Targeting Destruction of Mcl-1 by Activation of Protein Phosphatase 2A In CLL and B-Cell Lymphomas

Myeloid Cell Leukemia-1 (Mcl-1) is an anti-apoptotic protein that is elevated in fludarabine-resistant CLL patients. Expression levels of Mcl-1 correlate with time to first treatment and overall survival in CLL patients. Furthermore, elevated levels of Mcl-1 have also been reported in B-cell lymphomas. The regulation of Mcl-1 stability occurs through phosphorylation of several serine and threonine residues catalyzed by constitutively activated kinases. The tumor suppressor protein phosphatase 2A (PP2A) is important in deactivation of several kinases: Akt, the mitogen activated protein kinases (MAPK) p38, JNK, ERK, and NFkB (through IkK). We have discovered novel peptides that antagonize the SET oncoprotein, a potent physiological inhibitor of PP2A, and increase cellular PP2A activity. Importantly, we note that SET is overexpressed in CLL and B-cell lymphoma cells and that treatment of these cells with SET antagonist peptides is cytotoxic for the malignant cells both in vitro and in vivo. We show here that activation of PP2A by antagonism of SET results in reduced Mcl-1 levels and induction of apoptosis in CLL and B-cell lymphoma cells.

Patients were from the Duke University and V.A. Medical Centers, and normal controls were from the community. Blood CLL cells and control normal B-cells were purified using negative selection with antibodies. The human Ramos and Raji B-cell lymphoma cell lines were from ATCC. We determined cytotoxicity using the MTS colorimetric assay. SET antagonist peptides were prepared by chemical synthesis. Western blotting and immunoprecipitation were performed with antibodies to SET, Mcl-1, PP2Ac, c-Myc, Axin, Pin-1, GSK3beta, Bcl-2, Bcl-XL and beta-actin. Apoptosis assays were performed with the annexin-V/propidium iodide staining method.

We previously demonstrated SET overexpression in CLL cells and that SET antagonist peptides activate PP2A and are cytotoxic for malignant B-cells. Using freshly-isolated CLL cells and Raji and Ramos cell lines, cytotoxicity of the SET antagonist peptide COG449 was evaluated. We found the concentrations for 50% cytotoxicity (ED50) to be 77 nM for CLL cells, 125 nM for Ramos cells, 250 nM for Raji cells, and >10,000 nM for normal B-cells. Annexin-V staining indicated that apoptosis was induced at concentrations comparable to the ED50s for cytotoxicity of the compounds tested. COG449 treatment of NOD/SCID mice bearing tumors after xenografting with Ramos cells resulted in reduced tumor growth. After treatment for 8 days, there was a 61% reduction in final tumor mass at harvest on day 19 (p<0.001). To evaluate the induction of apoptosis, we treated CLL or Raji cells with COG449 and monitored the cellular levels of the anti-apoptotic Bcl-2, Bcl-XL, and Mcl-1 proteins. We found no significant changes in Bcl-2 or Bcl-XL levels, but Mcl-1 levels decreased in a dose dependent manner. Immunoprecipitation of Mcl-1 was performed to identify proteins that interact with Mcl-1 and regulate its stability. We identified a novel pattern of immunoprecipitating proteins and found that PP2A and SET are part of the Mcl-1 regulatory complex.

The observation that SET antagonist peptides induce apoptosis through dose-dependent reduction in the cellular level of Mcl-1 indicates that PP2A may modulate the stability of Mcl-1. Co-immunoprecipitation of SET and PP2A with Mcl-1 suggests that a novel complex exists for the regulation of Mcl-1 stability and that PP2A-mediated dephosphorylation of Mcl-1 may be a critical step in this process. Induction of Mcl-1 degradation with PP2A reactivation therapy may be a useful approach for the treatment of CLL and other B-cell malignancies.

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