ALBERT

Description: Splicing is a fundamental process by which introns are removed from primary RNA transcripts. Alternative splicing is a major mechanism of gene regulation by which eukaryotic cells expand their transcriptional repertoire. By contrast, aberrant splicing generates novel transcripts not found in normal cells. Heterozygous gain-of-function mutations in a core U2 spliceosome factor SF3B1 are present in ~25% of myelodysplastic syndrome (MDS) patients. Strikingly, SF3B1 mutations are present in ~80% of MDS with ring sideroblasts (MDS-RS) patients suggesting a causal connection between mutant SF3B1 and ring sideroblasts (RS), erythroid precursors with iron-laden mitochondria (Yoshida et al. Nature 2011, Papaemmanuil et al.NEJM 2011). However, the mechanism by which SF3B1 mutations cause RS formation remains poorly understood since existing models of SF3B1-mutant MDS do not recapitulate RS formation in vitro. We have established an induced pluripotent stem cell (iPSC) model of MDS-RS that recapitulates mutant SF3B1-mediated mis-splicing and in vitro ring sideroblast formation. We reprogrammed SF3B1-mutant and SF3B1-wild-type iPSCs from individual MDS-RS patients enabling internal normalization to the isogenic normal clone (Hsu et al. Blood 2019). We established expandable multipotential HPC lines by conditional expression of five transcription factors (5F-HPC), followed by an 18-day erythroid differentiation. We monitored RS formation during the erythroid differentiation of 5F-HPCs using Prussian blue histological staining. RS formation increased during terminal erythropoiesis and peaked at 25-40% on day 18 in SF3B1-mutant lines, whereas SF3B1-wild-type lines showed no detectable RS formation. The frequency of RS was similar in isogenic SF3B1-only and SF3B1/EZH2 co-mutant cells suggesting that the mutant SF3B1 is sufficient to drive RS formation. To identify mis-splicing events that contribute to RS formation, we performed RNA-sequencing and splicing analysis of SF3B1-mutant and SF3B1-wild-type iPSCs at three stages of erythroid differentiation: CD34+ progenitor, CD71+ early erythroblast, and CD71+Glycophorin A+ erythroblast. Global isoform usage was dramatically altered during erythropoiesis, but was more similar in stage-matched SF3B1-mutant and SF3B1-wild-type cells suggesting that mutant SF3B1 selectively mis-splices a subset of transcripts. We identified 2300 transcripts with 〉10% mis-splicing in SF3B1-mutant lines, and only 120 transcripts with more significant 〉40% mis-splicing. Of these, TMEM14C and PPOX, inner mitochondrial membrane components of the heme synthesis pathway were strongly mis-spliced throughout erythroid differentiation, consistent with previous studies (Conte et al.BJHaem 2015, Shiozawa et al. Nat Commun 2018). The transcript levels of PPOX but not TMEM14C were reduced in SF3B1-mutant lines as a result of mis-splicing. The expression of ABCB7, a mitochondrial iron sulfur cluster biogenesis component mutated in inherited X-linked sideroblastic anemia (Allikmets et al. Hum Mol Genet 1999), was also reduced in SF3B1-mutant cells as expected (Shiozawa et al. Nat Commun 2018, Dolatshad et al. Leukemia 2016). To investigate the role of these mis-splicing events in ring sideroblast formation, we performed lentiviral overexpression of TMEM14C, PPOX, and ABCB7, in SF3B1-mutant 5F-HPCs and quantified RS formation during late stages of erythroid differentiation. Overexpression of TMEM14C and ABCB7 in SF3B1-mutant cells partially but not completely rescued RS formation compared to luciferase control. These findings confirm the long-standing hypothesis that mis-splicing of mitochondrial iron metabolism genes causes RS formation. Furthermore, these findings suggest that RS formation in MDS is a multigenic event caused by coordinated but incomplete mis-splicing of several critical iron metabolism genes. Disclosures No relevant conflicts of interest to declare.

