BCL-2 Phosphorylation Modulates Sensitivity to the BH3-Mimetic GX15-070 (Obatoclax) and Reduces Its Synergistic Interaction with Bortezomib in Chronic Lymphocytic Leukemia Cells.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in Western countries and is characterized by the accumulation of CD5-positive monoclonal B cells. This clonal excess of B cells is caused by a concomitant defect in both cell death and proliferation. A key factor that explains this inappropriate cell survival is the imbalanced expression of BCL-2 family proteins, thus representing an attractive therapeutic target for the treatment of this neoplasm. The current strategies for BCL-2 antagonism are based on small molecules that target several antiapoptotic BCL-2 proteins by mimicking a BH3 domain. Among them, GX15-070/Obatoclax (GeminX Biotechnologies) is pan-BCL-2 inhibitor that binds to BCL-2, BCL-W, BCL-XL and MCL-1 with high affinity, and has shown efficacy against several hematologic malignancies and solid tumors. In the present work, we report how GX15-070 led to the disruption of BCL-2/BIM and MCL-1/BAK complexes in CLL cells after short incubation times (3h), followed by the activation of the mitochondrial apoptotic pathway. Ex vivo experiments in CLL primary cells showed that GX15-070 as a single agent induces apoptosis at pharmacological concentrations. GX15-070 is also effective in CLL cells presenting alterations in P53, ATM, 13q deletions or high levels of ZAP-70 expression. LD50 at 20h were significantly higher in CLL cells (5.95 + 2.8 μM) compared to those previously reported in mantle cell lymphoma (MCL) primary cells (2.93 + 2.48 μM) (P<0.01). Of interest, these differences correlated with higher levels of pBCL-2(Ser70) in CLL compared to MCL primary cells. In the same context, we also demonstrated that ZAP-70+ CLL cases, which showed higher LD50 values than ZAP-70- ones, also expressed higher levels of pBCL-2(Ser70). Considering that BCL-2 phosphorylation at serine 70 residue is required for its antiapoptotic function, and that limits its interaction with proapoptotic multidomain and BH3-only proteins, it is conceivable that high levels of phosphorylated BCL-2 could impede or reduce GX15-070 activity. Both ERK1 (p44) and ERK2 (p42) kinases have been proposed to be responsible for BCL-2 phosphorylation. Considering these studies, we have demonstrated that pharmacological inhibition of MEK1/ERK pathway by PD98059 is able to reduce pBCL-2(Ser70) levels, increasing GX15-070 activity in CLL primary cells. In addition, as the protein phosphatase PP2A has been found to be responsible for BCL-2 dephosphorylation, its inhibition by okadaic acid increased pBCL-2(Ser70) levels, reducing GX15-070 cytotoxic activity. GX15-070 activity was increased by cotreatment with the proteasome inhibitor bortezomib. However, as proteasome inhibition led to the accumulation of pBCL-2(Ser70), the degree of interaction between GX15-070 and bortezomib was also regulated by the levels of pBCL-2(Ser70). Accordingly, as ERK1/2 is responsible for this phosphorylation, we also demonstrated that ERK1/2 inhibition by PD98059 could reverse bortezomib-induced accumulation of pBCL-2(Ser70) and increased GX15-070 and bortezomib cytotoxic effect. These results support the role of BCL-2 phosphorylation as a mechanism of resistance to BH3 mimetic compounds, and demonstrate that combination approaches including ERK inhibitors could enhance BH3 mimetics activity both alone or in combination with proteasome inhibitors.