Preclinical Activity of Splicing Modulators in U2AF1 Mutant MDS/AML

Mutations in spliceosome genes are detectable in ~50% of patients with myelodysplastic syndromes (MDS), making this cellular pathway the most commonly mutated in MDS and providing a novel target for therapeutic intervention. Spliceosome gene mutations are mutually exclusive, implying they are either redundant in pathogenic function or are not tolerated in a cell when they co-occur. Our group and others identified recurrent heterozygous missense mutations in the splicing factor gene U2AF1 in 11% of MDS patient samples. The most common U2AF1 mutation results in a conversion of serine to phenylalanine at position 34 (S34F) of the U2AF1 protein. We previously reported that expression of mutant U2AF1(S34F) in vivo using doxycycline-inducible U2AF1(S34F) transgenic mice revealed an expansion of hematopoietic bone marrow progenitor cells and leukopenia following transgene induction; both phenotypes are seen in patients with MDS. We also identified mutant U2AF1-specific alterations in pre-mRNA splicing in transgenic mouse bone marrow progenitor cells, primary AML patient samples, and CD34+ cells by RNA sequencing. We hypothesize that cells harboring spliceosome gene mutations have increased sensitivity to pharmacological perturbation of the spliceosome by splicing modulator drugs, providing a new treatment approach for patients with U2AF1 mutations. For our studies, we utilize sudemycins, which are compounds that bind the SF3B1 spliceosome protein and modulate pre-mRNA splicing in non-hematopoietic tissues. We examined the effects of sudemycin treatment on pre-mRNA splicing in primary hematopoietic cells by treating CD34+ cells isolated from human umbilical cord blood with sudemycin in vitro. We performed whole transcriptome (RNA-seq) analysis following 6 hours of sudemycin treatment (1µM) of CD34+ cells and identified robustly altered pre-mRNA splicing patterns that are sudemycin-specific (26,120 splice junctions by DEXSeq, FDR<0.05, n=3), thereby validating that splicing is altered in hematopoietic cells treated with sudemycin. We confirmed a subset of these altered pre-mRNA splicing changes by RT-PCR and gel electrophoresis, as well as by Nanostring assay of RNA. We performed in vitro studies to examine the sensitivity of cells expressing U2AF1(S34F) to sudemycin treatment. Primary human MDS/AML cells with U2AF1(S34F) mutations display increased sensitivity to sudemycin, compared to non-mutant controls in a cell cycle (EdU incorporation) assay (n=3), while treatment with daunorubicin showed no specificity for mutant U2AF1(S34F) samples compared to non-mutant controls. Primary mouse c-Kit+ bone marrow cells transduced with a retrovirus expressing U2AF1(S34F) display a marked increase in apoptosis (by flow cytometry for Annexin V+ staining) in response to increasing concentrations of sudemycin, compared to controls (p<0.001, n=3-5). In addition, in vivo treatment of U2AF1(S34F) transgenic mice with sudemycin resulted in attenuation of hematopoietic progenitor cell expansion by colony forming unit (CFU-C) assay (p<0.01, n=6-11) and by flow cytometry for lineage-, c-Kit+, Sca-1+ (KLS) cells (p<0.001, n=6-11). Ongoing studies are examining the splicing alterations in U2AF1 mutant and wild-type transgenic mouse bone marrow cells treated with vehicle versus sudemycin. Taken together, these data suggest that we may be able to specifically treat hematological cancers with U2AF1 mutations using small molecule pre-mRNA splicing modulators such as sudemycin.