Unraveling a Novel Mechanism of RUNX1 Activity in Familial Platelet Disorder with Associated Myeloid Malignancy

Abstract Acute myeloid leukemia (AML) has a higher incidence and death rate than all other types of adult-onset acute leukemia in the USA, thus requiring a better understanding of the molecular mechanisms behind its progression. Since familial platelet disorder with associated myeloid malignancy (FPDMM) is closely related to AML and is caused by mutations in the RUNX1 gene, elucidation of RUNX1 in the development of FPDMM serves as a model for understanding the genesis of AML. FPDMM is a rare autosomal dominant disorder. FPDMM patients are characterized with defective megakaryopoiesis, abnormal platelet count and function, and bleeding disorders. Importantly, ~60% of patients develop hematological malignancies later on in their lives. FPDMM patients carry heterozygous, germline mutations in the RUNX1 gene. RUNX1 is a transcription factor that plays a critical role during early stages of definitive hematopoiesis, and megakaryopoiesis. Significantly, RUNX1 mutations have been reported in many cases of AML and myelodysplastic syndrome. RUNX1's C-terminal contains a VWRPY motif, which is a conserved binding site for transducin like enhancer of split1 (TLE1). TLE1 is a transcriptional corepressor that inhibits several transcription factors. Previous studies showed that RUNX1 missing the VWRPY motif could not bind TLE1, resulting in overexpression of RUNX1's target genes. However, the significance of RUNX1-TLE1 interaction was never investigated in regard to megakaryopoiesis, FPDMM pathogenesis, or leukemogenesis. Hence, there is a need to better understand the role of RUNX1-TLE1 interaction and their significance in megakaryopoiesis in general. A new FPDMM family has been identified carrying a GC insertion at the end of RUNX1's C-terminus. Genomic DNA sequencing of two patients from the family confirmed the mutation, which resulted in a frame shift mutation (L472fsX). As a result, the VWRPY motif is absent. Instead, the mutant protein contains additional, unrelated 123 amino acids, whose expression has been confirmed by western blot. Our hypothesis states that because the RUNX1 mutant lacks the TLE1 binding motif (VWRPY), its repression is defective which in turn affects normal megakaryopoiesis. Thereby, we are presenting a novel RUNX1 mutation in FPDMM and a possible novel mechanism that has never been studied before in FPDMM patients. To evaluate the effect of the mutation on RUNX1-TLE1 interaction, fluorescence resonance energy transfer (FRET) was performed in HEK293 cells. CFP-RUNX1 wild type (wt) and mutant co-transfected with YFP-CBFβ gave a FRET efficiency of 14% ± 2.5% and 16% ± 2.7%, respectively; suggesting that the mutation did not disrupt the physical binding between RUNX1 and its co-factor CBFβ. CFP-RUNX1 wt co- transfected with YFP-TLE1 gave an average of 10% ± 3.3% FRET efficiency, while CFP-RUNX1 mutant co-transfected with YFP-TLE1 gave an average of 0.65% ± 1.8% FRET efficiency, indicating no binding between the RUNX1 mutant and TLE1. These findings demonstrate that the existence of RUNX1’s C-terminus mutation abolished RUNX1’s interaction with TLE1. Furthermore, to assess the effect of the disrupted interaction between RUNX1 and TLE1 on RUNX1's activity, we performed a dual luciferase assay, which measures the promoter activity of a RUNX1's target, myeloid colony stimulating factor receptor (MCSFR). Results show that TLE1 was able to partially repress RUNX1 wt activity when co-transfected with CBFβ, consistent with previous data. On the contrary, TLE1 did not repress RUNX1 mutant activity, which resulted in increased RUNX1's target expression. Therefore, these preliminary results are consistent with the proposed regulatory role for RUNX1 and TLE1 during hematopoiesis. To corroborate these results, we have generated human induced pluripotent stem cells (iPSCs) from the FPDMM patient's blood cells containing the RUNX1 L472fsX mutation to model the defects in megakaryopoiesis. We are currently analyzing the hematopoietic differentiation of the mutated iPSCs and studying the mechanism through expression and pathway analysis of RNA-Seq data. Moreover, we have generated a mouse model closely representing the mutation using CRISPR-Cas9 system. Bothmodels will be used to provide a better understanding of megakaryopoiesis in general, and FPDMM pathogenesis and their progression to leukemia in particular. Disclosures No relevant conflicts of interest to declare.