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

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.

Figure 1. Distribution of exon 13–16 SF3B1 mutations within WHO 2016 classifications of MDS and AML.

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.

Figure 2. Asymmetric RNA missplicing by distinct SF3B1 mutation hotspots.

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.

Figure 3. Clinical parameters of MDS patients with the E592K variant of SF3B1.

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).

Figure 4. Co-mutation landscape of SF3B1-mutant MDS and AML.

All E592K-mutated cases are shown on the left, and all exon 14–16-mutated cases on the right. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 5. MDS patients with E592K have poor survival.

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 >10 years by Sex-GSS (right). D) Understaging of Pt #1, who developed AML 9 months after diagnosis.

Figure 6. E592K induces unique RNA missplicing events.

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.

Figure 7. The E592K variant preserves association of SF3B1 with SUGP1.

HEK293T cells were transfected with His6-FLAG-SF3B1 variants and subjected to affi nity 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.

Figure 8. E592K exhibits unique RNA missplicing in primary MDS samples.

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.