Splicing factor mutations in hematologic malignancies

Abstract

Mutations in genes encoding RNA splicing factors were discovered nearly 10 years ago and are now understood to be among the most recurrent genetic abnormalities in patients with all forms of myeloid neoplasms and several types of lymphoproliferative disorders, as well as subjects with clonal hematopoiesis. These discoveries implicate aberrant RNA splicing, the process by which precursor RNA is converted into mature messenger RNA, in the development of clonal hematopoietic conditions. Both the protein and the RNA components of the splicing machinery are affected by mutations at highly specific residues, and a number of these mutations alter splicing in a manner distinct from loss of function. Importantly, cells bearing these mutations have now been shown to generate mRNA species with novel aberrant sequences, some of which may be critical to disease pathogenesis and/or novel targets for therapy. These findings have opened new avenues of research to understand biological pathways disrupted by altered splicing. In parallel, multiple studies have revealed that cells bearing change-of-function mutation in splicing factors are preferentially sensitized to any further genetic or chemical perturbations of the splicing machinery. These discoveries are now being pursued in several early-phase clinical trials using molecules with diverse mechanisms of action. Here, we review the molecular effects of splicing factor mutations on splicing, the mechanisms by which these mutations drive clonal transformation of hematopoietic cells, and the development of new therapeutics targeting these genetic subsets of hematopoietic malignancies.

Graphical abstract

Figure 1.

RNA splicing catalysis, splicing regulation, and location of splicing factors mutated in hematologic malignancies in the splicing process. (A) Sequences embedded within premature RNA serve to recruit the spliceosome and include the 5' and 3' splice sites (which are most commonly GU and AG dinucleotides, respectively), the BPS, and polypyrimidine (poly Y) tract. The branch-point nucleotide is most commonly an adenosine nucleotide as shown, but other nucleotides can occasionally serve as branch points, and it is not uncommon for introns to have multiple branch points. (B) Although splicing requires several hundred proteins, the core steps of splicing catalysis consist of 2 sequential transesterification reactions as shown. RNA splicing is initiated when the branch nucleotide performs a nucleophilic attack of the 5'ss, resulting in the formation of an intron lariat intermediate with a 2', 5'-phosphodiester linkage. This is followed by a 5'ss-mediated attack on the 3'ss, leading to the removal of the intron lariat and the formation of the spliced RNA product. (C) The enzymatic steps of splicing are carried out by groups of proteins complexed with snRNAs termed snRNPs. Factors labeled in red in this diagram under recurrent mutations in patients with hematologic malignancies. Splicing is initiated with binding of the U1 snRNP binds the 5'ss, SF1 to the BPS, (iii) U2AF2 to the polypyrimidine tract, and (iv) U2AF1 to the 3ss. These interactions enhance recruitment of U2 snRNP to the BPS. SF3B1, a component of U2 snRNP, is involved in the binding to the BPS. The preassembled U4/U6.U5 tri-snRNP complex joins and the U1/U4 snRNPs are released to form a catalytically active complex of the spliceosome, which catalyze the first and second esterification reactions, respectively, and mediate excision of the intron and ligation of the proximal and distal exon to synthesize mature mRNA. (D) Beyond splice sites, BPS, and the poly Y tract, additional sequences located within introns and exons serve to recruit auxiliary splicing factors, that interact with the spliceosome and promote or repress spliceosome function. These are termed ESEs or exonic splicing silencers (ESSs), respectively) or intronic splicing enhancers or silencers (ISEs or ISSs, respectively). Splicing regulatory proteins known as SR or hnRNP proteins most commonly enhance or repress spliceosome recruitment, respectively, as illustrated.

Figure 2.

