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

Cellular stresses regulate transcriptional 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 myeloid 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, before gene expression and alternative splicing alterations emerge. Notably, imatinib-dependent messenger RNA (mRNA) isoform changes involved “readthrough chimeras,” in which exons from an upstream gene are alternatively spliced to exons in a downstream gene. Altered mRNA isoforms and chimera levels were detected in imatinib-resistant K562 cells as well as cells of patients with CML. Thus, imatinib can provoke a cascade of early changes to transcription and splicing fidelity that may lead to longer-term adjustments in gene expression, cancer cell differentiation, and the development of therapy resistance.