PRIMARY RESISTANCE TO AZACYTIDINE IN MDS: PRELIMINARY DATA ON THE POTENTIAL ROLE OF HIF-1A EXPRESSION
(Abstract release date: 05/19/16)
EHA Library. Pappa V. 06/09/16; 134807; PB1907
Dr. Vassiliki Pappa
Contributions
Contributions
Abstract
Abstract: PB1907
Type: Publication Only
Background
The hypomethylating agent azacytidine is the standard of care for patients with high-risk myelodysplastic syndromes (MDS) (IPSS intermediate-2 or high) and patients with AML with 20-30% bone marrow blasts. Nevertheless, approximately 40% of patients fail to respond, whereas even responders will inevitably relapse. Currently, the exact mechanisms of azacytidine failure are largely unknown and there is no approach to circumvent azacytidine resistance. Hematopoietic and leukemia stem cells (HSCs and LSCs, respectively) reside in a particularly hypoxic niche and several reports have demonstrated the significance of hypoxia in the regulation of both physiological and malignant hematopoiesis. In addition, Hif-1α regulates the expression of human equilibrative nucleoside transporters (ENTs) and ribonucleotide reductase (RR), both of which are involved in the transport and intracellular metabolism of hypomethylating agents.
Aims
The scope of this study is to investigate the potential role of Hif-1α in azacytidine resistance in MDS patients.
Methods
Bone marrow samples from 26 de novo MDS patients and 2 CMML patients (21 males and 7 females) with a median age of 75 (60-89) and 10 healthy donors were collected. MDS patients were classified according to WHO as RCMD (1/28), RAEB I (6/28) and RAEB II (19/28) and received treatment with azacytidine at the dose of 75mg/m2 x7 days SC. Before treatment, bone marrow mononuclear cells were isolated using the Ficol-paque method, followed by RNA extraction using TRIzol reagent and cDNA preparation using Superscript II reverse transcriptase. Hif-1α expression was estimated by real time PCR TaqMan gene expression assay, using the appropriate primers and probes. Relative gene expression was calculated by comparative threshold cycle (ΔΔCt) method. β-actin was used as a housekeeping gene.
Results
Out of the 28 patients used in our study, 17 responded to azacytidine-treatment (including CR, PR and HI) while 11 failed to respond. We found by real time PCR that the ΔΔCt ratio of Hif-1α/β-Actin expression for control samples was 0,72±0,13, for MDS patients who responded to azacytidine- treatment was 1,564±0,439, while for non-responders 0,839±0,17.
Conclusion
Our data suggest that increased pretreatment expression of Hif-1α might be associated with better response to azacytidine, indicating a potential role of hypoxia signaling in azacytidine resistance. These encouraging initial results need to be further confirmed in a larger number of patients. The identification of hypoxia related mechanisms in azacytidine resistance will provide novel insights regarding the links of hypoxia signaling with the epigenetic derangement in the pathobiology of MDS and can serve as a platform for the therapeutic targeting of the Hif-1α pathway.
Session topic: E-poster
Keyword(s): MDS
Type: Publication Only
Background
The hypomethylating agent azacytidine is the standard of care for patients with high-risk myelodysplastic syndromes (MDS) (IPSS intermediate-2 or high) and patients with AML with 20-30% bone marrow blasts. Nevertheless, approximately 40% of patients fail to respond, whereas even responders will inevitably relapse. Currently, the exact mechanisms of azacytidine failure are largely unknown and there is no approach to circumvent azacytidine resistance. Hematopoietic and leukemia stem cells (HSCs and LSCs, respectively) reside in a particularly hypoxic niche and several reports have demonstrated the significance of hypoxia in the regulation of both physiological and malignant hematopoiesis. In addition, Hif-1α regulates the expression of human equilibrative nucleoside transporters (ENTs) and ribonucleotide reductase (RR), both of which are involved in the transport and intracellular metabolism of hypomethylating agents.
Aims
The scope of this study is to investigate the potential role of Hif-1α in azacytidine resistance in MDS patients.
Methods
Bone marrow samples from 26 de novo MDS patients and 2 CMML patients (21 males and 7 females) with a median age of 75 (60-89) and 10 healthy donors were collected. MDS patients were classified according to WHO as RCMD (1/28), RAEB I (6/28) and RAEB II (19/28) and received treatment with azacytidine at the dose of 75mg/m2 x7 days SC. Before treatment, bone marrow mononuclear cells were isolated using the Ficol-paque method, followed by RNA extraction using TRIzol reagent and cDNA preparation using Superscript II reverse transcriptase. Hif-1α expression was estimated by real time PCR TaqMan gene expression assay, using the appropriate primers and probes. Relative gene expression was calculated by comparative threshold cycle (ΔΔCt) method. β-actin was used as a housekeeping gene.
Results
Out of the 28 patients used in our study, 17 responded to azacytidine-treatment (including CR, PR and HI) while 11 failed to respond. We found by real time PCR that the ΔΔCt ratio of Hif-1α/β-Actin expression for control samples was 0,72±0,13, for MDS patients who responded to azacytidine- treatment was 1,564±0,439, while for non-responders 0,839±0,17.
Conclusion
Our data suggest that increased pretreatment expression of Hif-1α might be associated with better response to azacytidine, indicating a potential role of hypoxia signaling in azacytidine resistance. These encouraging initial results need to be further confirmed in a larger number of patients. The identification of hypoxia related mechanisms in azacytidine resistance will provide novel insights regarding the links of hypoxia signaling with the epigenetic derangement in the pathobiology of MDS and can serve as a platform for the therapeutic targeting of the Hif-1α pathway.
