An inhibitor of UHM splicing factors exerts anti-leukemic activity by altering cell cycle progression and reducing lysosome acidification

Abstract

Mature mRNAs are generated by spliceosomes that recruit factors to aid RNA splicing in which introns are removed and exons joined. Among the splicing factors, a family of proteins contain a homologous U2 Auxiliary Factor (U2AF) Homology Motif (UHM) to bind with factors containing U2AF ligand motifs (ULM) and recruit them to regulate 3′ splice site selection. Mutations and overexpression of UHM splicing factors are frequently found in cancers. Although a few UHM inhibitors have been reported, their activities in cancer cells were not investigated. Here, we studied our recently discovered UHM inhibitor, SF-153, that targeted RBM39 and SPF45, and found SF-153 exerted anti-tumor activities in three leukemia cell lines. In the cell cycle and apoptosis analysis, SF-153 induces p53 mediated DNA damage response to give protracted S phase in NKM-1 but p53-independent G1 arrest in p53^null K562 cells. RNA-seq analysis of NKM-1 treated with SF-153 revealed downregulation of Myc target genes and genes involved in nucleotides and protein synthesis. Western blot analysis confirmed decreased proteins levels of Myc, EEF1A1, and MCM7 suggesting reduced DNA replication activity in NKM-1. Transcript analysis derived from the RNA-seq data indicated six genes changed isoform patterns but downregulation of only one DOCK2 isoform was confirmed in qRT-PCR. RNA-seq analysis additionally uncovered that SF-153 impaired lysosome acidification and inhibited autophagy to enhance the anti-leukemic activities. Taken together, our study characterized the multimodal mechanisms of inhibition by SF-153 in leukemia cells and laid the foundation for studying selective UHM inhibitors in future.