Abstract
Background: Mutations in splicing factor gene SRSF2 are recurrent drivers in 5-15 % of patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). A key property of SRSF2 mutations is that they occur early in the pathogenesis of MDS and are therefore present in all tumor cells in a patient. This property makes targetable vulnerabilities caused by SRSF2 mutations exceptionally important as they provide a way to inhibit the whole tumor. We previously demonstrated that splicing factor mutations induce R-loop-dependent activation of ATR, rendering cells sensitive to ATR inhibition. R-loops are transcription intermediates consisting of an RNA:DNA hybrid and a displaced single-stranded DNA. Accumulation of aberrant R-loops induces the ATR kinase, which activates the G2-M cell cycle checkpoint via CHK1- and WEE1-mediated signaling. In normal cells, activation of the G2-M checkpoint halts the cell cycle until R-loops have been resolved. When the ATR pathway is inhibited, checkpoint activation does not occur, causing cells with unresolved R-loops to proceed to mitosis, resulting in DNA damage and cell death. We, therefore, sought to assess primary human MDS/AML samples for sensitivity to perturbation of the ATR/CHK1/WEE1 pathway and identify mechanisms of resistance.
Methods: Sensitivity of 147 AML patient samples to 515 oncology drugs was tested ex vivo. Bone marrow mononuclear cells were incubated with 5 concentrations of each drug for 72 h followed by measurement of cell viability by CTG assay. Somatic mutations were identified by exome sequencing of matched leukemic bone marrow and skin biopsy samples.
Isogenic K562 cell line clones carrying SRSF2 P95H/L/R mutations were generated using CRISPR/Cas9 editing. The presence of SRSF2 mutation was confirmed by CRISPR-sequencing and expression by whole transcriptome RNA-sequencing. Drug sensitivity of the K562 clones with and without SRSF2 mutation was determined by incubating cells with 16 concentrations of prexasertib, SRA-737, adavosertib, or BAY-1895344, followed by determination of cell viability by the MTS assay.
Results: Analysis of ex vivo drug sensitivities in AML patient samples identified vulnerability to CHK1 and WEE1 inhibition in SRSF2-mutated AML: SRSF2 mutation is associated with sensitivity to the CHK1 inhibitors prexasertib (p = 0.006) (Fig 1 A and B) and PF-00477736 (p = 0.002) and the WEE1 inhibitor adavosertib (p = 0.003).
To establish whether the isogenic SRSF2-mutant K562 cell line models recapitulate known downstream aberrations associated with SRSF2 mutations in patients, we analyzed gene expression and splicing. SRSF2 contains an RNA binding domain with affinity to CCNG or GGNG exonic splicing enhancer sequences. Similar to what has been observed in patients, the K562 clones with SRSF2 mutation show reduced use of GGNG sequence motifs at skipped exons. These results demonstrate that isogenic K562 clones recapitulate known alterations caused by mutant SRSF2.
To determine whether SRSF2 mutations induce sensitivity to inhibition of ATR, CHK1, and WEE1, we tested 10 isogenic SRSF2 mutant and 4 wild-type K562 clones. Cells with SRSF2 mutation show increased sensitivity to ATR/CHK1/WEE1 inhibition (Fig 1C). We found no significant difference in drug sensitivity between clones carrying SRSF2 P95H/L/R substitutions. Clones with higher SRSF2 mutant allele dosage are more sensitive (Fig 1D).
We identified a subset of SRSF2 mutated AML samples that were resistant to CHK1 and WEE1 inhibition. All resistant AML have co-occurring RUNX1 mutations (Fig 1B). In AML, RUNX1 mutations are associated with therapy resistance, suggesting that these mutations contribute to drug resistance. To test whether RUNX1 mutations induce resistance to ATR/CHK1/WEE1 inhibition in SRSF2-mutant leukemia, we introduced RUNX1 loss-of-function mutations in isogenic K562 carrying SRSF2 mutations. Candidate resistance factors identified by ATAC and RNA-sequencing will be validated in functional assays.
Conclusions: Our results indicate that SRSF2-mutated leukemia harbor a vulnerability to the inhibition of ATR, CHK1, and WEE1 kinases. Cell line models indicate that sensitivity is similar across mutant alleles and dependent on allelic copy number. Several ATR/CHK1 and WEE1 inhibitors are in development, and our results suggest that these compounds could be effective treatments for SRSF2-mutated MDS and AML.
Figure 1 Figure 1.
Disclosures
Graubert: astrazeneca: Research Funding; Janssen: Research Funding; Calico: Research Funding.
Anticancer and Immunomodulatory Activities of a Novel Water-Soluble Derivative of Ellipticine
