Activation of Mitochondrial STAT3 Increases Mitochondrial Respiration and Inhibits Oxidative Stress in Chronic Lymphocytic Leukemic Cells

Abstract 287

Chronic lymphocytic leukemia (CLL) is a malignant disease occurring in the elderly and remains incurable. CLL is characterized by resistance to both spontaneous and induced apoptosis aided by changes induced by the tumor microenvironment. STAT3 is a signal responsive transcription factor that plays pivotal roles in tumorigensis in a number of malignancies including CLL. STAT3 resides in an inactive form in the cytoplasm of non-stimulated cells and in response to various cytokines and growth factors present in the microenvironment is activated through JAK-mediated phosphorylation of two residues, tyrosine 705 (Y705) and serine 727 (S727). Phosphorylation of this critical tyrosine residue (Y705) induces STAT3 dimerization through phosphotyrosine-SH2 domain interaction and. once dimerized, enters the nucleus and activates a broad array of target genes. The role of serine phosphorylation (S727) is less understood. It has been reported that STAT3 is constitutively phosphorylated on S727 and pS⁷²⁷-STAT3, not pY⁷⁰⁵-STAT3, binds DNA and activates transcription in CLL cells. However, it has also been reported that STAT3 is present in the mitochondria both in cell lines and primary liver and heart of mouse models, where it is one of the components of the mitochondrial electron transport chain (mETC) and plays an important role in mitochondrial respiration. The active form of mitochondrial STAT3 is pS⁷²⁷-STAT3 and it is crucial for Ras-dependent transformation by sustaining altered glycolytic and oxidative phosphorylation activities characteristic of cancer cells.

It is unknown whether STAT3 regulates mitochondrial function in CLL. We therefore investigated whether activated STAT3 regulates mitochondrial respiration in CLL and whether it is important for CLL cell survival. Screening by Western blotting in untreated CLL patients' samples (n=16 )revealed that both pS⁷²⁷-STAT3 and pY⁷⁰⁵-STAT3 were constitutively expressed and we demonstrated correlation of the expression levels between these two active forms. Using fluorescent microscopy and cellular protein fractionation, both pS⁷²⁷-STAT3 and pY⁷⁰⁵-STAT3 showed mitochondrial localization in CLL cells. Stimulation of CLL cells with IL-10 induced STAT3 activation and both active forms of STAT3 exhibited mitochondrial translocation. The JAK inhibitor AG490 prevented STAT3 translocation to the mitochondria and led to reduction of mitochondrial mass and expression of cytochrome c oxidase IV (COX IV), one of the components of mETC. Knockdown of STAT3 RNA also decreased COX IV expression. Flow cytometry studies demonstrated that activation of STAT3 by IL-10 prevented depolarization of mitochondrial membrane potential and free radical generation by CLL cells, but inhibition of STAT3 induced mitochondrial oxidative damage and CLL cell death. The role of STAT3 activation by IL-10 on mitochondrial respiration was determined using a Seahorse XF Extracellular Flux Analyzer and demonstrated significantly increased coupled and uncoupled mitochondrial respiration and ATP turnover. Inhibition of STAT3 by AG490 reduced mitochondrial respiration and ATP turnover. However, decreased mitochondrial respiration did not provoke glycolytic capacity in CLL cells, indicating that CLL cells mainly rely on mitochondria for energetic needs.

In summary, we demonstrate that activated STAT3 targets mitochondria and increases mitochondrial respiration and ATP turnover in CLL cells. This enables increased bioenergetic mitochondrial function and also prevents oxidative damage of CLL cells. Inhibition of STAT3 reduces mitochondrial mass and function but increases free radical generation and promotes CLL cell death. We therefore propose that mitochondrial STAT3 could be a therapeutic target for the treatment of CLL.