Recurrent Spliceosome Mutations in Cancer: Mechanisms and Consequences of Aberrant Splice Site Selection

Splicing alterations have been widely documented in tumors where the proliferation and dissemination of cancer cells is supported by the expression of aberrant isoform variants. Splicing is catalyzed by the spliceosome, a ribonucleoprotein complex that orchestrates the complex process of intron removal and exon ligation. In recent years, recurrent hotspot mutations in the spliceosome components U1 snRNA, SF3B1, and U2AF1 have been identified across different tumor types. Such mutations in principle are highly detrimental for cells as all three spliceosome components are crucial for accurate splice site selection: the U1 snRNA is essential for 3′ splice site recognition, and SF3B1 and U2AF1 are important for 5′ splice site selection. Nonetheless, they appear to be selected to promote specific types of cancers. Here, we review the current molecular understanding of these mutations in cancer, focusing on how they influence splice site selection and impact on cancer development.

Proteogenomic discovery of neoantigens facilitates personalized multi-antigen targeted T cell immunotherapy for brain tumors

Neoantigen discovery in pediatric brain tumors is hampered by their low mutational burden and scant tissue availability. Here we develop a proteogenomic approach combining tumor DNA/RNA sequencing and mass spectrometry proteomics to identify tumor-restricted (neoantigen) peptides arising from multiple genomic aberrations to generate a highly target-specific, autologous, personalized T cell immunotherapy. Our data indicate that aberrant splice junctions are the primary source of neoantigens in medulloblastoma, a common pediatric brain tumor. Proteogenomically identified tumor-specific peptides are immunogenic and generate MHC II-based T cell responses. Moreover, polyclonal and polyfunctional T cells specific for tumor-specific peptides effectively eliminate tumor cells in vitro. Targeting tumor-specific antigens obviates the issue of central immune tolerance while potentially providing a safety margin favoring combination with other immune-activating therapies. These findings demonstrate the proteogenomic discovery of immunogenic tumor-specific peptides and lay the groundwork for personalized targeted T cell therapies for children with brain tumors.

SARS-CoV-2 vaccine ChAdOx1 nCoV-19 infection of human cell lines reveals low levels of viral backbone gene transcription alongside very high levels of SARS-CoV-2 S glycoprotein gene transcription

Background ChAdOx1 nCoV-19 is a recombinant adenovirus vaccine against SARS-CoV-2 that has passed phase III clinical trials and is now in use across the globe. Although replication-defective in normal cells, 28 kbp of adenovirus genes is delivered to the cell nucleus alongside the SARS-CoV-2 S glycoprotein gene. Methods We used direct RNA sequencing to analyse transcript expression from the ChAdOx1 nCoV-19 genome in human MRC-5 and A549 cell lines that are non-permissive for vector replication alongside the replication permissive cell line, HEK293. In addition, we used quantitative proteomics to study over time the proteome and phosphoproteome of A549 and MRC5 cells infected with the ChAdOx1 nCoV-19 vaccine. Results The expected SARS-CoV-2 S coding transcript dominated in all cell lines. We also detected rare S transcripts with aberrant splice patterns or polyadenylation site usage. Adenovirus vector transcripts were almost absent in MRC-5 cells, but in A549 cells, there was a broader repertoire of adenoviral gene expression at very low levels. Proteomically, in addition to S glycoprotein, we detected multiple adenovirus proteins in A549 cells compared to just one in MRC5 cells. Conclusions Overall, the ChAdOx1 nCoV-19 vaccine’s transcriptomic and proteomic repertoire in cell culture is as expected. The combined transcriptomic and proteomics approaches provide a detailed insight into the behaviour of this important class of vaccine using state-of-the-art techniques and illustrate the potential of this technique to inform future viral vaccine vector design.

Only Hotspot SF3B1 K700E Mutation Is an Independent Predictor of Overall Survival in Myelodysplastic Syndromes

