Splicing Factor Mutations Research Articles

CCUS and Low-Risk MDS Are Inherently Similar, Sharing Clonal and Clinical Features

Background Idiopathic cytopenia of undetermined significance (ICUS) is a condition of unexplained blood cytopenia not meeting the diagnostic criteria of myelodysplastic syndromes (MDS). Recently, the newer designation of clonal cytopenia of undetermined significance (CCUS) has been proposed to characterize patients with ICUS in the presence of one or more somatic mutations or copy number abnormalities. Clinically, CCUS has been associated with an increased risk of developing MDS; however, the mechanism and factors associated with evolution remain unclear. We sought to determine the molecular similarity of cytopenic cases with equivocal morphological dysplasia to bona fide MDS, as well as whether serial sequencing of ICUS and CCUS cases could identify factors that predict evolution to MDS. Methods We compared the incidence and frequency of somatic variants in 48 genes recurrently mutated in myeloid neoplasms (MNs), using targeted Next-Generation Sequencing (NGS) of bone marrow genomic DNA from 193 individuals with confirmed or suspected MDS or MDS/myeloproliferative neoplasm (MDS/MPN), including sequential investigation for 28 individuals at the time of diagnosis and during follow-up. Cytopenias qualifying patients as ICUS were based on WHO laboratory diagnostic criteria: hemoglobin levels <130g/L for males or <120 g/L for females; platelet count <150x109/L; and neutrophils <1.8x109/L. MDS patients were further subclassified as low risk (MDS-LR), including "Very Low” and "Low” International Prognostic Scoring System-Revised (IPSS-R) categories; intermediate risk (MDS-IR), including "Intermediate” category; or high risk (MDS-HR), including "High” and "Very High” risk categories. Results NGS further facilitated the diagnosis of all suspicious cases, either as MN (21%), CCUS (34%), or ICUS (45%). We found that there was no significant difference in most measured clinical features between CCUS and MDS-LR. While we saw significantly increased hemoglobin and platelet counts in individuals with CCUS compared with MDS-HR (Mann-Whitney U test; p=0.003 and p=0.012, respectively), no significant differences in cell counts were seen between CCUS and the MDS-LR or MDS-IR subgroups (Table 1). There was also no difference in overall survival between CCUS and MDS-LR when adjusted for age and sex in multivariable models (HR=0.575; 95% CI=0.256-1.294; p=0.330), while MDS-HR was associated with worse overall survival when compared with CCUS (HR=2.759; 95% CI=1.274-5.975; p=0.010). We saw no significant difference in clonal phenotypes, including number of variants or variant allele frequencies (VAFs), when comparing CCUS and MDS-LR. We further classified genes mutated in at least one patient in our cohort into functional groups for further analysis. These groups included epigenetic regulators: ASXL1, BCOR, DNMT3A, EZH2, IDH1, IDH2, KDM6A, PHF6, RAD21, STAG2, TET2; splicing factors: DDX41, SF3B1, SRSF2, U2AF1, ZRSR2; genes involved in signal transduction: BRAF, CALR, CBL, CSF3R, FLT3, GNB1, JAK2, KIT, KRAS, MPL, NF1, NPM1, NRAS, PTPN11, SETBP1; transcription factors: CEBPA, ETV6, GATA2, RUNX1; and genes involved in DNA damage repair: PPM1D and TP53. A significantly higher number of transcription factor mutations was seen when comparing CCUS to the MDS-IR and MDS-HR groups (p=0.007 and p=0.008, respectively), but not the MDS-LR group (p=0.754). We did, however, see significant significantly lower number of splicing factor mutations in CCUS cases compared with MDS-LR (p<0.001; Figure 1). Serial sequencing revealed no significant associations between number, type, or VAF of variants initially present at ICUS or CCUS diagnosis compared to those later acquired at the time of MDS evolution. Of note, we saw an increased probability of evolution to MDS of individuals with CCUS compared to ICUS over the first 5 years (HR=3.569; 95% CI=1.029-12.380, p=0.045). Conclusions Our analyses revealed no conclusive pattern associating clonal expansion or number of variants with evolution of CCUS to MDS, perhaps further supporting the similarity of these diseases and the proposal that CCUS should be classified as low-risk MDS in the next revision of the WHO guidelines. If future studies can better associate variants in specific types of genes with progression (such as splicing factors), this may also assist in predicting the probability of evolution and thus inform management strategies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

DNA Replication Stress Due to Loss of R-Loops in Myelodysplastic Syndromes with SF3B1 Mutation

