The E592K variant of SF3B1 creates unique RNA missplicing and associates with high-risk MDS without ring sideroblasts

The E592K variant of SF3B1 creates unique RNA missplicing and associates with high-risk MDS without ring sideroblasts | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The E592K variant of SF3B1 creates unique RNA missplicing and associates with high-risk MDS without ring sideroblasts In Young Choi, Jonathan P. Ling, Jian Zhang, Eric Helmenstine, and 16 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2802265/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 May, 2024 Read the published version in Blood Advances → Version 1 posted You are reading this latest preprint version Abstract Among the most common genetic alterations in the myelodysplastic syndromes (MDS) are mutations in the spliceosome gene SF3B1 . Such mutations induce specific RNA missplicing events, directly promote ring sideroblast (RS) formation, generally associate with more favorable prognosis, and serve as a predictive biomarker of response to luspatercept. However, not all SF3B1 mutations are the same, and here we report that the E592K variant of SF3B1 associates with high-risk disease features in MDS, including a lack of RS, increased myeloblasts, a distinct co-mutation pattern, and decreased survival. Moreover, in contrast to canonical SF3B1 mutations, E592K induces a unique RNA missplicing pattern, retains an interaction with the splicing factor SUGP1 , and preserves normal RNA splicing of the sideroblastic anemia genes TMEM14C and ABCB7. These data expand our knowledge of the functional diversity of spliceosome mutations, and they suggest that patients with E592K should be approached differently from low-risk, luspatercept-responsive MDS patients with ring sideroblasts and canonical SF3B1 mutations. Health sciences/Diseases/Haematological diseases/Haematological cancer/Myelodysplastic syndrome Biological sciences/Cancer/Oncogenes Biological sciences/Genetics/Cancer genetics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction SF3B1 is the most mutated spliceosome gene in MDS, with a frequency of > 30% 1 . The mutations are primarily missense substitutions that induce neomorphic RNA missplicing in thousands of junctions, which in turn alter expression of hundreds of genes in diverse pathways 2 . This missplicing has been implicated in many MDS phenotypes, including dysfunctional iron metabolism, formation of ring sideroblasts, activation of innate immune signaling, and promotion of hematopoietic stem cell self-renewal 3 – 8 . SF3B1 mutations also contain prognostic value in MDS, associating with more indolent disease, though with important exceptions 9 – 12 . In MDS treatment, SF3B1 mutations are among the criteria used to determine eligibility for luspatercept, and they are the direct or indirect targets of investigational therapies 13 . Thus, SF3B1 mutations figure prominently in the research of—and clinical practice for—many MDS patients. Not well understood is whether—and how—distinct SF3B1 mutation hotspots differentially affect disease features and/or the RNA missplicing events that drive them. Previously, in an analysis of patients and cell models with SF3B1 exon 14–15 mutations, we found that the K666N variant was enriched in high-risk MDS and produced an asymmetrical lack of missplicing events that are induced by K700E and H662Q mutations 14 . Here we report the results of extending this approach to a larger cohort of patients that included exon 13–16 mutations and additional cell models. This analysis revealed a striking distinctiveness in the SF3B1 mutation E592K, which has implications for the understanding and management of SF3B1 -mutant MDS. Materials/subjects And Methods Patients Mutation-agnostic acquisition of SF3B1 mutations from MDS and AML cases and associated clinical parameters came from the Johns Hopkins Sidney Kimmel Cancer Center, the Vanderbilt-Ingram Cancer Center, the Chinese Academy of Medical Sciences, the Munich Leukemia Laboratory, the Allegheny Health Network Cancer Institute, Project Genie ( http://genie.cbioportal.org/ ), and manual extraction from 82 published studies (supplemental References) 15 . Additional E592K cases and their clinical parameters were deliberately obtained from The University of Manchester, Tong University Affiliated Sixth People's Hospital, Weill Cornell Medicine, and Memorial Sloan Kettering Cancer Center. Because breadth of gene panels varied among patients, co-mutation analysis included those cases in which at least a set of 35 genes were sequenced, representing a compromise between maximum sample inclusion and maximum gene inclusion. For EZH2 co-mutation analysis, cBioportal Oncoprinter ( https://www.cbioportal.org/oncoprinter ) was applied to all MDS cases from the MSK Myelodysplastic, Project GENIE, and IPSS-M cohorts 10 , 15 , 16 . For leukemia-free survival of E592K patients, the IPSS-M ( https://mds-risk-model.com ) and SEX-GSS ( https://mds.itb.cnr.it/#/mds/home ) calculators were used 10 , 11 . Use of deidentified patient data were approved by the Institutional Review Boards at the respective institutions. Cells HEK293T, TF1, and K562 cells were obtained from the ATCC. HEK293T cells were grown in DMEM/10% FBS, K562 cells were grown in RPMI/20% FBS, and TF1 cells were grown in RPMI/20% FBS with 2 ng/mL GM-CSF. STR cell line authentication and mycoplasma testing were done upon receipt and routinely thereafter, with last testing done 2/2022. Vectors WT and K700E FLAG- SF3B1 sequences were subcloned from Addgene plasmids 82576 and 82577 into pDONR-A-HYG (Addgene 29635) to make pENTR- SF3B1 -WT and pENTR- SF3B1 -K700E. Site-directed mutagenesis with overlap extension PCR then created pENTR plasmids for the E592K, E622D, K666R, K666N, R625H, K741N, and E902K variants of SF3B1 , and these were subcloned into lentiviral vector pLX301 (Addgene 25895). Plasmids used for SF3B1 affinity purification have been previously described 17 . Site-directed mutagenesis of the p3xFLAG-CMV-14-His6-FLAG-SF3B1 vector was done by overlap extension PCR to make the E592K vector. Transcriptome analysis For HEK293T cells, pLX301 plasmids were transfected using Lipofectamine 3000, 24h later cells were selected with puromycin for 48h, puro was washed out for 24h, and cells were harvested. For stable transduction, pLX301 lentivirus was produced as previously described 18 . TF1 cells were transduced and four independent clones per genotype were puro selected from single cells. For K562 cells, duplicate independent polyclonal populations per genotype were puro selected. RNA isolation, cDNA synthesis, endpoint PCR, and quantitative PCR were performed as described 18 . Primer sequences are in Supplementary Table 1. RNA-seq libraries from TF1 clones were constructed using TruSeq Stranded Total RNA Library Prep. Sequencing was performed on a NovaSeq S1 flowcell. Reads were aligned using STAR 19 . Splicing analysis was performed using ASCOT and gene expression using featureCounts 20 , 21 . Percent spliced in (PSI) values for junctions were determined by dividing inclusion split-read counts by the total split-read counts at the corresponding constitutive donor or acceptor sites, using a minimum coverage of 15 split-reads per junction. Junctions of interest were visualized from RNA-seq reads from primary MLL samples with the UCSC genome browser for 2 E592K, 10 E622D, 6 K666N, 9 K666R, 12 K700E, and 12 WT samples, using the ADD function (combining reads and normalizing track height for samples in each mutation group). Junction validation with endpoint PCR was done on independent bone marrow CD34 + MDS samples at Barts Cancer Institute. Affinity Purification of SF3B1 -Associated Proteins A small-scale protocol was applied to both HEK293T and TF1 cells as previously described, except that for TF1, 10 million cells were used and proteins were eluted with 30 µL (5 µg/µL) 3X FLAG peptide because cells had only one affinity tag (FLAG) attached to SF3B1 17 . Western Blotting For immunoblotting of SF3B1 /FLAG- SF3B1 proteins in transiently-transfected HEK293T and stably-transduced TF1 and K562 cells in which transcriptome analysis was done in parallel, Western blotting using a mouse anti-human- SF3B1 antibody (Abcam #172634) at 1:1000 dilution was used. Immunoblotting following affinity purification of SF3B1 in HEK293T and TF1 cells was performed as previously described, and primary antibodies were: anti-SF3B1 (Bethyl Laboratories, A300-996A, 1:1,000), anti-ACTIN (Sigma, A2066, 1:2,000), anti-DYKDDDDK (GenScript, A00187, 1:1,000), anti- SUGP1 (Bethyl Laboratories A304-675A-M, 1:1,000), and anti-PHF5A (Proteintech 15554-1-AP, 1:1000) 17 . Secondary antibodies were: Donkey anti-Rabbit IgG (LI-COR, 926-68073, 1:5,000) and Goat anti-Mouse IgG (LI-COR, 926-32210, 1:5,000). Results Combining patient data from five institutions, publicly available databases, and published literature, we established a dataset of 2,288 patients with SF3B1 -mutant MDS or AML in which exons 13 through 16 had been sequenced. We first determined how SF3B1 mutations partitioned into WHO 2016 classifications, as these data were available for virtually all patients. This distribution showed several asymmetries (Fig. 1 and Supplementary Fig. 1). Consistent with our previous report 14 , K666N was enriched in higher-risk disease types: only 2.1% (28/1327) in MDS-RS vs 8.7% (21/242) in MDS-SLD/MLD, 17.3% (46/266) in MDS-EB, and 25.8% (103/399) in AML (p < 0.0001 for all). With the inclusion of cases in which exon 13 had been sequenced, the distribution also revealed enrichment of a variant in this exon, E592K, in higher-risk disease: only 0.08% (1/1327) in MDS-RS vs 3.7% in (9/242) in MDS-SLD/MLD, 3.4% (9/266) in MDS-EB, and 2.5% (10/399) in AML (p < 0.0001 for all). Conversely, E622D was decreased in higher-risk disease: 6.9% (92/1327) in MDS-RS vs 2.9% (7/242) in MDS-SLD/MLD (p < 0.05), 0.4% (1/266) in MDS-EB (p < 0.0001), and 0.5% (2/399) in AML (p < 0.0001). K666R also decreased: 