Dissecting the tumorigenesis trajectory by single-cell RNA-seq reveals critical roles of RNA splicing

Abstract Background Leukemogenesis is proposed to be a multistep process by which normal hematopoietic stem and progenitor cells are transformed into full-blown leukemic cells, the details of which are not fully understood. Methods We performed serial single-cell transcriptome analyses(10X genomics) of preleukemic and leukemic cells (PLCs) and constructed the cellular and molecular transformation trajectory in a Myc-driven AML model in mice, which represented the transformation course in patients. Another single-cell approach, Smart Seq2, was used to analyze alternative splicing events in leukemic cell. Results We found that the Myc targets were gradually upregulated along the trajectory. Among them were splicing factors, which showed stage-specific prognosis for AML patients. Furthermore, we dissected the detailed gene network of a tipping point for HSPCs to generate initiating PLCs, which was characterized by dramatically increased splicing factors and unusual RNA velocity. In the late stage, PLCs acquired explosive heterogeneity through RNA alternative splicing. Among them, the Hsp90aahi subpopulation was conserved in both human and mouse AML and associated with poor prognosis. Exon 6 skipping of Tmem134 was identified in these cells. While the exon skipping product Tmem134β promoted the cell cycle, full-length Tmem134α delayed tumorigenesis. Conclusion Our study emphasized the critical roles of RNA splicing in the full process of leukemogenesis and identified the role of a novel tumor specific alternative splicing event, Exon 6 skipping of Tmem134, during the occurrence and maintenance of leukemia.