Introduction: Myelodysplastic syndromes (MDS) with SF3B1 mutation represent a subtype of hematopoietic stem cell disorders with bone marrow (BM) erythroid dysplasia and ring sideroblasts. This mutation in a splicing factor gene causes multiple alterations of mRNA maturation. Other mutated splicing factors SRSF2 or U2AF1, produce splicing alterations and formation of unscheduled R-loops, which trigger DNA damage response and genetic instability. Mechanistically, mutant SRSF2 impairs the transcription pause release downstream of TSS allowing invasion of relaxed DNA double helix by nascent RNA. Although previous studies suggested that R-loops inappropriately form in SF3B1 mutant cells, investigations are required to elucidate why MDS with SF3B1 mutation are less prone to genetic instability. Methods: A total of 96 patients including 53 MDS with SF3B1 mutation and 43 other low risk MDS was enrolled in this study. RNA-seq of BM mononuclear cells (MNC) was performed in 21 MDS with and 6 MDS w/o SF3B1 mutation. BM CD34+-derived erythroblasts were expanded from 69 MDS and 22 healthy controls to analyze transcriptome, proteome, R-loop landscape, and DNA replication stress. For global proteome analysis, after protein digestion, peptides were fractionated on SCX column and analyzed by mass spectrometry. R-loops were detected by DNA-RNA immunoprecipitation (DRIP-seq) using S9.6 antibody. Immunofluorescence labeling of pRPA32, 53BP1 and γH2AX, DNA combing, BrdU assay and metabolomics were used to investigate DNA replication stress and DNA biosynthesis in human primary erythroblasts or in murine proerythroblastic (proE) G1E-ER4 Crispr-cas9 Sf3b1K700E/+ cell line. expressing GATA1 under the control of estrogen receptor. Results: In SF3B1-mutated BM MNC samples, DeSeq2 analysis of RNA-seq data revealed 1,058 upregulated genes (log2(FC)>0.6, BH-adj P-value<0.05) involved in DNA replication, cell cycle, nucleotide biosynthesis, DNA repair, and erythroid differentiation. Analysis of splicing identified 3,937 differential splicing events (ΔPSI>0.10, BH-adj P-value<0.05) including 1,256 events of intron retention (IR). Among IR events, 384 loss of IR events correlated with altered expression of 293 genes. In SF3B1-mutated erythroblasts, loss of IR events were more frequent in polychromatophilic erythroblasts (polyE: 383) than in proE/early basophilic erythroblasts (eBasoE: 171) suggesting that gene extinction mediated by IR during normal differentiation was altered. Quantitative proteomics revealed 924 significantly deregulated proteins with little overlap with IR transcripts, consistently involved in DNA damage, DNA repair, and nucleotide biosynthesis pathways. DRIP-seq was performed in proE/eBasoE of 8 MDS w/o or with SF3B1 mutation to investigate R-loop formation. Stringent peak calling identified 2,208 shared peaks in wild-type samples compared to 159 shared peaks in SF3B1-mutated samples, suggesting a loss of R-loops. Integrative analysis and DRIP-qPCR demonstrated that R-loop losses overlapped IR loss events, consistent with the notion that intron excision prevents R-loop formation. Compared to SRSF2 or U2AF1-mutated erythroblasts, SF3B1-mutated cells exhibited a significant increase of DNA synthesis by BrdU labeling and of replication fork speed by DNA combing. Immunofluorescence analysis detected pRPA32 foci marking single strand DNA but no evidence of double strand breaks in contrast to SRSF2 mutant cells in which 53BP1 or γH2AX foci were detected. Compared to isogenic cell lines, Sf3b1K700E/+ proE G1E-ER4 cells demonstrated higher proliferation rate and fork speed in proliferation and in estradiol-induced differentiation conditions, and significantly lower amounts of deoxynucleotides (dATP, dCTP, TTP) suggesting persistence of DNA synthesis during differentiation. Targeting of DNA polymerase by aphidicolin or ribonucleotide reductase by low doses of hydroxyurea partially rescued erythroid differentiation. Conclusion: Our data demonstrated a highly specific signature of DNA replication stress at transcriptomic and proteomic level in SF3B1-mutant cells. High replication fork speed reflects the loss of R-loops in these cells, which may reduce the risk of genetic instability at the expense of normal differentiation. Pharmacological targeting of the DNA replication stress could improve erythropoiesis.

Emerging Roles of RNA-Binding Proteins in Inner Ear Hair Cell Development and Regeneration.

RNA-binding proteins (RBPs) regulate gene expression at the post-transcriptional level. They play major roles in the tissue- and stage-specific expression of protein isoforms as well as in the maintenance of protein homeostasis. The inner ear is a bi-functional organ, with the cochlea and the vestibular system required for hearing and for maintaining balance, respectively. It is relatively well documented that transcription factors and signaling pathways are critically involved in the formation of inner ear structures and in the development of hair cells. Accumulating evidence highlights emerging functions of RBPs in the post-transcriptional regulation of inner ear development and hair cell function. Importantly, mutations of splicing factors of the RBP family and defective alternative splicing, which result in inappropriate expression of protein isoforms, lead to deafness in both animal models and humans. Because RBPs are critical regulators of cell proliferation and differentiation, they present the potential to promote hair cell regeneration following noise- or ototoxin-induced damage through mitotic and non-mitotic mechanisms. Therefore, deciphering RBP-regulated events during inner ear development and hair cell regeneration can help define therapeutic strategies for treatment of hearing loss. In this review, we outline our evolving understanding of the implications of RBPs in hair cell formation and hearing disease with the aim of promoting future research in this field.

354 (PB134) - SpliceIO™ a novel AI platform for the discovery of splicing-derived immunotherapeutic targets

Alternative splicing (AS) plays a critical role in generating tumor-specific neoepitopes targetable through an arsenal of immunotherapeutic modalities. Previous studies have demonstrated tumor-promoting activity of deregulated AS isoforms and mutated splicing factors, both of which increase the yield of neoepitope-encoding AS isoforms. Therefore, the identification of AS isoforms has the potential to produce novel therapeutic targets, especially in tumor types where splicing deregulation is frequent, like breast cancer.