SF3B1 mutated (SF3B1mut) MDS is associated with increased ring sideroblasts (RS) and favorable outcome. IWG-PM proposed that SF3B1mut MDS be considered a distinct entity in the absence of other unfavorable features. However, not all SF3B1mut MDS have similar clinical course. Hotspot K700E SF3B1mut leads to aberrant splice junctions and large-scale mRNA downregulation by activating a cryptic splice site. The functional effects of SF3B1mut outside of K700E is not clear. The outcome can also be altered by concomitant gene mutations and karyotype. In this study, we studied the clinical-pathologic features and outcome in a single-institutional series of 94 SF3B1mut and 415 SF3B1wt MDS, and explored the differences between K700E and non-K700E subgroups. All untreated MDS patients diagnosed over a 3-year duration who underwent NGS were selected. Overall survival was calculated from diagnosis to death/ last follow-up. Univariate (UVA) Cox proportional hazards regression was used to identify any association of variables with outcome followed by multivariate modeling (MVA) (p-value 0.200 cutoff). Of 509 patients, 94 (19%) had SF3B1mut: 59 men, 35 women; median age: 74 (39-92) years. Baseline characteristics: Table 1. Compared to SF3B1wt, SF3B1mut had a significantly higher median age (74 vs. 70, p=0.0008), MCV (105 vs. 96, p<0.0001), platelet count (188 vs. 78, p<0.0001) and lower BM blasts (2 vs. 4, p=0.003). SF3B1mut were less frequently therapy-related (18% vs. 34%, p=0.002), significantly enriched in R-IPSS VL/L and WHO MDS-RS and MDS with iso del(5q). Majority (~66%) had concomitant mutations: TET2 (25%), DNMT3A (21%), RUNX1 (15%), TP53 (10%), ASXL1 (7%), BCOR (4%), IDH1/2 (4%), SRSF2 (3%), RAS (3%) and EZH2 (3%) (Fig 1B). ~10% showed complex karyotype (CK). Among SF3B1mut, hotspot K700E mutation was seen in 55 (~60%). Non K700E mutations (n=39, 40%) frequently involved codons: H662, K666 and R625, seen in 8 patients each (Fig 1A). SF3B1 K700E showed a higher median RS% (50% vs. 34%; p=0.038), ANC (2.4 vs. 1.8, p=0.005) and a trend for higher platelet (196 vs. 124, p=0.05). SF3B1mut were less likely MDS-EB than non-K700E (22% vs. 49%, p=0.008). All 4 SF3B1mut patients that fit WHO criteria for MDS with isolated del(5q) had K700E (Table). The frequency of RUNX1 mutation was significantly higher in non-K700E cases (26% vs. 7.3%, p=0.012); mutations in BCOR (p=0.02), IDH2 (p=0.07) and SRSF2 (p=0.07) were exclusively noted in non-K700E cases (Fig 1C). There was no significant difference in TP53mut or CHIP-associated mutations DNMT3A, ASXL1 and TET2 or SF3B1 VAF. There was no significant differences in diploid vs. CK. However, K700E had lower CCCS categories (0/1, n=39; 2/3/4, n=10 vs. 0/1, n=19; 2/3/4, n=17); p=0.011). Majority were treated with HMA [16/17 (94%) K700E; 15/19 (79%) non-K700, 217/ 277 (78%) SF3B1wt]. SF3B1mut had better OS than SF3B1wt in all MDS (NR vs. 25.2 months, p=0.0003; fig 1D), low-grade MDS (NR vs. 41.3 months, p=0.0015; fig 1E) and MDS-RS (NR vs. 22.3 months, p=0.0004; fig 1F). Four (7.3%) K700E died compared to 9 (23%; p=0.036) non-K700E. The outcome of non-K700E was similar to SF3B1wt, in all MDS, low-grade MDS and MDS-RS (median OS, NR for both; p=0.021). By UVA, the following associated with worse outcome: higher BM blasts, lower hemoglobin, platelet and MCV, prior chemo-radiation, CK, higher R-IPSS, absence of mutations in SF3B1 K700E, TET2 and U2AF1 and presence of TP53mut. Non-K700E did not associate with OS. By MVA, lower hemoglobin, higher R-IPSS, absence of SF3B1 K700E and presence of TP53mut were independent predictors of worse OS. Within MDS-RS categories, independent prognostic factors of worse OS included lower platelet, presence of mutations in non-K700E SF3B1mut, ASXL1, SRSF2 and TP53. TP53mut/CK was seen in 10% SF3B1mut MDS. No survival differences were noted between SF3B1mut with or without TP53mut/CK (median OS, NR) and SF3B1wt without TP53mut/CK (44.3 months), but TP53mut/CK with SF3B1wt MDS had a worse outcome (median OS, 12.9 months, HR 1.46, p=0.001; fig 1G). Same findings were noted within low-grade MDS and MDS-RS, suggesting SF3B1mut negates the poor prognostic effect of TP53mut/CK. About 40% SF3B1mut MDS show non K700E mutations. K700E and non K700E SF3B1mut MDS show distinct clinical and mutational profiles, with K700E showing a significantly better OS compared to non K700E and SF3B1wt. Only SF3B1 K700E independently predicted for worse OS in MDS. Figure Disclosures Sasaki: Pfizer Japan: Consultancy; Daiichi Sankyo: Consultancy; Novartis: Consultancy, Research Funding; Otsuka: Honoraria. Jabbour:Genentech: Other: Advisory role, Research Funding; BMS: Other: Advisory role, Research Funding; Pfizer: Other: Advisory role, Research Funding; AbbVie: Other: Advisory role, Research Funding; Takeda: Other: Advisory role, Research Funding; Adaptive Biotechnologies: Other: Advisory role, Research Funding; Amgen: Other: Advisory role, Research Funding. Kantarjian:Amgen: Honoraria, Research Funding; Ascentage: Research Funding; BMS: Research Funding; Daiichi-Sankyo: Honoraria, Research Funding; Immunogen: Research Funding; Jazz: Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Sanofi: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive biotechnologies: Honoraria; Aptitute Health: Honoraria; BioAscend: Honoraria; Delta Fly: Honoraria; Janssen: Honoraria; Oxford Biomedical: Honoraria; Abbvie: Honoraria, Research Funding. Garcia-Manero:Astex Pharmaceuticals: Consultancy, Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Merck: Research Funding; Onconova: Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Helsinn Therapeutics: Consultancy, Honoraria, Research Funding; Acceleron Pharmaceuticals: Consultancy, Honoraria; H3 Biomedicine: Research Funding; Amphivena Therapeutics: Research Funding; Novartis: Research Funding; Jazz Pharmaceuticals: Consultancy.

