EXPRESSION PROFILE OF EPIGENETIC MODULATORS IN ACUTE MYELOID LEUKEMIA OF INTERMEDIATE RISK
(Abstract release date: 05/18/17)
EHA Library. Sánchez Sosa S. 05/18/17; 182381; PB1667
Santiago Sánchez Sosa
Contributions
Contributions
Abstract
Abstract: PB1667
Type: Publication Only
Background
Whole-genome sequencing has revealed acute myeloid leukemia (AML) as a very complex and dynamic disease.Epigenetic modulation is among the functional categories of the mutational landscape in AML.According to recent reports, suppression of the epigenetic reader BRD4 with small-molecule inhibitors (BET-i) results in antileukemic activity. Clinical trials are being developed, however, so far, identification of those patients that may benefit from this therapy is not possible as changes in mRNA BRD4 levels seem to be very subtle. It has been recently suggested that antileukemic effect of BET-i could be due to c-myc suppression and also that high Bcl-2 levels may target those patients that would benefit of BET-i. We believe that establishing the expression profile of epigenetic modulators in AML may help in the identification of patients that could benefit from BET-i.
Aims
We wanted to get a better insight regarding the expression profile of epigenetic modulator in AML of intermediate risk by studying: 1)expression levels of EZH2, ASXL1, BRD4, c-myc and Bcl-2 in a consecutive series of AML patients; 2) correlation between mRNA and protein levels; 3) Determining BRD4 binding to the c-myc promoter through chromatin immunoprecipitation (CHIP).
Methods
Our series consisted of 104 consecutive patients with a mean age of 55.8 years (range 15-79 years) diagnosed and treated between 2005-2016 at the Hospital Universitario de Gran Canaria Dr. Negrín with a median follow up of 12 months. Gene expression analysis was carried out through real time PCR in a LightCycler 480 Instrument II (Roche) using GUS a control gene.Results were normalized with a cDNA pool from bone marrow of 10 healthy donors which was introduced as internal control in each experiment. Western blot were performed to determine protein levels for BRD4, c-myc and Bcl2. CHIP studies for BRD4 were carried out in HL60 cell line. For statistical analysis the SPSS (v.15.0) software was used.
Results
ASXL1 levels were positively associated with EZH2 (Spearman's= 0.285, p=0.021) and BRD4 withc-myc (Spearman's coefficient=0.420, p<0.001), Bcl2 (Spearman's= 0.471, p<0.001) EZH2 (Spearman's= 0.4565, p=0.008) and ASXL1 (Spearman's=0.949, p<0.001). Survival analysis considering 50th percentile as a cut-off value for BRD4 expression indicated that patients with higher levels shows better overall survival (median overall survival, OS, of 27 months, 95% IC 15.1-38.9) compared to those with low expression (median OS 12 months, 95% IC 0.4-23.7), although the association was not statistically significant (p=0.196) probably due to the limited series size. Protein levels of Bcl2 and c-myc correlated with those of mRNA, but not for BRD4, although other antibodies should be tested in order to confirm these results. CHIP analysis in HL60 cell lines confirmed the binding of BRD4 to c-myc promoter.
Conclusion
The positive association observed between EZH2 and ASXL1 agrees with the fact that both cooperate in the epigenetic repressive complex PRC2.The association of BRD4 expression levels with c-myc and Bcl2 is in accordance to the reported binding of BRD4 to the c-myc and Bcl2superenhancer regions and our CHIP analysis also support so. Further studies in a larger series are necessary to confirm the relationship between higher BRD4 levels and better overall survival. Finally, future analysis should be done to determine whether patients with higher BRD4 expression levels determine a subgroup with better response to BET-i.
Session topic: 3. Acute myeloid leukemia - Biology
Keyword(s): Expression profiling, Epigenetic, Acute Myeloid Leukemia
Abstract: PB1667
Type: Publication Only
Background
Whole-genome sequencing has revealed acute myeloid leukemia (AML) as a very complex and dynamic disease.Epigenetic modulation is among the functional categories of the mutational landscape in AML.According to recent reports, suppression of the epigenetic reader BRD4 with small-molecule inhibitors (BET-i) results in antileukemic activity. Clinical trials are being developed, however, so far, identification of those patients that may benefit from this therapy is not possible as changes in mRNA BRD4 levels seem to be very subtle. It has been recently suggested that antileukemic effect of BET-i could be due to c-myc suppression and also that high Bcl-2 levels may target those patients that would benefit of BET-i. We believe that establishing the expression profile of epigenetic modulators in AML may help in the identification of patients that could benefit from BET-i.
Aims
We wanted to get a better insight regarding the expression profile of epigenetic modulator in AML of intermediate risk by studying: 1)expression levels of EZH2, ASXL1, BRD4, c-myc and Bcl-2 in a consecutive series of AML patients; 2) correlation between mRNA and protein levels; 3) Determining BRD4 binding to the c-myc promoter through chromatin immunoprecipitation (CHIP).
Methods
Our series consisted of 104 consecutive patients with a mean age of 55.8 years (range 15-79 years) diagnosed and treated between 2005-2016 at the Hospital Universitario de Gran Canaria Dr. Negrín with a median follow up of 12 months. Gene expression analysis was carried out through real time PCR in a LightCycler 480 Instrument II (Roche) using GUS a control gene.Results were normalized with a cDNA pool from bone marrow of 10 healthy donors which was introduced as internal control in each experiment. Western blot were performed to determine protein levels for BRD4, c-myc and Bcl2. CHIP studies for BRD4 were carried out in HL60 cell line. For statistical analysis the SPSS (v.15.0) software was used.
Results
ASXL1 levels were positively associated with EZH2 (Spearman's= 0.285, p=0.021) and BRD4 withc-myc (Spearman's coefficient=0.420, p<0.001), Bcl2 (Spearman's= 0.471, p<0.001) EZH2 (Spearman's= 0.4565, p=0.008) and ASXL1 (Spearman's=0.949, p<0.001). Survival analysis considering 50th percentile as a cut-off value for BRD4 expression indicated that patients with higher levels shows better overall survival (median overall survival, OS, of 27 months, 95% IC 15.1-38.9) compared to those with low expression (median OS 12 months, 95% IC 0.4-23.7), although the association was not statistically significant (p=0.196) probably due to the limited series size. Protein levels of Bcl2 and c-myc correlated with those of mRNA, but not for BRD4, although other antibodies should be tested in order to confirm these results. CHIP analysis in HL60 cell lines confirmed the binding of BRD4 to c-myc promoter.
Conclusion
The positive association observed between EZH2 and ASXL1 agrees with the fact that both cooperate in the epigenetic repressive complex PRC2.The association of BRD4 expression levels with c-myc and Bcl2 is in accordance to the reported binding of BRD4 to the c-myc and Bcl2superenhancer regions and our CHIP analysis also support so. Further studies in a larger series are necessary to confirm the relationship between higher BRD4 levels and better overall survival. Finally, future analysis should be done to determine whether patients with higher BRD4 expression levels determine a subgroup with better response to BET-i.
Session topic: 3. Acute myeloid leukemia - Biology
Keyword(s): Expression profiling, Epigenetic, Acute Myeloid Leukemia
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