PS1139 P300 INDUCES STAT3 ACETYLATION AND PROVIDES CLL CELLS WITH SURVIVAL ADVANTAGE
Authors: U. Rozovski, D. Harris, P. Li, Z. Liu, P. Jain, A. Ferrajoli, J. Burger, P. Thompson, N. Jain, W. Wierda, M. Keating, Z. Estrov
Journal: HemaSphere (2019)

Background: The signal and transducer of activator (STAT)‐3 signaling protein is thought to promote oncogenesis by modulating the expression of genes that are required for tumor cell survival. Following translation STAT3 may undergo several modifications such as phosphorylation or acetylation which modulate its activity. These modifications are typically cytokine dependent but under unique conditions may be cytokine‐independent and constitutive. In chronic lymphocytic leukemia (CLL) STAT3 is constitutively phosphorylated on serine residues. Serine pSTAT3 forms heterodimers, shuttles to the nucleus and induces transcription of STAT3 targeted genes. The transcription activity of STAT3 is also increased if STAT3 undergoes acetylation 3 . The conserved bormodomain motif of p300 binds and acetylates lysine residues in several proteins including STAT3. Recently, we showed that STAT3 is acetylated in CLL cells. Aims: 1) To test whether p‐300 acetylates STAT3 in CLL cells. 2) To test to what extent acetylated STAT3 provides CLL cells with a survival advantage Methods: CLL cells were isolated from the peripheral blood of patients using Ficol gradient. Western immunoblotting and immunoprecipitation studies were used to detected acetylated and phosphorylated isoforms of STAT3. To silence p300 we used 300 small interfering (SI) RNA and by qRT‐PCR we quantified the expression of STAT3 target genes. To test DNA‐protein binding we used electromotility shift assay (EMSA) and chromatin immunoprecipitation and apoptosis was assessed by the annexin/PI assay. Results: By Western blot analysis we detected lysine acetSTAT3 in 16 patients with CLL and as previously reported by us and others we also detected serine pSTAT3 in all cases. To confirm, we immunoprecipitated the protein extract of CLL cells from 8 patients using antibody directed to detect all STAT3 isoforms (total STAT3 antibody) and detected in the precipitate lysine acetSTAT3 and serine pSTAT3. Because previous studies suggested that p300 mediates STAT3 acetylation we performed western blot analysis with anti‐p300 antibodies and detected p300 in 5 patients with CLL but not in B cells from 2 healthy individuals. To test whether p300 contributes to the transcriptional activity of STAT3 we transfected CLL cells with p300 (si)‐RNA. With transfection efficiency of 30%, levels of the p300 transcript and protein were significantly reduced. Using electormobility shift assay (EMSA) we found lower DNA‐STAT3 binding in CLL cells transfected with p300‐(si)RNA compared to controls and by chromatin immunoprecipitation (CHIP) assay we found a decrease in expression of the STAT3 target gene p21 in p300‐(si)RNA transfected cells. Furthermore, using Annexin V/PI staining we detected higher rates of apoptosis in p300‐(si)RNA transfected cells. Taken together these findings suggest that the transcriptional activity of STAT3 is p300 dependent and provides the cells with survival advantage. Summary/Conclusion: Our findings suggest that the an 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.