Frequency, genomic characteristics, and effects on splicing of RNA splicing factor mutations seen in hematologic malignancies. (A) Histogram of mutations in the most commonly mutated RNA splicing factors across hematologic malignancies. AML-MRC, acute myeloid leukemia with myelodysplasia-related changes; BPDCN, blastic plasmacytoid dendritic cell neoplasm; CLL: chronic lymphocytic leukemia; CMML, chronic myelomonocytic leukemia; RARS, refractory anemia with ring sideroblasts; RCMD-RS, refractory cytopenia with multilineage dysplasia and ring sideroblasts. (B) Location and relative frequency of mutations in SF3B1 and U2AF1 in myeloid neoplasms, CLL, and solid tumors. (C) Location and relative frequency of mutations in SRSF2 with indication of exact amino acid substitutions at mutated residues. (D) Location and relative frequency of mutations in ZRSR2 displaying known frameshift and insertion/deletion mutations only. (E) Location of recurrent mutations in the gene (or genes) encoding the U1 snRNA affect its third nucleotide, which makes critical base pairs with the 5' splice site. (F) The most frequent mutations in HNRNPH1 cluster in the introns surrounding exon 4 and promote inclusion of this exon. HNRNPH1 mutations in this region occur entirely as single-nucleotide point mutations (as indicated by the brown lollipops). Repression of exon 4 induces a NMD-inducing isoform of HNRNPH1, while inclusion of exon 4 promotes stable HNRNPH1 expression. WT, wild-type.

Figure 3.

Effect of splicing factor mutations on biological processes beyond RNA splicing. (A) The positive transcription elongation factor complex, P-TEFb, composed of cyclin-dependent kinase 9 (CDK9) and cyclin T1, stimulates synthesis of RNA through phosphorylation of RNA polymerase II (Pol II). However, when bound to 7SK snRNA, HEXIM,1 LARP7, and MePCE (7SK snRNA methyl phosphate capping enzyme), P-TEFb is catalytically inactive and represses transcription by suppressing the release of paused polymerase II. The transition of P-TEFb from repressive to active complexes depends on multiple factors including SRSF2. Mutant SRSF2, however, loses its ability to extract P-TEFb from the 7SK complex due to increased RNA binding. This results in elevated R-loops (nascent RNA-DNA hybrids formed during transcription leaving the nontemplate DNA strand looping out) and subsequent replication stress and activation of the ataxia telangiectasia and Rad3-related protein (ATR)-Chk1 pathway. (B) Mutations at proline 95 residue in SRSF2 change its RNA-binding affinity from G-rich (GGWG) to C-rich (C/GCWG) motifs (W = A/U) inducing transcriptome-wide missplicing events. Several mRNA isoforms promoted by SRSF2 mutants harbor a PTC and are therefore potential targets of NMD. Moreover, SRSF2 mutants further enhance NMD by promoting recruitment of EJC factors (eIF4A3, MAGOH, and Y14) to mRNAs downstream of PTCs within the nucleus. This subsequently enhances the association of several NMD factors (UPF3B, UPF2, and UPF1) to mRNA within the cytoplasm, thereby enhancing mRNA decay.

Figure 4.

Approaches to targeting RNA splicing. (A) SF3b binding agents physically interact with the branch-point binding pocket of SF3B1, thus blocking its binding with the branch point (i). Specific mutant residues in SF3B1 and PHF5A confer drug resistance to SF3b-binding agents (ii). (B) RBM39 degraders link the E3 ubiquitin ligase complex to RBM39 through the adaptor protein DCAF15, leading to polyubiquitination and subsequent proteosomal degradation of RBM39. Splicing factor–mutant leukemic cells are preferentially sensitive to RBM39 degradation. (C) PRMT5 inhibitors inhibiting PRMT5-mediated symmetric demethylation of arginines (SDMA) on Sm (D1, B/B, D3) proteins, which is required for spliceosome assembly (i). PRMT1 mediates asymmetric demethylation of arginines (ADMA) on RMB15, an RNA-binding protein regulating RNA splicing, among many additional splicing factors. Methylated RBM15 is targeted for polyubiquitination and proteosomal degradation, leading to aberrant splicing (ii). Type 1 PRMT inhibitors may prevent mis-splicing through dampening RBM15 degradation (ii). (D) Elevated R-loop formation in mutant splicing factor cells results in activation of ATR signaling pathway and DNA-damage response. Leukemic cells harboring splicing factor mutations preferentially respond to ATR inhibition. (E) ASOs complementary to the poison exon of Brd9 correct aberrant inclusion of the poison exon (i). ASOs block EJC deposition site on mRNA and prevent recruitment of the EJC downstream of a PTC, thereby preventing NMD induced by splicing factor mutations (ii).