The Mevalonate Pathway and Downstream Signal Transducers As Therapeutic Targets to Overcome Multidrug Resistance in Chronic Lymphocytic Leukemia (CLL)

Background: The mutational status of tumor immunoglobulin heavy chain variable region (IGHV) is a reliable prognosticator in chronic lymphocytic leukemia (CLL): patients with unmutated (UM) IGHV have a worse prognosis than patients with mutated (M) IGHV. The tumor microenvironment actively supports the survival of CLL cells and confers a multidrug resistance (MDR) phenotype to CLL cells. MDR is due to the over-expression of membrane transporters, like P-glycoprotein (Pgp), which actively extrudes several anticancer drugs. Pgp is under the positive control of the transcription factor Hypoxia-Inducible-Factor-1-alfa (HIF-1α) which is activated by isoprenylated Ras/Rho-dependent downstream signaling pathways. Ras and Rho isoprenylation are regulated by the mevalonate (Mev) pathway activity suggesting that this pathway can be exploited as a metabolic checkpoint to regulate chemresistance. Aim: The aim of this study was twofold: 1) to investigate the correlation between chemoresistance and the activity of the Mev pathway and Ras/Rho-A downstream signaling pathways in purified M and UM CLL cells under basal conditions and after incubation with stromal cells; 2) to evaluate the chemosensitizing effects of agents specifically targeting the Mev pathway and downstream signaling pathways under the same culture conditions. Methods: M and UM CLL cells were cultured in the presence and in the absence of murine stromal cells (M210B4) and exposed to Zoledronic acid (ZA) (1 μmol/L), Simvastatine (Sim) (1 μmol/L), ERK1/2 kinase inhibitor PD98059 (10 μmol/L), HIF-1α inhibitor YC-1 (10 μmol/L) and Doxorubicine (Doxo) (1 μmol/L). The Mev pathway activity was measured by cells radiolabelling with [14C]-mevalonic acid and thin layer chromatography. Ras, ERK1/2 and Akt activity were detected by Western blot. Rho, Rho Kinase and HIF-1α activity were assessed by ELISA. Mdr1 expression was measured by Real Time-PCR. PgP activity was evaluated by measuring Doxo intracellular accumulation. Doxo cytotoxicity was assessed by annexin V and propidium iodide staining. Results: The Mev pathway is significantly more active in UM than in M CLL cells. This hypermetabolic activity translates into a higher activation of Ras/Akt and Rho/Rho kinase signaling pathways and higher expression of the phosphorylated active form of HIF-1α. HIF-1α activation positively regulates mdr1 gene expression in UM CLL cells leading to a more effective Doxo extrusion and therefore better survival upon Doxo exposure. M210B4 stromal cells further protect UM CLL cells from Doxo induced cell death by upregulating Mev pathway activity, HIF-1α/mdr1/PgP axis activation, and Doxo extrusion. Targeting the Mev pathway of UM cells with ZA and Mev reduces the basal activity of HIF-1α/mdr1/PgP axis and significantly increases Doxo retention and cytotoxicity. Similar effects are obtained with PD85 and YC1–10 which are specific inhibitors of the downstream molecules ERK-1/2 and HIF-1α, respectively. All these agents are able to overcome the protective effect exerted by stromal cells by significantly increasing PgP activity and Doxo-induced cell death. Conclusions: Our data demonstrate that the Ras- and Rho-dependent HIF-1α/mdr1/PgP axis is more active and associated with higher levels of MDR in UM compared with M CLL cells. Targeting the Mev pathway and/or downstream signalling pathways is a promising strategy to circumvent basal and stroma-mediated chemoresistance especially in UM CLL cells.