STABLE IN VITRO BORTEZOMIB RESISTANCE OF MYELOMA CELLS IS CHARACTERIZED BY CHROMOSOME 1Q21 COPY NUMBER GAIN
(Abstract release date: 05/19/16)
EHA Library. Papanikolaou X. 06/09/16; 134837; PB1937
Dr. Xenofon Papanikolaou
Contributions
Contributions
Abstract
Abstract: PB1937
Type: Publication Only
Background
Bortezomib (Bz), the first agent of a new class of drugs in Multiple Myeloma (MM) -proteasome inhibitors (PI)-, has shown remarkable activity in MM and forms an important part of modern MM treatment. Nevertheless in the clinical setting refractoriness to the agent eventually develops in most cases with detrimental effects in their subsequent survival. Numerous publications have addressed this issue through in vitro developed models of acquired Bz resistance (BzR), however the results were quite different in each one and no common factor between them could be identified.
Aims
In order to address these issues a native effort was made for the development of an in vitro model of BzR that would resemble the clinical reality in the most accurate way.
Methods
Two MM cell lines were used, one resembling a multisensitive (JJN3) and the other a multiresistant (U266) drug behavior, that were both sensitive to Bz.Instead of a continuous increasing exposure to the agent, an intermittent one was chosen to resemble in a more accurate way the clinical pattern of drug exposure and subsequently the mechanisms of BzR.
Results
As a result of this method a 20 fold increase in the 48h Bortezomib IC50 was noted for both cell lines. The Bz resistant cell lines proved to be stable in the phenotype of resistance. The increase in the IC50 was able to be verified a year after the cell lines were cultured in normal medium. Bz resistant cell lines were cross-resistant with other PI, even with the PI Carfilzomib which belongs to a different drug category. A stable ratio of 3 fold increase in carfilzomib IC50 for every 10 fold increase in Bz IC50 was found in various different levels of BzR.Both basal activity and IC50 for the Bz specific β5-subunit of the proteasome was not statistical different in both Bz resistant and naive cell lines implying that the proteasome was not directly implemented in BzR and no pump flow mechanism was implicated.Interestingly enough the two MM cell lines through the various stages of their acquired BzR followed different phenotypical pathways. JJN3 through their evolution of their BzR and in accordance with various publications developed a more immature phenotype with loss of percentage and intensity of CD138 by flow cytometry, decreasing levels of the plasma cell differentiation factor XBP-1 and decreased immunoglobulin production both in terms of cell line specific immunoglobulin gene probe expression and cytoplasmic immunoglobulin content. On the contrary U266 retained percentage and intensity of CD138, while exhibiting increasing levels of XBP-1 and increased immunoglobulin production both in terms of cell line specific immunoglobulin gene probe expression and cytoplasmic immunoglobulin content. Driven from the stable nature of BzR of the cell lines, we applied metaphase cytogenetics, spectral karyotyping (SKY) and Fluoresence In situ Hybridization (FISH) to detect any structural genetic abnormalities. Both metaphase cytogenetics and SKY did not reveal any differences, while FISH was able to detect an increase in chromosome 1q21 copy number evident in both Bz resistant cell lines all in the form of jumping translocations.
Conclusion
In conclusion this work provides the in vitro verification of the linkage of 1q21 amplification and BzR. The fact that 1q21 amplification has been found to correlate with prognosis and a relapsed/refractory phenotype not only in MM but also in a multitude of solid tumors implies that at least part of BzR is linked with universal drug resistance mechanisms in cancer.
Session topic: E-poster
Keyword(s): Bortezomib, Myeloma, Resistance
Type: Publication Only
Background
Bortezomib (Bz), the first agent of a new class of drugs in Multiple Myeloma (MM) -proteasome inhibitors (PI)-, has shown remarkable activity in MM and forms an important part of modern MM treatment. Nevertheless in the clinical setting refractoriness to the agent eventually develops in most cases with detrimental effects in their subsequent survival. Numerous publications have addressed this issue through in vitro developed models of acquired Bz resistance (BzR), however the results were quite different in each one and no common factor between them could be identified.
Aims
In order to address these issues a native effort was made for the development of an in vitro model of BzR that would resemble the clinical reality in the most accurate way.