5.4% (72/1327) in MDS-RS vs 1.2% (3/242) in MDS-SLD/MLD, 1.1% (3/266) in MDS-EB, and 1.8% (7/399) in AML (p < 0.01 for all). These data confirm and extend the scope of asymmetrical partitioning of distinct SF3B1 hotspots in low- and high-risk disease. We next examined RNA splicing events produced by those SF3B1 mutations with the most significant asymmetric partitioning. We expressed FLAG-tagged codon-optimized constructs with the E592K, E622D, K666N, and K666R mutations in HEK293 cells, along with wild type SF3B1 and the dominant K700E mutation. For additional comparison, we expressed variants from solid tumors that are rare (R625H, K741N) or absent (E902K) in myeloid malignancies 22 . These transfections produced a comparable level of endogenous and exogenous SF3B1 protein (Fig. 2 ). Upon expression of most hotspots, we observed missplicing in junctions known to be affected by SF3B1 mutations, including SLTM , ZDHHC16 and MAP3K7 (Fig. 2 ) 2 , 6 . This included MDS mutations K700E, E622D, K666R, and K666N but also solid tumor hotspots R625H and K741N, the last of which produced lower magnitude missplicing as recently reported in the context of uveal melanoma 23 . In contrast, these junctions were not misspliced by E902K, which showed its own unique missplicing pattern as was noted in TCGA E902K bladder cancer samples (Supplementary Fig. 2) 22 . We also found that two other mutant SF3B1 -associated junctions, in DLST and UQCC1 , were misspliced to high magnitude by most mutations but were unaffected by K666N, confirming that the pattern of missplicing by K666N is different from that of other MDS/AML-associated hotspots 14 , 24 . Notably, this pattern occurred with K666N, but not K666R, demonstrating these variants are not functionally equivalent even though they affect the same starting amino acid, a phenomenon also observed with variants in the yeast homologue of SF3B1 25 . Finally, conspicuously absent was any missplicing of these events by E592K. Coupled with its enrichment in high-risk disease, this distinct missplicing pattern motivated us to investigate E592K further. We characterized the clinical features of E592K patients in more detail. The hotspot-agnostic collection of all patients with exon 13–16 sequencing had produced 29 cases of E592K. By specifically seeking out additional cases from multiple institutions, we gathered a total of 39 patients with E592K-mutated myeloid neoplasms, 35 of which were MDS or AML (Supplementary Table 2). This expanded E592K cohort also showed enrichment in higher-risk 2016 WHO classifications, and they had higher IPSS-R scores and lower platelets than cases with exon 14–16 mutations (Fig. 3 A-C). Hemoglobin and WBC were not significantly different (Fig. 3 D-E). E592K cases also had a notable lack of RS (Fig. 3 F), with only one instance of low-blast E592K MDS reporting any RS, at 8%, therein being the only E592K patient to meet WHO 2016 criteria for MDS-RS (> 5% RS if SF3B1 mutation present). By contrast, for low-blast MDS with exon 14–16 mutations in which exact RS percentages were available, the average was 37%, consistent with the known pathological and mechanistic links between mutant SF3B1 and RS in MDS (Fig. 3 G) 5 , 26 . E592K MDS/AML also had a different co-mutation profile (Fig. 4 and Supplementary Fig. 3–5). A striking distinction was the nearly ubiquitous co-mutation of ASXL1 (83%) in E592K cases, compared to only 9% in exon 14–16 disease (p < 0.0001). High ASXL1 co-mutation characterized both low and high blast E592K cases, suggesting this relationship occurs early in disease evolution (Supplementary Fig. 3–5). Also notable was the near absence of DNMT3A co-mutations (2.9%) with E592K, despite DNMT3A being the 2nd most commonly co-mutated gene (21%) in exon 14–16 disease (p < 0.01). In fact, the one DNMT3A mutation in E592K MDS was a VAF of 2.3% in a sample in which the VAFs for E592K and ASXL1 mutations were 20%, raising the possibility that the DNMT3A mutation was in a separate clone with wild type SF3B1 . Furthermore, E592K patients had increased co-mutations of RUNX1 (51% vs 11%) and STAG2 (29% vs 3%) (p < 0.0001 for both). Finally, consistent with these high-risk MDS clinical features, both overall survival and leukemia-free survival were markedly shorter in E592K patients (Fig. 5 A-B), including two patients (#9 and #1) who progressed from MDS to AML at 6 and 9 months, respectively, despite lower-risk prognostication scores from both the IPSS-M and SEX-GSS (Fig. 5 C-D) 10 , 11 . Because our initial splicing analysis merely showed the absence of known SF3B1 -mutant events in E592K cells, we next sought missplicing events specifically induced by this variant. To do so, we stably expressed the WT, K700E, and E592K mutations through lentiviral delivery in TF1 cells, isolated multiple independent clones for each genotype, and performed RNA-seq to quantify percent spliced in (PSI) values for all splice junctions (Fig. 6 A). K700E produced a characteristic pattern of increased expression of junctions using alternative 3’ acceptors, with high magnitude missplicing of genes like MAP3K7 and ZDHCC16 , as expected. In contrast, the E592K mutation produced a fundamentally different pattern of missplicing, with its affected genes nonoverlapping with those misspliced by K700E, and vice-versa. Western blotting showed that exogenous FLAG-tagged SF3B1 was equal or less than the endogenous SF3B1 form, not overexpressed above it (Fig. 6 B). We then validated several missplicing events with endpoint and quantitative PCR assays. For K700E-specific junctions, this included missplicing of ZDHHC16 , TMEM14C , and ABCB7 (Fig. 6 B-C). For E592K-specific junctions, this included RAVER2 , CEP43 , NUTM2A-AS1 , and EZH2 (Fig. 6 D). Interestingly, the EZH2 missplicing event was an alternate acceptor in intron 12, creating a premature termination codon predicted to activate nonsense-mediated decay (Fig. 6 E). We further validated the hotspot specificity of these events in two other cell contexts: transiently-transfected HEK293T and stably-transduced K562 cells (Supplementary Fig. 6). In all cases, K700E-dependent and E592K-dependent missplicing events were present and distinct. Of note, missplicing of TMEM14C and ABCB7 were recently shown to drive ring sideroblast formation in iPSCs derived from SF3B1 -mutant MDS 5 . Both genes were clearly misspliced by K700E, but not by E592K, consistent with the lack of sideroblastic anemia in E592K patients. Together, these data show that the E592K variant of SF3B1 has a unique pattern of RNA missplicing. In addition to shared RNA missplicing events, the most well-studied SF3B1 hotspot mutations also share a specific biochemical defect: disruption of the interaction between SF3B1 and SUGP1 17 . This disruption is not incidental to missplicing but directly mediates it; inactivation of SUGP1 recapitulates SF3B1 -mutant missplicing and overexpression of SUGP1 partially rescues it 17 . We therefore asked whether E592K, with its nonoverlapping missplicing events, might preserve the interaction of SF3B1 with SUGP1 . Indeed, when His6-FLAG- SF3B1 variants were introduced into HEK293T cells and affinity purified using anti-DYKDDDDK (FLAG) antibody and cobalt beads, the association of His6-FLAG- SF3B1 with endogenous SUGP1 was disrupted by K700E but not by wild type or E592K SF3B1 (Fig. 7 ). We then asked whether E592K might instead disrupt the interaction between SF3B1 and PHF5A , given that E592 is at the interface with PHF5A 2 7 . However, this interaction was preserved by each His6-FLAG-SF3B1 variant (Fig. 7 ). We also observed these effects in a second cell context, TF1 cells expressing FLAG- SF3B1 variants (Supplementary Fig. 7). Together, these data indicate that, consistent with its induction of unique RNA missplicing events, the E592K variant does not participate in the disruption of the SF3B1 - SUGP1 interaction that drives the cryptic splicing of other SF3B1 mutations. Finally, we identified and analyzed RNA-seq from two patients with E592K mutation, compared to other SF3B1 mutations, from the recent MLL cohort of spliceosome-mutant myeloid malignancy patients 28 . Inspection of the junctions validated in our cell models also demonstrated the same specificity of missplicing in these primary patient samples, when compared to other SF3B1 mutations (Fig. 8 A). In addition, endpoint PCR validation of TMEM14C and RAVER2 from a third primary E592K sample at a separate institution demonstrated the same hotspot specificity of RNA missplicing (Fig. 8 B), validating the findings from our cell models. Discussion Here we show the E592K variant of SF3B1 produces unique RNA missplicing and associates with high-risk MDS. These results have several implications. First, the distinctiveness of E592K informs the pathobiology of SF3B1 -mutant MDS. Clough et al elegantly showed that RS are formed by TMEM14C and ABCB7 missplicing in MDS-derived iPSCs with the G742D variant of SF3B1 5 . These missplicing events have been seen with other hotspots, including K700E, which we corroborated here 2 , 29 . Cells with E592K, on the other hand, preserved canonical splicing of these genes, and cases of E592K MDS lacked RS. Other events that have been implicated in SF3B1 -mutant MDS pathobiology include innate immune activation by missplicing of MAP3K7 and IRAK4, enhanced self-renewal by MECOM missplicing, impaired erythropoiesis by COASY missplicing, and hepcidin suppression by ERFE missplicing; all these genes were also canonically spliced in E592K cells (Supplementary Fig. 7) 3 , 4 , 6 – 8 . A distinct E592K pathobiology is also suggested by its high co-occurrence with ASXL1/RUNX1/STAG2 mutations and mutual exclusivity with DNMT3A mutations, a starkly different pattern than that of other SF3B1 hotspots. As with most co-mutation patterns in cancer, it is unclear whether these co-occurrences are driven by synergism or permissiveness—and whether the mutual exclusivity is driven by antagonism or redundancy. Interestingly, the same pattern