P300 Acetylates STAT3 and Increases Its Constitutive Transcriptional Activity in CLL Cells

Introduction: Signal transducer and activator of transcription (STAT)-3 is thought to promote oncogenesis by modulating the expression of genes that are required for tumor cell survival¹. Following translation STAT3 may undergo several modifications such as phosphorylation or acetylation which modulate its activity. These modifications are typically cytokine-dependent but may also be cytokine-independent or constitutive. In chronic lymphocytic leukemia (CLL) STAT3 is constitutively phosphorylated (p) on serine-727 residues². Serine pSTAT3 forms heterodimers, shuttles to the nucleus and induces transcription of STAT3 targeted genes. The transcriptional activity of STAT3 is also increased if STAT3 undergoes acetylation³. The conserved bormodomain motif of the acetyltransferase p300 binds and acetylates lysine residues of several proteins including STAT3⁴. Whether STAT3 is acetylated in CLL cells is currently unknown. We sought to determine whether p300 acetylates STAT3 and affects its transcriptional activity in CLL cells.

Methods and Results: Using western immunoblotting of the peripheral blood (PB) lymphocytes from 16 CLL patients we found that STAT3 is constitutively phosphorylated on lysine 685 residues. As previously reported^2,5 we also detected serine pSTAT3 in all samples. Flow cytometry analysis revealed that a significant number of CD19+ CLL cells are constitutively phosphorylated on serine 727 and acetylated on lysine 685 residues. Immunoprecipitation studies confirmed these data. Protein extract of PB from 8 CLL patients were immunoprecipitated either with STAT3 or serine pSTAT3 antibodies and acetyl-STAT3 was detected in all immunoprecipitates, suggesting that the activated form of STAT3 is also acetylated in CLL cells. Because previous studies suggested that p300 acetylates STAT3 we obtained PB samples from 5 CLL patients and using western immunobloting and detected high levels of p300 in all patients but not in B lymphocytes from 2 healthy individuals. Then, to determine whether p300 affects the transcriptional activity of STAT3, we transfected CLL cells with p300 small-interfering (si) RNA. With a transfection efficiency of 30%, p300 transcripts and protein levels and acetyl-STAT3 levels were significantly reduced. Transfection with p300-siRNA reduced STAT3-DNA binding, as assessed by an electormobility shift assay (EMSA), decrease the levels of the STAT3-target gene p21 as assessed by chromatin immunoprecipitation (ChIP), and the levels of several STAT3-induced genes including STAT3, VEGF-c, c-Myc and p21, as assessed by RT-PCR. In addition, using Annexin V/PI we detected high apoptosis rates in p300-siRNA- transfected cells. Taken together these data suggest that the transcriptional activity of STAT3 is enhanced by p300 and STAT3 acetylation provides the cells with survival advantage.

Conclusion: Our findings suggest that the aberrant expression of p300 induces acetylation and increases the transcriptional activity of STAT3 in CLL. Whether targeting STAT3 acetylation could be effective therapeutic modality in CLL remains to be determined.

References

1. Yu H, Pardoll D, Jove R. STATs in cancer inflammation and immunity: a leading role for STAT3. Nature reviews. Cancer. 2009;9(11):798-809.

2. Hazan-Halevy I, Harris D, Liu Z, et al. STAT3 is constitutively phosphorylated on serine 727 residues, binds DNA, and activates transcription in CLL cells. Blood. 2010;115(14):2852-2863.

3. Yuan ZL, Guan YJ, Chatterjee D, Chin YE. Stat3 dimerization regulated by reversible acetylation of a single lysine residue. Science. 2005;307(5707):269-273.

4. Wang R, Cherukuri P, Luo J. Activation of Stat3 sequence-specific DNA binding and transcription by p300/CREB-binding protein-mediated acetylation. J Biol Chem. 2005;280(12):11528-11534.

5. Frank DA, Mahajan S, Ritz J. B lymphocytes from patients with chronic lymphocytic leukemia contain signal transducer and activator of transcription (STAT) 1 and STAT3 constitutively phosphorylated on serine residues. J Clin Invest. 1997;100(12):3140-3148.