Microtubule modification defects underlie cilium degeneration in cell models of retinitis pigmentosa associated with pre-mRNA splicing factor mutations

Retinitis pigmentosa (RP) is the most common cause of hereditary blindness, and may occur in isolation as a non-syndromic condition or alongside other features in a syndromic presentation. Biallelic or monoallelic mutations in one of eight genes encoding pre-mRNA splicing factors are associated with non-syndromic RP. The molecular mechanism of disease remains incompletely understood, limiting opportunities for targeted treatment. Here we use CRISPR and base edited PRPF6 and PRPF31 mutant cell lines, and publicly-available data from human PRPF31 +/− patient derived retinal organoids and PRPF31 siRNA-treated organotypic retinal cultures to confirm an enrichment of differential splicing of microtubule, centrosomal, cilium and DNA damage response pathway genes in these cells. We show that genes with microtubule/centrosome/centriole/cilium gene ontology terms are enriched for weak 3′ and 5′ splice sites, and that subtle defects in spliceosome activity predominantly affect efficiency of splicing of these exons. We suggest that the primary defect in PRPF6 or PRPF31 mutant cells is microtubule and centrosomal defects, leading to defects in cilium and mitotic spindle stability, with the latter leading to DNA damage, triggering differential splicing of DNA damage response genes to activate this pathway. Finally, we expand understanding of “splicing factor RP” by investigating the function of TTLL3, one of the most statistically differentially expressed genes in PRPF6 and PRPF31 mutant cells. We identify that TTLL3 is the only tubulin glycylase expressed in the human retina, essential for monoglycylation of microtubules of the cilium, including the retinal photoreceptor cilium, to prevent cilium degeneration and retinal degeneration. Our preliminary data suggest that rescue of tubulin glycylation through overexpression of TTLL3 is sufficient to rescue cilium number in PRPF6 and PRPF31 mutant cells, suggesting that this defect underlies the cellular defect and may represent a potential target for therapeutic intervention in this group of disorders.

Activation of targetable inflammatory immune signaling is seen in myelodysplastic syndromes with SF3B1 mutations.

Mutations in the SF3B1 splicing factor are commonly seen in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), yet the specific oncogenic pathways activated by mis-splicing have not been fully elucidated. Inflammatory immune pathways have been shown to play roles in the pathogenesis of MDS, though the exact mechanisms of their activation in splicing mutant cases are not well understood. RNA-seq data from SF3B1 mutant samples was analyzed and functional roles of interleukin-1 receptor-associated kinase 4 (IRAK4) isoforms were determined. Efficacy of IRAK4 inhibition was evaluated in preclinical models of MDS/AML. RNA-seq splicing analysis of SF3B1 mutant MDS samples revealed retention of full-length exon 6 of IRAK4, a critical downstream mediator that links the Myddosome to inflammatory NF-kB activation. Exon 6 retention leads to a longer isoform, encoding a protein (IRAK4-long) that contains the entire death domain and kinase domain, leading to maximal activation of NF-kB. Cells with wild-type SF3B1 contain smaller IRAK4 isoforms that are targeted for proteasomal degradation. Expression of IRAK4-long in SF3B1 mutant cells induces TRAF6 activation leading to K63-linked ubiquitination of CDK2, associated with a block in hematopoietic differentiation. Inhibition of IRAK4 with CA-4948, leads to reduction in NF-kB activation, inflammatory cytokine production, enhanced myeloid differentiation in vitro and reduced leukemic growth in xenograft models. SF3B1 mutation leads to expression of a therapeutically targetable, longer, oncogenic IRAK4 isoform in AML/MDS models. This work was supported by Cincinnati Children's Hospital Research Foundation, Leukemia Lymphoma Society, and National Institute of Health (R35HL135787, RO1HL111103, RO1DK102759, RO1HL114582), Gabrielle's Angel Foundation for Cancer Research, and Edward P. Evans Foundation grants to DTS. AV is supported by Edward P. Evans Foundation, National Institute of Health (R01HL150832, R01HL139487, R01CA275007), Leukemia and Lymphoma Society, Curis and a gift from the Jane and Myles P. Dempsey family. AP and JB are supported by Blood Cancer UK (grants 13042 and 19004). GC is supported by a training grant from NYSTEM. We acknowledge support of this research from The Einstein Training Program in Stem Cell Research from the Empire State Stem Cell Fund through New York State Department of Health Contract C34874GG. MS is supported by a National Institute of Health Research Training and Career Development Grant (F31HL132420).

All-Atom Simulations Elucidate the Impact of U2AF2 Cancer-Associated Mutations on Pre-mRNA Recognition.

The U2AF2 splicing factor, made of two tandem RNA recognition motifs (RRMs) joined by a flexible linker, selects the intronic polypyrimidine sequence of premature mRNA, thus ensuring splicing fidelity. Increasing evidence links mutations of key splicing factors, including U2AF2, to a variety of cancers. Nevertheless, the impact of U2AF2 cancer-associated mutations on polypyrimidine recognition remains unclear. Here, we combined extensive (18 μs-long) all-atom molecular dynamics simulations and dynamical network theory analysis (NWA) of U2AF2, in its wild-type form and in the presence of the six most frequent cancer-associated mutations, bound to a poly-U strand. Our results reveal that the selected mutations affect the pre-mRNA binding at two hot spot regions, irrespectively of where these mutants are placed on the distinct U2AF2 domains. Complementarily, NWA traced the existence of cross-communication pathways, connecting each mutation site to these recognition hot spots, whose strength is altered by the mutations. Our outcomes suggest the existence of a structural/dynamical interplay of the two U2AF2's RRMs underlying the recognition of the polypyrimidine tract and reveal that the cancer-associated mutations affect the polypyrimidine selection by altering the RRMs' cooperativity. This mechanism may be shared by other RNA binding proteins hallmarked, like U2AF2, by multidomain architecture and high plasticity.

