Kathryn E. Pendleton

Modification of nucleocytoplasmic proteins with O-GlcNAc regulates a wide variety of cellular processes and has been linked to human diseases. The enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) add and remove O-GlcNAc, but the mechanisms regulating their expression remain unclear. Here, we demonstrate that retention of the fourth intron of OGT is regulated in response to O-GlcNAc levels. We further define a conserved intronic splicing silencer (ISS) that is necessary for OGT intron retention. Deletion of the ISS in colon cancer cells leads to increases in OGT, but O-GlcNAc homeostasis is maintained by concomitant increases in OGA protein. However, the ISS-deleted cells are hypersensitive to OGA inhibition in culture and in soft agar. Moreover, growth of xenograft tumors from ISS-deleted cells is compromised in mice treated with an OGA inhibitor. Thus, ISS-mediated regulation of OGT intron retention is a key component in OGT expression and maintaining O-GlcNAc homeostasis.

Transcriptome analysis of human cells has revealed that intron retention controls the expression of a large number of genes with diverse cellular functions. Detained introns (DI) constitute a subgroup of transcripts with retained introns that are not exported to the cytoplasm but instead remain in the nucleus. Previous studies reported that the splicing of DIs in the CLK1 transcript is post-transcriptionally induced to produce mature mRNA in the absence of new transcription. Thus, CLK1-DI serves as a precursor or “reservoir” for the CLK1 mRNA. However, whether this is a universal mechanism for gene regulation by intron detention remains unknown. The MAT2A gene encodes S -adenosylmethionine (SAM) synthetase and it contains a DI that is regulated in response to intracellular SAM levels. We used three independent assays to assess the precursor–product relationship between MAT2A-DI and MAT2A mRNA. In contrast to CLK1-DI, these data support a model in which the MAT2A-DI transcript is not a precursor to mRNA but is instead a “dead-end” RNA fated for nuclear decay. Additionally, we show that in SAM-deprived conditions the cotranscriptional splicing of MAT2A detained introns increases. We conclude that polyadenylated RNAs with DIs can have at least two distinct fates. They can serve as nuclear reservoirs of pre-mRNAs available for rapid induction by the cell, or they constitute dead-end RNAs that are degraded in the nucleus.

Abstract Tumor mutational burden (TMB) is currently of high interest in the field of immuno-oncology due to its correlation with patient response to checkpoint inhibitor chemotherapy. Traditionally, TMB is calculated using whole exome sequencing; however, targeted sequencing approaches provide better coverage of the genetic regions of interest at lower costs. While hybrid-capture based target enrichment methods are well-established, the 2-5 day workflows are time consuming and require specialized equipment and highly trained operators. Here we present an ultrafast, 4-hour method for preparing target enriched NGS libraries for assessing TMB that would streamline and lower the cost of immuno-oncology studies. This multiplex-PCR-based technology provides a highly efficient, accurate and robust method for unbiased enrichment of tens of thousands of target regions while minimizing non-specific primer-primer interactions and GC bias and maximizing coverage uniformity. We demonstrate this using a highly-multiplexed prototype NGS panel that contains ~20,000 amplicons and covers 355 genes for assessment of TMB. Sequencing-ready NGS libraries were prepared using the Paragon Genomics CleanPlex® target enrichment technology. The 3-step workflow combines target enrichment and NGS library preparation. The protocol includes an ultra-high multiplex PCR step to amplify regions of interest with target-specific primers, a background cleaning step to remove non-specific PCR products, and a final PCR to add Illumina sequencing adapter and sample indexes. Libraries were made using 20ng of genomic DNA and sequenced on an Illumina NextSeq® platform. Sequencing metrics such as on-target rates were calculated, and variants were identified using Paragon Genomics’ variant calling algorithm. Using the CleanPlex technology, this prototype TMB panel exhibits >95% uniformity at 0.2X mean, limited GC bias, and >94% detection rate for mutants with 5% allele frequencies. The CleanPlex background cleaning step was essential for removing the undesirable byproducts of the multiplexed reaction step. CleanPlex technology demonstrates that it is capable of creating high quality amplicon libraries with high uniformity, low GC bias, and sensitive variant calling even in ultra-multiplexed libraries with ~20,000 amplicons with a workflow under 4-hours. Citation Format: Kathryn E. Pendleton, Yang L. Liu, Lifeng Lin, Lucie S. Lee, Jeffery Liu, Guoying Liu, Zhitong Liu. Tumor mutational burden (TMB) assessment and variant detection using an ultra-high multiplexed 20,000 amplicons NGS panel via a rapid 4 hour workflow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3532.