Clonal evolution of mutation events in myeloproliferative neoplasms

Myeloproliferative disorders (MPD) are a heterogeneous group of diseases characterized by aberrant proliferation of the myeloid lineages. They represent clonal stem cell disorders with an inherent tendency towards leukemic transformation. Currently, MPD are subdivided into three disease entities: polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). A mutation in JAK2 (JAK2-V617F) is frequently found in all three entities. The discovery of this mutation had a major impact on the diagnostic workup of patients with suspected MPD, increased our understanding of the pathogenesis and has led to first clinical trials testing the effects of JAK2 inhibitors on late stages of MPD.
We now have convincing evidence that there are additional mutational events preceding JAK2-V617F. They are likely to be involved in initiating the disease or are acting as collaborating mutations to promote progression to myelofibrosis and acute leukemia. In this thesis, the clonal evolution of deletions on chromosome 20q (del20q) and TET2, a member of the Ten-Eleven-Translocation (TET) family of genes, were studied. In addition, the functional consequence of del20q was also analyzed by shRNA-mediated knockdown.
We developed a real time copy number PCR assay for deletions on chromosome 20q (del20q), screened peripheral blood granulocytes from 664 patients with myeloproliferative disorders (MPD) and identified 19 patients with del20q (2.9%), of which 14 (74%) were also positive for JAK2-V617F. We analyzed 8 patients with del20q using array comparative genomic hybridization (aCGH) and defined a 9 Mb common deleted region with 93 genes. To examine the temporal relationship between the occurrence of del20q and JAK2-V617F we performed colony assays in methylcellulose, picked individual BFU-E and CFU-G colonies and genotyped each colony individually for del20q and JAK2-V617F. In 2/9 patients we found del20q colonies with and without JAK2-V617F, suggesting that del20q preceded JAK2-V617F. 3/9 patients showed the inverse order of events, suggesting that del20q is not a general predisposing event for JAK2-V617F. Interestingly, 2 patients showed a complex pattern of mutation acquisition including several events of del20q affecting alleles of different parental origin and uniparental disomy on chromosome 9p (9p-UPD). The fact that rare somatic events, such as del20q or 9p-UPD, occurred more than once suggests that these patients carry a predisposition to acquire such genetic alterations.
In serial samples from patients with del20q, we observed an increase in the percentage of del20q positive cells, suggesting that del20q as the sole genetic alteration may provide a competitive advantage. To find genes involved in the del20q induced proliferation advantage, we analyzed changes in the gene expression pattern induced by del20q. Screening for such differences in expression patterns is most powerful if inter-individual differences in genetic background and differences in the composition of the tissues to be analyzed can be eliminated. Therefore we used pools of individually JAK2-V617F and del20q genotyped colonies grown in methylcellulose, which provided a pure enough cell population to detect small differences in expression and compared the expression pattern of colonies with and without del20q. We discovered 680 genes, which had significantly changes (p<0.05) in expression. Inside the CDR we found 28 genes downregulated, of which we selected three genes for further functional analysis: serin threonin kinase 4 (STK4), topoisomerase 1 (TOP1) and protein tyrosine phosphatase receptor T (PTPRT). A pool of shRNA targeting these genes was used to retrovirally transfect murin bone marrow (BM) cells before they were transplanted to lethally irradiated reciepient mice. Because del20q was preferentially found together with JAK2-V617F, we decided to use BM cells derived from transgenic MxCre;FF1 mice expressing JAK2-V617F. In addition, we also included two additional candiate genes, V-MYB myeloblastosis viral oncogene homolog (avian)-like 2 (MYBL2) and L3MBTL, a human homolog of the Drosophila lethal (3) malignant brain tumor (D-l(3)mbt) polycomb protein, in the bone marrow transplantation experiments. This is an ongoing project and we are monitoring the GFP expression in peripheral blood and the distribution of the integrated shRNAs. So far we did not see any proliferative advantage of the cells transfected with the shRNA pool targeting the del20q candidate genes.
Recently, a new gene was found to be mutated in MPN patients: TET2, a member of the Ten-Eleven-Translocation (TET) family of genes. Initial studies suggested, that TET2 mutations may represent a tumor suppressor gene and may precede the acquisition of JAK2-V617F. We therefore studied the clonal evolution of TET2 mutations by analyzing individually genotyped colonies of 8 patients. In 4/8 MPN patients we found that some colonies with mutated TET2 carried wild type JAK2, whereas others were JAK2-V617F positive, indicating that TET2 occurred before JAK2-V617F. One of these patients carried a 4 bp germline deletion, located in the C-terminal conserved region, leading to a frameshift and premature stop. This is the first report of a inherited mutation in TET2. In two other patients we observed the opposite order of events, with JAK2 exon 12 mutation preceding TET2 mutation in one case. These findings suggest, that mutations in TET2 do not represent a general predisposing event for acquiring mutations in JAK2. Two additional patients showed two separated clones for TET2 and JAK2- V617F, which further supports this theory.
The present work provided additional insights for understanding the complex clonal pattern in patients with MPN and the role of del20q and TET2 mutations in the clonal evolution of the disease.