Methods
Two MM cell lines were used, one resembling a multisensitive (JJN3) and the other a multiresistant (U266) drug behavior, that were both sensitive to Bz.Instead of a continuous increasing exposure to the agent, an intermittent one was chosen to resemble in a more accurate way the clinical pattern of drug exposure and subsequently the mechanisms of BzR.
Results
As a result of this method a 20 fold increase in the 48h Bortezomib IC50 was noted for both cell lines. The Bz resistant cell lines proved to be stable in the phenotype of resistance. The increase in the IC50 was able to be verified a year after the cell lines were cultured in normal medium. Bz resistant cell lines were cross-resistant with other PI, even with the PI Carfilzomib which belongs to a different drug category. A stable ratio of 3 fold increase in carfilzomib IC50 for every 10 fold increase in Bz IC50 was found in various different levels of BzR.Both basal activity and IC50 for the Bz specific β5-subunit of the proteasome was not statistical different in both Bz resistant and naive cell lines implying that the proteasome was not directly implemented in BzR and no pump flow mechanism was implicated.Interestingly enough the two MM cell lines through the various stages of their acquired BzR followed different phenotypical pathways. JJN3 through their evolution of their BzR and in accordance with various publications developed a more immature phenotype with loss of percentage and intensity of CD138 by flow cytometry, decreasing levels of the plasma cell differentiation factor XBP-1 and decreased immunoglobulin production both in terms of cell line specific immunoglobulin gene probe expression and cytoplasmic immunoglobulin content. On the contrary U266 retained percentage and intensity of CD138, while exhibiting increasing levels of XBP-1 and increased immunoglobulin production both in terms of cell line specific immunoglobulin gene probe expression and cytoplasmic immunoglobulin content. Driven from the stable nature of BzR of the cell lines, we applied metaphase cytogenetics, spectral karyotyping (SKY) and Fluoresence In situ Hybridization (FISH) to detect any structural genetic abnormalities. Both metaphase cytogenetics and SKY did not reveal any differences, while FISH was able to detect an increase in chromosome 1q21 copy number evident in both Bz resistant cell lines all in the form of jumping translocations.
Conclusion
In conclusion this work provides the in vitro verification of the linkage of 1q21 amplification and BzR. The fact that 1q21 amplification has been found to correlate with prognosis and a relapsed/refractory phenotype not only in MM but also in a multitude of solid tumors implies that at least part of BzR is linked with universal drug resistance mechanisms in cancer.
Session topic: E-poster
Keyword(s): Bortezomib, Myeloma, Resistance
Abstract: PB1937
Type: Publication Only
Background
Bortezomib (Bz), the first agent of a new class of drugs in Multiple Myeloma (MM) -proteasome inhibitors (PI)-, has shown remarkable activity in MM and forms an important part of modern MM treatment. Nevertheless in the clinical setting refractoriness to the agent eventually develops in most cases with detrimental effects in their subsequent survival. Numerous publications have addressed this issue through in vitro developed models of acquired Bz resistance (BzR), however the results were quite different in each one and no common factor between them could be identified.
Aims
In order to address these issues a native effort was made for the development of an in vitro model of BzR that would resemble the clinical reality in the most accurate way.
Methods
Two MM cell lines were used, one resembling a multisensitive (JJN3) and the other a multiresistant (U266) drug behavior, that were both sensitive to Bz.Instead of a continuous increasing exposure to the agent, an intermittent one was chosen to resemble in a more accurate way the clinical pattern of drug exposure and subsequently the mechanisms of BzR.
Results
As a result of this method a 20 fold increase in the 48h Bortezomib IC50 was noted for both cell lines. The Bz resistant cell lines proved to be stable in the phenotype of resistance. The increase in the IC50 was able to be verified a year after the cell lines were cultured in normal medium. Bz resistant cell lines were cross-resistant with other PI, even with the PI Carfilzomib which belongs to a different drug category. A stable ratio of 3 fold increase in carfilzomib IC50 for every 10 fold increase in Bz IC50 was found in various different levels of BzR.Both basal activity and IC50 for the Bz specific β5-subunit of the proteasome was not statistical different in both Bz resistant and naive cell lines implying that the proteasome was not directly implemented in BzR and no pump flow mechanism was implicated.Interestingly enough the two MM cell lines through the various stages of their acquired BzR followed different phenotypical pathways. JJN3 through their evolution of their BzR and in accordance with various publications developed a more immature phenotype with loss of percentage and intensity of CD138 by flow cytometry, decreasing levels of the plasma cell differentiation factor XBP-1 and decreased immunoglobulin production both in terms of cell line specific immunoglobulin gene probe expression and cytoplasmic immunoglobulin content. On the contrary U266 retained percentage and intensity of CD138, while exhibiting increasing levels of XBP-1 and increased immunoglobulin production both in terms of cell line specific immunoglobulin gene probe expression and cytoplasmic immunoglobulin content. Driven from the stable nature of BzR of the cell lines, we applied metaphase cytogenetics, spectral karyotyping (SKY) and Fluoresence In situ Hybridization (FISH) to detect any structural genetic abnormalities. Both metaphase cytogenetics and SKY did not reveal any differences, while FISH was able to detect an increase in chromosome 1q21 copy number evident in both Bz resistant cell lines all in the form of jumping translocations.
