Combination of the Histone Deacetylase Inhibitor Vorinostat and Dasatinib Increases Apoptosis in Bcr-abl+ Cells and Reverses Changes Associated with CML Progression.

Chronic myelogenous leukemia (CML) is associated with the bcr-abl fusion gene product, a constitutively active non-receptor tyrosine kinase driving cell division. The potent bcr-abl kinase inhibitor, dasatinib, is now FDA approved for imatinib-refractory patients, being active in most cases other than those with the T315I mutation. However, accelerated phase CML is typified by further genetic changes, including the suppression of various DNA damage response and apoptosis related proteins (Radich, et al PNAS 2006). Thus a novel approach which may reverse these changes, might provide better treatment of accelerated phase CML.

A new class of agents targeting epigenetic processes, the histone deacetylase inhibitors (HDACi), is believed to act upon chromatin allowing re-expression of tumor suppressor genes believed to be closed off to transcription by the tumor in its effort to grow in an uncontrolled manner. We reasoned that adding an HDACi to dasatinib treatment of CML cells could promote re-expression of genes responding to genetic instability in these cells. To test this hypothesis we tested dasatinib (D) and the HDACi Vorinistat (V, suberoylanilide hydroxamic acid, SAHA) alone and in combination, using K562 cells. Apoptosis was measured by Annexin V staining after 48 hours.

Apoptosis results: 1uM Vorinostat: 10%; 2 uM V: 16%. Dasatinib 0.5 nM: 28%, 1 nM D: 44% In combination, 1uM V + 0.5 nM D: 41%, 1 uM V + 1nM D: 64%, 2 uM V + 0.5 nM D: 65%, 2 uM V + 1 nM D: 67%, suggesting significant increase in apoptosis for the combination over either single agent treatment alone.

We therefore began surveying panels of DNA damage- and apoptosis-related genes by means of RT-PCR. Studying the combination of 2 uM V + 1 nM D, we found increased expression of several proteins, including GADD45G and FANCG, both DNA damage response proteins suppressed in the progression to accelerated phase CML. Other proapoptotic proteins were increased such as MAP2K6, SEMA4A, BIK, and TNF superfamily members 7 and 25. This data suggests that the changes associated with progression from chronic phase to accelerated and blast crisis CML may be epigenetic in nature, and that these changes may be reversed by the combination of vorinostat and dasatinib. A clinical trial of dasatanib and vorinostat in advanced phase CML would be of value.