of co-occurrence (ASXL1/RUNX1/STAG2) and mutual exclusivity (DNMT3A) occurs with loss-of-function EZH2 mutations in MDS (Supplementary Fig. 8) 10 , 15 . It is therefore intriguing that E592K missplicing produced a frameshifted EZH2 transcript in our studies here, and it is tempting to speculate this may create ‘mutant EZH2-ness’ in E592K cells, an area for future investigation. If so, mutual exclusivity due to redundancy might be expected between mutant EZH2 and E592K. While there were indeed no EZH2 mutations in the 39 E592K patients here, the low overall frequency (4%) of EZH2 in SF3B1 -mutant myeloid malignancies means a larger cohort would be needed for significance testing of this particular pair. Nonetheless, the distinctiveness of E592K here shows that certain MDS phenotypes associated with SF3B1 mutation, including sideroblastic anemia, are not inevitable pathobiological outcomes of RNA missplicing by all SF3B1 variants. Second, there are implications for MDS classification. Recently, both the International Consensus Classification (ICC) and the 5th edition of the World Health Organization (WHO) classification of myeloid neoplasms created nosologic entities for SF3B1 -mutant MDS 30 , 31 . These are based on the International Working Group (IWG) findings that SF3B1 mutation, low blasts, and lack of certain co-occurring genetic aberrations defined an indolent MDS with a shared pathobiology of idiosyncratic RNA missplicing that was more homogeneous than the MDS-RS classification 32 . This was due in part to exclusion of SF3B1 -unmutated MDS-RS which had greater myeloid/megakaryocyte dysplasia, more TP53 co-mutations, and poorer outcomes. Both the ICC and WHO criteria require SF3B1 mutation, low blasts, cytopenias, dysplasia, and lack of multi-hit TP53/del(5q)/-7/complex cytogenetics; the ICC also requires SF3B1 VAF > 10% and lack of RUNX1/del(7q)/abn3q26.2; and the WHO allows for > 15% RS to substitute for SF3B1 mutation. As we have seen here, E592K patients do not fit these groups: they have unique RNA missplicing, higher blasts, lack of RS, increased RUNX1 mutations, and poorer prognosis. These differences make an argument for excluding those E592K cases that would otherwise meet criteria for these entities. They also emphasize the need to consider specific hotspot mutations in future classification efforts. This should be the precise mutation, not just the affected amino acid, as we demonstrated different patterns of disease partitioning and missplicing between K666R and K666N. Third, our data have implications for MDS prognostication. Along with low blasts, shallow cytopenias, and certain cytogenetic abnormalities such as del(11q), SF3B1 mutations have been consistently associated with better MDS outcomes in multiple independent datasets 1 , 9 – 12 . Accordingly, new MDS prognosis scoring tools incorporating mutational data have generally weighted SF3B1 mutations favorably, although there is important context-dependence that overrides this favorability, such as increased blasts and aberrations such as del(5q), RUNX1 mutation, and others 10 – 12 . However, given the much larger patient cohorts that would be required, prognostication tools do not yet separately weight the myriad individual variants of mutated genes ( SF3B1 or otherwise) into their scores. By the same token, a comprehensive multivariate analysis of the prognosis of E592K MDS would require a larger patient cohort than ours here. Nonetheless, while increased blasts or co-occurring RUNX1 mutations would lead to high-risk scores for many E592K patients, others would be understaged by these tools, as we observed here for two patients with rapid progression to AML (Fig. 5 C-D). Thus, our data suggest caution in regarding any E592K patients as low risk. They also highlight the potential added value of incorporating specific hotspot mutations in the eventual next generation of prognostication tools. Fourth, these results have treatment implications. Luspatercept is indicated for treatment of anemia in ESA-refractory MDS-RS patients based on the phase III MEDALIST trial, which was conducted in this population due to strong associations of MDS-RS and SF3B1 mutation with response in the phase II PACE-MDS study 33 , 34 . However, response of MDS-RS patients was similar regardless of mutant SF3B1 allelic burden in MEDALIST, as well as in a recent real-world cohort 33 , 35 . These data would suggest that it is sideroblastic anemia (or the late-to-early erythroid progenitor ratio common in MDS-RS, according to long-term PACE-MDS follow up) that is predictive of luspatercept response, more so than SF3B1 mutation 36 . In the WHO 5th edition, MDS-RS has become MDS- SF3B1 and does not require RS if there is SF3B1 mutation, low blasts, and lack of high-risk cytogenetics 31 . Luspatercept would therefore be approved for many low-blast E592K patients, despite the sharp distinctions from MDS-RS patients that we have drawn here. Our data thus also recommend caution in approaching E592K patients like MDS-RS patients that are likely to respond to luspatercept. There are also implications for investigational therapies. Gene therapies that leverage missplicing in spliceosome-mutant cells are promising, but such vectors would need to be not only gene-specific (i.e. SF3B1 vs SRSF2 ) but, in the case of E592K, hotspot-specific due to its nonoverlapping missplicing 37 , 38 . Similarly, antisense oligonucleotide therapy aimed at rescuing BRD9 missplicing showed activity against SF3B1 -mutant tumors in vivo, but such a therapy would not apply to E592K, which does not missplice BRD9 (Supplementary Fig. 7) 39 . Finally, these findings add to our understanding of the functional diversity of spliceosome mutations. After these mutations were first discovered as conspicuously enriched in myeloid neoplasms in a mutually exclusive manner, efforts have sought unifying downstream functional effects that might explain this occurrence pattern 40 . This has revealed shared phenotypes, such as convergent disruption of certain pathways, creation of genotoxic R-loops, and sensitization to further disruption of the spliceosome 6 , 41 – 44 . At the same time, bulk and then single-cell analyses have shown that spliceosome mutations are not always mutually exclusive, with multiple mutations sometimes selected for in the same cells 24 . This indicates that different spliceosome mutations can confer different, even complementary, advantages to cancer cells, and the mutual exclusivity that does occur may be more from the splicing toxicity of combining certain mutations than from functional redundancy 24 . Consistent with this, studies have shown direct functional differences between spliceosome mutations: the missplicing events induced by mutations in different spliceosome genes (i.e. SRSF2 vs U2AF1 , etc) are nonoverlapping, the two dominant hotspot mutations in U2AF1 missplice different genes, and some less common SRSF2 and U2AF1 variants only partially or “dually” recapitulate the RNA missplicing of hotspot mutations 2 , 45 – 48 . For SF3B1 mutations, their asymmetric partitioning among cancer types (i.e. K700E/H662Q in MDS, R625C/R625H in uveal melanoma, G642D in CLL, etc.) first suggested the mutations may have functional differences 40 , 49 , 50 . Later, Seiler et al noted different missplicing events in TCGA RNA-seq from bladder tumors with the E902K variant, events that we experimentally confirmed here 22 . For other SF3B1 hotspots, studies have described differences of degree in RNA missplicing events, including K700E vs R625C/R625H in primary samples, R625H vs K741Q vs others in isogenic cells, and K666N vs K700E/H662Q in primary samples and isogenic cells 14 , 23 , 24 , 51 . To these we add E592K, whose missplicing events are differences of kind, nonoverlapping with those of other MDS-associated hotspots—and marked by a different underlying biochemistry that preserves the interaction between SUGP1 and SF3B1 . While it remains possible that the important underlying oncogenic effects of E592K are phenotypes still shared with other SF3B1 hotspots, the distinctiveness of E592K suggests to us that different SF3B1 variants can promote different kinds of leukemia by inducing different RNA missplicing events. Because these differences can impact our understanding and management of patients with myeloid neoplasms, additional studies of differences between other spliceosome variants are warranted. Declarations Acknowledgements This work was supported by grants from the NIH (HL159306), Edward P. Evans Foundation, Leukemia & Lymphoma Society, Maryland Stem Cell Research Fund, Gabrielle’s Angel Foundation for Cancer Research, Emerson Collective, and AbbVie (to WBD); NIH R35 GM118136 (to JLM); Lady Tata Memorial Trust (3218) and the Barts Charity (G-002167) (to CP and KR). Author Contributions AED and WBD conceived the study. IYC, JPL, JZ, EH, JLM, KRP, AED, and WBD designed research. IYC, JPL, JZ, EH, CP, and WBD performed experiments. JLM, KRP, and WBD supervised experiments. IYC, WW, RB, CP, BL, DHW, MO, EB, XL, TF, SF, CDG, TJ, AED, and WBD collected data. IYC, JPL, JZ, WW, JLM, KRP, AED, and WBD analyzed data. IYC, JPL, JZ, KRP, and WBD prepared figures. IYC and WBD wrote the manuscript. All authors critically reviewed and approved the final version of the manuscript. Competing Interests The authors declare no competing interests. Data Availability Statement Raw FASTQ files are deposited at the NCBI Sequence Read Archive (SRA) under accession number SRP##########. References Haferlach T, Nagata Y, Grossmann V, Okuno Y, Bacher U, Nagae G et al. Landscape of genetic lesions in 944 patients with myelodysplastic syndromes. Leukemia 2014; 28: 241–7. Darman RB, Seiler M, Agrawal AA, Lim KH, Peng S, Aird D et al. Cancer-Associated SF3B1 Hotspot Mutations Induce Cryptic 3′ Splice Site Selection through Use of a Different Branch Point. Cell Reports 2015; 13: 1033–1045. Bondu S, Alary A-S, Lefèvre C, Houy A, Jung G, Lefebvre T et al. A variant erythroferrone disrupts iron homeostasis in SF3B1-mutated myelodysplastic syndrome. Sci Transl Med 2019; 11: eaav5467. Mian SA, Philippe C, Maniati E, Protopapa P, Bergot T, Piganeau M et al. