Transcriptional readthrough precedes alternative splicing programs triggered in CML cells by imatinib

Cellular stresses induce transcription readthrough, whereby RNA polymerase II elongates past a gene's polyadenylation cleavage site without RNA cleavage. Readthrough has been reported in several cancer types. Here, we use long-read sequencing of nascent RNA to quantify transcriptional readthrough in chronic myelogenous leukemia (CML) cells and characterize early responses to the targeted therapeutic, imatinib. We show that the amount, length, and gene-specificity of readthrough increase within 1 hour, while gene expression and alternative splicing alterations emerge later. Strikingly, imatinib-dependent mRNA isoform changes involved "readthrough chimeras", in which exons from an upstream gene are alternatively spliced to exons in a downstream gene. Modifications in mRNA isoforms and chimera levels detected at 18 hours were also found in imatinib-resistant K562 as well as CML patient cells, suggesting a cascade of early changes in the fidelity of transcription and splicing, leading to long-term adjustment in gene expression and the development of therapy resistance.