MODELLING PONATINIB RESISTANCE IN BCR-ABL1+ CELL LINES: IMPLICATIONS FOR PONATINIB THERAPY
(Abstract release date: 05/19/16)
EHA Library. Poluskina L. 06/11/16; 135251; S495
Mrs. Liubov Poluskina
Contributions
Contributions
Abstract
Abstract: S495
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 17:00 - 17:15
Location: Hall C11
Background
Ponatinib overcomes TKI resistance (imatinib, nilotinib and dasatinib) that develops due to BCR-ABL1 kinase domain (KD) mutations including T315I. Besides KD mutations, there are several identified TKI resistance mechanisms, such as BCR-ABL1 overexpression, regulation of the expression of TKI transporters, and Bcr-Abl independent Axl kinase overexpression. However, little is known about the mechanisms that may cause ponatinib resistance. Here, modes of ponatinib resistance have been investigated in TKI naïve and pre-treated BCR-ABL1+ cell lines.
Aims
To characterise ponatinib resistance mechanisms in BCR-ABL1+ cell line models.
Methods
Ponatinib resistance was generated by using four BCR-ABL1+ cell-lines: 1) K562, 2) K562-DOX (ABCB1 overexpressing variant) 3) K562 DOX-55D (resistant to 55 nM dasatinib) and 4) K562 T315I (resistant to 200 nM dasatinib, harbouring the T315I mutation). The average steady state of ponatinib plasma concentration in patients is 101 nM for dosage at 45 mg/day. Hence, the resistant cells were exposed to increasing concentration of ponatinib to 100 nM (if higher concentration could not be achieved) or 200 nM, as these concentrations are close to clinically relevant. Parental ponatinib naïve controls were maintained in parallel. R indicates ponatinib resistant variants.
Results
Four resistant cell lines were established. All of the four cell lines were cross-resistant to other TKIs (2000 nM imatinib, 1000 nM nilotinib and 200 nM dasatinib). The two dasatinib pre-treated resistant cell lines developed BCR-ABL1 KD mutation(s). The level of T315I in the K562 T315I-R line (survived in 100 nM ponatinib) increased from 44% to 66%, and the expression level of BCR-ABL1 increased 6-fold compared to the naïve line (p<0.05). The K562 DOX 55D-R (survived in 200 nM ponatinib) developed compound mutations G250E/E255K (54%).However, KD mutations were not observed in the cell lines with no prior exposure to TKI. The ponatinib resistance in K562-R and K562 DOX-R cell lines (both survived in 200 nM ponatinib) were Bcr-Abl independent: active Bcr-Abl and CrkL (surrogate marker of Bcr-Abl activity) levels were reduced, while Axl protein and AXL mRNA expression levels were increased significantly (all p<0.05). After incubation with Axl inhibitors, 1 μM R428 or 12.5 μM BMS-777607, the K562-R and K562 DOX-R cell lines were re-sensitised (approximately 50% reduction in viability) to 200 nM ponatinib while the controls remained resistant to the drug (both p<0.05). Next, retroviral vector was used to reduce AXL gene expression in the K562-R and K562 DOX-R cell lines. This AXL gene reduction in the two cell lines resulted in re-sensitisation to 10 nM ponatinib (Figure 1A-B), while controls remained resistant to 200 nM ponatinib. These results suggested that Axl overexpression is critical for Bcr-Abl independent ponatinib resistance.
Conclusion
We show that in the setting of prior-TKI exposure, BCR-ABL1 KD mutations are likely to cause ponatinib resistance. In the TKI naïve setting, Bcr-Abl independent modes of resistance may be more likely, suggesting combination therapeutic approaches may be required in this setting. In addition, ponatinib resistance are unlikely to be overcome by the use of the first or second generation TKIs.
Session topic: Chronic myeloid leukemia - Biology
Keyword(s): Chronic myeloid leukemia, Kinase domain mutant, Resistance, Tyrosine kinase inhibitor
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 17:00 - 17:15
Location: Hall C11
Background
Ponatinib overcomes TKI resistance (imatinib, nilotinib and dasatinib) that develops due to BCR-ABL1 kinase domain (KD) mutations including T315I. Besides KD mutations, there are several identified TKI resistance mechanisms, such as BCR-ABL1 overexpression, regulation of the expression of TKI transporters, and Bcr-Abl independent Axl kinase overexpression. However, little is known about the mechanisms that may cause ponatinib resistance. Here, modes of ponatinib resistance have been investigated in TKI naïve and pre-treated BCR-ABL1+ cell lines.
Aims
To characterise ponatinib resistance mechanisms in BCR-ABL1+ cell line models.