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2802265","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":191197907,"identity":"802bf553-a5e5-49a9-83b9-d6255520b8bd","order_by":0,"name":"In Young Choi","email":"","orcid":"","institution":"The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"In","middleName":"Young","lastName":"Choi","suffix":""},{"id":191197908,"identity":"0bfa0464-75db-4a4f-9059-cb1ffe59d0e1","order_by":1,"name":"Jonathan P. 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Amino acid positions are shown along the x axis, and individual variants are counted along the y axis according to the legend above the graphs. E592K and K666N are increased, while E622D and K666R are decreased, in higher-risk disease types. RS = ring sideroblasts. SLD/MLD = single-lineage dysplasia/multi-lineage dysplasia. EB = excess blasts.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-2802265/v1/9867a5c1dff0d645b66a1bc0.png"},{"id":35771351,"identity":"63675dd4-75f8-4e68-ae08-1c6784f0120c","added_by":"auto","created_at":"2023-04-14 14:00:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":220721,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAsymmetric RNA missplicing by distinct SF3B1 mutation hotspots\u003c/strong\u003e. HEK293T cells were transfected with constructs expressing FLAG-SF3B1 variants. Top row is Western blotting with anti-SF3B1 antibody, showing FLAG-SF3B1 and endogenous SF3B1 at similar levels. Endpoint PCR used isoform-competitive primers, with arrows for canonical (blue), cryptic (red), and heteroduplex (green) forms. Cryptic vs canonical UQCC1 was quantified as a ratio between two separate isoform-specific qPCRs.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-2802265/v1/198b3a600164e0712c5dffc3.png"},{"id":35770797,"identity":"ae3ac39a-fa57-4fd6-beb3-c6c65c812adb","added_by":"auto","created_at":"2023-04-14 13:52:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":24052,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClinical parameters of MDS patients with the E592K variant of SF3B1\u003c/strong\u003e. Compared to cases with exon 14-16 mutations, patients with E592K have A) higher risk WHO 2016 classifications, B) higher IPSS-R, C) lower platelets, D-E) similar hemoglobin (Hb) and absolute neutrophil count (ANC), and E-F) nearly-absent ring sideroblasts (RS).\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-2802265/v1/1f0a4fb71c7e1be3b73202f0.png"},{"id":35770066,"identity":"9f197397-5373-4829-85ef-e864bc03f3f0","added_by":"auto","created_at":"2023-04-14 13:44:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":677927,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCo-mutation landscape of SF3B1-mutant MDS and AML\u003c/strong\u003e. All E592K-mutated cases are shown on the left, and all exon 14-16-mutated cases on the right. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-2802265/v1/32af05ed9e4a24e273d4556e.png"},{"id":35770799,"identity":"0d378b4c-161b-4ad0-a8f1-1bddf3f6f7a5","added_by":"auto","created_at":"2023-04-14 13:52:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":199827,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMDS patients with E592K have poor survival\u003c/strong\u003e. A) Overall and B) leukemia-free survival of all SF3B1-mutant MDS patients. p-values are for Log-rank (Mantel-Cox) tests. C) Example of understaging of Pt #9, who developed AML 6 months after diagnosis but is stratified as moderate low risk with median LFS of 4.5 years by the IPSS-M (left) and a median LFS of \u0026gt;10 years by Sex-GSS (right). D) Understaging of Pt #1, who developed AML 9 months after diagnosis.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-2802265/v1/3090d6cc2e949c89a29291b1.png"},{"id":35770801,"identity":"f02023bf-6c97-49c6-8819-56bc6542dc10","added_by":"auto","created_at":"2023-04-14 13:52:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":145503,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eE592K induces unique RNA missplicing events\u003c/strong\u003e. TF1 cells transduced with different SF3B1 variants were analyzed by RNA-seq, with A) highest-scoring ΔPSIs shown. B) Western blot with anti-SF3B1 antibody. C) Endpoint PCR/qPCR validation of K700E-specific missplicing events. D) Validation of E592K-specific events. E) RNA-seq reads from TF1 cells showing the cryptic event in EZH2. PTC = premature termination codon.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-2802265/v1/0782f8147c1666acb741006d.png"},{"id":35770802,"identity":"06e0a2ff-0896-44fe-8605-e0fe7dbb668e","added_by":"auto","created_at":"2023-04-14 13:52:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":234306,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe E592K variant preserves association of SF3B1 with SUGP1\u003c/strong\u003e. HEK293T cells were transfected with His6-FLAG-SF3B1 variants and subjected to affinity purification with anti-DYKDDDDK (FLAG) antibody. A) Silver-stained protein gel, with arrow pointing to the size of SUGP1, which is decreased in K700E but not E592K eluate. B) Western blot showing decreased SUGP1 in K700E, but not E592K, eluate. PHF5A is present with all SF3B1 variants. C) Reprobing with anti-SF3B1 shows native and His6-FLAG-tagged protein levels.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-2802265/v1/29b66d97412965f8414f7d41.png"},{"id":35770071,"identity":"95e59c4a-2811-4b59-8c6a-5bbb6c89b0e3","added_by":"auto","created_at":"2023-04-14 13:44:15","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":40004,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eE592K exhibits unique RNA missplicing in primary MDS samples\u003c/strong\u003e. RNA-seq read distribution in the MLL cohort shows that E592K exhibits A) canonical TMEM14C and ABCB7 missplicing, and B) cryptic RAVER2, NUTM2B-AS1, and EZH2 missplicing. C) Distinct TMEM14C and RAVER2 missplicing was validated in marrow CD34+ cells from an independent patient by endpoint PCR.\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-2802265/v1/80b5f9a3c57acb93d2d2b7c7.png"},{"id":61174638,"identity":"a3de7ca9-bef7-4abb-98a8-66b23697a2e2","added_by":"auto","created_at":"2024-07-26 15:21:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2330527,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2802265/v1/5241c6f8-bafd-4daf-8a2a-8a01f61f5d6a.pdf"},{"id":35770074,"identity":"bae6ec54-7d2c-4f01-98cb-e1f5959c09fd","added_by":"auto","created_at":"2023-04-14 13:44:15","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6713250,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Supplmaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2802265/v1/09c8d989edc8989cce89215b.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"The E592K variant of SF3B1 creates unique RNA missplicing and associates with high-risk MDS without ring sideroblasts","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eSF3B1\u003c/em\u003e is the most mutated spliceosome gene in MDS, with a frequency of \u0026gt;\u0026thinsp;30%\u003csup\u003e1\u003c/sup\u003e. The mutations are primarily missense substitutions that induce neomorphic RNA missplicing in thousands of junctions, which in turn alter expression of hundreds of genes in diverse pathways\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. This missplicing has been implicated in many MDS phenotypes, including dysfunctional iron metabolism, formation of ring sideroblasts, activation of innate immune signaling, and promotion of hematopoietic stem cell self-renewal\u003csup\u003e\u003cspan additionalcitationids=\"CR4 CR5 CR6 CR7\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eSF3B1\u003c/em\u003e mutations also contain prognostic value in MDS, associating with more indolent disease, though with important exceptions\u003csup\u003e\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In MDS treatment, \u003cem\u003eSF3B1\u003c/em\u003e mutations are among the criteria used to determine eligibility for luspatercept, and they are the direct or indirect targets of investigational therapies\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Thus, \u003cem\u003eSF3B1\u003c/em\u003e mutations figure prominently in the research of\u0026mdash;and clinical practice for\u0026mdash;many MDS patients.\u003c/p\u003e \u003cp\u003eNot well understood is whether\u0026mdash;and how\u0026mdash;distinct \u003cem\u003eSF3B1\u003c/em\u003e mutation hotspots differentially affect disease features and/or the RNA missplicing events that drive them. Previously, in an analysis of patients and cell models with \u003cem\u003eSF3B1\u003c/em\u003e exon 14\u0026ndash;15 mutations, we found that the K666N variant was enriched in high-risk MDS and produced an asymmetrical lack of missplicing events that are induced by K700E and H662Q mutations\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Here we report the results of extending this approach to a larger cohort of patients that included exon 13\u0026ndash;16 mutations and additional cell models. This analysis revealed a striking distinctiveness in the \u003cem\u003eSF3B1\u003c/em\u003e mutation E592K, which has implications for the understanding and management of \u003cem\u003eSF3B1\u003c/em\u003e-mutant MDS.