SARS-CoV-2 candidate vaccine ChAdOx1 nCoV-19 infection of human cell lines reveals a normal low range of viral backbone gene expression alongside very high levels of SARS-CoV-2 S glycoprotein expression.

Background: ChAdOx1 nCoV-19 is a recombinant adenovirus vaccine candidate against SARS-CoV-2. Although replication defective in normal cells, 28kbp of adenovirus genes are delivered to the cell nucleus alongside the SARS-CoV-2 S glycoprotein gene.Methods: We used direct RNA sequencing to analyse transcript expression from the ChAdOx1 nCoV-19 genome in human MRC-5 and A549 cell lines that are non-permissive for vector replication alongside the replication permissive cell line, HEK293. In addition, we used quantitative proteomics to study over time the proteome and phosphoproteome of A549 and MRC5 cells infected with the ChAdOx1 nCoV-19 vaccine candidate.Results: The expected SARS-CoV-2 S coding transcript dominated in all cell lines. We also detected rare S transcripts with aberrant splice patterns or polyadenylation site usage. Adenovirus vector transcripts were almost absent in MRC-5 cells but in A549 cells there was a broader repertoire of adenoviral gene expression at very low levels. Proteomically, in addition to S glycoprotein, we detected multiple adenovirus proteins in A549 cells compared to just one in MRC5 cells. Conclusions: Overall the ChAdOx1 nCoV-19 vaccine’s transcriptomic and proteomic repertoire is as expected. The combined transcriptomic and proteomics approaches provide an unparalleled insight into the behaviour of this important class of vaccine candidate and illustrate the potential of this technique to inform future viral vaccine vector design.

RBMX enables productive RNA processing of ultra-long exons important for genome stability

Previously we showed that the germline-specific RNA binding protein RBMXL2 is essential for male meiosis where it represses cryptic splicing patterns (1). Here we find that its ubiquitously expressed paralog RBMX helps underpin human genome stability by preventing non-productive splicing. In particular, RBMX blocks selection of aberrant splice and polyadenylation sites within some ultra-long exons that would interfere with genes needed for normal replication fork activity. Target exons include within the ETAA1 (Ewings Tumour Associated 1) gene, where RBMX collaborates with its interaction partner Tra2β to enable full-length exon inclusion by blocking selection of an aberrant 3’ splice site. Our data reveal a novel group of RNA processing targets potently repressed by RBMX, and help explain why RBMX is associated with gene expression networks in cancer, replication and sensitivity to genotoxic drugs.

2'-O-Methoxyethyl Splice-Switching Oligos to Reverse Splicing from IVS2-745 β-Thalassemia Patient Cells: A Foundation for Potential Therapies

