FIP1L1 knockdown in Kasumi-1 AML cells

Post-transcriptional regulation has emerged as a driver for leukemia development and an avenue for therapeutic targeting. Among post-transcriptional processes, alternative polyadenylation (APA) is globally dysregulated across cancer types. However, limited studies have focused on the prevalence and role of APA in 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. By performing 3'RNA sequencing on acute myeloid leukemia (AML) patient samples and healthy hematopoietic stem and progenitor cells (HSPCs), we show that patient cells exhibit global 3' untranslated region (UTR) shortening and coding sequence (CDS) lengthening due to differences in poly(A) site usage. Among APA regulators, FIP1L1 expression correlated with the degree of APA dysregulation and knockdown of this RNA-binding protein (RBP) 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 fusion oncoprotein AML1-ETO. In non-t(8;21) cells, knockdown also promoted differentiation by attenuating mTORC1 signaling and reducing MYC protein levels. Our study gives mechanistic insight into the role of APA in AML pathogenesis and provides evidence that targeting global APA patterns can overcome the differentiation block of AML patients. Overall design: RNA-sequencing and 3'RNA-sequencing (3'READS) of Kasumi-1 cells transduced with two independent FIP1L1-targeting shRNAs or a control shRNA. For RNA-sequencing, 4 biological replicates were prepared for each condition. For 3'READS, 3 biological replicates were prepared for shControl and shRNA #2; 4 biological replicates were prepared for shRNA #1.