Abstract 1509: Cost-effective, full-length transcriptome of chronic lymphocytic leukemia patient cd8 t cells reveals the impact of BTKi treatment on mRNA processing and gene expression

Abstract Ibrutinib, a Bruton’s tyrosine kinase inhibitor (BTKi), is used to treat chronic lymphocytic leukemia (CLL). It has effects not only on CLL cells but also on the immune microenvironment, particularly T cells. While T cell exhaustion and activation defects are well studied, the role of mRNA processing on the transcriptome and immunome needs investigation. We and others noted such events in CLL cells with shorter isoforms replacing the longer primary transcripts. They enable added regulations via altered mRNA stability, translational efficiency, and protein localization.To conserve precious patient time series samples, we developed a protocol that deposes 300 sorted live cells into cold lysis buffer containing 5’ template-switching primers and 3’ poly-dT primers to bypass RNA extraction and to capture full-length transcripts. Viewing our data in genome browser, we noted the aligned- and the junction-read tracks reveal exon dropout events as stretches of uncovered exons in a gene-specific manner within- and across patients. For isoform-informed gene expression analysis, we identify a known isoform for each gene that encompasses all the transcribed exons from 10 CLL patients (30 samples: baseline, 3y and 5y ibrutinib treatment) for length-normalized gene expression analysis. For this abstract, we performed global analysis and a subset analysis targeting 187 genes from immune pathways and CD8 T cell activation. For timepoint analysis, differentially expressed gene (DEG) analysis was performed separately for genes without exon dropout and those with exon dropout. The 187-gene timepoint analysis was performed in one list. Key findings are: i) DEGs from subset analysis are also found in the two global analyses; ii) similar DEG trends observed across timepoints in all 3 analyses: highest in BL > 3y and BL > 5y followed by BL > 3y and 5y > 3y with a few 3y > BL and 5y > BL events; iii) 88% of the expressed transcripts contain one or more exon dropout event; iv) STRING analysis using DEGs from subset analysis reveals CD8 activation genes such as ptprc and cd69 interact extensively with Perforin- and PD-1 pathway genes forming a dense network while genes from FAS-, mTOR- and TRAIL pathways form a separate network with few interactions. In summary, with a cost similar to bulk RNA-seq, our full-length data reveals mRNA processing events in both long genes (e.g., jak1, 26 exons, full exon coverage in 12/30 samples) and short genes (e.g., jun, part of the intronless gene not covered in some samples). An ultralow input of 300 pre- and post-treatment cells allow deep library sampling of both highly expressed genes with no exon dropout and lowly expressed genes with many dropouts. Lastly, DEGs from subset analysis lend support to the ability of the isoform-informed workflow to capture CD8 T cell functional genes in global DEG analysis. Citation Format: Shanmugapriya Thangavadivel, Altan Turkoglu, Lianbo Yu, Rosario Distefano, Alexander Pan, Logan Walker, Tina Wang, Shrilekha Misra, Tzung-Huei Lai, Britten Gordon, Samon Benrashid, Alexander He, Meixiao Long, rosa Lapalombella, John C.Byrd, Kerry A. Rogers, Bradley Blaser, Ralf Bundschuh, Jennifer A. Woyach, Pearlly S. Yan. Cost-effective, full-length transcriptome of chronic lymphocytic leukemia patient cd8 t cells reveals the impact of BTKi treatment on mRNA processing and gene expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1509.