Potent Inhibition of EEC Colony Formation in JAK2^V617F PV and ET by Low Doses of ITF2357, a New Histone Deacetylase Inhibitor.

Background

In Polycythemia Vera (PV) and Essential Thrombocythemia (ET) hematopoietic progenitor cells can proliferate in vitro in the absence of exogenous growth factors. A somatic point mutation in the JAK2 gene (JAK2^V617F) has been recently recognised as the key pathogenetic lesion of these diseases leading to constitutive tyrosine phosphorylation of JAK2, cytokine hypersensitivity and autonomous outgrowth of hematopoietic progenitor cells. Hystone-Deacetylase inhibitors (HDACi) are known inducers of cell differentiation, apoptosis and cell cycle arrest of neoplastic cells. ITF2357 is a new HDACi (Italfarmaco, Milano, Italy) which, at low micromolar concentration in vitro, inhibits the secretion of several cytokines such as IL-1, IL-6, VEGF and IFN-g and exerts a potent anti tumor activity against multiple myeloma (MM) and acute myeloid leukemia cells (AML) (Golay et al., submitted). ITF2357 is well tolerated when given to normal healthy volounteers and Phase II clinical trials are currently ongoing in AML and MM.

Aim

To investigate the ability of ITF2357 and the prototypic HDAC inhibitor Suberoyl Anilide Hydroxamic Acid (SAHA) used as control, to inhibit the spontaneous outgrowth of hematopoietic stem cells obtained from patients with PV (n= 6, all JAK2^V617F ), ET (n= 13, 7 JAK2^V617F ) and Idiopathic Erythrocytosis (IE, n= 6, all negative for JAK2^V617F ).

Results

Endogenous erythroid colonies (EEC) assays were performed using mononuclear cells (MNC) from peripheral blood samples obtained from patients at the time of regular follow-up visits in our clinic. MNC obtained from IE or ET patients negative for JAK2^V617F neither exhibited spontaneous EEC formation nor Epo hypersensitivity (from 0.1 UI/ml up to 10UI/ml). On the contrary, MNC from JAK2^V617F PV and ET patients invariably sustained the spontaneous EEC outgrowth with a marked Epo hypersensitivity. When ITF2357 was added to the colony assay (ranging from 0.001 to 0.75 μM), a 90% inhibition of EEC formation was observed in all JAK2^V617F PV and ET patients at 0.01 μM concentration, which corresponds to a blood level easily attained following oral administration of safe doses of ITF2357 to healthy individuals. By contrast, the prototypic HDAC inhibitor SAHA displayed a similar inhibitory activity on EEC formation only when used at 0.25 μM. By flow cytometry experiments performed on mature granulocytes isolated from PV patients we could show that ITF2357 does not modulate the overexpression of Leucocyte Alkaline Phospatase and CD177 (the PRV-1 gene product) thus suggesting that the inhibitory activity on hematopoietic cells is mainly due to a direct action on the stem cell compartment.

Conclusion

ITF2357, at concentration easily attained after low oral doses of the drug, show a potent inhibitory activity on the autonomous proliferation of hematopoietic stem cells of PV and TE carrying the JAK2 V617F mutation. This may provide the framework for a Phase II study of ITF2357 in these malignancies.