Splicing Factor 3B Subunit 1 Mutations Research Articles

SF3B1 K700E Alters the Splicing of Metabolism and Stress Response Genes, Attenuating the Effect of DNMT3A Mutations in MDS

Myelodysplastic syndromes (MDS) are clonal stem cell disorders associated with low blood counts and a high risk of leukemic transformation. Genomic sequencing studies have demonstrated that MDS develops due to the acquisition of somatic mutations in hematopoietic stem cells (HSCs). Splicing factor 3b subunit 1 ( SF3B1) mutations, one of the most frequent mutations in MDS, are associated with lower-risk disease characterized by ineffective erythropoiesis. Our group and others have shown that mutations in SF3B1 cause aberrant mRNA splicing in human and murine HSCs, an expansion of long-term HSCs (LT-HSCs) in primary mice, but a competitive disadvantage in transplantation assays. DNMT3A, de novo methyltransferase 3A, is an epigenetic regulator mutated in ~10-15% of MDS. DNMT3A mutations are associated with unfavorable outcomes in MDS including a higher risk of leukemia transformation and shorter overall survival. Murine models of Dnmt3a mutations revealed that Dnmt3a-mutant HSCs have increased self-renewal over lineage-specific differentiation. SF3B1 and DNMT3A mutations co-occur more frequently than expected by chance and arise early in MDS pathogenesis, making them attractive therapeutic targets. MDS patients with SF3B1 and DNMT3A mutations have shorter progression-free survival than those with solely SF3B1 mutations. However, the mechanisms by which these mutations cooperate to cause HSC dysfunction and their possible combined role in leukemogenesis are not well understood. To investigate how Dnmt3a and Sf3b1 mutations disrupt HSC function in vivo we crossed two conditional knock-in mouse models of common point mutations in SF3B1 (K700E) and DNMT3A (R787H, murine equivalent to human DNMT3A R882H). We followed monthly blood counts and evaluated stem and progenitor cell composition in the bone marrow at 6 months. We found that Sf3b1/Dnmt3a double mutant mice display a similar phenotype to Sf3b1K700E mice, with a progressive anemia and an expansion in LT-HSCs. However, when assayed for HSC self-renewal potential, whole bone marrow and LT-HSCs isolated from Sf3b1/Dnmt3a mutant mice display increased serial colony formation capacities similar to Dnmt3a mutant hematopoietic stem and progenitor cells (HSPCs, 5 to 6 passages), but have a competitive disadvantage in secondary and tertiary transplant assays that is less severe than Sf3b1K700E HSPCs but distinct from the competitive advantage of Dnmt3a mutant HSPCs. Transcriptionally, double mutant HSCs clustered closer to Sf3b1 HSCs with shared expression of 74% of upregulated genes and 64% of downregulated genes and have decreased expression of stress response and metabolism genes. These results indicate Sf3b1 mutations may mitigate the preleukemic phenotype due to Dnmt3a mutations in MDS. To identify factors regulated by SF3B1 mutations that may mitigate the effect of DNMT3A loss on leukemic progression, we generated isogenic DNMT3A KO and SF3B1 K700E single and double mutant K562 cell lines using CRISPR/Cas9. Transcriptome analysis demonstrated that SF3B1K700E/DNMT3AKO double mutant clones clustered with and shared similar upregulated (69%) and downregulated (62%) genes as SF3B1 single mutant clones. Similar to our findings in murine HSPCs, pathway analysis of SF3B1 and SF3B1/DNMT3A mutant clones displayed enrichment of stress response and metabolic pathways. Alternative mRNA splicing analysis identified 13 events (3 alternative 3' splice site, 9 intron retention, and 1 skipped exon) in 8 genes that were upregulated in DNMT3A KO clones and downregulated in SF3B1 and SF3B1/DNMT3A mutant clones. We confirmed decreased protein expression of these mis-spliced genes in SF3B1 single and double mutant clones, but not DNMT3A KO clones. These genes include components of the integrated stress response pathway, which are also mis-spliced in SF3B1 and SF3B1/DNMT3A mutant MDS patient samples. Taken together our data suggest that aberrant splicing by mutant SF3B1 prevents metabolic reprogramming and decreases leukemic progression caused by DNMT3A mutation in MDS.

SF3B1 mutation-mediated sensitization to H3B-8800 splicing inhibitor in chronic lymphocytic leukemia.

Splicing factor 3B subunit 1 (SF3B1) is involved in pre-mRNA branch site recognition and is the target of antitumor-splicing inhibitors. Mutations in SF3B1 are observed in 15% of patients with chronic lymphocytic leukemia (CLL) and are associated with poor prognosis, but their pathogenic mechanisms remain poorly understood. Using deep RNA-sequencing data from 298 CLL tumor samples and isogenic SF3B1 WT and K700E-mutated CLL cell lines, we characterize targets and pre-mRNA sequence features associated with the selection of cryptic 3' splice sites upon SF3B1 mutation, including an event in the MAP3K7 gene relevant for activation of NF-κB signaling. Using the H3B-8800 splicing modulator, we show, for the first time in CLL, cytotoxic effects in vitro in primary CLL samples and in SF3B1-mutated isogenic CLL cell lines, accompanied by major splicing changes and delayed leukemic infiltration in a CLL xenotransplant mouse model. H3B-8800 displayed preferential lethality towards SF3B1-mutated cells and synergism with the BCL2 inhibitor venetoclax, supporting the potential use of SF3B1 inhibitors as a novel therapeutic strategy in CLL.

Expression of circular RNAs in myelodysplastic neoplasms and their association with mutations in the splicing factor gene SF3B1.