Dhx38 is required for the maintenance and differentiation of erythro-myeloid progenitors and hematopoietic stem cells by alternative splicing.

Mutations that occur in RNA-splicing machinery may contribute to hematopoiesis-related diseases. How splicing factor mutations perturb hematopoiesis, especially in the differentiation of erythro-myeloid progenitors (EMPs), remains elusive. Dhx38 is a pre-mRNA splicing-related DEAH box RNA helicase, for which the physiological functions and splicing mechanisms during hematopoiesis currently remain unclear. Here, we report that Dhx38 exerts a broad effect on definitive EMPs as well as the differentiation and maintenance of hematopoietic stem and progenitor cells (HSPCs). In dhx38 knockout zebrafish, EMPs and HSPCs were found to be arrested in mitotic prometaphase, accompanied by a 'grape' karyotype, owing to the defects in chromosome alignment. Abnormal alternatively spliced genes related to chromosome segregation, the microtubule cytoskeleton, cell cycle kinases and DNA damage were present in the dhx38 mutants. Subsequently, EMPs and HSPCs in dhx38 mutants underwent P53-dependent apoptosis. This study provides novel insights into alternative splicing regulated by Dhx38, a process that plays a crucial role in the proliferation and differentiation of fetal EMPs and HSPCs.

Molecular Threat of Splicing Factor Mutations to Myeloid Malignancies and Potential Therapeutic Modulations.

Splicing factors are frequently mutated in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). These mutations are presumed to contribute to oncogenic transformation, but the underlying mechanisms remain incompletely understood. While no specific treatment option is available for MDS/AML patients with spliceosome mutations, novel targeting strategies are actively explored, leading to clinical trials of small molecule inhibitors that target the spliceosome, DNA damage response pathway, and immune response pathway. Here, we review recent progress in mechanistic understanding of splicing factor mutations promoting disease progression and summarize potential therapeutic strategies, which, if successful, would provide clinical benefit to patients carrying splicing factor mutations.

ACT1-CUP1 Assays Determine the Substrate-specific Sensitivities of Spliceosomal Mutants in Budding Yeast.

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. Whether disease-related or functionally selected, many of these mutations have been studied using growth assays in the model organism Saccharomyces cerevisiae (yeast). The splicing-specific copper growth assay, or ACT1-CUP1 assay, provides a comprehensive analysis of mutation at the phenotypic level. The ACT1-CUP1 assay utilizes reporters that confer copper tolerance when correctly spliced. Thus, in the presence of copper, changes in yeast viability correlate to changes in mRNA production through splicing. In a typical experiment, the yeast spliceosome is challenged with different non-consensus splicing reporters and the splicing factor mutation of interest to detect any synergetic or antithetical impact on splicing. Here a full description of copper plate preparation, plating of yeast cells, and data evaluation are given. A selection of complimentary experiments is described, highlighting the versatility of the ACT1-CUP1 reporters. The ACT1-CUP1 assay is a handy tool in the splicing toolbox thanks to the direct read-out of mutational effect(s) and the comparative possibilities from the continuing use in the field.

S161: ZRSR2 AND TET2 MUTATIONS PROMOTE MDS BY DYSREGULATING GENE EXPRESSION AND ABERRANT ALTERNATIVE SPLICING IN MICE

Background: Mutations in splicing factors and epigenetic regulators are the most frequent genetic alterations in patients with myelodysplastic syndromes (MDS). The minor spliceosome factor ZRSR2 and the epigenetic regulator TET2 appear significantly associated in MDS patients. However, the functional impact of such mutations in the hematopoietic system and MDS have been scarcely studied. To address this question, we established a murine model (Zrsr2m/mTet2−/−) carrying mutations in both genes, which exhibited signs compatible with MDS disease in mice. However, the molecular disease mechanism has not yet been elucidated. Aims: To interrogate the impact of ZRSR2 and TET2 mutations in gene expression and alternative splicing in the context of hematopoiesis and MDS. Methods: Whole transcriptome sequencing was performed to investigate changes in gene expression and alternative splicing patterns. Lin-Sca-1+c-kit+ (LSK) cells (50,000 per sample) were sorted from pools of 3 mice, and mRNA was sequenced in an Illumina NovaSeq 6000 platform. Differential gene expression was determined using DESeq2 with adjusted P-value ≤ 0.05, log2FoldChange ≥ 0. Functional enrichment from differentially spliced genes was identified with KEGG, and the enrichment cut-off p-value was set as adjusted P-value ≤ 0.05. Relevant alternative splicing events were validated by RT-PCR. Results: RNA-seq analysis of Zrsr2m/mTet2−/−cells revealed 2952 differentially expressed genes (DEG), compared to 571 in Zrsr2m/m and 1203 in Tet2−/−. From 2952, 1327 were up-regulated and 1625 were downregulated. Interestingly, relevant genes for hematopoietic lineage specification were significantly dysregulated. In particular, genes related to lymphoid lineage (Flt3, Notch1, and Tlr7) were downregulated, while those related to myeloid lineage (Ccl9, Mpo, Ms4a6b, and Prtn3) and megakaryocytic-erythroid lineage (Pdfgrb, Itgb3, Optn, and Tgfbr3) were upregulated. This suggests an early myeloid and megakaryocyte-erythroid priming of LSK towards the production of cells from these lineages in Zrsr2m/mTet2−/− mice. Enrichment analysis of DEG using Go enrichment analysis identified ribosome function, inflammation, and migration/motility processes as the most significantly altered. Further, KEGG pathway enrichment pointed ribosome, the MAPK family, and pro-inflammatory pathways as significantly enriched in Zrsr2m/mTet2−/− cells. Finally, KEGG pathway enrichment of alternative splicing targets identified the MAPK family and the Fanconi anemia pathway as the most altered targets in Zrsr2m/mTet2−/− cells. Importantly, a total of 9 genes related to the MAPK pathway were identified as mis-spliced, from which Dusp1, Tgfbr2, and Fgf11 were validated in this study. Summary/Conclusion: In this study, we broaden our previous report and show that concurrent mutations in Zrsr2 and Tet2 dysregulate normal gene expression and cause aberrant mRNA splicing. Gene expression analysis identified ribosome function, inflammation, and migration/motility as the most altered processes in Zrsr2m/mTet2−/− cells. Alternative splicing analysis identified the MAPK and the Fanconi Anemia pathway as key targets of aberrant splicing. All in all, gene expression dysregulation and aberrant mRNA splicing disturb important biological pathways and drive the molecular pathomechanism in Zrsr2m/mTet2−/− mice.

