Myeloproliferative neoplasms (MPNs) are caused by acquired mutations in hematopoietic stem and progenitor cells (HSPCs). The acquisition of additional mutations like TP53 and 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.
Grabek et al. (Fri,) studied this question.