Mutations in the splicing factor 3b subunit 1 (SF3B1) gene are frequent in myelodysplastic neoplasms (MDS). Because the splicing process is involved in the production of circular RNAs (circRNAs), we investigated the impact of SF3B1 mutations on circRNA processing. Using RNA sequencing, we measured circRNA expression in CD34+ bone marrow MDS cells. We defined circRNAs deregulated in a heterogeneous group of MDS patients and described increased circRNA formation in higher-risk MDS. We showed that the presence of SF3B1 mutations did not affect the global production of circRNAs; however, deregulation of specific circRNAs was observed. Particularly, we demonstrated that strong upregulation of circRNAs processed from the zinc finger E-box binding homeobox 1 (ZEB1) transcription factor; this upregulation was exclusive to SF3B1-mutated patients and was not observed in those with mutations in other splicing factors or other recurrently mutated genes, or with other clinical variables. Furthermore, we focused on the most upregulated ZEB1-circRNA, hsa_circ_0000228, and, by its knockdown, we demonstrated that its expression is related to mitochondrial activity. Using microRNA analyses,weproposed miR-1248 as a direct target of hsa_circ_0000228. To conclude, we demonstrated that mutated SF3B1 leads to deregulation of ZEB1-circRNAs, potentially contributing to the defects in mitochondrial metabolism observed in SF3B1-mutated MDS.

Co-mutation of ASXL1 and SF3B1 Predicts Poorer Overall Survival Than Isolated ASXL1 or SF3B1 Mutations.

Mutations in the ASXL transcriptional regulator 1 (ASXL1) and splicing factor 3b subunit 1(SF3B1) genes are commonly observed in myeloid neoplasms and are independent predicative factors for overall survival (OS). Only a few contradictory reports exist on the clinical significance of concurrent ASXL1 and SF3B1 mutations. Previous studies also did not exclude patients with mutations of other genes, which could be confounding factors. We identified 69 patients with mutation of only ASXL1, 89 patients with mutation of only SF3B1, and 17 patients with mutations exclusively of both ASXL1 and SF3B1 from our database of 8,285 patients and compared their clinical features and outcomes. Patients with ASXL1 mutations more frequently had acute myeloid leukemia (22.47%) or clonal cytopenia of unknown significance than patients with SF3B1 mutations (1.45%) or with ASXL1/SF3B1 mutations (11.76%). Patients with SF3B1 or ASXL1/SF3B1 mutations were more frequently diagnosed with myelodysplastic syndrome (75.36% and 64.71%, respectively) than patients with ASXL1 mutations (24.72%). Patients with ASXL1/SF3B1 (23.53%) mutations more frequently had myelodysplastic/myeloid proliferative neoplasm than did patients with ASXL1 mutations (5.62%) or with SF3B1 mutations (15.94%). OS of the ASXL1 mutation-only group was worse than that of the SF3B1 mutation-only group with a hazard ratio of 5.83 (p=0.017). Finally, and most importantly, the OS of the ASXL1/SF3B1 co-mutation group was poorer than that of both single-mutation groups (p=0.005). ASXL1/SF3B1 co-mutations portend worse OS than isolated ASXL1 or SF3B1 mutations, which might be due to abnormalities in both the epigenetic-regulatory and RNA-splicing pathways or because two genes instead of one are mutated.

Co-Existing ASXL1 and SF3B1 Mutations Carried Worse Outcome Than ASXL1 or SF3B1 Mutations Alone

Introduction: Somatic mutations in the ASXL1 gene (additional sex combs like 1) are frequently observed in myeloid malignancies, including Clonal cytopenia of unknown significance (CCUS), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), MDS/MPN, and acute myeloid leukemia (AML), and have been found to be independent predicative factor for poor overall survival. Mutations in SF3B1 (Splicing factor 3B subunit 1) were also frequently detected in the above hematopoietic disorders. It represents distinct molecular and clinical patient groups and shows particular association with ringed sideroblasts. Most studies have associated SF3B1 mutations with good clinical prognosis. Both ASXL1 and SF3B1 mutations have been reported to frequently coexist with the mutations in other genes. Co-mutation of ASXL1 with SF3B1 is less frequent and has even been reported to be mutually exclusive by some authors. There are very few and contradictory reports about the clinical significance of co-existing ASXL1 and SF3B1 mutations, which is the goal of this study. Previous studies were also confounded by patients with mutations in some other genes. Method: We identified 175 patients with only ASXL1 mutations, only SF3B1 mutations, or only ASXL1/SF3B1 co-mutations from our database of 8285 patients with NextGen sequencing (NGS) results, to study and compare their clinical features and outcomes. Baseline characteristics and other somatic mutation distribution were compared by chi square (categorical variables) and t-test (continuous variables). Overall survival was calculated using the Kaplan-Meier method log-rank test. Results: Approximately 8285 patients with diagnosed or suspected myeloid neoplasm were tested by NGS. There were1515 patients with ASXL1 mutations with or without other genes mutated, and 583 patients with SF3B1 mutations with or without other genes mutated. There were also 97 patients with concurrent ASXL1 and SF3B1 mutations, most with additional genes mutated. Out of these patients, we identified 69 patients with only ASXL1 mutations (ASXL1 group), 89 patients with only SF3B1 mutations (SF3B1 group), and 17 patients with only ASXL1 plus SF3B1 mutations (ASXL1/SF3B1 group). The median age of ASXL1 group (69) was much younger than SF3B1 (76) and ASXL1/SF3B1 group (p values 0.01208 and 0.0001, respectively). The female to male ratios were similar among these groups. ASXL1 mutations were more frequent in AML and CCUS (22.47% and 33.71%) than ASXL1/SF3B1 (11.76% and 0%) and SF3B1 (1.45% and 7.25%) (p values < 0.003) mutations. On the contrary, SF3B1 and ASXL1/SF3B1 mutations were more frequent in MDS patients (75.36% and 64.71%) than ASXL1 mutations. In MDS/MPN, ASXL1/SF3B1 mutations (23.53%) were more frequent than SF3B1 (15.94%) and ASXL1 mutations (5.62%, p value 0.03513). The three groups of patients showed similar cytogenetic changes, with similar percentages of patients with normal karyotype, del(5q), del(7/7q), del(17p/TP53), del(20q), or trisomy 8. Finally, we compared the overall survival of the three groups of patients with normal karyotype by Kaplan-Meier survival analysis. As expected, ASXL1 group showed worse overall survival than SF3B1 group with a hazard ratio of 5.834 (p value 0.0175). However, it is interesting to found that ASXL1/SF3B1 group showed poorer overall survival than both SF3B1 and ASXL1 groups with a hazard ratio of 9.840 (p value 0.0052) (Figure 1). Conclusion: Co-existing ASXL1 and SF3B1 mutations carried worse outcome than ASXL1 or SF3B1 mutation alone, likely due to abnormalities in both epigenetic regulatory pathway and RNA splicing pathway. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Exploring the mechanistic link between SF3B1 mutation and ring sideroblast formation in myelodysplastic syndrome