Conclusion
In conclusion this work provides the in vitro verification of the linkage of 1q21 amplification and BzR. The fact that 1q21 amplification has been found to correlate with prognosis and a relapsed/refractory phenotype not only in MM but also in a multitude of solid tumors implies that at least part of BzR is linked with universal drug resistance mechanisms in cancer.
Session topic: E-poster
Keyword(s): Bortezomib, Myeloma, Resistance
Type: Publication Only
Background
Bortezomib (Bz), the first agent of a new class of drugs in Multiple Myeloma (MM) -proteasome inhibitors (PI)-, has shown remarkable activity in MM and forms an important part of modern MM treatment. Nevertheless in the clinical setting refractoriness to the agent eventually develops in most cases with detrimental effects in their subsequent survival. Numerous publications have addressed this issue through in vitro developed models of acquired Bz resistance (BzR), however the results were quite different in each one and no common factor between them could be identified.
Aims
In order to address these issues a native effort was made for the development of an in vitro model of BzR that would resemble the clinical reality in the most accurate way.
Methods
Two MM cell lines were used, one resembling a multisensitive (JJN3) and the other a multiresistant (U266) drug behavior, that were both sensitive to Bz.Instead of a continuous increasing exposure to the agent, an intermittent one was chosen to resemble in a more accurate way the clinical pattern of drug exposure and subsequently the mechanisms of BzR.
Results
As a result of this method a 20 fold increase in the 48h Bortezomib IC50 was noted for both cell lines. The Bz resistant cell lines proved to be stable in the phenotype of resistance. The increase in the IC50 was able to be verified a year after the cell lines were cultured in normal medium. Bz resistant cell lines were cross-resistant with other PI, even with the PI Carfilzomib which belongs to a different drug category. A stable ratio of 3 fold increase in carfilzomib IC50 for every 10 fold increase in Bz IC50 was found in various different levels of BzR.Both basal activity and IC50 for the Bz specific β5-subunit of the proteasome was not statistical different in both Bz resistant and naive cell lines implying that the proteasome was not directly implemented in BzR and no pump flow mechanism was implicated.Interestingly enough the two MM cell lines through the various stages of their acquired BzR followed different phenotypical pathways. JJN3 through their evolution of their BzR and in accordance with various publications developed a more immature phenotype with loss of percentage and intensity of CD138 by flow cytometry, decreasing levels of the plasma cell differentiation factor XBP-1 and decreased immunoglobulin production both in terms of cell line specific immunoglobulin gene probe expression and cytoplasmic immunoglobulin content. On the contrary U266 retained percentage and intensity of CD138, while exhibiting increasing levels of XBP-1 and increased immunoglobulin production both in terms of cell line specific immunoglobulin gene probe expression and cytoplasmic immunoglobulin content. Driven from the stable nature of BzR of the cell lines, we applied metaphase cytogenetics, spectral karyotyping (SKY) and Fluoresence In situ Hybridization (FISH) to detect any structural genetic abnormalities. Both metaphase cytogenetics and SKY did not reveal any differences, while FISH was able to detect an increase in chromosome 1q21 copy number evident in both Bz resistant cell lines all in the form of jumping translocations.
Conclusion
In conclusion this work provides the in vitro verification of the linkage of 1q21 amplification and BzR. The fact that 1q21 amplification has been found to correlate with prognosis and a relapsed/refractory phenotype not only in MM but also in a multitude of solid tumors implies that at least part of BzR is linked with universal drug resistance mechanisms in cancer.
Session topic: E-poster
Keyword(s): Bortezomib, Myeloma, Resistance
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