposition (SVD) procedure and retaining only the left singular vectors whose corresponding singular values fall above a threshold. Next, in the conventional CBFM, we derive a reduced matrix by applying the Galerkin procedure and solve it directly if its size is manageable. However, when solving an array problem, which precludes the direct-solve option, we can utilize the adaptive integral method (AIM) algorithm, detailed below, not only to accelerate the solution but to reduce memory requirements as well. Numerical examples are included in this communication to demonstrate the accuracy and the numerical efficiency of the proposed technique.

  • characteristic basis function method for solving electromagnetic scattering problems over rough terrain profiles
    IEEE Transactions on Antennas and Propagation, 2010
    Co-Authors: Atacan Yagbasan, Celal Alp Tunc, Vakur B Erturk, Ayhan Altintas, R Mittra
    Abstract:

    A computationally efficient algorithm, which combines the characteristic basis function method (CBFM), the physical optics (PO) approach (when applicable) with the forward backward method (FBM), is applied for the investigation of electromagnetic scattering from-and propagation over-large-scale rough terrain problems. The algorithm utilizes high-level basis functions defined on macro-domains (blocks), called the characteristic basis functions (CBFs) that are constructed by aggregating low-level basis functions (i.e., conventional sub-domain basis functions). The FBM as well as the PO approach (when applicable) are used to construct the aforementioned CBFs. The conventional CBFM is slightly modified to handle large-terrain problems, and is further embellished by accelerating it, as well as reducing its storage requirements, via the use of an extrapolation procedure. Numerical results for the total fields, as well as for the path loss are presented and compared with either measured or previously published reference solutions to assess the efficiency and accuracy of the algorithm.