The Mevalonate Pathway As a Metabolic Target to Circumvent Multidrug-Resistance in Chronic Lymphocytic Leukemia Cells

Abstract 735

The mutational status of the tumor immunoglobulin heavy chain variable region (IGHV) is a very reliable prognosticator in chronic lymphocytic leukemia (CLL): patients with unmutated (UM) IGHV have a worse prognosis than patients with mutated (M) IGHV. We have recently shown that UM CLL cells are more dependent on microenvironment-mediated signals for survival (Coscia M. et al, Leukemia, 2011), but it is currently unknown whether this also translates into an enhanced chemotherapy resistance. Multi drug resistance (MDR) is mediated by the over-expression of membrane transporters, like P-glycoprotein (Pgp), which actively extrudes several anticancer drugs. The Pgp protein is the product of the mdr1 gene whose regulation is under the positive control of the mevalonate (Mev) metabolic pathway, the downstream Ras/RhoA dependent signaling pathways and the transcription factor Hypoxia-Inducible-Factor-1-alfa (HIF-1α). The aim of this study was to investigate the MDR phenotype and signaling cascade in M and UM CLL cells under basal conditions and after exposure to prosurvival microenvironment-mediated signals.

Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation from untreated CLL patients. More than 90% pure CLL cells were co-cultured with the murine stromal cell line M2-10B4. Culture conditions included: medium alone, Zoledronic acid (ZA) (1 μmol/L), ERK1/2 kinase inhibitor PD98059 (10 μmol/L) and the HIF-1α inhibitor YC-1 (10 μmol/L). The level of activity of the Mev pathway was evaluated by measuring the intermediate metabolite farnesyl pyrophosphate (FPP) and the final product cholesterol (Chol) as previously described (Coscia M. et al, J Cell Mol Med, 2010). The Ras and RhoA activity, was evaluated by measuring the GTP binding. Rho kinase activity was measured using the CycLex Rho Kinase Assay Kit (CycLex Co., Nagano). The following antibodies were used for Western Blot (WB) analyses: anti phospho-(Thr202/Tyr204, Thr185/Tyr187)-ERK1/2; anti-ERK 1/2; anti-HIF-1α; anti-Pgp; anti-GAPDH. The activity of HIF-1 in the nuclear extracts was assessed by the TransAM™ HIF-1 Transcription Factor Assay Kit (Active Motif). Mdr1 gene expression was evaluated by RT-PCR. Intracellular Doxorubicine (Doxo) accumulation was evaluated by cytofluorimetric analyses.

We have found that the Mev pathway is significantly accelerated in freshly isolated UM vs M CLL cells. This hypermetabolic activity translates into a higher activation of the Ras/ERK-1/2 and RhoA/Rho kinase signaling pathways, and to a significantly more active HIF-1α in UM compared to M CLL cells. HIF-1α activation positively regulates mdr1 gene expression in UM CLL cells leading to Pgp expression and more effective Doxo extrusion in these cells. The co-culture of CLL cells with the murine stromal cell line M2-10B4 further accelerated the Mev pathway and activated the HIF-1α/mdr1/PgP axis leading to even lower intracellular Doxo retention. This stroma-mediated effect was particularly evident in UM CLL cells. Targeting of the Mev pathway with ZA and statins, and targeting of ERK-1/2 and HIF-1α with specific inhibitors (PD85 and YC1-10, respectively) significantly reduced basal and stroma-induced activity of HIF-1α/mdr1/PgP axis, and significantly increased the intracellular concentrations of Doxo in UM CLL cells. These data indicate that the pharmacological inhibition of the Mev pathway and of the downstream HIF-1α/mdr1/PgP axis can be regarded as a novel potential strategy to circumvent basal and environment-mediated chemoresistance of CLL cells.