Acquired sideroblastic anemia, characterized by bone marrow ring sideroblasts (RS), is predominantly associated with myelodysplastic syndrome (MDS). Although somatic mutations in splicing factor 3b subunit 1 (SF3B1), which is involved in the RNA splicing machinery, are frequently found in MDS-RS, the detailed mechanism contributing to RS formation is unknown. To explore the mechanism, we established human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) cells stably expressing SF3B1K700E. SF3B1K700E expressing cells showed higher proportion of RS than the control cells along with erythroid differentiation, indicating the direct contribution of mutant SF3B1 expression in erythroblasts to RS formation. In SF3B1K700E expressing cells, ABCB7 and ALAS2, known causative genes for congenital sideroblastic anemia, were downregulated. Additionally, mis-splicing of ABCB7 was observed in SF3B1K700E expressing cells. ABCB7-knockdown HUDEP-2 cells revealed an increased frequency of RS formation along with erythroid differentiation, demonstrating the direct molecular link between ABCB7 defects and RS formation. ALAS2 protein levels were obviously decreased in ABCB7-knockdown cells, indicating decreased ALAS2 translation owing to impaired Fe–S cluster export by ABCB7 defects. Finally, RNA-seq analysis of MDS clinical samples demonstrated decreased expression of ABCB7 by the SF3B1 mutation. Our findings contribute to the elucidation of the complex mechanisms of RS formation in MDS-RS.

Impact of SF3B1 mutation on outcomes in myelofibrosis.

e19080 Background: Splicing factor 3B subunit 1 ( SF3B1) mutations have been shown to confer a unique phenotype in MDS and MDS/MPN overlap syndromes, with ring sideroblasts, thrombocytosis and favorable prognosis. In myelofibrosis (MF) the frequency of SF3B1 mutation is &lt;10% and may play a less important role in disease outcomes (Lasho et. al, Leukemia, 2011). Methods: We retrospectively analyzed all patients (pts) with WHO-defined MF (primary, including pre-fibrotic MF, or progressed from PV or ET) seen at our center from Jan. 2017 through Jul. 2021. We compared disease phenotypes, MPN driver and co-occurring mutations, cytogenetics, dynamic IPSS (DIPSS) score, transfusion requirements, treatment characteristics and survival outcomes between patients with SF3B1-mutated ( SF3B1+) and -wild type ( SF3B1-) MF. Results: A total of 381 pts were identified, 29 (8%) of whom were SF3B1+. There were similar frequencies of JAK2, CALR, MPL mutations and “triple negative” MF in the 2 groups (Table). The median number of SF3B1 mutations was 1 (range, 1-2); K666N being the most common (52%). The 2 groups were similar in regard to their baseline hemoglobin, white blood cell counts, platelets, co-occurring mutations, symptom burden and DIPSS but more SF3B1+ pts were PRBC transfusion-dependent at presentation than SF3B1- pts (38% vs. 15%, p=0.003). At a median follow up of 17.2 months for the entire cohort (range, 0.1- 52.2 mos., 22.6 mos. for SF3B1+ and 16.8 mos. for SF3B1-), 41 pts had died [14% SF3B1+ and 10% SF3B1-, p=0.5] and 7 pts (1 SF3B1+ and 6 SF3B1-) had leukemic transformation. The median estimated OS was 230 months for SF3B1- vs. not reached for SF3B1+ pts (p = 0.6). Conclusions: SF3B1 mutation is an uncommon event in MF and does not substantially affect disease phenotype and outcomes. MF pts with SF3B1 mutation are more likely to be PRBC transfusion dependent.[Table: see text]

Recent advances in the knowledge of sideroblastic anemia

Sideroblastic anemias (SAs) are a group of heterogeneous congenital and acquired disorders characterized by anemia and presence of ring sideroblasts in the bone marrow. Congenital SA is a rare condition caused by mutations of genes involved in heme biosynthesis, iron-sulfur cluster biosynthesis, and mitochondrial protein synthesis. SAs can also occur following exposure to certain drugs or alcohol or caused by copper deficiency (secondary SA). SAs have been found to be associated with myelodysplastic syndrome (idiopathic SA), which strongly correlates with specific somatic mutations in SF3B1 (splicing factor 3b subunit 1), involved in the RNA splicing machinery. The recent widespread use of genome-editing technology and next-generation sequencing has led to a better understanding of the molecular pathophysiology of SAs. This review discusses the current understanding of the pathophysiology of SAs.

