Biochemical and In Vivo Characterization of Amino Acid Substitutions in the Runx1 (AML1) Runt Domain Found in FPD/AML, AML M0, and Cleidocranial Dysplasia (CCD) Patients.
Authors: Matheny Christina J. , Corpora Takeshi , Speck Maren E. , Gu Ting-Lei , Bushweller John H. , Speck Nancy A. 
Published: April 2020

Abstract
Runx1 and CBF β are the DNA-binding and non DNA-binding subunits of a core-binding factor that is required for hematopoiesis, and that is frequently mutated in leukemia. Runx2 is the DNA-binding subunit of a core-binding factor required for bone formation. Mono-allelic deletion, nonsense, frameshift, and missense mutations have been found in RUNX1 in familial platelet disorder with predisposition for acute myelogenous leukemia (FPD/AML) and in myelodysplastic syndrome (MDS), and biallelic mutations in RUNX1 are found in 20% of AML M0 patients. Similar types of mono-allelic mutations have been found in RUNX2 in patients with cleidocranial dysplasia (CCD), an inherited skeletal syndrome. FPD/AML and CCD pedigrees have revealed varying degrees of disease severity depending on the nature of the specific mutation. Additionally, it has been observed that mutations involving amino acids in the DNA binding Runt domain that directly contact DNA are associated primarily with Runx1 and hematopoietic disorders, while mutations predicted to disrupt CBF β binding or the Runt domain structure are found only in Runx2 in CCD patients. We introduced 21 amino acid substitutions into the Runt domain of Runx1 identified in FPD/AML, AML M0, and CCD patients, and quantified their effects on DNA binding, heterodimerization with CBFβ, and the Runt domain structure using yeast one- and two-hybrid, quantitative electrophoretic mobility shift, heteronuclear single quantum correlation spectroscopy, and urea denaturation experiments. To address the impact on in vivo function, several of these point mutations were engineered into the endogenous Runx1 allele in mice. These five mutations include: R177X, R174Q, T149A, T161A, and L148F. R177X is found in FPD/AML patients and truncates Runx1 two amino acids before the C-terminal boundary of the Runt domain. R174Q (found in FPD/AML and CCD) disrupts DNA binding 1000-fold, but does not disrupt CBFb binding or perturb the Runt domain fold. T149A (found only in CCD) disrupts CBFβ binding 13-fold while T161A (not found in patients) disrupts CBFβ binding 40-fold. Both T149A and T161A slightly perturb the Runt domain fold, but do not alter DNA binding affinity. L148F (found in CCD) also disrupts the Runt domain fold, and decreases DNA binding. All animals heterozygous for these alleles are viable. Mice homozygous for R177X and R174Q die during gestation. Mice homozygous for the T149A and T161A mutations, on the other hand, are born at normal Mendelian frequencies, but 62% and 100%, respectively, die by or at three weeks of age from an undetermined cause. The effects of these mutations on hematopoietic progenitor and platelet numbers, both of which are affected in FPD/AML patients, will be presented. We conclude that mutations that affect CBFβ binding result in hypomorphic Runx1 alleles, while mutations involving DNA contacts result in more severe inactivation of Runx1 function. Thus FPD/AML, AML M0, and MDS require mutations that severely inactivate Runx1 function, while CCD can result from more subtle alterations in Runx2.