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Recurrent mutations in the spliceosome are observed in several human cancers, but their functional and therapeutic significance remains elusive. SF3B1, the most frequently mutated component of the spliceosome in cancer, is involved in the recognition of the branch point sequence (BPS) during selection of the 3′ splice site (ss) in RNA splicing. Here, we report that common and tumor-specific splicing aberrations are induced by SF3B1 mutations and establish aberrant 3′ ss selection as the most frequent splicing defect. Strikingly, mutant SF3B1 utilizes a BPS that differs from that used by wild-type SF3B1 and requires the canonical 3′ ss to enable aberrant splicing during the second step. Approximately 50% of the aberrantly spliced mRNAs are subjected to nonsense-mediated decay resulting in downregulation of gene and protein expression. These findings ascribe functional significance to the consequences of SF3B1 mutations in cancer.

Buonamici: H3 Biomedicine: Employment. Thomas:H3 Biomedicine: Employment. Seiler:H3 Biomedicine: Employment. Chan:H3 Biomedicine: Employment. Caleb:H3 Biomedicine: Employment. Darman:H3 Biomedicine: Employment. Fekkes:H3 Biomedicine: Employment. Karr:H3 Biomedicine: Employment. Liu:H3 Biomedicine: Employment. Meeske:H3 Biomedicine: Employment. Mizui:Eisai: Employment. Pazolli:H3 Biomedicine: Employment. Prajapati:H3 Biomedicine: Employment. Wang:Eisai: Employment. Warmuth:H3 Biomedicine: Employment. Yu:H3 Biomedicine: Employment. Zhu:H3 Biomedicine: Employment. Smith:H3 Biomedicine: Employment.

Mutations in RNA splicing factors confer an alteration of function and are common in patients with myelodysplastic syndrome (MDS, ~45%), chronic myelomonocytic leukemia (CMML, ~60%), and acute myeloid leukemia (AML) derived from these conditions. Recent data suggest that spliceosome-mutant cells are preferentially sensitive to genetic or pharmacologic splicing modulation compared with wildtype (WT) counterparts. Here, we describe the discovery of H3B-8800, a potent and orally bioavailable modulator of the SF3b complex, and demonstrate efficacy in models of spliceosome mutant myeloid malignancies including a novel xenograft system for CMML.

Buonamici: H3 Biomedicine: Employment. Darman:H3 Biomedicine: Employment. Perino:H3 Biomedicine: Employment. Agrawal:H3 Biomedicine: Employment. Peng:H3 Biomedicine: Employment. Seiler:H3 Biomedicine: Employment. Feala:H3 Biomedicine: Employment. Bailey:H3 Biomedicine: Employment. Chan:H3 Biomedicine: Employment. Fekkes:H3 Biomedicine: Employment. Keaney:H3 Biomedicine: Employment. Kumar:H3 Biomedicine: Employment. Kunii:H3 Biomedicine: Employment. Lee:H3 Biomedicine: Employment. Mackenzie:Eisai: Employment. Matijevic:Eisai: Employment. Mizui:H3 Biomedicine: Employment. Myint:Eisai: Employment. Park:H3 Biomedicine: Employment. Pazolli:H3 Biomedicine: Employment. Thomas:H3 Biomedicine: Employment. Wang:H3 Biomedicine: Employment. Warmuth:H3 Biomedicine: Employment. Yu:H3 Biomedicine: Employment. Zhu:H3 Biomedicine: Employment. Furman:Acerta Pharma BV: Research Funding; Gilead: Consultancy; Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria, Speakers Bureau. Ebert:Celgene: Consultancy; H3 Biomedicine: Consultancy; Genoptix: Consultancy, Patents & Royalties. Smith:H3 Biomedicine: Employment.

