Following CART-19 immunotherapy for B-cell acute lymphoblastic leukaemia (B-ALL), many patients relapse due to loss of the cognate CD19 epitope. Since epitope loss can be caused by aberrant CD19 exon 2 processing, we herein investigate the regulatory code that controls CD19 splicing. We combine high-throughput mutagenesis with mathematical modelling to quantitatively disentangle the effects of all mutations in the region comprising CD19 exons 1-3. Thereupon, we identify ~200 single point mutations that alter CD19 splicing and thus could predispose B-ALL patients to developing CART-19 resistance. Furthermore, we report almost 100 previously unknown splice isoforms that emerge from cryptic splice sites and likely encode non-functional CD19 proteins. We further identify cis-regulatory elements and trans-acting RNA-binding proteins that control CD19 splicing (e.g., PTBP1 and SF3B4) and validate that loss of these factors leads to pervasive CD19 mis-splicing. Our dataset represents a comprehensive resource for identifying predictive biomarkers for CART-19 therapy.

Background: Cancer is a set of diseases characterized by unchecked cell proliferation and invasion of surrounding tissues. The many genes that have been genetically associated with cancer or shown to directly contribute to oncogenesis vary widely between tumor types, but common gene signatures that relate to core cancer pathways have also been identified. It is not clear, however, whether there exist additional sets of genes or transcriptomic features that are less well known in cancer biology but that are also commonly deregulated across several cancer types. Results: Here, we agnostically identify transcriptomic features that are commonly shared between cancer types using 13,461 RNA-seq samples from 19 normal tissue types and 18 solid tumor types to train three feed-forward neural networks, based either on protein-coding gene expression, lncRNA expression, or splice junction use, to distinguish between normal and tumor samples. All three models recognize transcriptome signatures that are consistent across tumors. Analysis of attribution values extracted from our models reveals that genes that are commonly altered in cancer by expression or splicing variations are under strong evolutionary and selective constraints. Importantly, we find that genes composing our cancer transcriptome signatures are not frequently affected by mutations or genomic alterations and that their functions differ widely from the genes genetically associated with cancer. Conclusions: Our results highlighted that deregulation of RNA-processing genes and aberrant splicing are pervasive features on which core cancer pathways might converge across a large array of solid tumor types.

Significance: Acute myeloid leukemia (AML) is an aggressive hematologic cancer in which malignant myeloid precursor cells impair hematopoiesis and induce bone marrow failure. Most genes associated with AML are mutated in less than 10% of patients, suggesting that alternative mechanisms of gene disruption contribute to this disease. Here, we demonstrate that alternative splicing provides an additional layer of gene dysregulation and mechanism by which gene function is disrupted in AML. These results demonstrate that classical mutation analysis underestimates the burden of functional gene disruption in AML and highlight the importance of assessing the contribution of alternative splicing to gene dysregulation in human disease.

Quantification of RNA splicing variations based on RNA-Sequencing can reveal tissue- and disease-specific splicing patterns. To study such splicing variations, we introduce MAJIQlopedia, an encyclopedia of splicing variations that encompasses 86 human tissues and 41 cancer datasets. MAJIQlopedia reports annotated and unannotated splicing events for a total of 486 175 alternative splice junctions in normal tissues and 338 317 alternative splice junctions in cancer. This database, available at https://majiq.biociphers.org/majiqlopedia/, includes a user-friendly interface that provides graphical representations of junction usage quantification for each junction across all tissue or cancer types. To demonstrate case usage of MAJIQlopedia, we review splicing variations in genes WT1, MAPT and BIN1, which all have known tissue or cancer-specific splicing variations. We also use MAJIQlopedia to highlight novel splicing variations in FDX1 and MEGF9 in normal tissues, and we uncover a novel exon inclusion event in RPS6KA6 that only occurs in two cancer types. Users can download the database, request the addition of data to the webtool, or install a MAJIQlopedia server to integrate proprietary data. MAJIQlopedia can serve as a reference database for researchers seeking to understand what splicing variations exist in genes of interest, and those looking to understand tissue- or cancer-specific splice isoform usage.

Control over gene expression is exerted, in multiple stages of spermatogenesis, at the post-transcriptional level by RNA binding proteins (RBPs). We identify here an essential role in mammalian spermatogenesis and male fertility for ‘RNA binding protein 46’ (RBM46). A highly evolutionarily conserved gene,Rbm46is also essential for fertility in both flies and fish. We foundRbm46expression was restricted to the mouse germline, detectable in males in the cytoplasm of premeiotic spermatogonia and meiotic spermatocytes. To define its requirement for spermatogenesis, we generatedRbm46knockout (KO,Rbm46^-/-) mice; although maleRbm46^-/-mice were viable and appeared grossly normal, they were infertile. Testes from adultRbm46^-/-mice were small, with seminiferous tubules containing only Sertoli cells and few undifferentiated spermatogonia. Using genome-wide unbiased high throughput assays RNA-seq and ‘enhanced crosslinking immunoprecipitation’ coupled with RNA-seq (eCLIP-seq), we discovered RBM46 could bind, via a U-rich conserved consensus sequence, to a cohort of mRNAs encoding proteins required for completion of differentiation and subsequent meiotic initiation. In summary, our studies support an essential role for RBM46 in regulating target mRNAs during spermatogonia differentiation prior to the commitment to meiosis in mice.