Single-cell long-read genotyping of transcripts reveals discrete mechanisms of clonal evolution in post-MPN AML

ABSTRACT: Myeloproliferative neoplasms (MPNs) are caused by acquired mutations in hematopoietic stem and progenitor cells (HSPCs). The acquisition of additional mutations, such as TP53, and the overall mutational burden influence a patient's risk of disease progression to lethal post-MPN acute myeloid leukemias (AML). Recent technological advancements in linking single-cell gene expression with genotype have improved our understanding of tumor heterogeneity. However, current methodologies have limitations in simultaneously genotyping low-expression genes (such as JAK2) alongside other pathogenic loci. To address this, we developed a novel long-read genotyping pipeline of complementary DNA transcripts (long-read genotyping of transcripts [LOTR]-Seq), which can genotype the full length of expressed transcripts from 30 genes at once. Using LOTR-Seq, we genotyped HSPCs at the JAK2V617 locus in 9075 single cells from 8 patients with chronic-phase MPN (CP-MPN) and in 5016 cells from 4 patients with post-MPN AML. We then linked the mutations to the single-cell transcriptomes of 29 712 JAK2V617F-driven CP-MPN cells and 16 895 post-MPN AML cells. In our analysis of post-MPN AML, we identified 9 mutated loci across 6 genes (JAK2, IDH1/2, TP53, SRSF2, and U2AF1) and linked these mutations to specific transcriptional phenotypes. Overall, LOTR-Seq provides novel insights into the evolution of post-MPN AML.