To identify potential regulators of propagation and self-renewal of Acute Lymphoblastic Leukaemia (ALL), we performed an explorative genome-wide RNAi screen followed by CRISPR ex vivo and in vivo validation screens in the t(4;11)-positive ALL cell line SEM. These screens identified the splicing factor PHF5A as a crucial component of the leukemic program. PHF5A is a subunit of the SF3b protein complex, which directs alternative splicing by binding to the branchpoint of pre-mRNA. Mutations in members of this complex including SF3B1 have been implicated in several haematological malignancies.
Functional perturbation experiments demonstrated that PHF5A depletion impairs proliferation, viability and clonogenicity in a range of ALL and AML cell lines strongly suggesting that PHF5A is required for leukemic propagation and self-renewal. To identify genetic programs affected by PHF5A inhibition, we performed RNA-seq followed by analysis of differential gene expression and splicing events. We identified 473 genes with differential expression upon PHF5A knockdown. In addition, we performed in-depth analysis of splicing patterns by examining both differential exon/intron usage and exon junction formation. These analyses demonstrated that loss of PHF5A affects splicing of more than 2500 genes with exon skipping and intron retention being the most frequent splicing events.
In order to identify processes and pathways affected by PHF5A, we performed gene set enrichment analysis (GSEA) on both differential expression and splicing. While gene sets associated with RNA processing including splicing, turnover and translation were enriched in both data sets, the differential gene expression signature was also linked to DNA repair processes including base excision, mismatch and homologous recombination repair.
In line with these findings, knockdown of either PHF5A or its partner protein SF3B1 induced DNA strand breaks as indicated by comet assay and increased y-H2AX levels. Furthermore, both PHF5A and SF3B1 depletion sensitized ALL cells towards the DNA crosslinking agent mitomycin C. Closer inspection of RNA-seq datasets revealed reduced FANCD2 expression and skipping of exon 22 associated with impaired mono-ubiquitination of the FANCD2 protein as a consequence of PHF5A and SF3B1 knockdown. Furthermore, expression of RAD51, a key component of double strand break repair, also decreased upon PHF5A and SF3B1 knockdown. Notably, in vitro pharmacological inhibition of SF3b complex activity using H3B-8800 (or Pladienolide B) showed a very similar effect on FANCD2 expression, and ubiquitination as well as decrease of RAD51 and an increase in y-H2AX levels on a dose and time-dependent manner. This strongly suggests a mechanistic link between impaired RNA splicing and the repair of DNA double-strand breaks.
These combined data show that leukemic cells are highly dependent on a functional SF3b splicing complex. Interference with its function results in DNA damage and also sensitizes towards DNA damaging agents pointing towards a possible benefit of the combined application of inhibitors targeting the SF3b complex with more conventional chemotherapy.
Disclosures
Ponthan: Epistem Ltd: Employment. Zwaan:Sanofi: Consultancy; Incyte: Consultancy; BMS: Research Funding; Roche: Consultancy; Janssen: Consultancy; Daiichi Sankyo: Consultancy; Servier: Consultancy; Jazz Pharmaceuticals: Other: Travel support; Pfizer: Research Funding; Celgene: Consultancy, Research Funding. Vormoor:Abbvie (uncompensated): Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria, Research Funding; AstraZeneca: Research Funding.
RankerGUI: A Computational Framework to Compare Differential Gene Expression Profiles Using Rank Based Statistics
