Selective Targeting of the JAK2V617F Mutation in Polycythemia Vera and Essential Thrombocythemia by ITF2357, a Novel Histone Deacetylase Inhibitor

Autor: Tiziano Barbui, Martino Introna, Vittoria Guerini, Valentina Barbui, Orietta Spinelli, Alessandro Rambaldi, Anna Salvi, Josée Golay, Chiara Dellacasa
Rok vydání: 2007
Předmět:
Zdroj: Blood. 110:555-555
ISSN: 1528-0020
0006-4971
Popis: A somatic point mutation in the JAK2 gene (JAK2V617F) is the key pathogenetic lesion of Polycythemia Vera (PV) and Essential Thrombocythemia (ET) and a significant effort is now paid to identify drugs which may be able to interfere with the JAK2V617Fmutated protein. Among others, one potentially interesting drug family is represented by the Histone Deacetylase Inhibitors (HDACi), which may modify the chromatin structure and ultimately the transcription of many genes, the cell cycle progression and the programmed cell death. ITF2357 is a new HDACi (Italfarmaco, Milan, SpA) that shows a potent anti-proliferative and pro-apoptotic activity against acute myeloid leukemia and multiple myeloma cells and little toxicity against normal hematopoietic and mesenchymal stem cells (Golay J et al.: Leukemia 2007). The most common side effects after its administration to normal volounteers and MM patients are represented by thrombocytopenia and gastrointestinal toxicity. These observations prompted us to investigate the inhibitory activity played by ITF2357 on the autonomous proliferation of cells obtained by PV and ET patients carrying the JAK2V617F mutation and to elucidate the mechanism of action of this inhibition. We first investigated the effect of ITF2357 on the clonogenic activity of cell lines carrying or not the JAK2V617F mutation. ITF2357 inhibited colony formation of HEL cells (an erythroleukemia cell line carrying a JAK2V617F homozygous mutation) with an IC50 of about 0.001 μM. In contrast, the doses of drug required to block colony formation by K562, KG1, NB4 and GF-D8 (all negative for the JAK2V617F mutation) were 100–500 fold higher (IC50 ranging from 0.1 to 0.5 μM). Clonogenic assays were then performed using blood mononuclear cells obtained from 4 PV and 7 ET patients, all carrying the JAK2V617F mutation. Either in the presence or absence (EEC assay) of exogenous growth factors, colonies obtained from JAK2V617F mutated progenitor cells were inhibited at much lower doses of ITF2357 (IC50 0.001 μM) as compared to colonies obtained from JAK2 wild type progenitor cells (IC50 0.1–0.25 μM). When single colonies were picked randomly and analyzed by PCR for the presence of wild type or mutated JAK2V617F alleles, a striking reduction of mutated colonies was detected when ITF2357 was added at 0.001 μM and 0.01 μM, confirming that low doses of ITF357 allow the preferential outgrowth of unmutated over mutated colonies from the peripheral blood mononuclear cells of PV patients bearing JAK2V617F. By Western blotting we also showed that ITF2357 treatment for 24 hours, led to virtual disappearance of total and phosphorylated JAK2V617F in HEL cells whereas it did not affect the wild type JAK2 protein in the control K562 cell line, even after 48 hours in the same conditions. Down-modulation of mutated JAK2V617F was accompanied by specific disappearance of p-STAT5 protein. Finally, by Real time PCR analysis of PV cells treated with ITF2357 for 24 hours, we could demonstrate that this drug does not affect JAK2 mRNA but rather it induces a significant decrease of the PRV1 gene, a known JAK2 target. These data suggest that ITF2357 down-modulates the mutated JAK2V617F protein by post-transcriptional mechanisms and that is followed by inhibition of p-STAT5 protein and PRV1 gene expression. The specific inhibition induced by ITF2357 on cells bearing the JAK2V617F mutation underlines its therapeutic potential as a new drug for PV and ET patients.
Databáze: OpenAIRE
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