Screening of drugs that selectively inhibit uveal melanoma cells with SF3B1 mutations

To screen compounds that can selectively inhibit uveal melanoma cells with splicing factor 3B subunit 1 (SF3B1) mutations in comparison with isogenic SF3B1 wild-type counterparts in a cell model of SF3B1 mutant allele knockout. Principal component analysis was used to analyze transcriptome alternative splicing in TCGA cohorts of uveal melanoma with wild-type SF3B1 and SF3B1 mutations, and abnormal alternative splicing events derived from SF3B1 mutations were identified. The SF3B1 mutant allele in Mel202 cells was knocked out using CRISPR-Cas9 technology, and Sanger sequencing was used to verify the edited sequence. MTT and colony formation assays were used to assess the proliferation of Mel202 and Mut-KO cells. RT-PCR agarose electrophoresis combined with Sanger sequencing was used to determine alternative splicing events in Mel202 and Mut-KO cells. MTT assay was performed to screen the compounds that showed selective inhibitory effect against Mel202 cells with SF3B1 mutation. Specific knockout of SF3B1 mutant allele in Mel202 cells obviously promoted the cell proliferation and caused changes in alternative splicing of ZDHHC16 and DYNLL1 transcripts. The screening data showed that 13 compounds had selective inhibitory activity against Mel202 cells with SF3B1 mutation (Fold change≥2), and among them, tetrandrine and lapatinib showed good dose-effect curves. This study provides a cell screening model for identification of potential individualized treatment drugs for patients with uveal melanoma with SF3B1 mutation.

Co-occurrence of BAP1 and SF3B1 mutations in uveal melanoma induces cellular senescence.

Uveal melanoma (UM) is the most common intraocular tumor in adults. Recurrent mutations in BRCA1‐associated protein 1 (BAP1) and splicing factor 3B subunit 1 (SF3B1) display a mutually exclusive pattern in UM, but the underlying mechanism is unknown. We show that combined BAP1 deficiency and SF3B1 hotspot mutation lead to senescence and growth arrest in human UM cells. Although p53 protein expression is induced, deletion of TP53 (encoding p53) only modestly rescues the observed senescent phenotype. UM cells with BAP1 loss or SF3B1 mutation are more sensitive to chemotherapeutic drugs compared with their isogenic parental cells. Transcriptome analysis shows that DNA‐repair genes are downregulated upon co‐occurrence of BAP1 deletion and SF3B1 mutation, thus leading to impaired DNA damage response and the induction of senescence. The co‐occurrence of these two mutations reduces invasion of UM cells in zebrafish xenograft models and suppresses growth of melanoma xenografts in nude mice. Our findings provide a mechanistic explanation for the mutual exclusivity of BAP1 and SF3B1 mutations in human UM.

Single-Cell Multi-Omics Defines the Cell-Type Specific Impact of SF3B1 Splicing Factor Mutations on Hematopoietic Differentiation in Human Clonal Hematopoiesis and Myelodysplastic Syndromes

