The myelodysplastic syndromes (MDS) are common myeloid malignancies. Mutations in genes involved in pre-mRNA splicing (SF3B1, SRSF2, U2AF1 and ZRSR2) are the most common mutations found in MDS. There is evidence that some spliceosomal components play a role in the maintenance of genomic stability. Splicing is a transcription coupled process; splicing factor mutations affect transcription and may lead to the accumulation of R-loops (RNA-DNA hybrids with a displaced single stranded DNA). Mutations in the splicing factors SRSF2 and U2AF1 have been recently shown to increase R-loops formation in leukemia cell lines, resulting in increased DNA damage, replication stress and activation of the ATR-Chk1 pathway. SF3B1 is the most frequently mutated splicing factor gene in MDS, but a role for mutated SF3B1 in R-loop accumulation and DNA damage has not yet been reported in hematopoietic cells. We have investigated the effects of the common SF3B1 K700E mutation on R-loop formation and DNA damage response in MDS and leukemia cells.
R-loop signals and the DNA damage response were measured by immunofluorescence staining using S9.6 and anti-γ-H2AX antibodies respectively. Firstly, we studied K562 (myeloid leukemia) cells with the SF3B1 K700E mutation and isogenic SF3B1 K700K wildtype (WT) K562 cells. K562 cells with SF3B1 mutation showed a significant increase in the number of S9.6 foci [Fold change (FC) 2.01, p<0.001] and in the number of γ-H2AX foci (FC 2.32, p<0.001), indicating increased R-loops and DNA damage, compared to SF3B1 WT K562 cells. Moreover, we observed increased Chk1 phosphorylation at Ser345, a hallmark of activation of the ATR pathway, in SF3B1 mutant K562 cells. Next, we analyzed induced pluripotent stem cells (iPSCs) that we generated from the bone marrow cells of one SF3B1 mutant MDS patient and of one healthy control. A significant increase in R-loops and DNA damage response was observed in an iPSC clone harboring SF3B1 mutation compared to another iPSC clone without SF3B1 mutation obtained from same MDS patient (S9.6 mean fluorescence intensity - FC 1.72, p<0.001; γ-H2AX foci - FC 1.34, p=0.052) and to iPSCs from the healthy control (S9.6 mean fluorescence intensity - FC 1.53, p<0.001; γ-H2AX foci - FC 1.61, p=0.006). In addition, bone marrow CD34+ cells from a SF3B1 mutant MDS patient showed increased R-loops (as measured by number of S9.6 foci) compared to CD34+ cells from a MDS patient without splicing factor mutations (FC 1.9) and from a healthy control (FC 2.6).
To investigate whether the observed DNA damage and ATR activation in SF3B1 mutant K562 cells result from induced R-loops, we overexpressed RNASEH1 (encoding an enzyme that degrades the RNA in RNA:DNA hybrids) to resolve R-loops in these cells. RNASEH1 overexpression significantly reduced the number of S9.6 (FC 0.51, p<0.001) and γ-H2AX foci (FC 0.63, p=0.035) in SF3B1 mutant K562 cells compared to SF3B1 WT K562 cells. RNASEH1 overexpression also resulted in decreased Chk1 phosphorylation, indicating suppression of ATR pathway activation in SF3B1 mutant K562 cells. To determine the functional importance of ATR activation associated with SF3B1 mutation, we evaluated the sensitivity of SF3B1 mutant cells towards the ATR inhibitor VE-821. SF3B1 mutant K562 cells showed preferential sensitivity towards VE-821 compared to SF3B1 WT K562 cells. Chk1 is a critical substrate of ATR, and we next investigated the effects of Chk1 inhibition in SF3B1 mutant cells. Interestingly, SF3B1 mutant K562 cells demonstrated preferential sensitivity towards the Chk1 inhibitor UCN-1 (IC50 61.8 nM) compared to SF3B1 WT K562 cells (IC50 267 nM), suggesting that ATR activation is important for the survival of SF3B1 mutant cells. SF3B1 mutant K562 cells were preferentially sensitive to the splicing modulator Sudemycin D6 (IC50 53.2 nM) compared to SF3B1 WT K562 cells (IC50 130.7 nM). The effects of VE-821 and UCN-1 on SF3B1 mutant K562 cells were enhanced by Sudemycin D6 (Combination index <1), indicating synergy.
In summary, our results show that the SF3B1 mutation leads to accumulation of R-loops and associated DNA damage resulting in activation of the ATR pathway in MDS and leukemia cells. Thus different mutated splicing factors have convergent effects on R-loop elevation leading to DNA damage. Moreover, our data suggest that Chk1 inhibition, alone or in combination with splicing modulators, may represent a novel therapeutic strategy to target SF3B1 mutant cells.
Schuh:Janssen: Speakers Bureau; Verastem: Speakers Bureau; Kite: Speakers Bureau; Gilead: Speakers Bureau; Seattle Genetics: Speakers Bureau; Jazz Pharmaceuticals: Speakers Bureau; Bristol-Myers Squibb: Research Funding; AbbVie: Consultancy, Speakers Bureau; Genentech: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy, Speakers Bureau. Wiseman:Novartis, Celgene: Consultancy, Honoraria.
Asterisk with author names denotes non-ASH members.