Vorinostat Impairs Cellular Viability, Differentiation, Redox Homeostasis and Gene Expression in BCR-ABL-Negative Myeloproliferative Neoplasms

Background: The classical BCR-ABL-negative myeloproliferative neoplasms (MPN) are characterized by increased proliferation of hematopoietic precursors in the bone marrow resulting in an elevated number of terminally differentiated cells. Despite the recent description of JAK2 activating mutations and other mutations, these do not completely explain the pathophysiology and clinical heterogeneity of MPN.

Epigenetic modifications, particularly histone acetylation, play pivotal roles in the pathogenesis of several hematological malignancies, and treatment of such disorders with histone deacetylase inhibitors results cell death and proliferation arrest. Importantly, epigenetic agents have proven to be effective in several hematological malignancies.

Aims: HDAC inhibition has demonstrated some efficacy in patients with MPN. In order to investigate the effects of HDAC inhibitors in MPN, we analyzed the impact of Vorinostat on the cellular biology of MPN cell lines and primary bone marrow samples.

Material and Methods: MPN bone marrow samples were collected at diagnosis following informed consent in the course of routine clinical laboratory tests. Mononuclear cells were isolated by gradient separation were used for culture experiments and lysed for RNA extraction. RNA extracted from MPN primary samples was used to synthetize cDNA and the transcript levels of genes associated with Apoptosis, Proliferation, Epigenetic modifications and several Signaling pathways were analyzed by quantitative-PCR. MPN primary cells and MPN derived cell lines were incubated with Vorinostat and at different time points the cells were harvest, lysed for gene expression analysis and stained with different antibodies, Annexin-V/PI and DCF-DA to analyze cellular differentiation, apoptosis and Reactive Oxygen Species (ROS) respectively.

Results: We performed a targeted-genome wide screen and compared the transcript levels of a defined set of genes between normal bone marrow and MPN primary samples. We identified 9 genes (BIRC3, TNFRSF9, DLL4, IL1B, CDKN1A, FOSL1, CREL, SERPINB9 and EGR1) whose expression increased for at least 4 fold and 2 genes (HIP1 and DTX1) whose expression decreased by at least 0.5 fold in MPN patients relative to normal bone marrow samples. Interestingly, incubation of Vorinostat in MPN cell lines at physiological concentrations increases the expression of such genes, and also the expression of genes associated with apoptosis and growth arrest while decreasing the expression of genes associated with proliferation, growth arrest and JAK-STAT signaling pathway. Regarding cellular physiology, Vorinostat induces apoptosis in MPN cultured cell lines in a time- and dose-dependent manner. Furthermore, incubation of primary MPN bone marrow samples with Vorinostat induced apoptosis, blocked differentiation and also diminished ROS levels in a dose dependent manner. These effects were most marked in the monocytic lineage, a population which expresses the highest levels of ROS. Vorinostat also reduced the levels of GPA and CD61, markers of erythroid and megakaryocytic differentiation, respectively.

Summary/Conclusions: Here, we show that Vorinostat incubation impairs MPN cellular differentiation and reduces ROS and cellular viability, possibly through the down-regulation of genes associated with cellular proliferation, particularly the JAK-STAT target genes, and up-regulation of genes important for apoptosis and growth arrest. Interestingly, the genes that we identified to be up-regulated in MPN primary samples relative to normal controls, are further increased by Vorinostat treatment, suggesting that these could act as potential biomarkers for Vorinostat effectiveness in the MPN context. Furthermore, these results hold therapeutic promise as Vorinostat reduced differentiation markers associated with Polycythemia Vera and Essential Thrombocytosis. The observation that Vorinostat is particularly effective against the monocytic lineage is interesting in the context of the recently described role of bone marrow monocytes in the pathogenesis of Polycythemia Vera in mouse models.

Our results point towards the potential role of Vorinostat (and possibly other HDAC inhibitors) in the treatment of MPN. This potential would require clinical trials to investigate its efficacy.