Program: Oral and Poster Abstracts
Session: 602. Disordered Gene Expression in Hematologic Malignancy, including Disordered Epigenetic Regulation: Poster II
The sequence-based algorithm AVISPA (Barash et al., 2013) predicts several splicing factors (SF) to bind near exon 2. We used RNA crosslink immunoprecipitation (CLIP) in nuclear lysates from Nalm-6 B-ALL cells to test the direct binding to exon 2 of 9 AVISPA-predicted SFs and 6 SFs commonly involved in exon skipping. This allowed us to identify SRSF3, hnRNP-A, and hnRNP-C as CD19 exon 2-bound proteins. Subsequent siRNA knockdown experiments reveled that downregulation of SRSF3, but not hnRNP-C, increases the frequency of exon 2 skipping in a dose dependent manner, suggesting that SRSF3 promotes the inclusion of exon 2. To further validate the role of SRSF3 in CD19 splicing we mined the publicly available GSE52834 dataset where 22 RNA binding proteins were knocked down in the GM19238 lymphoblastoid cell line. Of all siRNAs tested, only the anti-SRSF3 siRNA caused an increase in exon 2 skipping, suggesting that SRSF3 is indeed the key regulator of CD19 splicing.
Interestingly, SRSF3 has been shown to interact with PSIP1, a cofactor known to “read” modified histone H3K36me3 (Pradeepa et al., 2012), suggesting a convergence of splicing-based and epigenetics mechanisms. Indeed, exonic regions in genomic DNA are enriched for H3K36me3, and knockdown of Setd2, the H3K36 methyltransferase, results in changes in exon inclusion (Luco et al., 2010; Brown et al., 2012; Hnilicova and Stanek, 2011). Thus, we are currently investigating the connection between the H3K36me3 marks in the CD19 locus and alternative splicing of CD19. Our data could suggest a method of restoring full-length CD19 expression in immunotherapy-resistant cancers using epigenetic drugs.
Maude, S L, Noelle, F, Shaw, PA, Aplenc, R, Barrett, DM, Bunin, NJ, Chew, A, Gonzalez, VE, Zheng, Z, Lacey, SF, Mahnke, YD, Melenhorst, JJ, Rheingold, SR, Shen, A, Teachey, DT, Levine, BL, June CH, Porter, DL, and Grupp, SA. Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med 2014; 371: 1507-1517.
Sotillo, E, Barrett, D, Bagashev, A, Black, K, Lanauze, C, Oldridge, D, Sussman, R, Harrington, C, Chung, EY, Hofmann, TJ, Maude, SL, Martinez, NM, Raman, P, Ruella, M, Allman, D, Jacoby, E, Fry, T, Barash, Y, Lynch, KW, Mackall, C, Maris, J, Grupp, SA, and Thomas-Tikhonenko, A. Alternative splicing of CD19 mRNA in leukemias escaping CART-19 immunotherapy eliminates the cognate epitope andcontributes to treatment failure. 2015AACR Annual Meeting, Philadelphia.
Barash Y, Vaquero-Garcia J, González-Vallinas J, Xiong HY, Gao W, Lee LJ, and Frey BJ. AVISPA: a web tool for the prediction and analysis of alternative splicing. Genome Biol 2013; 14(10):R114.
Pradeepa, MM, Sutherland, HG, Ule, J, Grimes, GR, and Bickmore, WA. Psip1/Ledgf p52 binds methylated histone H3K36 and splicing factors and contributes to the regulation of alternative splicing. PLOS Genets 2012; 8:e1002717.
Luco, RF, Pan, Q, Tominaga, K, Blencowe, BJ, Pereira-Smith, OM, Misteli, T. Regulation of alternative splicing by histone modifications. Science 2010; 327: 996-1000.
Brown, SJ, Stoilov, P, and Xing, Y. Chromatin and epigenetic regulation of pre-mRNA processing. Human Mol Genets 2012; 21:R90-R96.
Hnilicova, J, and Stanek, D. Where splicing joins chromatin. Nucleus 2011; 2:182-188.
Disclosures: No relevant conflicts of interest to declare.
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