The β thalassemia trait is associated with over 300 mutations in the β-globin gene that lead to reduced (β+ allele) or absent (β0 allele) synthesis of the β globin chain. A subset of these mutations affect the canonic splicing of the β globin mRNA. Such mutations activate aberrant splice sites, which lead to an altered splicing pathway and consequently affects protein synthesis. The (C>G) IVS-2-745 mutation is common in South Eastern Europe, Cyprus, Lebanon, India, Malaysia, and Indonesia. This mutation, located within intron 2 of the β-globin gene, creates an aberrant 5' splice site at nucleotide 745 of intron 2 and activates a cryptic 3' splice site within the same intron. Portions of the intronic sequence are incorrectly retained in the spliced mutant mRNA. The mutation results in a premature stop codon that prevents proper mRNA translation and causes a β‐globin deficiency, resulting in β‐thalassemia. The IVS-2-745 allele has the functional splice sites preserved, but produces a significantly reduced level of correctly spliced β-globin mRNA and results in only marginal synthesis of HbA. Therefore, the IVS-2-745 mutation in homozygosity leads to severe transfusion-dependent thalassemia major. Taking advantage of conserved canonical splice sites in defective β‐globin genes, such as IVS-2-745, recently developed approaches show that by targeting the aberrant splice sites it is possible to circumvent the aberrant splice site and restore the normal β-globin splicing pattern. We sought to use uniform 2'-O-methoxyethyl (2'-MOE) splice switching oligos (SSOs) to reverse aberrant splicing in the pre-mRNA for the IVS-2-745 mutation. Using these SSOs, we show effective aberrant-to-wild-type splice switching. This leads to an increase in adult hemoglobin (HbA) by up to 80% in erythroid cells from patients with the IVS-2-745 mutation. Furthermore, we demonstrate a restoration of the balance between β-like- and α-globin chains, and up to an 87% reduction in α-heme aggregates. While examining the potential benefit of 2'-MOE-SSOs in a sickle/IVS-2-745-thalassemic genotype setting, we found that use of these oligos restored production of HbA and reduced HbS synthesis, which ultimately lessened cell sickling under hypoxic conditions. We confirmed increased WT β-globin expression in specimens treated with 2'-MOE-SSOs with semi- and quantitative methods (RT and Q-PCR), and further supported this evidence using a direct quantification method (ddPCR). Compared to treated specimens heterozygous for IVS-2-745 , homozygous specimens showed elevated WT HbA, reflecting the additive effect of targeting the aberrant splicing of both alleles as opposed to a single IVS-2-745 allele. In fact, while 2'-MOE-SSOs significantly reduced aberrant splicing, leading to a consequent 60% increase in HbA levels in specimens from patients with a β0/IVS-2-745 genotype, the same oligos produced a more robust effect in specimens with a homozygous IVS-2-745 genotype, resulting in an 80% increase in HbA levels. This level of increase could potentially be curative for patients with this particular genotype. Moreover, we compared the effect of 2'-MOE-SSOs treatment to a lentiviral vector carrying a WT β-globin gene. In this comparative assay, β0/IVS-2-745 cells treated with 2'-MOE-SSOs or the lentivector (with 1.13 copies integrated per genome) lead to a similar increase in HbA (50%). This suggests that the oligo-based technology is a competitive approach and a viable alternative to gene addition therapy to overcome anemia in IVS-2-745 β-thalassemia. In summary, 2'-MOE-SSOs are promising therapeutic tools for certain forms of β-thalassemia caused by aberrant splicing. Their ability to correct the underlying splicing defect offers a pharmacological treatment that is direct, specific, and accessible. In comparison, gene therapy approaches utilizing gene addition or editing are primarily available in advanced medical care environment resulting in an unfulfilled demand in regions where such conditions are not readily available. The restoration of target gene activity reported here suggests that this treatment strategy could be applicable to other forms of thalassemia resulting from mutations affecting splicing. This could have, with an effective method of delivery, potential clinical utility in helping patients reduce their transfusion dependence or even achieving transfusion independence. Disclosures Dong: Aruvant Sciences INC: Employment. Motta:Sanofi-Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees. Guo:Ionis Pharmaceutical, INC: Employment, Other: shareholders. Peralta:Ionis Pharmaceutical, Inc: Employment. Freier:Ionis Pharmaceuticals: Employment. Watt:Ionis Pharmaceuticals: Employment. Manwani:Novartis: Consultancy; Pfizer: Consultancy; GBT: Consultancy, Research Funding. Cappellini:Genzyme/Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Honoraria; Vifor Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; CRISPR Therapeutics: Membership on an entity's Board of Directors or advisory committees. Abdulmalik:The Children's Hospital of Philadelphia: Patents & Royalties: Provisional Patent. Rivella:Meira GTx, Ionis Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Disc medicine, Protagonist, LIPC, Meira GTx: Consultancy.

Editing aberrant splice sites efficiently restores β-globin expression in β-thalassemia

The thalassemias are compelling targets for therapeutic genome editing in part because monoallelic correction of a subset of hematopoietic stem cells (HSCs) would be sufficient for enduring disease amelioration. A primary challenge is the development of efficient repair strategies that are effective in HSCs. Here, we demonstrate that allelic disruption of aberrant splice sites, one of the major classes of thalassemia mutations, is a robust approach to restore gene function. We target the IVS1-110G>A mutation using Cas9 ribonucleoprotein (RNP) and the IVS2-654C>T mutation by Cas12a/Cpf1 RNP in primary CD34+ hematopoietic stem and progenitor cells (HSPCs) from β-thalassemia patients. Each of these nuclease complexes achieves high efficiency and penetrance of therapeutic edits. Erythroid progeny of edited patient HSPCs show reversal of aberrant splicing and restoration of β-globin expression. This strategy could enable correction of a substantial fraction of transfusion-dependent β-thalassemia genotypes with currently available gene-editing technology.