Methods
Ponatinib resistance was generated by using four BCR-ABL1+ cell-lines: 1) K562, 2) K562-DOX (ABCB1 overexpressing variant) 3) K562 DOX-55D (resistant to 55 nM dasatinib) and 4) K562 T315I (resistant to 200 nM dasatinib, harbouring the T315I mutation). The average steady state of ponatinib plasma concentration in patients is 101 nM for dosage at 45 mg/day. Hence, the resistant cells were exposed to increasing concentration of ponatinib to 100 nM (if higher concentration could not be achieved) or 200 nM, as these concentrations are close to clinically relevant. Parental ponatinib naïve controls were maintained in parallel. R indicates ponatinib resistant variants.
Results
Four resistant cell lines were established. All of the four cell lines were cross-resistant to other TKIs (2000 nM imatinib, 1000 nM nilotinib and 200 nM dasatinib). The two dasatinib pre-treated resistant cell lines developed BCR-ABL1 KD mutation(s). The level of T315I in the K562 T315I-R line (survived in 100 nM ponatinib) increased from 44% to 66%, and the expression level of BCR-ABL1 increased 6-fold compared to the naïve line (p<0.05). The K562 DOX 55D-R (survived in 200 nM ponatinib) developed compound mutations G250E/E255K (54%).However, KD mutations were not observed in the cell lines with no prior exposure to TKI. The ponatinib resistance in K562-R and K562 DOX-R cell lines (both survived in 200 nM ponatinib) were Bcr-Abl independent: active Bcr-Abl and CrkL (surrogate marker of Bcr-Abl activity) levels were reduced, while Axl protein and AXL mRNA expression levels were increased significantly (all p<0.05). After incubation with Axl inhibitors, 1 μM R428 or 12.5 μM BMS-777607, the K562-R and K562 DOX-R cell lines were re-sensitised (approximately 50% reduction in viability) to 200 nM ponatinib while the controls remained resistant to the drug (both p<0.05). Next, retroviral vector was used to reduce AXL gene expression in the K562-R and K562 DOX-R cell lines. This AXL gene reduction in the two cell lines resulted in re-sensitisation to 10 nM ponatinib (Figure 1A-B), while controls remained resistant to 200 nM ponatinib. These results suggested that Axl overexpression is critical for Bcr-Abl independent ponatinib resistance.
Conclusion
We show that in the setting of prior-TKI exposure, BCR-ABL1 KD mutations are likely to cause ponatinib resistance. In the TKI naïve setting, Bcr-Abl independent modes of resistance may be more likely, suggesting combination therapeutic approaches may be required in this setting. In addition, ponatinib resistance are unlikely to be overcome by the use of the first or second generation TKIs.
Session topic: Chronic myeloid leukemia - Biology
Keyword(s): Chronic myeloid leukemia, Kinase domain mutant, Resistance, Tyrosine kinase inhibitor
Abstract: S495
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 17:00 - 17:15
Location: Hall C11
Background
Ponatinib overcomes TKI resistance (imatinib, nilotinib and dasatinib) that develops due to BCR-ABL1 kinase domain (KD) mutations including T315I. Besides KD mutations, there are several identified TKI resistance mechanisms, such as BCR-ABL1 overexpression, regulation of the expression of TKI transporters, and Bcr-Abl independent Axl kinase overexpression. However, little is known about the mechanisms that may cause ponatinib resistance. Here, modes of ponatinib resistance have been investigated in TKI naïve and pre-treated BCR-ABL1+ cell lines.
Aims
To characterise ponatinib resistance mechanisms in BCR-ABL1+ cell line models.
Methods
Ponatinib resistance was generated by using four BCR-ABL1+ cell-lines: 1) K562, 2) K562-DOX (ABCB1 overexpressing variant) 3) K562 DOX-55D (resistant to 55 nM dasatinib) and 4) K562 T315I (resistant to 200 nM dasatinib, harbouring the T315I mutation). The average steady state of ponatinib plasma concentration in patients is 101 nM for dosage at 45 mg/day. Hence, the resistant cells were exposed to increasing concentration of ponatinib to 100 nM (if higher concentration could not be achieved) or 200 nM, as these concentrations are close to clinically relevant. Parental ponatinib naïve controls were maintained in parallel. R indicates ponatinib resistant variants.
Results
Four resistant cell lines were established. All of the four cell lines were cross-resistant to other TKIs (2000 nM imatinib, 1000 nM nilotinib and 200 nM dasatinib). The two dasatinib pre-treated resistant cell lines developed BCR-ABL1 KD mutation(s). The level of T315I in the K562 T315I-R line (survived in 100 nM ponatinib) increased from 44% to 66%, and the expression level of BCR-ABL1 increased 6-fold compared to the naïve line (p<0.05). The K562 DOX 55D-R (survived in 200 nM ponatinib) developed compound mutations G250E/E255K (54%).However, KD mutations were not observed in the cell lines with no prior exposure to TKI. The ponatinib resistance in K562-R and K562 DOX-R cell lines (both survived in 200 nM ponatinib) were Bcr-Abl independent: active Bcr-Abl and CrkL (surrogate marker of Bcr-Abl activity) levels were reduced, while Axl protein and AXL mRNA expression levels were increased significantly (all p<0.05). After incubation with Axl inhibitors, 1 μM R428 or 12.5 μM BMS-777607, the K562-R and K562 DOX-R cell lines were re-sensitised (approximately 50% reduction in viability) to 200 nM ponatinib while the controls remained resistant to the drug (both p<0.05). Next, retroviral vector was used to reduce AXL gene expression in the K562-R and K562 DOX-R cell lines. This AXL gene reduction in the two cell lines resulted in re-sensitisation to 10 nM ponatinib (Figure 1A-B), while controls remained resistant to 200 nM ponatinib. These results suggested that Axl overexpression is critical for Bcr-Abl independent ponatinib resistance.
