PF660 TRANSCRIPTIONAL PROFILES OF PRIMARY CULTURES OF ERYTHROID AND MEGAKARYOCYTIC CELLS IN ESSENTIAL THROMBOCYTHEMIA UNCOVER A HIGH DEGREE OF GENE DEREGULATION IN THE MEGAKARYOCYTIC LINEAGE

Abstract

Background: Essential Thrombocythemia (ET) is a myeloproliferative neoplasm (MPN) characterized by megakaryocytic hyperplasia in the bone marrow and elevated number of platelets in the circulation. The driver mutations (JAK2V617F, MPLW515K/L, CALR Type I &II) in MPN have led to a better understanding of pathways involved in MPN, in particular the role of the JAK/STAT pathway. However, not much is known about the transcriptional programs in the pathogenesis of ET. Aims: We wanted to characterize the transcriptional signature of ET megakaryocytes to understand the molecular cues that drive the disease in the presence of different driver mutations. Methods: Progenitor cells were cultured from peripheral blood of 13 healthy donors (HD) and 16 untreated ET patients (JAK2V617F: n = 8; CALRType I: n = 4; triple-negative (TN-no JAK2, MPL, CALRmutations): n = 4). We obtained human erythroid (ERYs-proerythroblasts) and mature CD41+megakaryocytes (MKs) from each patient and healthy donor as described previously (Leberbauer, C. et al, 2005 and Salunkhe, V. et al, 2015) and RNA-seq was performed. Gene expression analysis data were selectively validated with qPCR for several targets. Results: Gene expression profiles from erythroid cultures and mature megakaryocytes (CD41+) from ET patients were grouped according to cell type (ERYs or MKs), type of driver mutations (JAK2, CALR) and compared with HD as controls. The expression profile of CALR mut ERYs was characterized by 60 significantly deregulated genes, 56 of which were upregulated (adjusted P-value 0.05) including non-erythroid targets (e.g. MPL, VWF, PF4, GP5, GP6). No major differences were identified between the transcriptional profile of HD ERY, JAK2V617F ERY or TN ERY. Similar comparisons were also made for the MKs. In a total of 4302 significantly deregulated genes (adjusted P-value 0.05) more than half (2348 genes) were uniquely up- or downregulated in the CALR mutant ET. Only 75 and 135 unique targets corresponded to JAK2V617F- and TN-MKs respectively and 925 deregulated genes were shared among the ET genetic groups. Principal component analysis (PCA) indicated that the MKs are more heterogeneous than ERYs and the JAK2V617F MK samples were the most variable of all. Gene ontology (GO) analysis showed that deregulated genes belong to the class of master regulators and hematopoietic cell fate, immune response, apoptosis, metabolic pathways and signal transduction. Some of these biological processes are shared by JAK2V617F and CALRmut-MKs (i.e. immune response). However, JAK-STAT and MAPK signaling pathways and apoptosis together form part of the unique transcriptional signature of CALRmut MKs. Summary/Conclusion: For the first time to our knowledge, paired cultures of committed erythroid cells and mature megakaryocytes from ET patients have been analyzed by RNA-seq. Comparison of ERY and MK transcriptional profiles show major differences between the two cell lineages, and between the driver mutations of CALR and JAK2. Our findings suggest that, in addition to JAK-STAT signaling, other mechanisms are involved in the transcriptional control in CALRmutated ET.