Session: 602. Disordered Gene Expression in Hematologic Malignancy, including Disordered Epigenetic Regulation: Altered Splicing and Transcriptional Regulation
Hematology Disease Topics & Pathways:
Biological, Leukemia, Diseases, Therapies, CLL, Biological Processes, epigenetics, Clinically relevant, Lymphoid Malignancies, genomics, molecular interactions
Regulatory network analysis of differentially expressed genes in SF3B1 mutated CLL identified MYC as the top master regulator (Figure C). MYC activation in SF3B1 mutated CLL was also verified by differential expression analyses (Figure D) and was common to SF3B1K700E mutant cancers while absent in cancers with mutations affecting R625. Taken together, these observations suggested that tumors harboring SF3B1K700E mutations activate the MYC transcriptional program.
We next sought to verify the effects of c-Myc activation by mutant Sf3b1 in the B-cell lineage in vivo. We crossed Cd19-cre Sf3b1K700E/+ mice with Eμ-Myc transgenic mice to generate Cd19-cre+ control, Sf3b1K700E/+, Eμ-MycTg/+, and Sf3b1K700E/+ Eμ-MycTg/+ double-mutant mice. While control or single mutant primary mice did not develop disease over one year, double-mutant mice developed a lethal B-cell malignancy. This effect was consistent in serial transplantation, where mice transplanted with double-mutant cells had shorter survival compared to single-mutant controls (Figure E). These data provide the first evidence that SF3B1 mutations contribute to tumorigenesis in vivo.
To understand the molecular mechanism for MYC activation across SF3B1 mutant human and mouse cells, we analyzed RNA-seq data from CLL patients, isogenic Nalm-6 cells, and splenic B-cells from the mouse models. This revealed a significant overlap in aberrant (3’ss) events across SF3B1 mutant samples. Interestingly, mis-spliced events across mouse and human SF3B1K700E mutant samples identified aberrant 3’ss usage and decay of PPP2R5A (Figure F), a gene whose product has previously been shown to regulate c-MYC protein stability and the only gene whose aberrant splicing was most prominent in K700E compared with R625 mutant SF3B1. PPP2R5A is a subunit of the PP2A phosphatase complex that dephosphorylates Serine 62 (S62) of c-MYC, resulting in an unstable form of c-MYC that is a substrate for proteasomal degradation. Consistent with this, SF3B1K700E mutant cells exhibited dramatic increase in S62-phosphorylated c-MYC and increased stability of c-MYC protein. MYC expression, stability, and S62 phosphorylation could be abrogated in SF3B1 mutant cells by restoring PPP25RA expression. In addition to c-MYC S62 phosphorylation, PPP2R5A-containing PP2A reduced S70 phosphorylation of BCL2 (a modification important for apoptosis induction) in SF3B1 mutant cells. To functionally evaluate the importance of impaired PP2A enzymatic activity in SF3B1 mutant cells further, we assessed the therapeutic potential of the FDA-approved oral PP2A activator, FTY-720. SF3B1 mutant cells were more sensitive to FTY-720 treatment than SF3B1 WT counterparts, experiencing growth arrest at lower concentration (Figure G). Moreover, both S62-phosphorylated c-MYC and S70-phosphorylated BCL2 decreased in a dose-dependent manner upon treatment with FTY-720 (Figure H).
Here through combined evaluation of the effects of the SF3B1 mutation on splicing, gene expression, and transcriptional networks across cancer types, we identify a novel mechanism by which mutant SF3B1-mediated alterations in RNA splicing contribute to activation of oncogenic MYC through effects on MYC proteolysis. Moreover, these data highlight a novel therapeutic approach targeting the impact of mutant SF3B1 on post-translational modification of MYC.
Disclosures: Mato: Janssen: Consultancy, Honoraria; Celgene: Consultancy; Prime Oncology: Speakers Bureau; TG Therapeutics: Research Funding; Regeneron: Research Funding; Abbvie: Consultancy; Sunesis: Honoraria, Research Funding; Acerta: Research Funding; AstraZeneca: Consultancy; Pharmacyclics: Consultancy, Honoraria, Research Funding.
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