Description: Myeloid malignancies are susceptible to immunologic destruction but antigen-specific T cell immunotherapies for these diseases are currently limited. Immunotherapies targeting neoantigens created from recurrent protein-coding mutations should selectively eradicate malignant cells and spare their normal counterparts to limit the risk of myeloablation. In particular, the paucity of neoplastic cells in MDS specimens impedes the identification and validation of targetable neoantigens for the development of immunotherapies for this disorder. Induced pluripotent stem cells (iPSC), generated from primary patient cells and differentiated into hematopoietic lines, can recapitulate the genotype and phenotype of the original disease (Hsu et al., Blood 2019) and could provide a renewable source of MDS progenitor cells for the development of novel T cell immunotherapy. SF3B1 mutations serve as a neoplastic driver for myeloid neoplasms (Papaemmanuil et al., Blood 2013), occuring in 20-30% of all MDS and 〉60% of MDS with ringed sideroblasts (Cazzola et al., Blood 2013). We hypothesized that SF3B1 mutations produce HLA-presented neoantigens, which can be targeted to eliminate primary MDS cells and iPSC with SF3B1 mutations. We interrogated the amino acid sequences from protein-coding mutations in SF3B1 using HLA-binding prediction algorithms (Immune Epitope Database Stabilized Matrix Method and Artificial Neural Network, netMHCpan 4.0) to predict binding to 20 prevalent HLA-A, -B, and -C alleles, and identified a candidate SF3B1mut epitope with strong predicted binding to HLA-B*40:01. HLA binding is necessary but not sufficient for T cell recognition of an epitope. We thus next evaluated the immunogenicity of the candidate SF3B1mut neoantigen epitope by stimulating CD8+ T cells isolated from healthy volunteer donors with peptide epitope-loaded autologous monocyte-derived dendritic cells, and isolated SF3B1mut -specific T cell clones, confirming the candidate epitope's immunogenicity. Two clones showed high functional avidity for the epitope in cytotoxicity assays (Figure 1A). To determine whether the epitope was naturally processed and presented by primary malignant myeloid cells, high-avidity SF3B1mut-specific clones were co-cultured with hematopoietic cells from patients with active MDS or acute myeloid leukemia (AML), then assessed for antigen recognition in a CD107a degranulation assay. High-avidity clones showed increased degranulation in response to a primary SF3B1mut HLA-B*40:01+ sample compared to samples lacking either the mutation or restricting HLA (Figure 1B), indicating that the SF3B1mut epitope is naturally processed and presented. We then evaluated whether SF3B1mut-specific clones could recognize SF3B1mut iPSC-derived hematopoietic cells. We reprogrammed hematopoietic stem/progenitor cells (HSPC) from SF3B1mut MDS patient bone marrow and established two iPSC lines: SF3B1mut and an isogenic control with wild-type SF3B1. Multipotent hematopoietic progenitor lines (MPP-5F) were then generated from iPSC lines by doxycycline-dependent expression of five HSPC transcription factors (Hsu et al., Blood 2019). In a CD107a degranulation assay, high-avidity clones showed ~30% degranulation with SF3B1mut MPP-5F, compared to ~5% with the isogenic control (Figure 1C), indicating that the iPSC-derived MPP-5F line recapitulated presentation of the neoantigen. We present a novel neoantigen with promise as an immunotherapy target for MDS, and for other hematologic malignancies with SF3B1 mutations including secondary AML, MDS/myeloproliferative neoplasm overlap syndrome, and advanced chronic lymphocytic leukemia (Cazzola et al., Blood 2013). Future experiments will assess the efficacy of SF3B1mut-specific T cell immunotherapy in vivo in a murine model and evaluate transfer of SF3B1mut-specific T cell receptors as a potential therapeutic approach. Our studies also indicate that iPSC-derived MPP-5F lines have potential to substitute for primary patient cells in neoantigen validation studies, a finding with broad utility for immunotherapy development in hematologic malignancies. Ongoing studies will assess MPP-5F presentation of a diverse panel of antigens, including minor histocompatibility antigens, non-mutated cancer antigens, and other neoantigens. Disclosures Oehler: Pfizer, Inc: Research Funding; Takeda: Consultancy; BMS: Consultancy. Bleakley:HighPass Bio: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.