RUNX1 C-terminal mutations impair blood cell differentiation by perturbing specific enhancer-promoter networks.

ABSTRACT: The transcription factor RUNX1 is a master regulator of hematopoiesis and is frequently mutated in myeloid malignancies. Mutations in its runt homology domain (RHD) frequently disrupt DNA binding and result in loss of RUNX1 function. However, it is not clearly understood how other RUNX1 mutations contribute to disease development. Here, we characterized RUNX1 mutations outside of the RHD. Our analysis of the patient data sets revealed that mutations within the C-terminus frequently occur in hematopoietic disorders. Remarkably, most of these mutations were nonsense or frameshift mutations and were predicted to be exempt from nonsense-mediated messenger RNA decay. Therefore, this class of mutation is projected to produce DNA-binding proteins that contribute to the pathogenesis in a distinct manner. To model this, we introduced the RUNX1R320∗ mutation into the endogenous gene locus and demonstrated the production of RUNX1R320∗ protein. Expression of RUNX1R320∗ resulted in the disruption of RUNX1 regulated processes such as megakaryocytic differentiation, through a transcriptional signature different from RUNX1 depletion. To understand the underlying mechanisms, we used Global RNA Interactions with DNA by deep sequencing (GRID-seq) to examine enhancer-promoter connections. We identified widespread alterations in the enhancer-promoter networks within RUNX1 mutant cells. Additionally, we uncovered enrichment of RUNX1R320∗ and FOXK2 binding at the MYC super enhancer locus, significantly upregulating MYC transcription and signaling pathways. Together, our study demonstrated that most RUNX1 mutations outside the DNA-binding domain are not subject to nonsense-mediated decay, producing protein products that act in concert with additional cofactors to dysregulate hematopoiesis through mechanisms distinct from those induced by RUNX1 depletion.

Alternative polyadenylation dysregulation contributes to the differentiation block of acute myeloid leukemia.

Posttranscriptional regulation has emerged as a driver for leukemia development and an avenue for therapeutic targeting. Among posttranscriptional processes, alternative polyadenylation (APA) is globally dysregulated across cancer types. However, limited studies have focused on the prevalence and role of APA in myeloid leukemia. Furthermore, it is poorly understood how altered poly(A) site usage of individual genes contributes to malignancy or whether targeting global APA patterns might alter oncogenic potential. In this study, we examined global APA dysregulation in patients with acute myeloid leukemia (AML) by performing 3' region extraction and deep sequencing (3'READS) on a subset of AML patient samples along with healthy hematopoietic stem and progenitor cells (HSPCs) and by analyzing publicly available data from a broad AML patient cohort. We show that patient cells exhibit global 3' untranslated region (UTR) shortening and coding sequence lengthening due to differences in poly(A) site (PAS) usage. Among APA regulators, expression of FIP1L1, one of the core cleavage and polyadenylation factors, correlated with the degree of APA dysregulation in our 3'READS data set. Targeting global APA by FIP1L1 knockdown reversed the global trends seen in patients. Importantly, FIP1L1 knockdown induced differentiation of t(8;21) cells by promoting 3'UTR lengthening and downregulation of the fusion oncoprotein AML1-ETO. In non-t(8;21) cells, FIP1L1 knockdown also promoted differentiation by attenuating mechanistic target of rapamycin complex 1 (mTORC1) signaling and reducing MYC protein levels. Our study provides mechanistic insights into the role of APA in AML pathogenesis and indicates that targeting global APA patterns can overcome the differentiation block in patients with AML.

A CRISPR RNA-binding protein screen reveals regulators of RUNX1 isoform generation.

The proper balance of hematopoietic stem cell (HSC) self-renewal and differentiation is critical for normal hematopoiesis and is disrupted in hematologic malignancy. Among regulators of HSC fate, transcription factors have a well-defined central role, and mutations promote malignant transformation. More recently, studies have illuminated the importance of posttranscriptional regulation by RNA-binding proteins (RBPs) in hematopoiesis and leukemia development. However, the RBPs involved and the breadth of regulation are only beginning to be elucidated. Furthermore, the intersection between posttranscriptional regulation and hematopoietic transcription factor function is poorly understood. Here, we studied the posttranscriptional regulation of RUNX1, a key hematopoietic transcription factor. Alternative polyadenylation (APA) of RUNX1 produces functionally antagonistic protein isoforms (RUNX1a vs RUNX1b/c) that mediate HSC self-renewal vs differentiation, an RNA-processing event that is dysregulated in malignancy. Consequently, RBPs that regulate this event directly contribute to healthy and aberrant hematopoiesis. We modeled RUNX1 APA using a split GFP minigene reporter and confirmed the sensitivity of our model to detect changes in RNA processing. We used this reporter in a clustered regularly interspaced short palindromic repeats (CRISPR) screen consisting of single guide RNAs exclusively targeting RBPs and uncovered HNRNPA1 and KHDRBS1 as antagonistic regulators of RUNX1a isoform generation. Overall, our study provides mechanistic insight into the posttranscriptional regulation of a key hematopoietic transcription factor and identifies RBPs that may have widespread and important functions in hematopoiesis.