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

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.