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Chromosomal translocations involving the human CBFB gene, which codes for the non-DNA binding subunit of CBF (CBFβ), are associated with a large percentage of human leukemias. The translocation inv(16) that disrupts the CBFB gene produces a chimeric protein composed of the heterodimerization domain of CBFβ fused to the C-terminal coiled-coil domain from smooth muscle myosin heavy chain (CBFβ-SMMHC). Isothermal titration calorimetry results show that this fusion protein binds the Runt domain from Runx1 (CBFα) with higher affinity than the native CBFβ protein. NMR studies identify interactions in the CBFβ portion of the molecule, as well as the SMMHC coiled-coil domain. This higher affinity provides an explanation for the dominant negative phenotype associated with a knock-in of the CBFB-MYH11 gene and also helps to provide a rationale for the leukemia-associated dysregulation of hematopoietic development that this protein causes.

No relevant conflicts of interest to declare.

Abstract 763

AML1/ETO is the chimeric fusion protein resulting from the t(8;21) found in AML of the M2 subtype. It contains the N-terminal 177 amino acids of RUNX1 and virtually all (575aa) of ETO. The RUNX1 component includes the Runt domain, which mediates both DNA binding and heterodimerization with CBFβ, but lacks the more C-terminal sequences required for transactivation. AML1/ETO occupies RUNX target genes in vivo and is associated with a repressive chromatin structure characterized by reduced levels of acetylated histone H3. AML1/ETO is thought to repress transcription by recruiting a SMRT (N-CoR)/Sin3A/HDAC complex to chromatin via sequences in ETO. ETO is the human homologue of the Drosophila Nervy protein and shares 4 regions of homology with Nervy called Nervy Homology Regions (NHR) 1–4. Deletion studies have shown that three of the AML1/ETO domains essential for its repressive function are the Runt domain, NHR2, and NHR4. The NHR2 domain is a hydrophobic heptad repeat that mediates oligomerization of AML1/ETO, interaction with ETO family members, and also with mSin3A and HDACs. We recently solved an x-ray structure of the NHR2 domain and found it to be an alpha-helical tetramer. Based on this structure we have introduced amino acid substitutions into the NHR2 domain that disrupt tetramer formation but not AML1/ETO stability. These mutations impair the ability of AML1/ETO to inhibit the differentiation of GR−1+/Mac−1+ cells following retroviral transduction into primary mouse bone marrow cells, and also inhibit the serial replating ability of AML1/ETO expressing bone marrow cells in vitro. We also assessed the outcome of disrupting oligomerization on a variety of previously described protein-protein interactions, and found that neither deleting the NHR2 domain nor disrupting oligomerization affected the ability of HDAC1, HDAC2, HDAC3, N-CoR, SMRT, PKA RIIα, PLZF, or HEB, to co-immunoprecipitate AML1/ETO from cell extracts. Deletion of the NHR2 domain reduced binding of mSin3a as shown previously, but disruption of oligomerization did not. To investigate the contribution of oligomerization to AML1/ETO-mediated transcriptional modulation, we amplified RNA from retrovirally-transduced, lineage depleted primary mouse bone marrow cells and performed Real Time Quantitative PCR of genes whose expression is known to be regulated by AML1/ETO or RUNX1. We show that the requirement for oligomerization is target gene dependent, with several classes of genes resulting from our study. We also introduced mutations in the Runt domain of AML1/ETO that disrupt CBFβ binding by defined amounts (40-fold, 200-fold, 500-fold), and demonstrated that CBFβ binding by AML1/ETO is essential for its dominant negative activity. The latter results suggest that small molecules designed to selectively impair heterodimerization of AML1/ETO with CBFβ could potentially block AML1/ETO’s dominant negative activity.

Core-binding factors (CBFs) are heterodimeric transcriptional factors consisting of a DNA-binding Runx1 (CBFα) subunit and a CBFβ subunit. Cbfβ allosterically increases the affinity of Runx1 for DNA ~2.5 fold. CBF subunits are encoded by four genes in mammals. RUNX1 (AML1), RUNX2, and RUNX3 encode for CBFα subunits, and CBFB encodes the CBFβ subunit. Homozygous disruption of either the Runx1 or the Cbfb genes in mice results in essentially identical phenotypes: midgestation embryonic lethality accompanied by extensive hemorrhaging and a profound block at the fetal liver stage of hematopoiesis. In humans, chromosomal rearrangements that disrupt the Runx1 and CBFB genes are associated with a significant percentage of leukemias. CBFβ is disrupted in acute myeloid leukemia by inv(16)(p13;q22), t(16;16), and del(16)(q22). These translocations result in the production of novel fusion proteins containing most of the CBFβ protein fused to the C-terminal coiled-coil domain from smooth muscle myosin heavy chain (SMMHC) encoded by the MYH11 gene. A knock-in of the CBFB-MYH11 allele in mice resulted in embryonic lethality with a profound block in hematopoietic development, the same phenotype observed for the Runx1 and Cbfb knockouts. We recently demonstrated that the CBFβ-SMMHC fusion protein binds to the DNA binding Runt domain from Runx1 with both higher affinity and altered stoichiometry relative to native CBFβ. We also provided NMR-based evidence for multiple sites of contact between Runx1 and CBFβ-SMMHC, proving the role of the SMMHC sequence in creating this altered affinity. Here we demonstrate that CBFβ-SMMHC inhibits DNA binding of the Runx1 Runt domain by ~6-fold for the CD4 dual-site silencer element. Cross-saturation NMR mapping on the Runt domain in complex with CBFβ-SMMHC reveals that the SMMHC portion of the oncoprotein makes contacts with β-strands 1 and 2 in the Runt domain. We propose that the inhibition of DNA-binding and increased affinity combine to mediate the dysregulation of Runx-regulated genes caused by CBFβ-SMMHC. These results also clearly suggest that targeting of the CBFβ-SMMHC protein for drug development may well be a viable approach for the treatment of the associated leukemia.