Splicing factor mutations are recurrent genetic alterations in blood disorders, highlighting the importance of alternative splicing regulation in hematopoiesis. Specifically, mutations in splicing factor 3B subunit 1 (SF3B1) are implicated in the pathogenesis of myelodysplastic syndromes (MDS) and linked to a high-risk of leukemic transformation in clonal hematopoiesis (CH). SF3B1 mutations are associated with aberrant RNA splicing, leading to increased cryptic 3' splice site (ss) usage and MDS with ring sideroblasts phenotype.The study of mutant SF3B1-driven splicing aberrations in humans has been hampered by the inability to distinguish mutant and wildtype single cells in patient samples and the inadequate coverage of short-read sequencing over splice junctions. To overcome these limitations, we developed GoT-Splice by integrating Genotyping of Transcriptomes (GoT; Nam et al. 2019) with Nanopore long-read single-cell transcriptome profiling and CITE-seq (Fig. A). This allowed for the simultaneous single-cell profiling of protein and gene expression, somatic mutation status, and alternative splicing. Our method selectively enriched full-length sequencing reads with the accurate structure, enabling the capture of higher number of junctions per cell and greater coverage uniformity vs. short-read sequencing (10x Genomics; Fig. B, C).We applied GoT-Splice to CD34+ bone marrow progenitor cells from MDS (n = 15,436 cells across 3 patients; VAF: [0.38-0.4]) to study how SF3B1 mutations corrupt human hematopoiesis (Fig. D). High-resolution mapping of SF3B1 mut vs. SF3B1 wt hematopoietic progenitors revealed an increasing fitness advantage of SF3B1 mut cells towards the megakaryocytic-erythroid lineage, resulting in an expansion of SF3B1 muterythroid progenitor (EP) cells (Fig. E, F). Accordingly, SF3B1 mutEP cells displayed higher protein expression of erythroid lineage markers, CD71 and CD36, vs. SF3B1 wt cells (Fig. G). In these SF3B1 mutEP cells, we identified up-regulation of genes involved in regulation of cell cycle and checkpoint controls (e.g., CCNE1, TP53), and mRNA translation (eIFs gene family; Fig. H).Next, while SF3B1 mut cells showed the expected increase of cryptic 3' splicing vs. SF3B1 wt cells (Fig. I), they exhibited distinct cryptic 3' ss usage as a function of hematopoietic progenitor cell identity, displaying stage-specific aberrant splicing during erythroid maturation (Fig. J). In less differentiated EP cells, we observed mis-splicing of genes involved in iron homeostasis, such as the hypoxia-inducible factor HIF1A, and key regulators of erythroid cell growth, such as SEPT2. At later stages, we observed mis-splicing of BAX, a pro-apoptotic member of the Bcl-2 gene family and transcriptional target of p53, and erythroid-specific genes (e.g., PPOX). We further predicted 54% of the aberrantly spliced mRNAs to introduce premature stop codons, promoting RNA degradation through nonsense-mediated decay (NMD). In line with this notion, we observed a significant decrease in expression of NMD-inducing genes in SF3B1 mut vs . SF3B1 wt EP cells (Fig. K).Lastly, splicing factor mutations observed in CH subjects provide an opportunity to interrogate the downstream impact of SF3B1 mutations prior to development of disease. Like MDS, by applying GoT-splice to CD34+ progenitor cells from SF3B1 mut CH subjects (n = 9,007 cells across 2 subjects; VAF: [0.15-0.22]; Fig. L), we revealed increased mutant cell frequency in EP cells (Fig. M) with concomitant increased expression of genes involved in mRNA translation (Fig. N), consistent with SF3B1 mutation causing mis-splicing injury to translational machinery and ineffective erythropoiesis. Notably, CH patients already exhibited cell-type specific cryptic 3' ss usage in SF3B1 mut cells (Fig. O).In summary, we developed a novel multi-omics single-cell toolkit to examine the impact of splicing factor mutations on cellular fitness directly in human samples. With this approach, we showed that, while SF3B1 mutations arise in uncommitted HSCs, their effect on fitness increases with differentiation into committed EPs, in line with the mutant SF3B1-driven dyserythropoiesis phenotype. We revealed that SF3B1 mutations exert cell-type specific mis-splicing that leads to abnormal erythropoiesis. Finally, we demonstrated that the impact of SF3B1 mutations on EP cells begins before disease onset, as observed in CH subjects. [Display omitted] DisclosuresDai: Oxford Nanopore Technologies: Current Employment. Beaulaurier: Oxford Nanopore Technologies: Current Employment. Drong: Oxford Nanopore Technologies: Current Employment. Hickey: Oxford Nanopore Technologies: Current Employment. Juul: Oxford Nanopore Technologies: Current Employment. Wiseman: Astex: Research Funding; Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Takeda: Consultancy; StemLine: Consultancy. Harrington: Oxford Nanopore Technologies: Current Employment. Ghobrial: AbbVie, Adaptive, Aptitude Health, BMS, Cellectar, Curio Science, Genetch, Janssen, Janssen Central American and Caribbean, Karyopharm, Medscape, Oncopeptides, Sanofi, Takeda, The Binding Site, GNS, GSK: Consultancy. Abdel-Wahab: H3B Biomedicine: Consultancy, Research Funding; Foundation Medicine Inc: Consultancy; Merck: Consultancy; Prelude Therapeutics: Consultancy; LOXO Oncology: Consultancy, Research Funding; Lilly: Consultancy; AIChemy: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Envisagenics Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Circular RNAs in Myelodysplastic Syndromes and Impact of SF3B1 Mutations on Their Expression

Myelodysplastic syndromes (MDS) are a heterogeneous group of diseases with a high risk of transformation to acute myeloid leukemia (AML). One of the processes implicated in MDS pathogenesis is RNA splicing. Its alterations are caused by somatic mutations in splicing factor genes. Mutations in SF3B1 (Splicing Factor 3b Subunit 1) gene are the most frequently found mutations in MDS. Circular RNAs (circRNAs) are covalently closed RNAs that are produced by back-splicing process. CircRNAs can regulate multiple biological processes through various molecular mechanisms, such as microRNA sponging. Their deregulation is frequently found in cancer. It is likely that they also contribute to the development of MDS, however, their role in MDS has not been researched yet. Therefore, our aim was to explore circRNA levels in MDS and analyze their association with patient prognosis. We further hypothesized that mutations in splicing factor genes can affect production of circRNAs and thus, we examined circRNA levels with respect to the mutational status. We explored transcriptome of 78 MDS patients, 7 AML patients, and 13 healthy donors using Illumina RNA-seq of total RNA isolated from CD34+ bone marrow cells. To associate circRNA levels with mutational status, Illumina TruSight Myeloid Sequencing Panel Kit examining 54 genes was applied. Of 8,620 circRNAs identified by RNA-seq, 204 circRNAs were deregulated in MDS (e.g., MENTRNL, EBF1, and PPM1L-derived circRNAs) and 246 circRNAs were altered between lower- and higher-risk patients (e.g., CHST15, TMTC2, and PDE3B-derived circRNAs). Most of the progression-related circRNAs (n = 234) showed elevated levels in higher-risk patients, suggesting that the back-splicing process might be stimulated during the disease progression. In MDS patients with SF3B1 mutations, other 40 circRNAs were deregulated (e.g., ZNF91, ZEB1, and ZNF124-derived circRNAs). This circRNA profile was substantially different from the profiles associated with the rest of recurrently mutated splicing factor genes (SRSF2, U2AF1, and ZRSR2). To study alterations in forward- and back-splicing in SF3B1-mutated patients, we examined transcriptional differences on the levels of whole genes, transcript variants, and circRNAs and searched for specificities in transcription within individual gene loci. A set of circRNAs whose levels differed specifically without affecting expression of corresponding forward-spliced mRNAs included several oncology/hematopoiesis-relevant genes (e.g., ATM, CBL, ERCC5, ETV6, FLT3, and MAPK6). Gene loci with changed expression of both, alternative mRNA transcripts and circRNAs, but stable transcription on whole gene level included for example CDK14, KDM1A, and ZEB1. Because ZEB1 (Zinc Finger E-Box Binding Homeobox 1) serves as an essential hematopoietic transcription factor, we focused on ZEB1-derived circRNAs (hsa_circ_0000228 and hsa_circ_0003793) in more detail. We demonstrated that upregulation of these circRNAs is SF3B1-specific and not related to any other clinical or molecular characteristics. Finally, using RNA-seq data from CRISPR/Cas9 edited K562 cells (Liberante FG, et al., Sci Rep. 2019; 9:2678), we confirmed that SF3B1 K700E mutation leads to strong upregulation of ZEB1 circRNAs. To conclude, this is an early report showing for the first time that the levels of specific circRNAs are altered in MDS. We demonstrated that particular circRNAs may have potential to become markers that would contribute to more accurate prognosis of MDS patients. Further, we identified circRNAs with deregulated levels specifically in MDS patients with SF3B1 mutation, suggesting that this mutation affects circRNA production.Supported by GA CR (N20-19162S) and MH CZ-DRO (UHKT, 00023736). DisclosuresNo relevant conflicts of interest to declare.

