Contributions
Abstract: PB1613
Type: Publication Only
Background
B cell acute lymphoblastic leukemia (B-ALL) is the most frequent hematologic neoplasia in children and is characterized by the accumulation of blast cells that are phenotypically reminiscent of normal stages of B-cell differentiation. Epigenetic alterations, namely DNA methylation and histone modifications, are involved in B-ALL development and progression. Unlike chromosomal translocations or gene mutations, which induce permanent changes in the DNA sequence, hypermethylation of gene promoters is a reversible event that could be targeted with therapeutic agents designed to alter aberrant epigenetic events.
Aims
The aim of this study was to evaluate the therapeutic potential of hypomethylating agents ([DNMTi: Azacitidine (5-AC) and Decitabine (DAC)]) and histone deacetylase inhibitors [HDACi: Panobinostat (PAN) and Vorinostat (SAHA)], in monotherapy and in combination therapy, in in vitro models of B-ALL.
Methods
Two B-ALL cell lines, the KOPN-8 and 697 cells, were incubated in the absence and presence of DNMTis and/or HDACis, in monotherapy and in therapeutic combination. Cell viability was determined by the FMCA assay (Fluorometric Microculture Cytotoxicity Assay). Cell death was evaluated by optical microscopy (May-Grünwald-Giemsa staining) and by Flow Cytometry (FC; Annexin V/7-AAD double staining). The hypomethylating effect was evaluated through 5-methylcytosine (5-mC) levels analyzed by FC and the methylation status of 24 tumor suppressor genes was carried out by MS-MLPA. The results were analyzed statistically considering a significance level of 95% (p <0.05).
Results
The results showed that 697 cells have more tumor suppressor genes methylated (15/24) than KOPN8 cells (12/24). The studied drugs reduced cell viability in a time-, dose- and cell line-dependent manner, being the 697 cells more sensitive than KOPN-8 cells. B-ALL cells were found to be more sensitive to DAC (IC50 697 = 10 μM; IC50 KOPN-8 > 15 μM) than to 5-AC (IC50 697 = 15 μM; IC50 KOPN-8 > 20 μM), after 72h of treatment. They were also more sensitive to PAN (IC50 697 = 7.5 nM; IC50 KOPN-8 > 20 nM) than to SAHA (IC50 697 = 750 nM; IC50 KOPN-8 = 1000 nM). In addition, therapeutic associations of DAC with PAN or SAHA reduced more effectively the cell viability when compared to monotherapy treatments. These drugs induced predominantly apoptosis in 697 cells and apoptosis and necrosis in KOPN-8 cells. Cell death by apoptosis was confirmed by morphological aspects such as blebbing, cellular contraction, and nuclear fragmentation. Moreover, SAHA induced cell cycle arrest in phase S and G0/G1, respectively in 697 and KOPN-8 cells. Finally, both DNMTi and their association with PAN induced a decrease in 5-mC levels and induced demethylation of at least two tumor suppressor genes (697: TP73, KLLN, MGMT, and CD44; KOPN8: MGMT and STK11).
Conclusion
Our results suggest the therapeutic potential of epigenetic modulators in the treatment of B-ALL in monotherapy and in the usefulness of the association of DNMTi with panobinostat. However, the therapeutic efficacy may depend on the methylation profile.
This work was supported by CIMAGO and by the FCT through the fellowship SFRH/BD/51994/2012.
Session topic: 1. Acute lymphoblastic leukemia – Biology & Translational Research
Keyword(s): histone deacetylase inhibitor, Methylation, Therapy, ALL
Abstract: PB1613
Type: Publication Only
Background
B cell acute lymphoblastic leukemia (B-ALL) is the most frequent hematologic neoplasia in children and is characterized by the accumulation of blast cells that are phenotypically reminiscent of normal stages of B-cell differentiation. Epigenetic alterations, namely DNA methylation and histone modifications, are involved in B-ALL development and progression. Unlike chromosomal translocations or gene mutations, which induce permanent changes in the DNA sequence, hypermethylation of gene promoters is a reversible event that could be targeted with therapeutic agents designed to alter aberrant epigenetic events.
Aims
The aim of this study was to evaluate the therapeutic potential of hypomethylating agents ([DNMTi: Azacitidine (5-AC) and Decitabine (DAC)]) and histone deacetylase inhibitors [HDACi: Panobinostat (PAN) and Vorinostat (SAHA)], in monotherapy and in combination therapy, in in vitro models of B-ALL.
Methods
Two B-ALL cell lines, the KOPN-8 and 697 cells, were incubated in the absence and presence of DNMTis and/or HDACis, in monotherapy and in therapeutic combination. Cell viability was determined by the FMCA assay (Fluorometric Microculture Cytotoxicity Assay). Cell death was evaluated by optical microscopy (May-Grünwald-Giemsa staining) and by Flow Cytometry (FC; Annexin V/7-AAD double staining). The hypomethylating effect was evaluated through 5-methylcytosine (5-mC) levels analyzed by FC and the methylation status of 24 tumor suppressor genes was carried out by MS-MLPA. The results were analyzed statistically considering a significance level of 95% (p <0.05).
Results
The results showed that 697 cells have more tumor suppressor genes methylated (15/24) than KOPN8 cells (12/24). The studied drugs reduced cell viability in a time-, dose- and cell line-dependent manner, being the 697 cells more sensitive than KOPN-8 cells. B-ALL cells were found to be more sensitive to DAC (IC50 697 = 10 μM; IC50 KOPN-8 > 15 μM) than to 5-AC (IC50 697 = 15 μM; IC50 KOPN-8 > 20 μM), after 72h of treatment. They were also more sensitive to PAN (IC50 697 = 7.5 nM; IC50 KOPN-8 > 20 nM) than to SAHA (IC50 697 = 750 nM; IC50 KOPN-8 = 1000 nM). In addition, therapeutic associations of DAC with PAN or SAHA reduced more effectively the cell viability when compared to monotherapy treatments. These drugs induced predominantly apoptosis in 697 cells and apoptosis and necrosis in KOPN-8 cells. Cell death by apoptosis was confirmed by morphological aspects such as blebbing, cellular contraction, and nuclear fragmentation. Moreover, SAHA induced cell cycle arrest in phase S and G0/G1, respectively in 697 and KOPN-8 cells. Finally, both DNMTi and their association with PAN induced a decrease in 5-mC levels and induced demethylation of at least two tumor suppressor genes (697: TP73, KLLN, MGMT, and CD44; KOPN8: MGMT and STK11).
Conclusion
Our results suggest the therapeutic potential of epigenetic modulators in the treatment of B-ALL in monotherapy and in the usefulness of the association of DNMTi with panobinostat. However, the therapeutic efficacy may depend on the methylation profile.
This work was supported by CIMAGO and by the FCT through the fellowship SFRH/BD/51994/2012.
Session topic: 1. Acute lymphoblastic leukemia – Biology & Translational Research
Keyword(s): histone deacetylase inhibitor, Methylation, Therapy, ALL