Contributions
Abstract: PB1662
Type: Publication Only
Background
Halofuginone (HF) is a halogenated derivative of Febrifugine, which is a molecule isolated from the plant Dichroa febrifuga. It has been demonstrated that Halofuginone exhibits anti-fibrotic, anti-cancerogenic, anti-inflammatory and pro-apoptotic effects. Previously, we have reported that treatment with HF has anti-leukemic properties in-vitro and in-vivo in acute promyelocytic leukemia (APL), reducing tumor growth through the induction of apoptosis and by stimulating the synthesis of the TGF-β protein and activating its downstream targets. In addition, HF presented anti-angiogenic effects by modulating the level of pro and anti-angiogenic factors including VEGF. However, it is unknown whether HF has anti-leukemic activity against other subtypes of acute myeloid leukemia (AML) and HF targets were not determined yet.
Aims
Evaluate the anti-leukemic effect of HF on other AML subtypes than APL and investigate its targets using a proteomic approach.
Methods
AML cell lines Kasumi-1, THP-1, MV4-11, U937 and OCI-AML3 were treated in- vitro with HF at concentrations ranging from 25 to 1000 ng/ml. The % of apoptotic cells, the distribution of cells in different cell cycle phases, and the HF IC50 was determined for each cell line. We used the Proteome Profiler TM Array – HumanPhospho-Kinase Array to verify the possible tyrosine kinases and signaling pathways that could be modulated by HF. To analyze the in-vivo effect of HF, we transplanted the cell lines Kasumi-1 and THP-1 into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice, which were then treated by intra-peritoneal injections of HF at a dosage of 150 mg/Kg daily for 14 days. The leukemic infiltration of the peripheral blood was quantified by flow cytometry every 2 weeks (using a anti-human CD45+).
Results
HF IC50 values ranged from 125.58 ng/ml in Kasumi-1 to 786.15 ng/ml in THP-1 cells. Kasumi-1 cells halted in the S phase of the cell cycle when treated with HF, displaying a significant decrease in proliferation, while no effect was observed for THP-1 cells. Corroborating our in-vitro observation indicating resistant of THP-1 cells towards HF, we did not detect significant differences in the overall survival (OS) of NSG mice transplanted with THP-1 cells treated with vehicle or HF (mean OS of 70,5 and 68 days, respectively; p= 0,24). In contrast, the mean OS for NSG mice transplanted with Kasumi-1 cells treated with HF was significantly prolonged compared to the control group (144 versus 94.5 days; p= 0.007). The proteomic analysis identified significant decrease upon treatment with HF of four phosphorylated-proteins in both cell lines: Phospholipase C gamma 1 ( PLCγ1), Proline-rich tyrosine kinase 2 (PYK2), Endothelial nitric oxied syntase (eNos) and Signal transducer and activator of transcription 3 (STAT3 Y705), thus suggesting that these proteins are primary targets of HF. In addition, the protein target of rapamycin (TOR) was down-regulated only in THP-1, while the levels of STAT3 S727 and STAT5a/b were significantly decreased by HF treatment only in Kasumi-1 cells. This comparative analysis suggests that the sensitivity to HF may be dependent on inhibition of STAT3/5 pathway.
Conclusion
In summary, our results suggest that HF may be effective against core binding factor leukemias and, that the methodology based on a Phospho-Kinase Array is useful to identify drug molecular targets.
Session topic: 3. Acute myeloid leukemia - Biology
Keyword(s): proteomics, Acute Myeloid Leukemia
Abstract: PB1662
Type: Publication Only
Background
Halofuginone (HF) is a halogenated derivative of Febrifugine, which is a molecule isolated from the plant Dichroa febrifuga. It has been demonstrated that Halofuginone exhibits anti-fibrotic, anti-cancerogenic, anti-inflammatory and pro-apoptotic effects. Previously, we have reported that treatment with HF has anti-leukemic properties in-vitro and in-vivo in acute promyelocytic leukemia (APL), reducing tumor growth through the induction of apoptosis and by stimulating the synthesis of the TGF-β protein and activating its downstream targets. In addition, HF presented anti-angiogenic effects by modulating the level of pro and anti-angiogenic factors including VEGF. However, it is unknown whether HF has anti-leukemic activity against other subtypes of acute myeloid leukemia (AML) and HF targets were not determined yet.
Aims
Evaluate the anti-leukemic effect of HF on other AML subtypes than APL and investigate its targets using a proteomic approach.
Methods
AML cell lines Kasumi-1, THP-1, MV4-11, U937 and OCI-AML3 were treated in- vitro with HF at concentrations ranging from 25 to 1000 ng/ml. The % of apoptotic cells, the distribution of cells in different cell cycle phases, and the HF IC50 was determined for each cell line. We used the Proteome Profiler TM Array – HumanPhospho-Kinase Array to verify the possible tyrosine kinases and signaling pathways that could be modulated by HF. To analyze the in-vivo effect of HF, we transplanted the cell lines Kasumi-1 and THP-1 into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice, which were then treated by intra-peritoneal injections of HF at a dosage of 150 mg/Kg daily for 14 days. The leukemic infiltration of the peripheral blood was quantified by flow cytometry every 2 weeks (using a anti-human CD45+).
Results
HF IC50 values ranged from 125.58 ng/ml in Kasumi-1 to 786.15 ng/ml in THP-1 cells. Kasumi-1 cells halted in the S phase of the cell cycle when treated with HF, displaying a significant decrease in proliferation, while no effect was observed for THP-1 cells. Corroborating our in-vitro observation indicating resistant of THP-1 cells towards HF, we did not detect significant differences in the overall survival (OS) of NSG mice transplanted with THP-1 cells treated with vehicle or HF (mean OS of 70,5 and 68 days, respectively; p= 0,24). In contrast, the mean OS for NSG mice transplanted with Kasumi-1 cells treated with HF was significantly prolonged compared to the control group (144 versus 94.5 days; p= 0.007). The proteomic analysis identified significant decrease upon treatment with HF of four phosphorylated-proteins in both cell lines: Phospholipase C gamma 1 ( PLCγ1), Proline-rich tyrosine kinase 2 (PYK2), Endothelial nitric oxied syntase (eNos) and Signal transducer and activator of transcription 3 (STAT3 Y705), thus suggesting that these proteins are primary targets of HF. In addition, the protein target of rapamycin (TOR) was down-regulated only in THP-1, while the levels of STAT3 S727 and STAT5a/b were significantly decreased by HF treatment only in Kasumi-1 cells. This comparative analysis suggests that the sensitivity to HF may be dependent on inhibition of STAT3/5 pathway.
Conclusion
In summary, our results suggest that HF may be effective against core binding factor leukemias and, that the methodology based on a Phospho-Kinase Array is useful to identify drug molecular targets.
Session topic: 3. Acute myeloid leukemia - Biology
Keyword(s): proteomics, Acute Myeloid Leukemia