Effect of Valproic Acid (VPA) on Fludarabine Activity In Chronic Lymphocytic Leukemia (CLL) Patients

Abstract 4626

Valproic acid (VPA) is a first-generation anti-epileptic drug that has been shown to inhibit proliferation and induce apoptosis in various hematological malignancies, including multiple myeloma and chronic lymphocytic leukemia (CLL). VPA-induced apoptosis is thought to be due to its ability to inhibit histone deacetylases (HDACs), resulting in hyper-acetylation of histones and altered expression levels of pro- and anti-apoptotic genes. We show here that the mechanism of VPA-induced apoptosis in CLL is associated with activation of the TNF-related apoptosis-inducing ligand (TRAIL) apoptotic pathway in vitro and ex vivo. TRAIL induces cell death by binding to death receptors 4 and 5 (DR4, DR5), which in turn activate the extrinsic apoptotic pathway. We have previously shown that 30–40% of the activities of fludarabine and chlorambucil are mediated through the TRAIL pathway, and this is related to up-regulation of the expressions of DR4 and DR5. We confirm here that VPA induces apoptosis in two CLL-like cell lines, I-83 and BJAB. Co-treatment of the two cell lines with VPA and activating antibodies against either DR4 (ETR1) or DR5 (ETR2) enhanced VPA-induced apoptosis in both cell lines after 24 hours. Conversely, blocking the TRAIL apoptotic pathway with DR4:F_C fusion protein decreased the cytotoxicity of VPA, as well as another HDAC inhibitor, trichostatin A, in BJAB cells. These results confirmed the importance of activation of the TRAIL apoptotic pathway for the antitumor activity of VPA. Treatment with VPA also induced formation of reactive oxygen species (ROS) and enhanced fludarabine-induced apoptosis in cell lines and primary CLL cells. Having shown the effects of VPA in vitro, a phase II clinical trial was initiated at CancerCare Manitoba to determine the activity of VPA in CLL, either when used alone or in combination with fludarabine. Five patients who had received at least one prior therapy with fludarabine have to date been examined. Three out of 5 patients were fludarabine-resistant, as defined as no response to fludarabine or relapse < 6-months after completion of treatment with fludarabine. Patients received VPA at a starting dose of 15 mg/kg/day orally in divided doses, with the goal of reaching a serum level of >1 mM. Various side effects were observed with higher doses of VPA, including anaemia, thrombocytopaenia, GI toxicity and fatigue. No responses were seen after 28 days using VPA alone. However, in four patients who continued on VPA with fludarabine, 3 patients showed a >50% fall in lymphocyte/lymph node size after receiving 5 cycles of the combination. Peripheral blood samples were obtained prior to each treatment cycle, and mononuclear cells were isolated for analysis by immunoblotting and immunocytochemistry. Levels of both histone 3-acetyl and histone 4-acetyl initially increased and then fluctuated during the course of treatment. When DR4 and DR5 levels were examined by immunoblotting, DR4 levels increased during the course of therapy, while no significant changes in DR5 levels were observed. Expression of mRNA levels of genes involved in apoptosis was examined in one patient before and 28 days following VPA. There was a global up-regulation of both pro- and anti-apoptotic genes (as categorized by their gene ontology) which likely explains the effect of VPA on fludarabine sensitivity in CLL. In summary, VPA induces hyper-acetylation of histones in CLL and activates the TRAIL apoptotic pathway through upregulation of DR4 levels. While VPA is ineffective as a single agent in CLL, it can sensitize CLL cells to fludarabine and could be used as an adjuvant in fludarabine-based treatment regimens. Ongoing studies are evaluating the effectiveness of lower and less toxic doses of VPA in combination with fludarabine in CLL.