Chk1 Inhibition Shows Efficacy for Splicing Factor Mutant MDS and Leukemia

A new study found Chk1 inhibition with or without splicing modulators may be an effective targeted treatment approach for splicing factor mutant myelodysplastic syndromes (MDS) and leukemia cells. When DNA is replicated in a cell, the two DNA-strands are separated and pre-mRNA binds to the template DNA while the other DNA strand, the so-called nontemplate DNA, remains free in a loop-like structure called R-loop. Thus, the R-loop consists of a 3-stranded nucleic acid structure formed by RNA-DNA hybrids and the displaced nontemplate single stranded DNA. The study investigators found that Chk1 inhibition exploits the R-loop-associated vulnerability induced by SF3B1 mutations in MDS and other types of cancers and could potentially be used as a therapeutic strategy in other cancer types with this mutation.

As lead author Shalini Singh, PhD, and colleagues from the University of Oxford explained in the paper, the most common mutated genes found in MDS that are associated with pre-mRNA splicing include SF3B1, SRSF2, U2AF1, and ZRSR2. Mutations in the splicing factor genes SRSF2 and U2AF1 have been shown to result in increased R-loops. With this analysis, the investigators examined the impact of SF3B1 mutations on R-loop formation in MDS and leukemia cells, as well as the impact of SF3B1 mutations on associated DNA damage response in MDS and leukemia cells.

When the researchers looked at cells from 3 patients with MDS and an SF3B1 mutation, 3 patients with splicing factor wild-type MDS, and 3 healthy controls, they observed a 2.4-fold increase in R-loops in CD34+ cells from patients with MDS who harbored the mutation, compared with the same cells from patients with MDS but without splicing factor mutations. In addition, a 2.6-fold increase in R-loops were observed in cells of patients with an SF3B1 mutation versus healthy controls.

Another goal of the study was to test the impact of SF3B1 mutations on the DNA damage response, which the researchers measured via immunofluorescence staining. K562 cells with the SF3B1K700E mutation displayed increased DNA damage compared with the nonmutated control cells and were associated with increased R-loop levels. This DNA damage was also seen in bone marrow cells from MDS patients with SF3B1-mutations compared with cells from the same MDS patient without SF3B1 mutation.

Further, the investigators examined two important signaling pathways frequently activated after DNA damage, namely the ATR and ATM pathways. ATR signaling was activated in SF3B1K700E mutated cells while ATM wasn’t. ATR activation results in Chk1 phosphorylation. When the researchers suppressed R-loop formation by RNaseH1 overexpression, Chk1 phosphorylation was reduced, indicating suppression of ATR activation in SF3B1 mutated cells once R-loop formation was decreased. ATR activation could also be reversed using the ATR inhibitor VE-821.

The study investigators then examined the effects of the inhibition of Chk1 in SF3B1K700E K562 cells. Using the Chk1 inhibitor UCN-01, the authors found that SF3B1K700E K562 showed preferential sensitivity to UCN-01 when compared with unmutated K562 cells. According to the authors, these findings suggest that the survival of SF3B1 mutant cells partly relies on the activation of ATR-Chk1 pathway.

Next, a splicing modulator, sudemycin D6, was tested in combination with ATR or Chk1 inhibitors (VE-821 and UCN-01). Compared with isogenic SF3B1K700K K562 cells, SF3B1K700E K562 cells demonstrated preferential sensitivity to sudemycin D6. In addition, CD34+ cells from patients with MDS and SF3B1 mutation were preferentially sensitive to sudemycin D6, compared with CD34+ cells from healthy controls and patients with MDS but without mutations. The addition of sudemycin D6 enhanced the effects of VE-821 and UCN-01 on bone marrow CD34+ cells from patients with SF3B1-mutant MDS patients and on SF3B1 mutant K562 cells.

“We found that the sensitivity of SF3B1 mutant primary MDS patient bone marrow cells and SF3B1 mutant K562 cells to the splicing modulator sudemycin D6 was enhanced by treatment with the Chk1 inhibitor UCN-01, indicative of synergy between these drugs,” study corresponding authors Jacqueline Boultwood, PhD, and Andrea Pellagatti, PhD, told ASH Clinical News. In addition, Dr. Pellagatti noted that this finding has translational implications and warrants further preclinical studies.

Drs. Boultwood and Pellagatti also suggested Chk1 inhibition with or without splicing modulators could potentially be extended to therapeutically target other types of cancers with SF3B1 mutations, including chronic lymphocytic leukemia, uveal melanoma, breast cancer, and pancreatic cancer. However, Dr. Boultwood noted that “it would first be necessary to demonstrate that the presence of SF3B1 mutations in these other cancers results in increased R-loops and DNA damage and increased phosphorylation of Chk1.”

According to Dr. Pellagatti, future studies are needed to investigate the efficacy of spliceosome inhibitors plus Chk1 inhibitors in SF3B1 mutant, MDS-derived xenograft models. “It would also be interesting to compare R-loop levels in the CD34+ cells of SF3B1-mutant MDS cases with those in CD34+ cells of patients with MDS and mutations of other splicing factors, including SRSF2, U2AF1, and ZRSR2,” concluded Dr. Pellagatti.

The authors report no relevant conflicts of interest.

Reference

Singh S, Ahmed D, Dolatshad H, et al. SF3B1 mutations induce R-loop accumulation and DNA damage in MDS and leukemia cells with therapeutic implications. Leukemia. 2020 February 19. [Epub ahead of print]