\u003c/p\u003e"},{"header":"Materials/subjects And Methods","content":"\u003ch2\u003ePatients\u003c/h2\u003e\n\u003cp\u003eMutation-agnostic acquisition of \u003cem\u003eSF3B1\u003c/em\u003e mutations from MDS and AML cases and associated clinical parameters came from the Johns Hopkins Sidney Kimmel Cancer Center, the Vanderbilt-Ingram Cancer Center, the Chinese Academy of Medical Sciences, the Munich Leukemia Laboratory, the Allegheny Health Network Cancer Institute, Project Genie (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://genie.cbioportal.org/\u003c/span\u003e\u003c/span\u003e), and manual extraction from 82 published studies (supplemental References)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Additional E592K cases and their clinical parameters were deliberately obtained from The University of Manchester, Tong University Affiliated Sixth People\u0026apos;s Hospital, Weill Cornell Medicine, and Memorial Sloan Kettering Cancer Center. Because breadth of gene panels varied among patients, co-mutation analysis included those cases in which at least a set of 35 genes were sequenced, representing a compromise between maximum sample inclusion and maximum gene inclusion. For EZH2 co-mutation analysis, cBioportal Oncoprinter (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cbioportal.org/oncoprinter\u003c/span\u003e\u003c/span\u003e) was applied to all MDS cases from the MSK Myelodysplastic, Project GENIE, and IPSS-M cohorts\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. For leukemia-free survival of E592K patients, the IPSS-M (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://mds-risk-model.com\u003c/span\u003e\u003c/span\u003e) and SEX-GSS (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://mds.itb.cnr.it/#/mds/home\u003c/span\u003e\u003c/span\u003e) calculators were used\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Use of deidentified patient data were approved by the Institutional Review Boards at the respective institutions.\u003c/p\u003e\n\u003ch2\u003eCells\u003c/h2\u003e\n\u003cp\u003eHEK293T, TF1, and K562 cells were obtained from the ATCC. HEK293T cells were grown in DMEM/10% FBS, K562 cells were grown in RPMI/20% FBS, and TF1 cells were grown in RPMI/20% FBS with 2 ng/mL GM-CSF. STR cell line authentication and mycoplasma testing were done upon receipt and routinely thereafter, with last testing done 2/2022.\u003c/p\u003e\n\u003ch2\u003eVectors\u003c/h2\u003e\n\u003cp\u003eWT and K700E FLAG-\u003cem\u003eSF3B1\u003c/em\u003e sequences were subcloned from Addgene plasmids 82576 and 82577 into pDONR-A-HYG (Addgene 29635) to make pENTR-\u003cem\u003eSF3B1\u003c/em\u003e-WT and pENTR-\u003cem\u003eSF3B1\u003c/em\u003e-K700E. Site-directed mutagenesis with overlap extension PCR then created pENTR plasmids for the E592K, E622D, K666R, K666N, R625H, K741N, and E902K variants of \u003cem\u003eSF3B1\u003c/em\u003e, and these were subcloned into lentiviral vector pLX301 (Addgene 25895). Plasmids used for \u003cem\u003eSF3B1\u003c/em\u003e affinity purification have been previously described\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Site-directed mutagenesis of the p3xFLAG-CMV-14-His6-FLAG-SF3B1 vector was done by overlap extension PCR to make the E592K vector.\u003c/p\u003e\n\u003ch2\u003eTranscriptome analysis\u003c/h2\u003e\n\u003cp\u003eFor HEK293T cells, pLX301 plasmids were transfected using Lipofectamine 3000, 24h later cells were selected with puromycin for 48h, puro was washed out for 24h, and cells were harvested. For stable transduction, pLX301 lentivirus was produced as previously described\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. TF1 cells were transduced and four independent clones per genotype were puro selected from single cells. For K562 cells, duplicate independent polyclonal populations per genotype were puro selected. RNA isolation, cDNA synthesis, endpoint PCR, and quantitative PCR were performed as described\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Primer sequences are in Supplementary Table\u0026nbsp;1. RNA-seq libraries from TF1 clones were constructed using TruSeq Stranded Total RNA Library Prep. Sequencing was performed on a NovaSeq S1 flowcell. Reads were aligned using STAR\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Splicing analysis was performed using ASCOT and gene expression using featureCounts\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Percent spliced in (PSI) values for junctions were determined by dividing inclusion split-read counts by the total split-read counts at the corresponding constitutive donor or acceptor sites, using a minimum coverage of 15 split-reads per junction. Junctions of interest were visualized from RNA-seq reads from primary MLL samples with the UCSC genome browser for 2 E592K, 10 E622D, 6 K666N, 9 K666R, 12 K700E, and 12 WT samples, using the ADD function (combining reads and normalizing track height for samples in each mutation group). Junction validation with endpoint PCR was done on independent bone marrow CD34\u0026thinsp;+\u0026thinsp;MDS samples at Barts Cancer Institute.\u003c/p\u003e\n\u003ch2\u003eAffinity Purification of \u003cem\u003eSF3B1\u003c/em\u003e-Associated Proteins\u003c/h2\u003e\n\u003cp\u003eA small-scale protocol was applied to both HEK293T and TF1 cells as previously described, except that for TF1, 10\u0026nbsp;million cells were used and proteins were eluted with 30 \u0026micro;L (5 \u0026micro;g/\u0026micro;L) 3X FLAG peptide because cells had only one affinity tag (FLAG) attached to SF3B1\u003csup\u003e17\u003c/sup\u003e.\u003c/p\u003e\n\u003ch2\u003eWestern Blotting\u003c/h2\u003e\n\u003cp\u003eFor immunoblotting of \u003cem\u003eSF3B1\u003c/em\u003e/FLAG-\u003cem\u003eSF3B1\u003c/em\u003e proteins in transiently-transfected HEK293T and stably-transduced TF1 and K562 cells in which transcriptome analysis was done in parallel, Western blotting using a mouse anti-human-\u003cem\u003eSF3B1\u003c/em\u003e antibody (Abcam #172634) at 1:1000 dilution was used. Immunoblotting following affinity purification of SF3B1 in HEK293T and TF1 cells was performed as previously described, and primary antibodies were: anti-SF3B1 (Bethyl Laboratories, A300-996A, 1:1,000), anti-ACTIN (Sigma, A2066, 1:2,000), anti-DYKDDDDK (GenScript, A00187, 1:1,000), anti-\u003cem\u003eSUGP1\u003c/em\u003e (Bethyl Laboratories A304-675A-M, 1:1,000), and anti-PHF5A (Proteintech 15554-1-AP, 1:1000)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Secondary antibodies were: Donkey anti-Rabbit IgG (LI-COR, 926-68073, 1:5,000) and Goat anti-Mouse IgG (LI-COR, 926-32210, 1:5,000).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eCombining patient data from five institutions, publicly available databases, and published literature, we established a dataset of 2,288 patients with \u003cem\u003eSF3B1\u003c/em\u003e-mutant MDS or AML in which exons 13 through 16 had been sequenced. We first determined how \u003cem\u003eSF3B1\u003c/em\u003e mutations partitioned into WHO 2016 classifications, as these data were available for virtually all patients. This distribution showed several asymmetries (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and Supplementary Fig. 1). Consistent with our previous report\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, K666N was enriched in higher-risk disease types: only 2.1% (28/1327) in MDS-RS vs 8.7% (21/242) in MDS-SLD/MLD, 17.3% (46/266) in MDS-EB, and 25.8% (103/399) in AML (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 for all). With the inclusion of cases in which exon 13 had been sequenced, the distribution also revealed enrichment of a variant in this exon, E592K, in higher-risk disease: only 0.08% (1/1327) in MDS-RS vs 3.7% in (9/242) in MDS-SLD/MLD, 3.4% (9/266) in MDS-EB, and 2.5% (10/399) in AML (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 for all). Conversely, E622D was decreased in higher-risk disease: 6.9% (92/1327) in MDS-RS vs 2.9% (7/242) in MDS-SLD/MLD (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), 0.4% (1/266) in MDS-EB (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and 0.5% (2/399) in AML (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). K666R also decreased: 5.4% (72/1327) in MDS-RS vs 1.2% (3/242) in MDS-SLD/MLD, 1.1% (3/266) in MDS-EB, and 1.8% (7/399) in AML (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 for all). These data confirm and extend the scope of asymmetrical partitioning of distinct \u003cem\u003eSF3B1\u003c/em\u003e hotspots in low- and high-risk disease.\u003c/p\u003e\n\u003cp\u003eWe next examined RNA splicing events produced by those \u003cem\u003eSF3B1\u003c/em\u003e mutations with the most significant asymmetric partitioning. We expressed FLAG-tagged codon-optimized constructs with the E592K, E622D, K666N, and K666R mutations in HEK293 cells, along with wild type \u003cem\u003eSF3B1\u003c/em\u003e and the dominant K700E mutation. For additional comparison, we expressed variants from solid tumors that are rare (R625H, K741N) or absent (E902K) in myeloid malignancies\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. These transfections produced a comparable level of endogenous and exogenous \u003cem\u003eSF3B1\u003c/em\u003e protein (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Upon expression of most hotspots, we observed missplicing in junctions known to be affected by \u003cem\u003eSF3B1\u003c/em\u003e mutations, including \u003cem\u003eSLTM\u003c/em\u003e, \u003cem\u003eZDHHC16\u003c/em\u003e and \u003cem\u003eMAP3K7\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. This included MDS mutations K700E, E622D, K666R, and K666N but also solid tumor hotspots R625H and K741N, the last of which produced lower magnitude missplicing as recently reported in the context of uveal melanoma\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In contrast, these junctions were not misspliced by E902K, which showed its own unique missplicing pattern as was noted in TCGA E902K bladder cancer samples (Supplementary Fig.\u0026nbsp;2)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. We also found that two other mutant \u003cem\u003eSF3B1\u003c/em\u003e-associated junctions, in \u003cem\u003eDLST\u003c/em\u003e and \u003cem\u003eUQCC1\u003c/em\u003e, were misspliced to high magnitude by most mutations but were unaffected by K666N, confirming that the pattern of missplicing by K666N is different from that of other MDS/AML-associated hotspots\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Notably, this pattern occurred with K666N, but not K666R, demonstrating these variants are not functionally equivalent even though they affect the same starting amino acid, a phenomenon also observed with variants in the yeast homologue of \u003cem\u003eSF3B1\u003c/em\u003e\u003csup\u003e25\u003c/sup\u003e. Finally, conspicuously absent was any missplicing of these events by E592K. Coupled with its enrichment in high-risk disease, this distinct missplicing pattern motivated us to investigate E592K further.