Heterozygous mutations in several core members of the spliceosome complex have been reported in Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML). In particular high frequency SF3B1 hotspot mutations, a component of the U2 complex involved in the interaction with the branch point (BP) and recognition of the 3' splice sites (ss) during splicing, have been identified in Refractory Anemia with Ringed Sideroblasts (RARS) a subtype of MDS. Using computational analyses of RNAseq from several cancer types including RARS, we identified that SF3B1 hotspot mutations induce aberrant 3'ss selection by recognizing a cryptic AG located between 15 to 24 nucleotides upstream of the canonical AG. Experimental confirmation of these motif features was performed using minigenes in SF3B1 mutant cells. Furthermore, we discovered that SF3B1 mutant utilized a different BP from that used by SF3B1 wild-type providing novel mechanistic insights into changes in function induced by the hotspot mutations. The induction of aberrant splicing can introduce premature termination codons thus targeting mRNA for degradation by Nonsense Mediated Decay (NMD). We predicted that close to 50% of the aberrantly spliced genes would be subject to NMD and showed (using isogenic Nalm-6 cells engineered by AAV homology to express SF3B1K700Eor SF3B1K700K) that several of these genes were downregulated at the transcript and protein levels. These downregulated genes/proteins might be involved in the pathogenesis of SF3B1 mutant cancers. Interestingly, pathway analysis of genes differentially expressed or aberrantly spliced in SF3B1 mutant compared to wild-type in RARS samples identified cell differentiation and epigenetics as the primary misregulated pathways. To study the impact of SF3B1 mutations on differentiation, we used the TF-1 differentiation cell model where erythroid differentiation is induced by treatment with erythropoietin (EPO). EPO treatment, as expected, induced erythroid differentiation in TF-1 cells transduced with SF3B1WT, but a block in erythroid differentiation was observed in TF-1 cells transduced with SF3B1K700E(the most common mutation in MDS and chronic lymphocytic leukemia (CLL)). Intriguingly, SF3B1G742D, which is found mutated in CLL but not MDS, did not block differentiation in this myeloid differentiation model, implying that specific SF3B1 mutations and splicing aberrations have important context dependent effects. Pathway analysis comparing SF3B1K700Evs. SF3B1WTor SF3B1G742Didentified several genes involved in heme biosynthesis or downstream of GATA1 to be downregulated (such as, AHSP, ALAS2, CCL5, CD36, EPOR, GP1BB, HBB, HBE1, HBG1, PRG2) in SF3B1K700Ecells only. This is consistent with the role of SF3B1K700Ein RARS. In our analyses, we also identified that ABCB7 is aberrantly spliced and that the aberrant transcript is subject to NMD, causing downregulation of the canonical transcript and protein. ABCB7 is a mitochondrial transporter important in cellular iron metabolism and in heme production; moreover, partial loss of function mutation in ABCB7 has been identified in X-linked sideroblastic anemia and ataxia, demonstrating an iron overload phenotype in cells with defective ABCB7. Interestingly, when ABCB7 was knocked down in TF-1 cells we observed block in differentiation similar to that observed in SF3B1K700Ecells suggesting a link between SF3B1 mutation and ABCB7 levels and impaired differentiation. Taken together, these data suggest that SF3B1 mutations induce aberrant splicing and as a consequence downregulation of several genes that contribute to the block in erythroid differentiation, one of the key biological defects observed in MDS.

Buonamici: H3 Biomedicine: Employment. Perino:H3 Biomedicine: Employment. Lim:H3 Biomedicine: Employment. Feala:H3 Biomedicine: Employment. Aicher:H3 Biomedicine: Employment. Aird:H3 Biomedicine: Employment. Bailey:H3 Biomedicine: Employment. Berkenblit:H3 Biomedicine: Employment. Chan:H3 Biomedicine: Employment. Erik:H3 Biomedicine: Employment. Corson:H3 Biomedicine: Employment. Darman:H3 Biomedicine: Employment. Fekkes:H3 Biomedicine: Employment. Furman:Pharmacyclics: Consultancy, Speakers Bureau. Keaney:H3 Biomedicine: Employment. Kumar:Eisai: Employment. Kunii:H3 Biomedicine: Employment. Lee:H3 Biomedicine: Employment. Mackenzie:Eisai: Employment. Park:H3 Biomedicine: Employment. Puyang:H3 Biomedicine: Employment. Selvaraj:H3 Biomedicine: Employment. Thomas:H3 Biomedicine: Employment. Wang:H3 Biomedicine: Employment. Warmuth:H3 Biomedicine: Employment. Yu:H3 Biomedicine: Employment. Zhu:H3 Biomedicine: Employment. Mizui:H3 Biomedicine: Employment. Smith:H3 Biomedicine: Employment.

Recently, heterozygous mutations in several spliceosome genes have been observed in hematological and solid cancers, but their functional role in these diseases is not well understood. Among these, SF3B1 is the most commonly mutated spliceosome gene in myelodysplastic syndromes (MDS) and chronic lymphocytic leukemia (CLL). SF3B1 is part of the U2 complex involved in the recognition of the 3’ splice sites (3’ss) during early spliceosome assembly.

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