Application of AI-Based Analytical Approaches for Multiplex Immunophenotyping of SF3B1-Mutant Myelodysplastic Syndrome Subtype

Application of AI-Based Analytical Approaches for Multiplex Immunophenotyping of SF3B1-Mutant Myelodysplastic Syndrome Subtype

Altered DNA Methylation Profiles in SF3B1 Mutated CLL Patients.

Mutations in splicing factor genes have a severe impact on the survival of cancer patients. Splicing factor 3b subunit 1 (SF3B1) is one of the most frequently mutated genes in chronic lymphocytic leukemia (CLL); patients carrying these mutations have a poor prognosis. Since the splicing machinery and the epigenome are closely interconnected, we investigated whether these alterations may affect the epigenomes of CLL patients. While an overall hypomethylation during CLL carcinogenesis has been observed, the interplay between the epigenetic stage of the originating B cells and SF3B1 mutations, and the subsequent effect of the mutations on methylation alterations in CLL, have not been investigated. We profiled the genome-wide DNA methylation patterns of 27 CLL patients with and without SF3B1 mutations and identified local decreases in methylation levels in SF3B1mut CLL patients at 67 genomic regions, mostly in proximity to telomeric regions. These differentially methylated regions (DMRs) were enriched in gene bodies of cancer-related signaling genes, e.g., NOTCH1, HTRA3, and BCL9L. In our study, SF3B1 mutations exclusively emerged in two out of three epigenetic stages of the originating B cells. However, not all the DMRs could be associated with the methylation programming of B cells during development, suggesting that mutations in SF3B1 cause additional epigenetic aberrations during carcinogenesis.

Dynamic BH3 Profiling to Assess the Effects of Novel Agents on Anti-Apoptotic Protein Dependence of CLL Cells

IntroductionOur group previously developed dynamic BH3 profiling (DBP) (Montero el al, 2015), which measures early changes in net pro-apoptotic signaling of mitochondria in response to various drug combinations. The degree of change in the propensity to undergo apoptosis is known as “delta priming”. We recently used DBP to demonstrate that BTK inhibition with ibrutinib leads to increased BCL-2 dependence and enhanced sensitivity to the BCL-2 inhibitor venetoclax in CLL cells (Deng et al., 2017). The effects of other novel agents on BCL-2 dependency in CLL cells relative to other anti-apoptotic family members such as MCL-1 and BCL-XL have yet to be systematically explored.Pathways that are recurrently mutated in CLL represent a logical next area in which to explore the potential of DBP to study the effects of novel agents on the BCL-2 family. One such gene is splicing factor 3B subunit 1 (SF3B1), which is recurrently mutated in a subset of patients with CLL, and confers an adverse prognosis. Here, we treated CLL cells ex vivo with a panel of novel agents including the splice modulator E7107, and utilized DBP to evaluate the functional effects of drug treatments on BCL-2 family proteins.MethodsMononuclear cells were isolated from 13 CLL patients and treated ex vivo with E7107 (H3 Biomedicine) and/or one of several other drugs (all obtained commercially). Cells were co-cultured with the human stroma NKTert cell line (1:10 ratio) with 20-hour drug treatments. DBP was performed as previously reported by measuring the release of cytochrome C (cyto-C) in gently permeabilized CLL cells in response to BH3-only peptides using a BD FACS Fortessa. Cyto-C release in response to BAD BH3 peptide was used as a measure of BCL-2 dependence, while cyto-C release in response to HRK and MS-1 BH3 peptide was used as a measure of BCL-XL and MCL-1 dependence, respectively. Western blot analyses were performed by standard protocols with signals captured by an LAS 4000 imager. Statistical analyses were by Welch t-test and one-sample t-test, with a two-tailed nominal p≤0.05 considered as significant.ResultsAs an initial screen, we treated cells from two different CLL patients in duplicate with a panel of 13 drugs and found that 5 agents resulted in increased BCL-2 dependence (defined as at least 35% cyto C release in both patients) (Fig. 1). These included E7107, ibrutinib, fludarabine, LY2784544 (JAK2 inhibitor), and AZD8055 (AKT inhibitor). As the greatest increase in BCL-2 dependence was observed with E7107, we next explored its effects on 11 additional CLL patient samples (6 SF3B1mut and 5 SF3B1wt). Significantly higher baseline BCL-2 dependence was observed in untreated SF3B1mut cells compared to SF3B1wt (p=0.02) (Fig. 2A). Treatment with E7107 alone or in combination with venetoclax increased BCL-2 dependence of CLL cells irrespective of SF3B1 mutation status (p values between 0.01-0.02). Venetoclax treatment alone had no effect on BCL-2 dependence in either group (p=0.39, p=0.68). In all samples, E7107 treatment made CLL cells significantly more BCL-2 dependent (BAD BH3 peptide, p=0.0009) and less MCL-1 dependent (MS-1 BH3 peptide, p<0.0001), but did not affect BCL-XL dependence (HRK BH3 peptide, p=0.11) (Fig. 2B). Consistent with these findings, CLL cells treated with E7107 had uniformly decreased MCL-1 protein expression on Western blot compared to untreated samples across all patient samples.ConclusionWe demonstrate the feasibility of using DBP to examine the effects of a panel of novel agents on BCL-2 family anti-apoptotic protein dependence of CLL cells. We identified several candidate drugs to explore in future studies, including the splice modulator E7107, which consistently increased BCL-2 dependence while reciprocally decreasing MCL-1 dependence. These preliminary results support the exploration of DBP in a high throughput format on a larger panel of drugs across more patient samples, with a goal of prioritizing the combinations that appear most promising to explore in clinical trials, to develop more personalized treatment for patients with CLL. [Display omitted] DisclosuresValentin:Abbvie: Other: Travel reimbursement; Roche: Other: Travel reimbursement. Buonamici:H3 Biomedicine, Inc.: Employment. Neuberg:Synta Pharmaceuticals: Other: Stock shares. Wu:Neon Therapeutics: Consultancy. Letai:AbbVie, AstraZeneca, Novartis: Consultancy, Research Funding. Davids:Merck: Consultancy; InCyte: Membership on an entity's Board of Directors or advisory committees; Infinity: Consultancy, Research Funding; Celgene: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra-Zeneca: Consultancy.