P411: SF3B1 MUTATIONS IN AML ARE STRONGLY ASSOCIATED WITH MECOM REARRANGEMENTS AND MAY BE INDICATIVE OF AN MDS PRE-PHASE

Background: Mutations in the splicing factor gene SF3B1 are most frequent in myelodysplastic syndrome (MDS) or myelodysplastic/myeloproliferative neoplasm (MDS/MPN) but are also recurrently found in acute myeloid leukemia (AML). Splicing factor mutations have been suggested to have additive value if incorporated into the current risk classification of AML (van der Werf et al., Blood Advances 2021). Aims: Explore the genetic landscape of SF3B1 mutated AML and determine the prognostic impact of SF3B1 mutations. Methods: We analyzed 735 patients diagnosed with AML based on cytomorphology, cytogenetics, and molecular genetics according to WHO classification 2017. Reads from amplification-free whole genome sequencing (WGS, median coverage 103x, Illumina, San Diego, CA) were aligned to the human reference genome (GRCh37, Ensembl annotation, Isaac aligner) and variants were called using Strelka Somatic Variant Caller v2.4.7. Results: We identified SF3B1 mutations in 41 of 735 (6%) AML patients. SF3B1 mutations were found in AML with recurrent genetic abnormalities (24/471; 5%), AML with myelodysplasia-related changes (MRC; 11/158; 7%) and AML not otherwise specified (NOS; 6/106; 6%). When focussing on SF3B1mut cases, AML-MRC (11/41; 27%) and AML with GATA2::MECOM (10/41; 24%) were most frequent. 9 additional cases showed another MECOM rearrangement (-rear) resulting in 46% (19/41) of patients with SF3B1 mutations accompanied by a MECOM-rear. A prior history of MDS or MDS/MPN was documented in 20% (8/41) of SF3B1mut patients. Thereof, 63% (5/8) harbored a MECOM rearrangement and 25% RUNX1mut. On average, SF3B1mut patients harbored 2.85 mutations (including SF3B1). The most frequent additional mutations of SF3B1mut patients were RUNX1 (9/41; 22%) and NRAS (8/41; 20%), and NPM1, TET2, or DNMT3A mutations or FLT3-ITD were detected in 15% (6/41) each. SF3B1 showed an average variant allelic frequency (VAF) of 41%. Notably, the VAF of SF3B1 in AML with NPM1 or RUNX1 was higher than the VAFs of NPM1 or RUNX1 in 9/11 (82%) cases, suggesting that SF3B1 preceded these mutations. In 16/41 (39%) cases molecular follow-up data was available. The SF3B1 mutation persisted during the entire disease course in 8 cases (including 1 with relapse). In particular, in two of these AML patients with mutated NPM1, the SF3B1 mutation remained detectable by NGS, despite complete hematologic remission and undetectable NPM1 mutation by high-sensitive qPCR. In all 5 cases with relapse the SF3B1 mutation re-occurred at relapse. In 4 patients, the VAF of SF3B1 decreased, similar to accompanying mutations. In contrast to MDS, SF3B1 mutations did not affect the overall survival (median OS: 16.9 months in SF3B1mut; 5.7 months in unmutated group; p=0.822) in the total AML cohort. When stratified for AML sub-entities, the prognosis of the SF3B1mut AML seems to be dominated by the sub-entity. Image:Summary/Conclusion: Within AML, SF3B1 mutations are enriched in AML patients with GATA2::MECOM and AML-MRC. This association with poor risk subtypes dominates the prognosis of SF3B1mut AML. The high VAF of SF3B1 mutations and the persistence of SF3B1 mutations in AML patients in complete remission suggest the early acquisition of SF3B1 mutations in a pre-leukemic clone in a number of patients. While an antecedent MDS or MDS/MPN has been documented in some cases of SF3B1mut AML, it might be unidentifiable in others. Alternatively, SF3B1mut clonal hematopoiesis of indeterminate potential (CHIP) may represent a relevant pre-phase of SF3B1mut AML.

