We investigated the activity of ITF2357, a novel histone deacetylase inhibitor (HDACi) with antitumor activity, on cells carrying the JAK2 V617F mutation obtained from polycythemia vera (PV) and essential thrombocythemia (ET) patients as well as the HEL cell line. The clonogenic activity of JAK2 V617F mutated cells was inhibited by low concentrations of ITF2357 (IC 50 0.001-0.01 lM), 100-to 250-fold lower than required to inhibit growth of normal or tumor cells lacking this mutation. Under these conditions, ITF2357 allowed a seven fold increase in the outgrowth of unmutated over mutated colonies. By western blotting we showed that in HEL cells, ITF2357 led to the disappearance of total and phosphorylated JAK2 V617F as well as pSTAT5 and pSTAT3, but it did not affect the wild-type JAK2 or STAT proteins in the control K562 cell line. By real-time PCR, we showed that, upon exposure to ITF2357, JAK2 V617F mRNA was not modified in granulocytes from PV patients while the expression of the PRV-1 gene, a known target of JAK2, was rapidly downmodulated. Altogether, the data presented suggest that ITF2357 inhibits proliferation of cells bearing the JAK2 V617F mutation through a specific downmodulation of the JAK2 V617F protein and inhibition of its downstream signaling.

We have investigated the activity of ITF2357, a novel hydroxamate histone deacetylase inhibitor, on multiple myeloma (MM) and acute myelogenous leukemia (AML) cells in vitro and in vivo. ITF2357 induced apoptosis in 8/9 MM and 6/7 AML cell lines, as well as 4/4 MM and 18/20 AML freshly isolated cases, with a mean IC 50 of 0.2 lM. ITF2357 activated the intrinsic apoptotic pathway, upregulated p21 and downmodulated Bcl-2 and Mcl-1. The drug induced hyperacetylation of histone H3, H4 and tubulin. When studied in more physiological conditions, ITF2357 was still strongly cytotoxic for the interleukin-6 (IL-6)-dependent MM cell line CMA-03, or for AML samples maximally stimulated by co-culture on mesenchymal stromal cells (MSCs), but not for the MSCs themselves. Interestingly, ITF2357 inhibited the production of IL-6, vascular endothelial growth factor (VEGF) and interferon-c by MSCs by 80-95%. Finally, the drug significantly prolonged survival of severe combined immunodeficient mice inoculated with the AML-PS in vivo passaged cell line already at the 10 mg/kg oral dose. These data demonstrate that ITF2357 has potent anti-neoplastic activity in vitro and in vivo through direct induction of leukemic cell apoptosis. Furthermore, the drug inhibits production of growth and angiogenic factors by bone marrow stromal cells, in particular IL-6 and VEGF.

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The online version of this article has a supplementary appendix. BackgroundThe histone deacetylase inhibitor ITF2357 has potent cytotoxic activity in multiple myeloma in vitro and has entered clinical trials for this disease. Design and MethodsIn order to gain an overall view of the activity of ITF2357 and identify specific pathways that may be modulated by the drug, we performed gene expression profiling of the KMS18 multiple myeloma cell line treated with the drug. The modulation of several genes and their biological consequence were verified in a panel of multiple myeloma cell lines and cells freshly isolated from patients by using polymerase chain reaction analysis and western blotting. ResultsOut of 38,500 human genes, we identified 140 and 574 up-regulated genes and 102 and 556 down-modulated genes at 2 and 6 h, respectively, with a significant presence of genes related to transcription regulation at 2 h and to cell cycling and apoptosis at 6 h. Several of the identified genes are particularly relevant to the biology of multiple myeloma and it was confirmed that ITF2357 also modulated their encoded proteins in different multiple myeloma cell lines. In particular, ITF2357 down-modulated the interleukin-6 receptor α (CD126) transcript and protein in both cell lines and freshly isolated patients' cells, whereas it did not significantly modify interleukin-6 receptor β (CD130) expression. The decrease in CD126 expression was accompanied by decreased signaling by interleukin-6 receptor, as measured by STAT3 phosphorylation in the presence and absence of interleukin-6. Finally, the drug significantly down-modulated the MIRHG1 transcript and its associated microRNA, miR-19a and miR-19b, known to have oncogenic activity in multiple myeloma. ConclusionsITF2357 inhibits several signaling pathways involved in myeloma cell growth and survival.Key words: pleiotropic anti-myeloma, ITF2357, interleukin-6 receptor, miR-19a, miR-19b. -19a and miR-19b. Haematologica. 2010; 95:260-269. doi:10.3324/haematol.2009 This is an open-access paper.Pleiotropic anti-myeloma activity of ITF2357: inhibition of interleukin-6 receptor signaling and repression of miR-19a and miR-19b

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.