Single Cell Gene Expression Array Analysis in Patients with Myeloproliferative Neoplasms

Significant phenotypic heterogeneity exists in patients with all subtypes of Philadelphia (-) myeloproliferative neoplasms (MPN) which include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Three driver mutations (JAK2, CALR and MPL) have been reported to be disease initiators, yet none of these mutations has been proved to be subtype-specific. It remains a major mystery why the same driver mutation could lead to discrepant phenotypes and why patients with the same disease subtype will run different clinical trajectories. Single-cell RNA sequencing (scRNA-Seq) holds the promise of unraveling the biology of MPN in an unprecedented level of resolution. To understand the clonal heterogeneity in MPN, we first employed this approach to dissect the transcriptomes in the CD34+ cells from the peripheral blood of two previously untreated JAK2-mutated ET patients and one healthy adult. Our preliminary data reveal substantial heterogeneity within these samples. Activation of JAK/STAT signaling is recognized in discrepant progenitor lineages among different samples. Significantly disparate molecular profiling is identified in the comparison not only between ET patients and the control individual but also between patients harboring the same driver mutation. Also, intra-individual clonal diversity is also found in the CD34+ progenitor population of a patient, possibly indicating the presence of multiple clones in this case. Moreover, one of the enrolled patients whose disease progressed into secondary MF exhibited a distinct gene profiling in her progenitors. We further identified TGFb/miR-21 signaling pathway dys-regulation in her pre-transformed progenitors as well as the buffy coats of 12 additional JAK2-mutated MF cases. We speculate that aberrant TGF/miR-21 signaling could well represent a novel mechanism in ET-to-MF transformation. Therefore, we bring forward this proposal to address these pressing questions with regards to the pathobiology of MPN by aiming to achieve the following goals: 1) Dissection of the transcriptomic heterogeneity in hematopoietic progenitors from ET patients stratified by different driver mutations. 2) Discrimination of potentially “lineage-biased” differentiation in ET progenitors. 3) Integration of data from scRNA-Seq and results on targeted sequencing to comprehensively characterize the molecularly distinct hematopoietic cell fractions that harbor “disease-initiating” potentials. 4) Elucidation of the causal relationship between miR-21 and TGFb in MF transformation through in vitro and in vivo studies. With the detail pathogenesis of MPN subtype specification and the disease-initiating mechanism of hematopoietic progenitors never fully appreciated, we propose this work to unravel some of the puzzling issues in MPN. The obtained information could potentially leapfrog our efforts in the elucidation of the pathogenesis of the disease.

Project ID:PC10901-1365
External Project ID:MOST108-2314-B182-045-MY3