\u003c/p\u003e\n\u003cp\u003eWe characterized the clinical features of E592K patients in more detail. The hotspot-agnostic collection of all patients with exon 13\u0026ndash;16 sequencing had produced 29 cases of E592K. By specifically seeking out additional cases from multiple institutions, we gathered a total of 39 patients with E592K-mutated myeloid neoplasms, 35 of which were MDS or AML (Supplementary Table 2). This expanded E592K cohort also showed enrichment in higher-risk 2016 WHO classifications, and they had higher IPSS-R scores and lower platelets than cases with exon 14\u0026ndash;16 mutations (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). Hemoglobin and WBC were not significantly different (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD-E). E592K cases also had a notable lack of RS (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF), with only one instance of low-blast E592K MDS reporting any RS, at 8%, therein being the only E592K patient to meet WHO 2016 criteria for MDS-RS (\u0026gt;\u0026thinsp;5% RS if \u003cem\u003eSF3B1\u003c/em\u003e mutation present). By contrast, for low-blast MDS with exon 14\u0026ndash;16 mutations in which exact RS percentages were available, the average was 37%, consistent with the known pathological and mechanistic links between mutant \u003cem\u003eSF3B1\u003c/em\u003e and RS in MDS (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eG)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. E592K MDS/AML also had a different co-mutation profile (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e and Supplementary Fig. 3\u0026ndash;5). A striking distinction was the nearly ubiquitous co-mutation of ASXL1 (83%) in E592K cases, compared to only 9% in exon 14\u0026ndash;16 disease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). High ASXL1 co-mutation characterized both low and high blast E592K cases, suggesting this relationship occurs early in disease evolution (Supplementary Fig. 3\u0026ndash;5). Also notable was the near absence of DNMT3A co-mutations (2.9%) with E592K, despite DNMT3A being the 2nd most commonly co-mutated gene (21%) in exon 14\u0026ndash;16 disease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In fact, the one DNMT3A mutation in E592K MDS was a VAF of 2.3% in a sample in which the VAFs for E592K and ASXL1 mutations were 20%, raising the possibility that the DNMT3A mutation was in a separate clone with wild type \u003cem\u003eSF3B1\u003c/em\u003e. Furthermore, E592K patients had increased co-mutations of RUNX1 (51% vs 11%) and STAG2 (29% vs 3%) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 for both). Finally, consistent with these high-risk MDS clinical features, both overall survival and leukemia-free survival were markedly shorter in E592K patients (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA-B), including two patients (#9 and #1) who progressed from MDS to AML at 6 and 9 months, respectively, despite lower-risk prognostication scores from both the IPSS-M and SEX-GSS (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC-D)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eBecause our initial splicing analysis merely showed the absence of known \u003cem\u003eSF3B1\u003c/em\u003e-mutant events in E592K cells, we next sought missplicing events specifically induced by this variant. To do so, we stably expressed the WT, K700E, and E592K mutations through lentiviral delivery in TF1 cells, isolated multiple independent clones for each genotype, and performed RNA-seq to quantify percent spliced in (PSI) values for all splice junctions (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). K700E produced a characteristic pattern of increased expression of junctions using alternative 3\u0026rsquo; acceptors, with high magnitude missplicing of genes like \u003cem\u003eMAP3K7\u003c/em\u003e and \u003cem\u003eZDHCC16\u003c/em\u003e, as expected. In contrast, the E592K mutation produced a fundamentally different pattern of missplicing, with its affected genes nonoverlapping with those misspliced by K700E, and vice-versa. Western blotting showed that exogenous FLAG-tagged \u003cem\u003eSF3B1\u003c/em\u003e was equal or less than the endogenous \u003cem\u003eSF3B1\u003c/em\u003e form, not overexpressed above it (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB). We then validated several missplicing events with endpoint and quantitative PCR assays. For K700E-specific junctions, this included missplicing of \u003cem\u003eZDHHC16\u003c/em\u003e, \u003cem\u003eTMEM14C\u003c/em\u003e, and \u003cem\u003eABCB7\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB-C). For E592K-specific junctions, this included \u003cem\u003eRAVER2\u003c/em\u003e, \u003cem\u003eCEP43\u003c/em\u003e, \u003cem\u003eNUTM2A-AS1\u003c/em\u003e, and \u003cem\u003eEZH2\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD). Interestingly, the EZH2 missplicing event was an alternate acceptor in intron 12, creating a premature termination codon predicted to activate nonsense-mediated decay (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE). We further validated the hotspot specificity of these events in two other cell contexts: transiently-transfected HEK293T and stably-transduced K562 cells (Supplementary Fig. 6). In all cases, K700E-dependent and E592K-dependent missplicing events were present and distinct. Of note, missplicing of \u003cem\u003eTMEM14C\u003c/em\u003e and \u003cem\u003eABCB7\u003c/em\u003e were recently shown to drive ring sideroblast formation in iPSCs derived from \u003cem\u003eSF3B1\u003c/em\u003e-mutant MDS\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Both genes were clearly misspliced by K700E, but not by E592K, consistent with the lack of sideroblastic anemia in E592K patients. Together, these data show that the E592K variant of \u003cem\u003eSF3B1\u003c/em\u003e has a unique pattern of RNA missplicing.\u003c/p\u003e\n\u003cp\u003eIn addition to shared RNA missplicing events, the most well-studied \u003cem\u003eSF3B1\u003c/em\u003e hotspot mutations also share a specific biochemical defect: disruption of the interaction between \u003cem\u003eSF3B1\u003c/em\u003e and \u003cem\u003eSUGP1\u003c/em\u003e\u003csup\u003e17\u003c/sup\u003e. This disruption is not incidental to missplicing but directly mediates it; inactivation of \u003cem\u003eSUGP1\u003c/em\u003e recapitulates \u003cem\u003eSF3B1\u003c/em\u003e-mutant missplicing and overexpression of \u003cem\u003eSUGP1\u003c/em\u003e partially rescues it\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. We therefore asked whether E592K, with its nonoverlapping missplicing events, might preserve the interaction of \u003cem\u003eSF3B1\u003c/em\u003e with \u003cem\u003eSUGP1\u003c/em\u003e. Indeed, when His6-FLAG-\u003cem\u003eSF3B1\u003c/em\u003e variants were introduced into HEK293T cells and affinity purified using anti-DYKDDDDK (FLAG) antibody and cobalt beads, the association of His6-FLAG-\u003cem\u003eSF3B1\u003c/em\u003e with endogenous \u003cem\u003eSUGP1\u003c/em\u003e was disrupted by K700E but not by wild type or E592K \u003cem\u003eSF3B1\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). We then asked whether E592K might instead disrupt the interaction between \u003cem\u003eSF3B1\u003c/em\u003e and \u003cem\u003ePHF5A\u003c/em\u003e, given that E592 is at the interface with \u003cem\u003ePHF5A\u003c/em\u003e\u003csup\u003e2\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. However, this interaction was preserved by each His6-FLAG-SF3B1 variant (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). We also observed these effects in a second cell context, TF1 cells expressing FLAG-\u003cem\u003eSF3B1\u003c/em\u003e variants (Supplementary Fig. 7). Together, these data indicate that, consistent with its induction of unique RNA missplicing events, the E592K variant does not participate in the disruption of the \u003cem\u003eSF3B1\u003c/em\u003e-\u003cem\u003eSUGP1\u003c/em\u003e interaction that drives the cryptic splicing of other \u003cem\u003eSF3B1\u003c/em\u003e mutations.\u003c/p\u003e\n\u003cp\u003eFinally, we identified and analyzed RNA-seq from two patients with E592K mutation, compared to other \u003cem\u003eSF3B1\u003c/em\u003e mutations, from the recent MLL cohort of spliceosome-mutant myeloid malignancy patients\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Inspection of the junctions validated in our cell models also demonstrated the same specificity of missplicing in these primary patient samples, when compared to other \u003cem\u003eSF3B1\u003c/em\u003e mutations (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA). In addition, endpoint PCR validation of TMEM14C and RAVER2 from a third primary E592K sample at a separate institution demonstrated the same hotspot specificity of RNA missplicing (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eB), validating the findings from our cell models.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere we show the E592K variant of \u003cem\u003eSF3B1\u003c/em\u003e produces unique RNA missplicing and associates with high-risk MDS. These results have several implications. First, the distinctiveness of E592K informs the pathobiology of \u003cem\u003eSF3B1\u003c/em\u003e-mutant MDS. Clough et al elegantly showed that RS are formed by TMEM14C and ABCB7 missplicing in MDS-derived iPSCs with the G742D variant of \u003cem\u003eSF3B1\u003c/em\u003e\u003csup\u003e5\u003c/sup\u003e. These missplicing events have been seen with other hotspots, including K700E, which we corroborated here\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Cells with E592K, on the other hand, preserved canonical splicing of these genes, and cases of E592K MDS lacked RS. Other events that have been implicated in \u003cem\u003eSF3B1\u003c/em\u003e-mutant MDS pathobiology include innate immune activation by missplicing of MAP3K7 and IRAK4, enhanced self-renewal by MECOM missplicing, impaired erythropoiesis by COASY missplicing, and hepcidin suppression by ERFE missplicing; all these genes were also canonically spliced in E592K cells (Supplementary Fig.