BCL-2 Phosphorylation Modulates Sensitivity to the BH3-Mimetic GX15-070 (Obatoclax) and Reduces Its Synergistic Interaction with Bortezomib in Chronic Lymphocytic Leukemia Cells.
Authors: Galan Patricia Perez , Roue Gael , Guerra Monica Lopez , Villamor Neus , Montserrat Emili , Campo Elias , Colomer Dolors 
Published: October 2019

Abstract
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.