P758: INHIBITION OF ATR WITH CERALASERTIB (AZD6738) FOR THE TREATMENT OF PROGRESSIVE OR RELAPSED MYELODYSPLASTIC SYNDROMES AND CHRONIC MYELOMONOCYTIC LEUKEMIA: A PHASE IB/II STUDY

Background: Myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) are enriched for mutations in the spliceosome complex, including the genes SF3B1, SRSF2, ZRSR2, and U2AF1. Ceralasertib (AZD6738) is a selective and potent inhibitor of Ataxia Telangiectasia and Rad3 Related (ATR) kinase. We have previously shown that cells with splicing factor (SF) mutations have increased R loops, which require ATR for resolution and preferentially undergo apoptosis after ATR inhibition. Aims: We hypothesized that inhibition of ATR may be useful in patients with MDS or CMML, particularly those who harbor SF gene mutations. Methods: This 2-part study evaluated ceralasertib monotherapy in adult patients with MDS or CMML progressing on or not responsive to front-line therapy (DNMTI for HR MDS, ESA for LR MDS). Enrollment cohorts included SF mutant (SF3B1, SRSF2, U2AF1, or ZRSR2) and SF wildtype (wt). Part 1 enrolled 3-6 patients at de-escalating dose levels: 160mg BID days 1-14 of 28 d cycles, 120mg BID d1-14, and 80mg BID d1-14. DLTs were assessed the first 30d and de-escalation would occur for DLTs in >1/6 patients. Higher-risk MDS (HR-MDS, IPSS-R > 3.5) and CMML patients were enrolled first; after a dose was deemed safe, lower-risk MDS (LR-MDS, IPSS-R 3.5, transfusion-dependent post/ineligible for ESA or severe neutropenia/thrombocytopenia) was evaluated. Part 2 enrolled up to 20 SF mut patients and 20 SF wt patients for efficacy. A Simon 2-stage design mandated that at least 1/10 patients in each group needed to respond (by IWG 2006 criteria) to enroll the final 10 patients. Results: At the data cut-off (12/15/21) 32 patients have been enrolled with 30 evaluable: 22 with HR-MDS or CMML, and 8 with LR-MDS. The median age was 73 (range 43-88) and predominantly male (26/30). 21 patients harbored SF mut disease while 9 had SF wt MDS/CMML. The most frequently seen ≥grade 3 adverse events (AEs) for higher-risk patients (n=20) included anemia (n=6), febrile neutropenia (n=4), neutropenia (n=5), thrombocytopenia (n=6). In LR-MDS (n=8), ≥grade 3 AEs included thrombocytopenia (n=2), and neutropenia (n=2). 5 deaths occurred within 30d of study treatment; 1 patient with active CMML had an intracranial bleed with thrombocytopenia, possibly related to study treatment, while 4 deaths were attributed to underlying disease (3 with progression to AML, 1 MDS patient with sepsis). No DLTs were observed in the HR or LR groups and 160mg BID days 1-14 of 28 days was deemed the RP2D. Responses were assessed across the cohort as well as according to SF mutant status (Table). A total of 8 patients had an IWG response (BOR: CR, n=1; mCR, n=3; HI, n=4), including 6/21 SF mut patients (ORR 29%), and 2/9 SF wt patients (ORR 22%). The responses in SF wt patients included one mCR (5% to 1% marrow blasts in a patient with DNMT3A and ETV6 mut and del(chr7)), and one HI-P (82 BL to 234 max in a patient with DNMT3A, IDH2, ETV6 mut and del(chr7)). Responses in SF mut included CR (n=1), mCR (n=2, one with associated HI-N), SD with HI (n=3). An additional 3 SF mut patients had improvements in blood counts but not lasting 8 weeks (ANC n=2, platelets n=1) and one with platelet improvement but a baseline not meeting IWG criteria (plt avg=108k prior to C1D1). Image:Summary/Conclusion: Ceralasertib is an oral ATR inhibitor with preliminary activity in patients with progressive/refractory MDS and CMML at doses of 160mg BID on days 1-14 of a 28 day cycle. The study is ongoing and will evaluate additional dosing schedules.

Abstract 868: Splicing factor mutant lung cancers: A comprehensive molecular and clinicopathologic characterization

Abstract Background: Splicing factor mutations can drive oncogenesis in different cancer types. More importantly, strategies to target splicing-dependent cancers have been developed, including PRMT5 inhibition which regulates alternative splicing and targets posttranslational modification; PRMT5 inhibitors are in ongoing phase I clinical trials. The characterization of splicing alterations in non-small cell lung cancer (NSCLC) has not been extensively studied and represents an unmet need. Methods: Based on previously known genes associated with splicing alterations, genomic and clinicopathologic data from NSCLC patients with hotspot or loss of function mutations identified by next-generation sequencing in the splicing factors SF3B1, SRSF2, U2AF1, ZRSR2, RBM10, and FUBP1 were explored. Results: Among 626 NSCLC patients with at least one splicing mutation, 85% (529/626) also harbored a co-occurring oncogenic driver, such as alterations involving KRAS (60%; 319/529), EGFR (25%;132/529), MET (5%; 27/529), BRAF(4%; 24/529), and ERBB2 (3%; 17/529). After excluding patients with concurrent drivers, 97 splicing-factor-mutant NSCLCs were identified, with 80 (82%) adenocarcinomas and 6 (6%) squamous cell carcinomas. The median age was 71 years (43-89). Forty-nine patients (51%) were female. Sixty patients (62%) were diagnosed with stage I-III disease. Eleven (11%) were never smokers, while 86 (89%) were former/current smokers, with a median of pack years of 30. The median tumor mutational burden was 7.9 mut/Mb (0.9-90.4). Regarding splicing factor mutations, sixty-two patients (64%) had nonsense or splice-site RBM10 mutations, 17 (17%) had missense hotspot SF3B1 mutations, 16 (16%) had hotspot missense U2AF1 mutations, and 7 (7%) had nonsense, splice-site, or hotspot missense FUBP1 mutation. No patients with ZRSR2 or SRSF2 were identified. Only three patients presented concurrent splicing mutations: one with two SF3B1 mutations; one with RBM10 and SF3B1 mutations; and one with U2AF1 and SF3B1 mutations. Conclusion: Most patients with splicing factor mutant NSCLCs are smokers whose cancers commonly harbor RBM10 mutations and higher TMB compared to other driver-positive cancers. The characterization of this profile is key to identifying appropriate patients for ongoing targeted therapy trials for this population. Citation Format: Guilherme Harada, Christina J. Falcon, Fernando C. Santini, Jason C. Chang, Soo R. Yang, Maria E. Arcila, Natasha Rekhtman, Alexander Drilon. Splicing factor mutant lung cancers: A comprehensive molecular and clinicopathologic characterization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 868.

