Modulation of splicing catalysis for therapeutic targeting of leukemia with mutations in genes encoding spliceosomal proteins

Mutations in genes encoding splicing factors (which we refer to as spliceosomal genes) are commonly found in patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML)(1-3). These mutations recurrently affect specific amino acid residues, leading to perturbed normal splice site and exon recognition(4-6). Spliceosomal gene mutations are always heterozygous and rarely occur together with one another, suggesting that cells may tolerate only a partial deviation from normal splicing activity. To test this hypothesis, we engineered mice to express a mutated allele of serine/arginine-rich splicing factor 2 (Srsf2(P95H))-which commonly occurs in individuals with MDS and AML-in an inducible, hemizygous manner in hematopoietic cells. These mice rapidly succumbed to fatal bone marrow failure, demonstrating that Srsf2-mutated cells depend on the wild-type Srsf2 allele for survival. In the context of leukemia, treatment with the spliceosome inhibitor E7107 (refs. 7,8) resulted in substantial reductions in leukemic burden, specifically in isogenic mouse leukemias and patient-derived xenograft AMLs carrying spliceosomal mutations. Whereas E7107 treatment of mice resulted in widespread intron retention and cassette exon skipping in leukemic cells regardless of Srsf2 genotype, the magnitude of splicing inhibition following E7107 treatment was greater in Srsf2-mutated than in Srsf2-wild-type leukemia, consistent with the differential effect of E7107 on survival. Collectively, these data provide genetic and pharmacologic evidence that leukemias with spliceosomal gene mutations are preferentially susceptible to additional splicing perturbations in vivo as compared to leukemias without such mutations. Modulation of spliceosome function may thus provide a new therapeutic avenue in genetically defined subsets of individuals with MDS or AMLclos

Intronic cleavage and polyadenylation regulates gene expression during DNA damage response through U1 snRNA

The DNA damage response involves coordinated control of gene expression and DNA repair. Using deep sequencing, we found widespread changes of alternative cleavage and polyadenylation site usage on ultraviolet-treatment in mammalian cells. Alternative cleavage and polyadenylation regulation in the 3ʹ untranslated region is substantial, leading to both shortening and lengthening of 3ʹ untranslated regions of genes. Interestingly, a strong activation of intronic alternative cleavage and polyadenylation sites is detected, resulting in widespread expression of truncated transcripts. Intronic alternative cleavage and polyadenylation events are biased to the 5ʹ end of genes and affect gene groups with important functions in DNA damage response and cancer. Moreover, intronic alternative cleavage and polyadenylation site activation during DNA damage response correlates with a decrease in U1 snRNA levels, and is reversible by U1 snRNA overexpression. Importantly, U1 snRNA overexpression mitigates ultraviolet-induced apoptosis. Together, these data reveal a significant gene regulatory scheme in DNA damage response where U1 snRNA impacts gene expression via the U1-alternative cleavage and polyadenylation axis

Molecular basis of differential 3' splice site sensitivity to anti-tumor drugs targeting U2 snRNP

Several splicing-modulating compounds, including Sudemycins and Spliceostatin A, display anti-tumor properties. Combining transcriptome, bioinformatic and mutagenesis analyses, we delineate sequence determinants of the differential sensitivity of 3' splice sites to these drugs. Sequences 5' from the branch point (BP) region strongly influence drug sensitivity, with additional functional BPs reducing, and BP-like sequences allowing, drug responses. Drug-induced retained introns are typically shorter, displaying higher GC content and weaker polypyrimidine-tracts and BPs. Drug-induced exon skipping preferentially affects shorter alternatively spliced regions with weaker BPs. Remarkably, structurally similar drugs display both common and differential effects on splicing regulation, SSA generally displaying stronger effects on intron retention, and Sudemycins more acute effects on exon skipping. Collectively, our results illustrate how splicing modulation is exquisitely sensitive to the sequence context of 3' splice sites and to small structural differences between drugs

H3B-8800, an orally available small-molecule splicing modulator, induces lethality in spliceosome-mutant cancers

Genomic analyses of cancer have identified recurrent point mutations in the RNA splicing factor-encoding genes SF3B1, U2AF1, and SRSF2 that confer an alteration of function(1-6). Cancer cells bearing these mutations are preferentially dependent on wild-type (WT) spliceosome function(7-11), but clinically relevant means to therapeutically target the spliceosome do not currently exist. Here we describe an orally available modulator of the SF3b complex, H3B-8800, which potently and preferentially kills spliceosome-mutant epithelial and hematologic tumor cells. These killing effects of H3B-8800 are due to its direct interaction with the SF3b complex, as evidenced by loss of H3B-8800 activity in drug-resistant cells bearing mutations in genes encoding SF3b components. Although H3B-8800 modulates WT and mutant spliceosome activity, the preferential killing of spliceosome-mutant cells is due to retention of short, GC-rich introns, which are enriched for genes encoding spliceosome components. These data demonstrate the therapeutic potential of splicing modulation in spliceosome-mutant cancers