UC San Diego

Post-transcriptional regulation by RNA-binding proteins (RBPs) is an important layer of gene regulation implicated in both healthy hematopoiesis and hematologic malignancy. Among post-transcriptional mechanisms, alternative polyadenylation (APA) regulates gene expression and function, mediating normal cellular differentiation and malignant transformation across cellular systems. In hematology, APA plays a critical role in lymphocyte maturation and dysregulation contributes to multiple myeloma and lymphocytic leukemia. Despite its documented importance in immune cells, it is unknown whether APA plays a critical role in myeloid malignancy or in healthy hematopoietic stem cell (HSC) maintenance. Furthermore, RBPs that regulate APA in hematologic systems have not been identified.

Here, we first addressed the prevalence and global function of APA in myeloid malignancy. We compared poly(A) site usage in acute myeloid leukemia (AML) blasts to usage in healthy hematopoietic stem and progenitor cells (HSPCs), uncovering global patterns and individual leukemia-promoting genes altered in malignancy. By targeting the RBP and APA regulator FIP1L1, we reversed the global trends in patients and observed cellular differentiation across diverse AML subtypes by disrupting leukemogenic signaling networks. In t(8;21) AML, we validated APA regulation of AML1-ETO, showing for the first time that expression of a prominent oncofusion is sensitive to this mode of post-transcriptional regulation. Altogether, our work defines a critical role for APA in AML and illuminates a new pathway that may be exploited for differentiation therapy in patients.

We also studied the role of APA in healthy HSC pool maintenance by focusing on APA regulation of the critical hematopoietic transcription factor RUNX1. Polyadenylation upstream of the most distal 3'UTR produces a C-terminally truncated protein that antagonizes the pro-differentiation function of full-length RUNX1 in HSCs. We modeled this relevant APA event using a dual fluorescent minigene reporter and used this reporter in a CRISPR screen targeting RBPs. We identified HNRNPA1 and KHDRBS1 as regulators of RUNX1 APA, assigning a new role to these RBPs in HSC fate. Overall, our work highlights the intersection between post-transcriptional regulation and transcription factor function in healthy hematopoiesis.