Contributions
Abstract: PB1678
Type: Publication Only
Background
The hypoxic microenvironment plays a crucial role in survival and chemoresistance of leukemia stem cells (LSC) in acute myeloid leukemia (AML) and shapes cell metabolism. The inhibitor of bromodomain–containing (BRD) proteins (BETi), which is effective against AML cells in normoxia, targets the transcriptional program controlled by MYC, a crucial gene controlling LSC biology and metabolic functions under hypoxia.
Aims
To investigate the role of the hypoxic microenvironment on the selective pressure of BETi treatment on AML models with different genomic background.
Methods
AML cell lines (OCI-AML3: NPM1 and DNMT3A mutated, Kasumi-1: t(8;21), H-60: MYC-amplified, MOLM-13, NOMO-1: MLL-driven, KG-1) were treated for 16 or 48h with the BETi GSK1215101A (250 nM or 500 nM) after 4h-adaptation to hypoxia. Downstream analyses were performed on Kasumi-1 and OCI-AML3 cells after 16h of treatment in order to allow cell adaption to the pharmacological pressure, while avoiding massive cell death. Gene expression profiling was carried out on actively translated mRNAs isolated by polysome profiling (Affymetrix) and enrichment analysis was performed by GSEA (Broad Insititute). The metabolic profile was obtained by Liquid Chromatography-Tandem Mass Spectroscopy (Metabolon).
Results
BETi induced a dose-dependent reduction of cell viability at 48h in AML cell lines (15%>35% decrease at 250 nM and 25%>65% decrease at 500 nM) except for HL60. Kasumi-1 was the most sensitive model. The treatment caused a significant arrest in the G0/G1 phase of the cell cycle in OCI-AML3, Kasumi-1, HL-60 and KG-1 cells and induction of apoptosis in NOMO-1 and Kasumi-1. Kasumi1 and OCI-AML3 showed a different translational profile, the latter being more active under steady state conditions. Hypoxia and BETi reduced the translational rate of both lines, as determined by a decrease of disome-polysome peaks height. This associated with BETi-mediated downregulation of a ribosome pathway signature (p<0.001). Downregulation of the MYC transcriptional program was enriched in the translatome of both lines under hypoxia (p<0.001). The two models shared a core translational program of 86 differentially expressed genes. Additional 881 and 168 genes were altered at translational level in Kasumi-1 and OCI-AML3 cells, respectively. Moreover, the cell type strongly influenced the metabolic profile in response to hypoxia and drug treatment. Hypoxic Kasumi-1 cells showed reduced lactate levels upon treatment, which may be due to a drug-dependent decrease in lactate dehydrogenase activity and decreased asparagine levels, along with downregulation of a gene signature of alanine, aspartate and glutamate metabolism (p<0.001), including asparagine synthetase (1.5-fold decrease, p<0.01). OCI-AML3 showed a significant increase of both reduced (3.9-fold) and oxidized (2.6-fold) forms of glutathione under hypoxia, which were confirmed by colorimetric assay.
Conclusion
AML cell lines are sensitive to BETi under hypoxia, which strengthens the drug effects on MYC expression and highlights novel potential dependencies. The consequences on leukemic cell metabolism suggest pathways to be exploited for combination therapies in genomic-driven approaches.
Supported by: EHA research fellowship award, ELN, AIL, AIRC, project Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project, HARMONY project, Fondazione del Monte BO e RA project.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): Acute Myeloid Leukemia, Genetic, Inhibitor, MYC
Abstract: PB1678
Type: Publication Only
Background
The hypoxic microenvironment plays a crucial role in survival and chemoresistance of leukemia stem cells (LSC) in acute myeloid leukemia (AML) and shapes cell metabolism. The inhibitor of bromodomain–containing (BRD) proteins (BETi), which is effective against AML cells in normoxia, targets the transcriptional program controlled by MYC, a crucial gene controlling LSC biology and metabolic functions under hypoxia.
Aims
To investigate the role of the hypoxic microenvironment on the selective pressure of BETi treatment on AML models with different genomic background.
Methods
AML cell lines (OCI-AML3: NPM1 and DNMT3A mutated, Kasumi-1: t(8;21), H-60: MYC-amplified, MOLM-13, NOMO-1: MLL-driven, KG-1) were treated for 16 or 48h with the BETi GSK1215101A (250 nM or 500 nM) after 4h-adaptation to hypoxia. Downstream analyses were performed on Kasumi-1 and OCI-AML3 cells after 16h of treatment in order to allow cell adaption to the pharmacological pressure, while avoiding massive cell death. Gene expression profiling was carried out on actively translated mRNAs isolated by polysome profiling (Affymetrix) and enrichment analysis was performed by GSEA (Broad Insititute). The metabolic profile was obtained by Liquid Chromatography-Tandem Mass Spectroscopy (Metabolon).
Results
BETi induced a dose-dependent reduction of cell viability at 48h in AML cell lines (15%>35% decrease at 250 nM and 25%>65% decrease at 500 nM) except for HL60. Kasumi-1 was the most sensitive model. The treatment caused a significant arrest in the G0/G1 phase of the cell cycle in OCI-AML3, Kasumi-1, HL-60 and KG-1 cells and induction of apoptosis in NOMO-1 and Kasumi-1. Kasumi1 and OCI-AML3 showed a different translational profile, the latter being more active under steady state conditions. Hypoxia and BETi reduced the translational rate of both lines, as determined by a decrease of disome-polysome peaks height. This associated with BETi-mediated downregulation of a ribosome pathway signature (p<0.001). Downregulation of the MYC transcriptional program was enriched in the translatome of both lines under hypoxia (p<0.001). The two models shared a core translational program of 86 differentially expressed genes. Additional 881 and 168 genes were altered at translational level in Kasumi-1 and OCI-AML3 cells, respectively. Moreover, the cell type strongly influenced the metabolic profile in response to hypoxia and drug treatment. Hypoxic Kasumi-1 cells showed reduced lactate levels upon treatment, which may be due to a drug-dependent decrease in lactate dehydrogenase activity and decreased asparagine levels, along with downregulation of a gene signature of alanine, aspartate and glutamate metabolism (p<0.001), including asparagine synthetase (1.5-fold decrease, p<0.01). OCI-AML3 showed a significant increase of both reduced (3.9-fold) and oxidized (2.6-fold) forms of glutathione under hypoxia, which were confirmed by colorimetric assay.
Conclusion
AML cell lines are sensitive to BETi under hypoxia, which strengthens the drug effects on MYC expression and highlights novel potential dependencies. The consequences on leukemic cell metabolism suggest pathways to be exploited for combination therapies in genomic-driven approaches.
Supported by: EHA research fellowship award, ELN, AIL, AIRC, project Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project, HARMONY project, Fondazione del Monte BO e RA project.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): Acute Myeloid Leukemia, Genetic, Inhibitor, MYC