\u0026nbsp;7)\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. A distinct E592K pathobiology is also suggested by its high co-occurrence with ASXL1/RUNX1/STAG2 mutations and mutual exclusivity with DNMT3A mutations, a starkly different pattern than that of other \u003cem\u003eSF3B1\u003c/em\u003e hotspots. As with most co-mutation patterns in cancer, it is unclear whether these co-occurrences are driven by synergism or permissiveness\u0026mdash;and whether the mutual exclusivity is driven by antagonism or redundancy. Interestingly, the same pattern of co-occurrence (ASXL1/RUNX1/STAG2) and mutual exclusivity (DNMT3A) occurs with loss-of-function EZH2 mutations in MDS (Supplementary Fig.\u0026nbsp;8)\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. It is therefore intriguing that E592K missplicing produced a frameshifted EZH2 transcript in our studies here, and it is tempting to speculate this may create \u0026lsquo;mutant EZH2-ness\u0026rsquo; in E592K cells, an area for future investigation. If so, mutual exclusivity due to redundancy might be expected between mutant EZH2 and E592K. While there were indeed no EZH2 mutations in the 39 E592K patients here, the low overall frequency (4%) of EZH2 in \u003cem\u003eSF3B1\u003c/em\u003e-mutant myeloid malignancies means a larger cohort would be needed for significance testing of this particular pair. Nonetheless, the distinctiveness of E592K here shows that certain MDS phenotypes associated with \u003cem\u003eSF3B1\u003c/em\u003e mutation, including sideroblastic anemia, are not inevitable pathobiological outcomes of RNA missplicing by all \u003cem\u003eSF3B1\u003c/em\u003e variants.\u003c/p\u003e \u003cp\u003eSecond, there are implications for MDS classification. Recently, both the International Consensus Classification (ICC) and the 5th edition of the World Health Organization (WHO) classification of myeloid neoplasms created nosologic entities for \u003cem\u003eSF3B1\u003c/em\u003e-mutant MDS\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. These are based on the International Working Group (IWG) findings that \u003cem\u003eSF3B1\u003c/em\u003e mutation, low blasts, and lack of certain co-occurring genetic aberrations defined an indolent MDS with a shared pathobiology of idiosyncratic RNA missplicing that was more homogeneous than the MDS-RS classification\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. This was due in part to exclusion of \u003cem\u003eSF3B1\u003c/em\u003e-unmutated MDS-RS which had greater myeloid/megakaryocyte dysplasia, more TP53 co-mutations, and poorer outcomes. Both the ICC and WHO criteria require \u003cem\u003eSF3B1\u003c/em\u003e mutation, low blasts, cytopenias, dysplasia, and lack of multi-hit TP53/del(5q)/-7/complex cytogenetics; the ICC also requires \u003cem\u003eSF3B1\u003c/em\u003e VAF\u0026thinsp;\u0026gt;\u0026thinsp;10% and lack of RUNX1/del(7q)/abn3q26.2; and the WHO allows for \u0026gt;\u0026thinsp;15% RS to substitute for \u003cem\u003eSF3B1\u003c/em\u003e mutation. As we have seen here, E592K patients do not fit these groups: they have unique RNA missplicing, higher blasts, lack of RS, increased RUNX1 mutations, and poorer prognosis. These differences make an argument for excluding those E592K cases that would otherwise meet criteria for these entities. They also emphasize the need to consider specific hotspot mutations in future classification efforts. This should be the precise mutation, not just the affected amino acid, as we demonstrated different patterns of disease partitioning and missplicing between K666R and K666N.\u003c/p\u003e \u003cp\u003eThird, our data have implications for MDS prognostication. Along with low blasts, shallow cytopenias, and certain cytogenetic abnormalities such as del(11q), \u003cem\u003eSF3B1\u003c/em\u003e mutations have been consistently associated with better MDS outcomes in multiple independent datasets\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Accordingly, new MDS prognosis scoring tools incorporating mutational data have generally weighted \u003cem\u003eSF3B1\u003c/em\u003e mutations favorably, although there is important context-dependence that overrides this favorability, such as increased blasts and aberrations such as del(5q), RUNX1 mutation, and others\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. However, given the much larger patient cohorts that would be required, prognostication tools do not yet separately weight the myriad individual variants of mutated genes (\u003cem\u003eSF3B1\u003c/em\u003e or otherwise) into their scores. By the same token, a comprehensive multivariate analysis of the prognosis of E592K MDS would require a larger patient cohort than ours here. Nonetheless, while increased blasts or co-occurring RUNX1 mutations would lead to high-risk scores for many E592K patients, others would be understaged by these tools, as we observed here for two patients with rapid progression to AML (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-D). Thus, our data suggest caution in regarding any E592K patients as low risk. They also highlight the potential added value of incorporating specific hotspot mutations in the eventual next generation of prognostication tools.\u003c/p\u003e \u003cp\u003eFourth, these results have treatment implications. Luspatercept is indicated for treatment of anemia in ESA-refractory MDS-RS patients based on the phase III MEDALIST trial, which was conducted in this population due to strong associations of MDS-RS and \u003cem\u003eSF3B1\u003c/em\u003e mutation with response in the phase II PACE-MDS study\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. However, response of MDS-RS patients was similar regardless of mutant \u003cem\u003eSF3B1\u003c/em\u003e allelic burden in MEDALIST, as well as in a recent real-world cohort\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. These data would suggest that it is sideroblastic anemia (or the late-to-early erythroid progenitor ratio common in MDS-RS, according to long-term PACE-MDS follow up) that is predictive of luspatercept response, more so than \u003cem\u003eSF3B1\u003c/em\u003e mutation\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In the WHO 5th edition, MDS-RS has become MDS-\u003cem\u003eSF3B1\u003c/em\u003e and does not require RS if there is \u003cem\u003eSF3B1\u003c/em\u003e mutation, low blasts, and lack of high-risk cytogenetics\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Luspatercept would therefore be approved for many low-blast E592K patients, despite the sharp distinctions from MDS-RS patients that we have drawn here. Our data thus also recommend caution in approaching E592K patients like MDS-RS patients that are likely to respond to luspatercept. There are also implications for investigational therapies. Gene therapies that leverage missplicing in spliceosome-mutant cells are promising, but such vectors would need to be not only gene-specific (i.e. \u003cem\u003eSF3B1\u003c/em\u003e vs \u003cem\u003eSRSF2\u003c/em\u003e) but, in the case of E592K, hotspot-specific due to its nonoverlapping missplicing\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Similarly, antisense oligonucleotide therapy aimed at rescuing \u003cem\u003eBRD9\u003c/em\u003e missplicing showed activity against \u003cem\u003eSF3B1\u003c/em\u003e-mutant tumors in vivo, but such a therapy would not apply to E592K, which does not missplice \u003cem\u003eBRD9\u003c/em\u003e (Supplementary Fig.\u0026nbsp;7)\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFinally, these findings add to our understanding of the functional diversity of spliceosome mutations. After these mutations were first discovered as conspicuously enriched in myeloid neoplasms in a mutually exclusive manner, efforts have sought unifying downstream functional effects that might explain this occurrence pattern\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. This has revealed shared phenotypes, such as convergent disruption of certain pathways, creation of genotoxic R-loops, and sensitization to further disruption of the spliceosome\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR42 CR43\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. At the same time, bulk and then single-cell analyses have shown that spliceosome mutations are not always mutually exclusive, with multiple mutations sometimes selected for in the same cells\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. This indicates that different spliceosome mutations can confer different, even complementary, advantages to cancer cells, and the mutual exclusivity that does occur may be more from the splicing toxicity of combining certain mutations than from functional redundancy\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Consistent with this, studies have shown direct functional differences between spliceosome mutations: the missplicing events induced by mutations in different spliceosome genes (i.e. \u003cem\u003eSRSF2\u003c/em\u003e vs \u003cem\u003eU2AF1\u003c/em\u003e, etc) are nonoverlapping, the two dominant hotspot mutations in \u003cem\u003eU2AF1\u003c/em\u003e missplice different genes, and some less common \u003cem\u003eSRSF2\u003c/em\u003e and \u003cem\u003eU2AF1\u003c/em\u003e variants only partially or \u0026ldquo;dually\u0026rdquo; recapitulate the RNA missplicing of hotspot mutations\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR46 CR47\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. For \u003cem\u003eSF3B1\u003c/em\u003e