Session topic: E-poster
Keyword(s): MDS
Abstract: PB1907
Type: Publication Only
Background
The hypomethylating agent azacytidine is the standard of care for patients with high-risk myelodysplastic syndromes (MDS) (IPSS intermediate-2 or high) and patients with AML with 20-30% bone marrow blasts. Nevertheless, approximately 40% of patients fail to respond, whereas even responders will inevitably relapse. Currently, the exact mechanisms of azacytidine failure are largely unknown and there is no approach to circumvent azacytidine resistance. Hematopoietic and leukemia stem cells (HSCs and LSCs, respectively) reside in a particularly hypoxic niche and several reports have demonstrated the significance of hypoxia in the regulation of both physiological and malignant hematopoiesis. In addition, Hif-1α regulates the expression of human equilibrative nucleoside transporters (ENTs) and ribonucleotide reductase (RR), both of which are involved in the transport and intracellular metabolism of hypomethylating agents.
Aims
The scope of this study is to investigate the potential role of Hif-1α in azacytidine resistance in MDS patients.
Methods
Bone marrow samples from 26 de novo MDS patients and 2 CMML patients (21 males and 7 females) with a median age of 75 (60-89) and 10 healthy donors were collected. MDS patients were classified according to WHO as RCMD (1/28), RAEB I (6/28) and RAEB II (19/28) and received treatment with azacytidine at the dose of 75mg/m2 x7 days SC. Before treatment, bone marrow mononuclear cells were isolated using the Ficol-paque method, followed by RNA extraction using TRIzol reagent and cDNA preparation using Superscript II reverse transcriptase. Hif-1α expression was estimated by real time PCR TaqMan gene expression assay, using the appropriate primers and probes. Relative gene expression was calculated by comparative threshold cycle (ΔΔCt) method. β-actin was used as a housekeeping gene.
Results
Out of the 28 patients used in our study, 17 responded to azacytidine-treatment (including CR, PR and HI) while 11 failed to respond. We found by real time PCR that the ΔΔCt ratio of Hif-1α/β-Actin expression for control samples was 0,72±0,13, for MDS patients who responded to azacytidine- treatment was 1,564±0,439, while for non-responders 0,839±0,17.
Conclusion
Our data suggest that increased pretreatment expression of Hif-1α might be associated with better response to azacytidine, indicating a potential role of hypoxia signaling in azacytidine resistance. These encouraging initial results need to be further confirmed in a larger number of patients. The identification of hypoxia related mechanisms in azacytidine resistance will provide novel insights regarding the links of hypoxia signaling with the epigenetic derangement in the pathobiology of MDS and can serve as a platform for the therapeutic targeting of the Hif-1α pathway.
Session topic: E-poster
Keyword(s): MDS
Type: Publication Only
Background
The hypomethylating agent azacytidine is the standard of care for patients with high-risk myelodysplastic syndromes (MDS) (IPSS intermediate-2 or high) and patients with AML with 20-30% bone marrow blasts. Nevertheless, approximately 40% of patients fail to respond, whereas even responders will inevitably relapse. Currently, the exact mechanisms of azacytidine failure are largely unknown and there is no approach to circumvent azacytidine resistance. Hematopoietic and leukemia stem cells (HSCs and LSCs, respectively) reside in a particularly hypoxic niche and several reports have demonstrated the significance of hypoxia in the regulation of both physiological and malignant hematopoiesis. In addition, Hif-1α regulates the expression of human equilibrative nucleoside transporters (ENTs) and ribonucleotide reductase (RR), both of which are involved in the transport and intracellular metabolism of hypomethylating agents.
Aims
The scope of this study is to investigate the potential role of Hif-1α in azacytidine resistance in MDS patients.
Methods
Bone marrow samples from 26 de novo MDS patients and 2 CMML patients (21 males and 7 females) with a median age of 75 (60-89) and 10 healthy donors were collected. MDS patients were classified according to WHO as RCMD (1/28), RAEB I (6/28) and RAEB II (19/28) and received treatment with azacytidine at the dose of 75mg/m2 x7 days SC. Before treatment, bone marrow mononuclear cells were isolated using the Ficol-paque method, followed by RNA extraction using TRIzol reagent and cDNA preparation using Superscript II reverse transcriptase. Hif-1α expression was estimated by real time PCR TaqMan gene expression assay, using the appropriate primers and probes. Relative gene expression was calculated by comparative threshold cycle (ΔΔCt) method. β-actin was used as a housekeeping gene.
Results
Out of the 28 patients used in our study, 17 responded to azacytidine-treatment (including CR, PR and HI) while 11 failed to respond. We found by real time PCR that the ΔΔCt ratio of Hif-1α/β-Actin expression for control samples was 0,72±0,13, for MDS patients who responded to azacytidine- treatment was 1,564±0,439, while for non-responders 0,839±0,17.
Conclusion
Our data suggest that increased pretreatment expression of Hif-1α might be associated with better response to azacytidine, indicating a potential role of hypoxia signaling in azacytidine resistance. These encouraging initial results need to be further confirmed in a larger number of patients. The identification of hypoxia related mechanisms in azacytidine resistance will provide novel insights regarding the links of hypoxia signaling with the epigenetic derangement in the pathobiology of MDS and can serve as a platform for the therapeutic targeting of the Hif-1α pathway.
Session topic: E-poster
Keyword(s): MDS
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