Abstract 778: Dysregulation of alternative mRNA splicing by oncogenic KRAS in lung adenocarcinoma

Abstract Alternative mRNA splicing is dysregulated in many cancers including lung adenocarcinoma. These aberrant splicing events can sometimes be explained by mutations in splice sites or splicing factors. However, the majority of mis-splicing in cancers remains unexplained. We hypothesize that oncogenic signaling pathways play a role in regulating alternative splicing. In this study, we focus on mRNA splicing activity regulated by the Ras signaling cascade, which is frequently mutated in cancers. These pathways offer avenues for therapeutic modulation through small molecule inhibitors.We took a global proteomic and transcriptomic approach to study the effects of oncogenic Ras signaling on RNA splicing in vitro. Specifically, we performed LC-MS/MS and RNA-seq profiling on normal human lung airway epithelial (AALE) cells overexpressing wild-type or mutant KRAS or RIT1 (N = 3 replicates per allele). Phosphorylation of splicing factors was preferentially downregulated in KRASmut cells compared to KRASWT cells, suggesting that oncogenic KRAS signaling regulates splicing factor activity. Additionally, of the 2227 and 2452 skipped exon (SE) events in KRASG12V and KRASQ61H cells, respectively, compared to KRASWT overexpressing cells, 1013 events were shared between the two mutants. This statistically significant overlap (p &amp;lt; 0.001, hypergeometric test) indicates that KRAS has a role in splicing regulation that is disrupted when the protein is mutated. To determine how KRAS-regulated splicing activity compared to other signaling proteins and oncogenes, we also profiled the whole transcriptomes of isogenic A549 lung adenocarcinoma cell lines overexpressing 86 wild-type or variant alleles across 27 genes implicated in lung cancers (N = 4 to 8 replicates per allele). Of all pairs of mutant and wild-type alleles tested, KRASmut cells compared to KRASWT overexpressing cells exhibited the second highest levels of alternative splicing, behind only RNA binding protein RBM45 and its mutant allele RBM45M126I. In particular, ten SE events were observed to be differentially regulated in a KRAS dependent manner in both AALE and A549 cells. One of these 10 events is the differential splicing of the Myc Associated Zinc Finger (MAZ) protein to increase the expression of its splice variant that acts as a negative regulator of transcription. As MAZ regulates expression of KRAS, this splice variant may be a mechanism for the cell to modulate wild-type KRAS levels in the presence of KRAS mutants. Our proteomic and transcriptomic profiling in lung epithelial and lung adenocarcinoma cells uncovers splicing factor activity and mRNA splicing events regulated by oncogenic KRAS. With further studies of the alternative splicing events described, it will be possible to design or repurpose therapies in order to target aberrant splicing caused by disrupted Ras signaling and its downstream effectors. Citation Format: April Lo, Maria McSharry, Alice H. Berger. Dysregulation of alternative mRNA splicing by oncogenic KRAS in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 778.

Abstract 883: Defining the observed mutual exclusivity between SF3B1 and TP53 mutations in cancer

Abstract SF3B1 is the most commonly mutated splicing factor in cancer, occurring in a broad variety of hematologic and solid malignancies, including: breast, melanoma, pancreatic, bladder, colorectal, leukemia, and more. SF3B1 is a protein component of the spliceosome responsible for recognizing intronic splice sites and stabilizing the interaction between the spliceosome and pre-mRNA transcripts. Hotspot mutations in SF3B1 result in a neomorphic protein that causes aberrant splicing of hundreds of transcripts. These transcriptional alterations have several proposed impacts on known cancer pathways; however, the mechanisms by which SF3B1 mutations contribute to tumorigenesis are incompletely understood. Interestingly, systematic analysis of online cancer databases shows SF3B1 mutations and TP53 alterations are mutually exclusive in cancers with predominant SF3B1 hotspots, suggesting either synthetic lethality or overlapping roles in promoting tumorigenesis. To address this question, TP53 was knocked-out using CRISPR-Cas9 in AAV-generated SF3B1 K700E mutant knock-in and wild type MCF-10A breast epithelial cells. Successful generation of TP53 knock-outs in both the SF3B1 mutant and wild type groups suggests the mutations are unlikely to be synthetic lethal. Instead, this finding may direct us to novel mechanisms by which SF3B1 mutations contribute to cancer development and progression. Two overarching areas of interest based on preliminary data include changes in mitochondrial metabolism and metabolic reprogramming and alterations in DNA damage, repair, and genomic stability. Further defining the relationship between mutant SF3B1 and TP53 may reveal new mechanisms of tumorigenesis and reveal therapeutic vulnerabilities that can be additionally leveraged against the large subset of cancers with TP53 mutations. Citation Format: Riley Bergman, Ben Park, Sarah Croessmann. Defining the observed mutual exclusivity between SF3B1 and TP53 mutations in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 883.

