Chromatin Regulator Mutations Are Drivers of Clonal Evolution in the Early Multipotent HSC Compartment of Primary Myelofibrosis

The definition of initiating genetic events remains up to date the main research quest in MPN disease biology. Identification of characteristic mutational clusters has shed more light on the complexity of various MPN genotypes, even though no specific genetic lesion or mutational order can be exclusively linked to the different MPN subtypes. Our aim is to determine the sequence of mutational clusters that orchestrate the expansion of aberrant clones in MPN with respect to the prime hematopoietic stem cell compartment.

Genotyping for 54 MPN related gene mutations (100 MPN Patient cohort with myelofibrosis) indicated high incidence of mutations in chromatin regulator genes ASXL1 (40%) and EZH2 (14%). In 11% of the patients both ASXL1 and EZH2 gene mutations were detected, which correlated with significantly lower hemoglobin levels, higher leukocyte counts, higher numbers of additional mutations and higher risk score.

In our former work we reported the identification of an expanded Lin-CD133+CD34+ neoplastic HSC population in patient peripheral blood that reproduces PMF parameters and fibrosis in vivo. Analysis of this aberrant HSC in the single cell level revealed the heterogeneity of the malignant clones and the mutational hierarchies that propagate myeloproliferation. From this analysis we report that ASXL1 and/or EZH2 mutations precede JAK2 signalling related genetic lesions (JAK2, CALR, MPL) or other epigenetic modifier mutational groups (DMT3A, TET2). Neoplastic stem cells bearing both ASXL1/EZH2 mutations exhibit higher levels of engraftment in vivo and limited erythroid colony output in vitro .

In order to determine the exact contribution of chromatin regulator mutations with regard to clonal hematopoiesis determined by changes in prime HSC dynamics we established a sorting strategy for the isolation of early LT-HSC circulating in MF patient peripheral blood. By usage of 27 lineage specific and 6 additional HSC specific markers we sorted HSC on the apex of the hierarchy with the phenotype; 27xLin-CD34+CD38-CD90+CD93+CD49f+CD133+ and all classes of early and more committed progenitors from 11 patients with ASXL1 and/or EZH2 mutations. Our data indicate that both mutations emerge in the LT-HSC fraction (10/11 patients) dominate aberrant myelopoiesis and can be also detected in mature T cells (3/11 Patients). JAK2-V617F mutation was not detected in the early HSC fraction.

The incidence of EZH2 mutations with or without the synergistic effect of ASXL1 mutations, is correlated with the expansion of morphologically aberrant erythroid progenitors in patient peripheral blood, which are found to be arrested in G0 phase of the cell cycle.

Our results suggest that ASXL1 and EZH2 mutations are the drivers of clonal hematopoiesis in MPN and occur early at the multipotent HSC compartment. EZH2 mutations in PMF represent the first genetic cluster that correlates with aberrant erythropoiesis in these patients.