SF3B1 mutation in pancreatic cancer contributes to aerobic glycolysis and tumor growth through a PP2A-c-Myc axis.

Hot spot gene mutations in splicing factor 3b subunit 1 (SF3B1) are observed in many types of cancer and create abundant aberrant mRNA splicing, which is profoundly implicated in tumorigenesis. Here, we identified that the SF3B1 K700E (SF3B1K700E) mutation is strongly associated with tumor growth in pancreatic ductal adenocarcinoma (PDAC). Knockdown of SF3B1 significantly retarded cell proliferation and tumor growth in a cell line (Panc05.04) with the SF3B1K700E mutation. However, SF3B1 knockdown had no notable effect on cell proliferation in two cell lines (BxPC3 and AsPC1) carrying wild‐type SF3B1. Ectopic expression of SF3B1K700E but not SF3B1WT in SF3B1‐knockout Panc05.04 cells largely restored the inhibitory role induced by SF3B1 knockdown. Introduction of the SF3B1K700E mutation in BxPC3 and AsPC1 cells also boosted cell proliferation. Gene set enrichment analysis demonstrated a close correlation between SF3B1 mutation and aerobic glycolysis. Functional analyses showed that the SF3B1K700E mutation promoted tumor glycolysis, as evidenced by glucose consumption, lactate release, and extracellular acidification rate. Mechanistically, the SF3B1 mutation promoted the aberrant splicing of PPP2R5A and led to the activation of the glycolytic regulator c‐Myc via post‐translational regulation. Pharmacological activation of PP2A with FTY‐720 markedly compromised the growth advantage induced by the SF3B1K700E mutation in vitro and in vivo. Taken together, our data suggest a novel function for SF3B1 mutation in the Warburg effect, and this finding may offer a potential therapeutic strategy against PDAC with the SF3B1K700E mutation.

The Effect of SF3B1 Mutation on the DNA Damage Response and Nonsense-Mediated mRNA Decay in Cancer.

Recurrent mutations in splicing factor 3B subunit 1 (SF3B1) have been identified in several malignancies and are associated with an increased expression of 3’ cryptic transcripts as a result of alternative branchpoint recognition. A large fraction of cryptic transcripts associated with SF3B1 mutations is expected to be sensitive for RNA degradation via nonsense-mediated mRNA decay (NMD). Several studies indicated alterations in various signaling pathways in SF3B1-mutated cells, including an impaired DNA damage response (DDR) in chronic lymphocytic leukemia (CLL). In this study, we investigated isogenic cell lines and treatment naïve primary CLL samples without any TP53 and/or ATM defect, and found no significant effects of SF3B1 mutations on the ATM/p53 response, phosphorylation of H2AX and sensitivity to fludarabine. Cryptic transcripts associated with SF3B1 mutation status were observed at relatively low levels compared to the canonical transcripts and were validated as target for mRNA degradation via NMD. Expression of cryptic transcripts increased after NMD inhibition. In conclusion, our results confirm involvement of NMD in the biological effects of SF3B1 mutations. Further studies may elucidate whether SF3B1-mutant patients could benefit from NMD modulatory agents.

SF3B1 modulators affect key genes in metastasis and drug influx: a new approach to fight pancreatic cancer chemoresistance

Aim: Because mutations of splicing factor 3B subunit-1 (SF3B1) have been identified in 4% of pancreatic ductal adenocarcinoma (PDAC) patients, we investigated the activity of new potential inhibitors of SF3B1 in combination with gemcitabine, one of the standard drugs, in PDAC cell lines. Methods: One imidazo[2,1-b][1,3,4]thiadiazole derivative (IS1) and three indole derivatives (IS2, IS3 and IS4), selected by virtual screening from an in-house library, were evaluated by the sulforhodamine-B and wound healing assay for their cytotoxic and antimigratory activity in the PDAC cells SUIT-2, Hs766t and Panc05.04, the latter harbouring the SF3B1 mutations. The effects on the splicing pattern of proto-oncogene recepteur d’origine nantais (RON) and the gemcitabine transporter human equilibrative nucleoside transporter-1 (hENT1) were assessed by PCR, while the ability to reduce tumour volume was tested in spheroids of primary PDAC cells. Results: The potential SF3B1 modulators inhibited PDAC cell proliferation and prompted induction of cell death. All compounds showed an interesting anti-migratory ability, associated with splicing RON/ΔRON shift in SUIT-2 cells after 24 h exposure. Moreover, IS1 and IS4 potentiated the sensitivity to gemcitabine in both conventional 2D monolayer and 3D spheroid cultures, and these results might be explained by the statistically significant increase in hENT1 expression (P < 0.05 vs. untreated control cells), potentially reversing PDAC chemoresistance. Conclusion: These results support further studies on new SF3B1 inhibitors and the role of RON/hENT1 modulation to develop effective drug combinations against PDAC.

