Single cell long-read genotyping of transcriptomes reveals discrete mechanisms of clonal evolution in post-myeloproliferative neoplasm acute myeloid leukemia.

Myeloproliferative neoplasms (MPNs) are caused by acquired mutations in hematopoietic stem and progenitor cells (HSPCs). The acquisition of additional mutations like TP53and the overall mutational burden influence a patient's risk of disease progression toward lethal post-MPN acute myeloid leukemia (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 of transcriptomes pipeline called LOTR-Seq, which can genotype the full length of 30 expressed genes at once. Using LOTR-Seq, we genotyped HSPCs at the JAK2V617F locus in 9,075 single cells from eight patients with chronic phase MPN (CP-MPN) and in 5,016 cells from four patients with post-MPN AML. We then linked the mutations to the single cell transcriptome of 29,712 JAK2V617F-driven CP-MPN cells and 16,895 post-MPN AML cells. In our analysis of post-MPN AMLs, we identified nine mutated loci across six genes (JAK2, IDH1/2, TP53, SRSF2, U2AF1) and linked these mutations to specific transcriptional phenotypes. Overall, LOTR-Seq has provided us with unparalleled insights into the pathogenesis of the JAK2V617F mutation in HSPCs in CP-MPN and the effects of secondary mutations in post-MPN AML.