Conclusion
We show that in the setting of prior-TKI exposure, BCR-ABL1 KD mutations are likely to cause ponatinib resistance. In the TKI naïve setting, Bcr-Abl independent modes of resistance may be more likely, suggesting combination therapeutic approaches may be required in this setting. In addition, ponatinib resistance are unlikely to be overcome by the use of the first or second generation TKIs.
Session topic: Chronic myeloid leukemia - Biology
Keyword(s): Chronic myeloid leukemia, Kinase domain mutant, Resistance, Tyrosine kinase inhibitor
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 17:00 - 17:15
Location: Hall C11
Background
Ponatinib overcomes TKI resistance (imatinib, nilotinib and dasatinib) that develops due to BCR-ABL1 kinase domain (KD) mutations including T315I. Besides KD mutations, there are several identified TKI resistance mechanisms, such as BCR-ABL1 overexpression, regulation of the expression of TKI transporters, and Bcr-Abl independent Axl kinase overexpression. However, little is known about the mechanisms that may cause ponatinib resistance. Here, modes of ponatinib resistance have been investigated in TKI naïve and pre-treated BCR-ABL1+ cell lines.
Aims
To characterise ponatinib resistance mechanisms in BCR-ABL1+ cell line models.
Methods
Ponatinib resistance was generated by using four BCR-ABL1+ cell-lines: 1) K562, 2) K562-DOX (ABCB1 overexpressing variant) 3) K562 DOX-55D (resistant to 55 nM dasatinib) and 4) K562 T315I (resistant to 200 nM dasatinib, harbouring the T315I mutation). The average steady state of ponatinib plasma concentration in patients is 101 nM for dosage at 45 mg/day. Hence, the resistant cells were exposed to increasing concentration of ponatinib to 100 nM (if higher concentration could not be achieved) or 200 nM, as these concentrations are close to clinically relevant. Parental ponatinib naïve controls were maintained in parallel. R indicates ponatinib resistant variants.
Results
Four resistant cell lines were established. All of the four cell lines were cross-resistant to other TKIs (2000 nM imatinib, 1000 nM nilotinib and 200 nM dasatinib). The two dasatinib pre-treated resistant cell lines developed BCR-ABL1 KD mutation(s). The level of T315I in the K562 T315I-R line (survived in 100 nM ponatinib) increased from 44% to 66%, and the expression level of BCR-ABL1 increased 6-fold compared to the naïve line (p<0.05). The K562 DOX 55D-R (survived in 200 nM ponatinib) developed compound mutations G250E/E255K (54%).However, KD mutations were not observed in the cell lines with no prior exposure to TKI. The ponatinib resistance in K562-R and K562 DOX-R cell lines (both survived in 200 nM ponatinib) were Bcr-Abl independent: active Bcr-Abl and CrkL (surrogate marker of Bcr-Abl activity) levels were reduced, while Axl protein and AXL mRNA expression levels were increased significantly (all p<0.05). After incubation with Axl inhibitors, 1 μM R428 or 12.5 μM BMS-777607, the K562-R and K562 DOX-R cell lines were re-sensitised (approximately 50% reduction in viability) to 200 nM ponatinib while the controls remained resistant to the drug (both p<0.05). Next, retroviral vector was used to reduce AXL gene expression in the K562-R and K562 DOX-R cell lines. This AXL gene reduction in the two cell lines resulted in re-sensitisation to 10 nM ponatinib (Figure 1A-B), while controls remained resistant to 200 nM ponatinib. These results suggested that Axl overexpression is critical for Bcr-Abl independent ponatinib resistance.
Conclusion
We show that in the setting of prior-TKI exposure, BCR-ABL1 KD mutations are likely to cause ponatinib resistance. In the TKI naïve setting, Bcr-Abl independent modes of resistance may be more likely, suggesting combination therapeutic approaches may be required in this setting. In addition, ponatinib resistance are unlikely to be overcome by the use of the first or second generation TKIs.
Session topic: Chronic myeloid leukemia - Biology
Keyword(s): Chronic myeloid leukemia, Kinase domain mutant, Resistance, Tyrosine kinase inhibitor
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