Clinico-Genomic Profiling and Clonal Dynamic Modeling of Spliceosome-Mutant Myelodysplastic Syndrome and Acute Myeloid Leukemia

Introduction: The identification of driver mutations has dramatically changed the management of myeloid malignancies. More recently, mutations have been identified in RNA splicing machinery in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) (Yoshida et al. 2011). Recurring missense mutations have been observed in the RNA splicing factor 3B subunit 1 (SF3B1), serine and arginine-rich splicing factor (SRSF2) and U2 small nuclear RNA auxiliary factor 1 (U2AF1). In this study, we sought to decipher the disease-specific features and clinical outcomes of individual spliceosome mutations in patients with MDS and AML. Methods: We conducted a retrospective observational study of 40 patients with one or more mutations in 3 spliceosome genes (SRSF2, U2AF1, SF3B1) from the University of Massachusetts (UMass) Leukemia Registry. Demographics, peripheral blood data, clinical parameters such as treatment offered including bone marrow transplant and response to chemotherapy was longitudinally recorded. We performed clonal dynamic modeling to identify clonal evolution in individual patients with spliceosome-mutant MDS or AML. Results: Longitudinal data was available for 83% (33/40) patients with an average follow up duration of 91 weeks. Average age at diagnosis was 75±4 years which was consistent across the 3 mutational cohorts. A mortality of ~35% was observed in our cohort (Table 2). MDS was diagnosed in 48% (19/40), AML and AML-MRC were diagnosed in 33% (13/40) and 13% (5/40), respectively (Table 1). SRSF2 was the most common spliceosome mutation (50%) and SF3B1 and U2AF1 were equally divided in the remainder. Cytogenetic abnormalities were found in 5/33 (15%) in SRSF2, 4/33 (12%) in U2AF1, and 1/33 (3%) in the SF3B1 groups. Of the 16 patients with SRSF2-mutant hematological conditions, 7/16 (40%) had persistent disease (PD) which was similar across the other two spliceosome genes. Compared to U2AF1- and SF3B1-mutant patients, SRSF2-mutant patients presented with a higher WBC count (mean 45.9 x 103/mcl ± 13.6 x 103/mcl), higher bone marrow (BM) cellularity (73% ± 4.4%), and higher BM blast percentage (24% ± 6.9%) at the time of diagnosis (Table 3), which was independent of the FLT3 status. The most commonly recurring co-occurring mutants with SRSF2 were TET2 (occurred 8/16 times along with SRSF2 with a rate of 50%), IDH2 (31%), STAG2 (25%) and RUNX1 (25%). In the U2AF1-mutant cohort, RUNX1 (30%) and TET2 (20%) were the most frequent co-mutants. In the SF3B1 cohort, BCOR (29%) and KRAS (29%) were the most frequently mutated and shared few co-occurring mutations with SFSR2-mutant and U2AF1-mutant cohorts as demonstrated in the circosplots (Figure 3). The heatmap further elaborates this association and alludes to a possible genetic signature associated with favorable or adverse clinical outcomes (Figure 2). Conclusion: This study deciphers among SRSF2-, U2AF1-, and SF3B1-mutant MDS/AML and demonstrates that SRSF2-mutant MDS/AML presents with higher WBC count, BM cellularity, and BM blast burden. SRSF2-mutant MDS/AML carries the most dismal prognosis amongst the spliceosome mutations. The co-mutational landscape of SF3B1-mutant MDS/AML is distinctly different from SRSF2 and U2AF1 cohort. While we only identified 1 individual with SF3B1 mutation with longitudinal data and hence no meaningful conclusion is possible, SRSF2 cohort shows poor response to standard chemotherapy in 2/3 individuals (Figure 1). We also found that BRAF and CEBPA are not co-occurring mutations within the spliceosome-mutant cohort, suggesting mutual exclusivity (Figure 2). While our understanding of MDS/AML expands, new drugs are constantly under development and have led to significant improvements in the disease outcome in younger patients. Seiler et al recently studied a new oral drug H3B-8800, which binds the SF3B complex in a competitive manner and has demonstrated potent inhibition and lethality of the spliceosome-mutant cells (Seiler et al. 2018). After demonstration of survival advantage in vivo in murine models, the drug is now in Phase 1 clinical trial which is pending completion soon. Our dataset provides clinical correlates of spliceosome-mutant AML/MDS in a longitudinal fashion and also alludes to a possible genetic signature associated with favorable or adverse outcomes in these patients. Disclosures No relevant conflicts of interest to declare.

The metabolic reprogramming and vulnerability of SF3B1 mutations

ABSTRACTMutations in the splicing factor 3b subunit 1 (SF3B1) gene create a neomorphic protein that disrupts RNA splicing, but the downstream consequences of this missplicing are unclear. Our recent study of isogenic human cells demonstrated that SF3B1MUT induces reprogramming of energy metabolism and a targetable vulnerability to deprivation of the nonessential amino acid serine.