mutations, their asymmetric partitioning among cancer types (i.e. K700E/H662Q in MDS, R625C/R625H in uveal melanoma, G642D in CLL, etc.) first suggested the mutations may have functional differences\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Later, Seiler et al noted different missplicing events in TCGA RNA-seq from bladder tumors with the E902K variant, events that we experimentally confirmed here\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. For other \u003cem\u003eSF3B1\u003c/em\u003e hotspots, studies have described differences of degree in RNA missplicing events, including K700E vs R625C/R625H in primary samples, R625H vs K741Q vs others in isogenic cells, and K666N vs K700E/H662Q in primary samples and isogenic cells\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. To these we add E592K, whose missplicing events are differences of kind, nonoverlapping with those of other MDS-associated hotspots\u0026mdash;and marked by a different underlying biochemistry that preserves the interaction between \u003cem\u003eSUGP1\u003c/em\u003e and \u003cem\u003eSF3B1\u003c/em\u003e. While it remains possible that the important underlying oncogenic effects of E592K are phenotypes still shared with other \u003cem\u003eSF3B1\u003c/em\u003e hotspots, the distinctiveness of E592K suggests to us that different \u003cem\u003eSF3B1\u003c/em\u003e variants can promote different kinds of leukemia by inducing different RNA missplicing events. Because these differences can impact our understanding and management of patients with myeloid neoplasms, additional studies of differences between other spliceosome variants are warranted.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThis work was supported by grants from the NIH (HL159306), Edward P. Evans Foundation, Leukemia \u0026amp; Lymphoma Society, Maryland Stem Cell Research Fund, Gabrielle\u0026rsquo;s Angel Foundation for Cancer Research, Emerson Collective, and AbbVie (to WBD); NIH R35 GM118136 (to JLM); Lady Tata Memorial Trust (3218) and the Barts Charity (G-002167) (to CP and KR).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eAED and WBD conceived the study. IYC, JPL, JZ, EH, JLM, KRP, AED, and WBD designed research. IYC, JPL, JZ, EH, CP, and WBD performed experiments. JLM, KRP, and WBD supervised experiments. IYC, WW, RB, CP, BL, DHW, MO, EB, XL, TF, SF, CDG, TJ, AED, and WBD collected data. IYC, JPL, JZ, WW, JLM, KRP, AED, and WBD analyzed data. IYC, JPL, JZ, KRP, and WBD prepared figures. IYC and WBD wrote the manuscript. All authors critically reviewed and approved the final version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eRaw FASTQ files are deposited at the NCBI Sequence Read Archive (SRA) under accession number SRP##########.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHaferlach T, Nagata Y, Grossmann V, Okuno Y, Bacher U, Nagae G \u003cem\u003eet al.\u003c/em\u003e Landscape of genetic lesions in 944 patients with myelodysplastic syndromes. \u003cem\u003eLeukemia\u003c/em\u003e 2014; 28: 241\u0026ndash;7.\u003c/li\u003e\n\u003cli\u003eDarman RB, Seiler M, Agrawal AA, Lim KH, Peng S, Aird D \u003cem\u003eet al.\u003c/em\u003e Cancer-Associated SF3B1 Hotspot Mutations Induce Cryptic 3\u0026prime; Splice Site Selection through Use of a Different Branch Point. \u003cem\u003eCell Reports\u003c/em\u003e 2015; 13: 1033\u0026ndash;1045.\u003c/li\u003e\n\u003cli\u003eBondu S, Alary A-S, Lef\u0026egrave;vre C, Houy A, Jung G, Lefebvre T \u003cem\u003eet al.\u003c/em\u003e A variant erythroferrone disrupts iron homeostasis in SF3B1-mutated myelodysplastic syndrome. \u003cem\u003eSci Transl Med\u003c/em\u003e 2019; 11: eaav5467.\u003c/li\u003e\n\u003cli\u003eMian SA, Philippe C, Maniati E, Protopapa P, Bergot T, Piganeau M \u003cem\u003eet al.\u003c/em\u003e Vitamin B5 and succinyl-CoA improve ineffective erythropoiesis in SF3B1-mutated myelodysplasia. \u003cem\u003eSci Transl Med\u003c/em\u003e 2023; 15: eabn5135.\u003c/li\u003e\n\u003cli\u003eClough CA, Pangallo J, Sarchi M, Ilagan JO, North K, Bergantinos R \u003cem\u003eet al.\u003c/em\u003e Coordinated mis-splicing of TMEM14C and ABCB7 causes ring sideroblast formation in SF3B1-mutant myelodysplastic syndrome. \u003cem\u003eBlood\u003c/em\u003e 2021; 139: 2038\u0026ndash;2049.\u003c/li\u003e\n\u003cli\u003eLee SC-W, North K, Kim E, Jang E, Obeng E, Lu SX \u003cem\u003eet al.\u003c/em\u003e Synthetic Lethal and Convergent Biological Effects of Cancer-Associated Spliceosomal Gene Mutations. \u003cem\u003eCancer Cell\u003c/em\u003e 2018; 34: 225-241.e8.\u003c/li\u003e\n\u003cli\u003eChoudhary GS, Pellagatti A, Agianian B, Smith MA, Bhagat TD, Gordon-Mitchell S \u003cem\u003eet al.\u003c/em\u003e Activation of targetable inflammatory immune signaling is seen in myelodysplastic syndromes with SF3B1 mutations. \u003cem\u003eElife\u003c/em\u003e 2022; 11: e78136.\u003c/li\u003e\n\u003cli\u003eTanaka A, Nakano TA, Nomura M, Yamazaki H, Bewersdorf JP, Mulet-Lazaro R \u003cem\u003eet al.\u003c/em\u003e Aberrant EVI1 splicing contributes to EVI1-rearranged leukemia. \u003cem\u003eBlood\u003c/em\u003e 2022; 140: 875\u0026ndash;888.\u003c/li\u003e\n\u003cli\u003eMalcovati L, Papaemmanuil E, Bowen DT, Boultwood J, Porta MGD, Pascutto C \u003cem\u003eet al.\u003c/em\u003e Clinical significance of SF3B1 mutations in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms. \u003cem\u003eBlood\u003c/em\u003e 2011; 118: 6239\u0026ndash;6246.\u003c/li\u003e\n\u003cli\u003eBernard E, Tuechler H, Greenberg PL, Hasserjian RP, Ossa JEA, Nannya Y \u003cem\u003eet al.\u003c/em\u003e Molecular International Prognostic Scoring System for Myelodysplastic Syndromes. \u003cem\u003eNejm Évid\u003c/em\u003e 2022; 1. doi:10.1056/evidoa2200008.\u003c/li\u003e\n\u003cli\u003eBersanelli M, Travaglino E, Meggendorfer M, Matteuzzi T, Sala C, Mosca E \u003cem\u003eet al.\u003c/em\u003e Classification and Personalized Prognostic Assessment on the Basis of Clinical and Genomic Features in Myelodysplastic Syndromes. \u003cem\u003eJ Clin Oncol\u003c/em\u003e 2021; 39: 1223\u0026ndash;1233.\u003c/li\u003e\n\u003cli\u003eNazha A, Komrokji R, Meggendorfer M, Jia X, Radakovich N, Shreve J \u003cem\u003eet al.\u003c/em\u003e Personalized Prediction Model to Risk Stratify Patients With Myelodysplastic Syndromes. \u003cem\u003eJ Clin Oncol\u003c/em\u003e 2021; 39: 3737\u0026ndash;3746.\u003c/li\u003e\n\u003cli\u003eKubasch AS, Fenaux P, Platzbecker U. 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Recurrent mutations at codon 625 of the splicing factor SF3B1 in uveal melanoma. \u003cem\u003eNature Genetics\u003c/em\u003e 2013; 45: 133.\u003c/li\u003e\n\u003cli\u003eRossi D, Bruscaggin A, Spina V, Rasi S, Khiabanian H, Messina M \u003cem\u003eet al.\u003c/em\u003e Mutations of the SF3B1 splicing factor in chronic lymphocytic leukemia: association with progression and fludarabine-refractoriness. \u003cem\u003eBlood\u003c/em\u003e 2011; 118: 6904\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eLiu Z, Yoshimi A, Wang J, Cho H, Lee SC-W, Ki M \u003cem\u003eet al.\u003c/em\u003e Mutations in the RNA Splicing Factor SF3B1 Promote Tumorigenesis through MYC Stabilization. \u003cem\u003eCancer Discov\u003c/em\u003e 2020; 10: 806\u0026ndash;821.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2802265/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2802265/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAmong the most common genetic alterations in the myelodysplastic syndromes (MDS) are mutations in the spliceosome gene \u003cem\u003eSF3B1\u003c/em\u003e. Such mutations induce specific RNA missplicing events, directly promote ring sideroblast (RS) formation, generally associate with more favorable prognosis, and serve as a predictive biomarker of response to luspatercept. However, not all \u003cem\u003eSF3B1\u003c/em\u003e mutations are the same, and here we report that the E592K variant of \u003cem\u003eSF3B1\u003c/em\u003e associates with high-risk disease features in MDS, including a lack of RS, increased myeloblasts, a distinct co-mutation pattern, and decreased survival. Moreover, in contrast to canonical SF3B1 mutations, E592K induces a unique RNA missplicing pattern, retains an interaction with the splicing factor \u003cem\u003eSUGP1\u003c/em\u003e, and preserves normal RNA splicing of the sideroblastic anemia genes \u003cem\u003eTMEM14C\u003c/em\u003e and ABCB7. These data expand our knowledge of the functional diversity of spliceosome mutations, and they suggest that patients with E592K should be approached differently from low-risk, luspatercept-responsive MDS patients with ring sideroblasts and canonical SF3B1 mutations.\u003c/p\u003e","manuscriptTitle":"The E592K variant of SF3B1 creates unique RNA missplicing and associates with high-risk MDS without ring sideroblasts","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-04-14 13:44:10","doi":"10.21203/rs.3.rs-2802265/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4a1964e3-5f8a-40d3-8621-d23b9705b237","owner":[],"postedDate":"April 14th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":20651713,"name":"Health sciences/Diseases/Haematological diseases/Haematological cancer/Myelodysplastic syndrome"},{"id":20651714,"name":"Biological sciences/Cancer/Oncogenes"},{"id":20651715,"name":"Biological sciences/Genetics/Cancer genetics"}],"tags":[],"updatedAt":"2024-07-26T15:21:12+00:00","versionOfRecord":{"articleIdentity":"rs-2802265","link":"https://doi.org/10.1182/bloodadvances.2023011260","journal":{"identity":"blood-advances","isVorOnly":true,"title":"Blood Advances"},"publishedOn":"2024-05-17 15:21:12","publishedOnDateReadable":"May 17th, 2024"},"versionCreatedAt":"2023-04-14 13:44:10","video":"","vorDoi":"10.1182/bloodadvances.2023011260","vorDoiUrl":"https://doi.org/10.1182/bloodadvances.2023011260","workflowStages":[]},"version":"v1","identity":"rs-2802265","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2802265","identity":"rs-2802265","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}