Abstract IA6: Misregulation of pre-mRNA splicing in cancer

Alternative splicing occurs in transcripts produced from ~95% of human genes. Under normal conditions, this process is tightly regulated, but it can go awry in numerous diseases. Cancer cells, for example, take advantage of this mechanism to produce aberrant proteins with added, deleted, or altered functional domains that contribute to tumorigenesis. Misregulation in cancer occurs frequently by overexpression of splicing regulatory factors, but recent studies have for the first time identified mutated components of the splicing machinery in certain cancers. Here I describe our studies relevant to both these pathways. HnRNP proteins are an example of proteins overexpressed in many cancers. Our previous work defined a pathway whereby expression of three of these, hnRNPA1, A2 and PTB, is upregulated by the cMyc oncogenic transcription factor, which leads to a switch in splicing of the pyruvate kinase M (PKM) pre-mRNA such that a form of PKM necessary for tumor cell proliferation is made. Our more recent studies have identified other pathways that contribute to overexpression of the hnRNPs, and begun to elucidate the role that these proteins play in glioblastoma (GBM). In human GBM patients, expression of these proteins inversely correlates with survival, while in rat and mouse models of GBM, the proteins are significantly overexpressed, and reducing their expression inhibits cell proliferation and tumor formation. Comparing RNA-seq data we obtained following knockdown of the proteins in human U87 GBM cells with known changes in alternative splicing in human GBM brain samples, we identified a number of transcripts, which include PKM, that are direct targets of the hnRNP proteins and relevant to GBM. Examples of these proteins, and the pathways they contribute to, will be discussed. Mutations in the genes encoding components of the RNA splicing machinery have been described in myelodysplastic syndromes (MDS), a group of neoplasms characterized by an abnormality in myeloid blood cell production and a propensity for progression into acute myeloid leukemia. The most frequently mutated genes encode splicing factors SF3B1, a U2 snRNP associated protein, U2AF1, SRSF2 and ZRSR2, all of which function in 3' splice site recognition. Mutational frequencies for SF3B1 are particularly high in refractory anemia with ring sideroblasts, and SF3B1 is also frequently mutated in Chronic Lymphocytic Leukemia as well as in uveal melanoma. All SF3B1 mutations described to date are heterozygous missense mutations. We are studying how mutations in SF3B1 affect the protein's function in splicing, and contribute to MDS. Wild-type and mutant SF3B1 behave identically in a number of splicing-related assays, indicating that MDS mutation does not grossly affect protein function. We are also analyzing RNA samples obtained from MDS patients to identify changes in splicing. Remarkably, we observe a sharp increase in intron retention in patient samples, which will lead to production of truncated proteins and frequently mRNA decay by the NMD pathway. We have identified several candidate genes that may contribute to MDS, including one that produces an enzyme in the heme synthesis pathway that when defective may lead to anemia, and another that encodes a protein important for controlling expansion of hematopoietic stem cells, perhaps resulting in clonal expansion of mutant cells. Citation Format: Jian Zhang, Ritam Neupane, Peter Canoll, Yen Lieu, Siddhartha Mukherjee, Raul Rabadan, Azra Raza, James L. Manley. Misregulation of pre-mRNA splicing in cancer. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr IA6.