Effects of spliceosomal mutations on brain patterning and morphogenesis

The major spliceosome consists of U1, U2, U5, and U4/U6 small nuclear ribonucleoprotein (snRNPs), and each snRNP has distinct and sequential roles during the mRNA splicing process. Mutations in the core components of the spliceosome are associated with brain and neurological defects. The goal of this study is to examine the role of three core spliceosomal components, SNRPB, SF3B4, and EFTUD2, in brain patterning and morphogenesis, and whether or not they are required in the neural tube or neural crest cells for brain development. Previously, we identified brain defects associated with the mutation of these spliceosomal components in the neural tube and neural crest cells. Structures derived from the midbrain and hindbrain region, such as the diencephalon, pons, and cerebellum, were found to be absent in E12.5 and E14.5 mutant embryos. In this study, we aim to use mutant mouse models to identify shared transcripts and pathways disrupted by mutations in these spliceosomal genes. Through RNA‐seq analysis, we identified a number of mis‐expressed RNA‐binding proteins that may contribute to the malformationis found in these mutants. We plan to examine level and expression of these RNA binding proteins in wild type and mutant embryos to evaluate how they contribute to the phenotypes found. Furthermore, to assess the potential clinical application of our findings, we will use human embryonic stem cells (hESC) to generate human neural crest cells with mutatioin in these splicing factors; shared transcripts and pathways identified in the mouse model will be compared to those found in induced human neural crest cells. Distinct spliceosomophathies are difficult to diagnose due to phenotypic overlaps. The significance of this work lies in its potential to identify a disrupted pathway shared by mutations in various splicing factors, a therapeutic alternative across multiple spliceosomopathies.

Molecular mechanisms by which splice modulator GEX1A inhibits leukaemia development and progression.

Splice modulators have been assessed clinically in treating haematologic malignancies exhibiting splice factor mutations and acute myeloid leukaemia. However, the mechanisms by which such modulators repress leukaemia remain to be elucidated. The primary goal of this assessment was to assess the molecular mechanism by which the natural splice modulator GEX1A kills leukaemic cells in vitro and within in vivo mouse models. Using human leukaemic cell lines, we assessed the overall sensitivity these cells have to GEX1A via EC50 analysis. We subsequently analysed its effects using in vivo xenograft mouse models and examined whether cell sensitivities were correlated to genetic characteristics or protein expression levels. We also utilised RT-PCR and RNAseq analyses to determine splice change and RNA expression level differences between sensitive and resistant leukaemic cell lines. We found that, in vitro, GEX1A induced an MCL-1 isoform shift to pro-apoptotic MCL-1S in all leukaemic cell types, though sensitivity to GEX1A-induced apoptosis was negatively associated with BCL-xL expression. In BCL-2-expressing leukaemic cells, GEX1A induced BCL-2-dependent apoptosis by converting pro-survival BCL-2 into a cell killer. Thus, GEX1A + selective BCL-xL inhibition induced synergism in killing leukaemic cells, while GEX1A + BCL-2 inhibition showed antagonism in BCL-2-expressing leukaemic cells. In addition, GEX1A sensitised FLT3-ITD+ leukaemic cells to apoptosis by inducing aberrant splicing and repressing the expression of FLT3-ITD. Consistently, in in vivo xenografts, GEX1A killed the bulk of leukaemic cells via apoptosis when combined with BCL-xL inhibition. Furthermore, GEX1A repressed leukaemia development by targeting leukaemia stem cells through inhibiting FASTK mitochondrial isoform expression across sensitive and non-sensitive leukaemia types. Our study suggests that GEX1A is a potent anti-leukaemic agent in combination with BCL-xL inhibitors, which targets leukaemic blasts and leukaemia stem cells through distinct mechanisms.

Precision analysis of mutant U2AF1 activity reveals deployment of stress granules in myeloid malignancies

Splicing factor mutations are common among cancers, recently emerging as drivers of myeloid malignancies. U2AF1 carries hotspot mutations in its RNA-binding motifs; however, how they affect splicing and promote cancer remain unclear. The U2AF1/U2AF2 heterodimer is critical for 3' splice site (3'SS) definition. To specifically unmask changes in U2AF1 function invivo, we developed a crosslinking and immunoprecipitation procedure that detects contacts between U2AF1 and the 3'SS AG at single-nucleotide resolution. Our data reveal that the U2AF1 S34F and Q157R mutants establish new 3'SS contacts at -3 and+1 nucleotides, respectively. These effects compromise U2AF2-RNA interactions, resulting predominantly in intron retention and exon exclusion. Integrating RNA binding, splicing, and turnover data, we predicted that U2AF1 mutations directly affect stress granule components, which was corroborated by single-cell RNA-seq. Remarkably, U2AF1-mutant cell lines and patient-derived MDS/AML blasts displayed a heightened stress granule response, pointing to a novel role for biomolecular condensates in adaptive oncogenic strategies.

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