AML1/ETO is the chimeric fusion protein resulting from the t(8;21) found in AML of the M2 subtype. It contains the N-terminal 177 amino acids of RUNX1 and virtually all (575aa) of ETO. The RUNX1 component includes the Runt domain, which mediates both DNA binding and heterodimerization with CBFβ, but lacks the more C-terminal sequences required for transactivation. AML1/ETO occupies RUNX target genes in vivo and is associated with a repressive chromatin structure characterized by reduced levels of acetylated histone H3. AML1/ETO is thought to repress transcription by recruiting a SMRT (N-CoR)/Sin3A/HDAC complex to chromatin via sequences in ETO. ETO is the human homologue of the Drosophila Nervy protein and shares 4 regions of homology with Nervy called Nervy Homology Regions (NHR) 1–4. Deletion studies have shown that three of the AML1/ETO domains essential for its repressive function are the Runt domain, NHR2, and NHR4. The NHR2 domain is a hydrophobic heptad repeat that mediates oligomerization of AML1/ETO, interaction with ETO family members, and also with mSin3A and HDACs. We recently solved an x-ray structure of the NHR2 domain and found it to be an alpha-helical tetramer. Based on this structure we have introduced amino acid substitutions into the NHR2 domain that disrupt tetramer formation but not AML1/ETO stability. These mutations impair the ability of AML1/ETO to inhibit the differentiation of GR−1+/Mac−1+ cells following retroviral transduction into primary mouse bone marrow cells, and also inhibit the serial replating ability of AML1/ETO expressing bone marrow cells in vitro. We also assessed the outcome of disrupting oligomerization on a variety of previously described protein-protein interactions, and found that neither deleting the NHR2 domain nor disrupting oligomerization affected the ability of HDAC1, HDAC2, HDAC3, N-CoR, SMRT, PKA RIIα, PLZF, or HEB, to co-immunoprecipitate AML1/ETO from cell extracts. Deletion of the NHR2 domain reduced binding of mSin3a as shown previously, but disruption of oligomerization did not. To investigate the contribution of oligomerization to AML1/ETO-mediated transcriptional modulation, we amplified RNA from retrovirally-transduced, lineage depleted primary mouse bone marrow cells and performed Real Time Quantitative PCR of genes whose expression is known to be regulated by AML1/ETO or RUNX1. We show that the requirement for oligomerization is target gene dependent, with several classes of genes resulting from our study. We also introduced mutations in the Runt domain of AML1/ETO that disrupt CBFβ binding by defined amounts (40-fold, 200-fold, 500-fold), and demonstrated that CBFβ binding by AML1/ETO is essential for its dominant negative activity. The latter results suggest that small molecules designed to selectively impair heterodimerization of AML1/ETO with CBFβ could potentially block AML1/ETO's dominant negative activity.

Core-binding factors (CBFs) are heterodimeric transcriptional factors consisting of a DNA-binding Runx1 (CBFα) subunit and a CBFβ subunit. Cbfβ allosterically increases the affinity of Runx1 for DNA ~2.5 fold. CBF subunits are encoded by four genes in mammals. RUNX1 (AML1), RUNX2,and RUNX3encode for CBFα subunits, and CBFBencodes the CBFβ subunit. Homozygous disruption of either the Runx1or the Cbfbgenes in mice results in essentially identical phenotypes: midgestation embryonic lethality accompanied by extensive hemorrhaging and a profound block at the fetal liver stage of hematopoiesis. In humans, chromosomal rearrangements that disrupt the Runx1and CBFBgenes are associated with a significant percentage of leukemias. CBFβ is disrupted in acute myeloid leukemia by inv(16)(p13;q22), t(16;16), and del(16)(q22). These translocations result in the production of novel fusion proteins containing most of the CBFβ protein fused to the C-terminal coiled-coil domain from smooth muscle myosin heavy chain (SMMHC) encoded by the MYH11gene. A knock-in of the CBFB-MYH11allele in mice resulted in embryonic lethality with a profound block in hematopoietic development, the same phenotype observed for the Runx1and Cbfbknockouts. We recently demonstrated that the CBFβ-SMMHC fusion protein binds to the DNA binding Runt domain from Runx1 with both higher affinity and altered stoichiometry relative to native CBFβ. We also provided NMR-based evidence for multiple sites of contact between Runx1 and CBFβ-SMMHC, proving the role of the SMMHC sequence in creating this altered affinity. Here we demonstrate that CBFβ-SMMHC inhibits DNA binding of the Runx1 Runt domain by ~6-fold for the CD4 dual-site silencer element. Cross-saturation NMR mapping on the Runt domain in complex with CBFβ-SMMHC reveals that the SMMHC portion of the oncoprotein makes contacts with β-strands 1 and 2 in the Runt domain. We propose that the inhibition of DNA-binding and increased affinity combine to mediate the dysregulation of Runx-regulated genes caused by CBFβ-SMMHC. These results also clearly suggest that targeting of the CBFβ-SMMHC protein for drug development may well be a viable approach for the treatment of the associated leukemia.