EXPRESSION OF HUMAN CRBN SENSITIZES MOUSE MULTIPLE MYELOMA CELLS TO LENALIDOMIDE
(Abstract release date: 05/19/16)
EHA Library. Serdar D. 06/09/16; 132795; E1246
Mrs. Dilan Serdar
Contributions
Contributions
Abstract
Abstract: E1246
Type: Eposter Presentation
Background
The immunomodulatory drugs (IMiDs) lenalidomide, thalidomide, and pomalidomide are highly active in the treatment of multiple myeloma. We and others have shown that IMiDs inhibit growth of multiple myeloma cells by inducing ubiquitination and degradation of the lymphoid transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) by the cereblon (CRBN) E3 ubiquitin ligase. Recently, we identified another lenalidomide-induced substrate of the CRBN E3 ligase, Casein Kinase 1A1 (CK1α), the degradation of which is responsible for lenalidomide’s activity in del(5q) myelodysplastic syndrome. However, in contrast to human cells, mouse cells are resistant to lenalidomide-induced degradation of CK1α due to a single amino acid change in the mouse CRBN (mCRBN) protein at position 391 (valine in human CRBN (hCRBN)), isoleucine in mCRBN) that can be overcome by expression of the human variant.
Aims
To determine whether expression of hCRBN and mCrbnI391V sensitizes mouse multiple myeloma cells to lenalidomide.
Methods
We studied the mouse multiple myeloma cell line MOPC-315.BM that has been previously shown to induce multiple myeloma in BALB/c mice after transplantation. The different CRBN forms were expressed by retroviral vectors.
Results
Western blot analyses showed that MOPC-315.BM multiple myeloma cells had high protein levels of IKZF1 and IKZF3 as compared to non-myeloma mouse cells such as Ba/F3 pro-B cells and myeloblast-like 32D cells. However, lenalidomide treatment of MOPC-315.BM neither induced degradation of IKZF1 and IKZF3 nor had an impact on proliferation. Using retroviral transduction we expressed mCrbn, hCRBN, and mCrbnI391V in MOPC-315.BM cells. After lenalidomide treatment, we observed effective degradation of IKZF1 and IKZF3 proteins by western blot analysis in hCRBN and mCrbnI391V expressing MOPC-315.BM cells but not in those expressing mCrbn or empty vector. Immunoprecipitation analysis revealed that in the presence of lenalidomide IKZF3 bound to hCRBN and mCrbnI391V but not mCRBN, indicating that the isoleucine present in mCRBN at position 391 prevents direct interaction with IKZF3. Next, we examined the effect of expressing different CRBN forms on drug sensitivity of MOPC315.BM cells. As in the parental cells, mCrbn-expressing cells were insensitive to lenalidomide. In contrast, lenalidomide inhibited proliferation of hCRBN and mCrbnI391V expressing cells in a dose-dependent fashion. Similar results were obtained for pomalidomide and thalidomide. Expression of a dominant negative IKZF3 isoform that inactivates IKZF3 and IKZF1 inhibited proliferation of MOPC315.BM cells, demonstrating that these cells depend on both transcription factors as their human counterparts. Expression of hCRBN and mCrbnI391V did not sensitize Ba/F3 or 32D cells to lenalidomide, demonstrating the multiple myeloma-specific effect of IMiDs. In MOPC315.BM cells, expression of hCRBN or mCrbnI391V had no effect on the sensitivity of other potent anti-multiple myeloma drugs including bortezomib and dexamethasone, confirming the specific role of CRBN as a drug target for IMiDs.
Conclusion
These results demonstrate that mouse multiple myeloma cells can be specifically sensitized to IMiDs by expression of hCRBN or mCrbnI391V and provide a basis for further development of in vivo models for IMiDs.
Session topic: E-poster
Keyword(s): Ikaros, Multiple myeloma
Type: Eposter Presentation
Background
The immunomodulatory drugs (IMiDs) lenalidomide, thalidomide, and pomalidomide are highly active in the treatment of multiple myeloma. We and others have shown that IMiDs inhibit growth of multiple myeloma cells by inducing ubiquitination and degradation of the lymphoid transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) by the cereblon (CRBN) E3 ubiquitin ligase. Recently, we identified another lenalidomide-induced substrate of the CRBN E3 ligase, Casein Kinase 1A1 (CK1α), the degradation of which is responsible for lenalidomide’s activity in del(5q) myelodysplastic syndrome. However, in contrast to human cells, mouse cells are resistant to lenalidomide-induced degradation of CK1α due to a single amino acid change in the mouse CRBN (mCRBN) protein at position 391 (valine in human CRBN (hCRBN)), isoleucine in mCRBN) that can be overcome by expression of the human variant.
Aims
To determine whether expression of hCRBN and mCrbnI391V sensitizes mouse multiple myeloma cells to lenalidomide.
Methods
We studied the mouse multiple myeloma cell line MOPC-315.BM that has been previously shown to induce multiple myeloma in BALB/c mice after transplantation. The different CRBN forms were expressed by retroviral vectors.
Results
Western blot analyses showed that MOPC-315.BM multiple myeloma cells had high protein levels of IKZF1 and IKZF3 as compared to non-myeloma mouse cells such as Ba/F3 pro-B cells and myeloblast-like 32D cells. However, lenalidomide treatment of MOPC-315.BM neither induced degradation of IKZF1 and IKZF3 nor had an impact on proliferation. Using retroviral transduction we expressed mCrbn, hCRBN, and mCrbnI391V in MOPC-315.BM cells. After lenalidomide treatment, we observed effective degradation of IKZF1 and IKZF3 proteins by western blot analysis in hCRBN and mCrbnI391V expressing MOPC-315.BM cells but not in those expressing mCrbn or empty vector. Immunoprecipitation analysis revealed that in the presence of lenalidomide IKZF3 bound to hCRBN and mCrbnI391V but not mCRBN, indicating that the isoleucine present in mCRBN at position 391 prevents direct interaction with IKZF3. Next, we examined the effect of expressing different CRBN forms on drug sensitivity of MOPC315.BM cells. As in the parental cells, mCrbn-expressing cells were insensitive to lenalidomide. In contrast, lenalidomide inhibited proliferation of hCRBN and mCrbnI391V expressing cells in a dose-dependent fashion. Similar results were obtained for pomalidomide and thalidomide. Expression of a dominant negative IKZF3 isoform that inactivates IKZF3 and IKZF1 inhibited proliferation of MOPC315.BM cells, demonstrating that these cells depend on both transcription factors as their human counterparts. Expression of hCRBN and mCrbnI391V did not sensitize Ba/F3 or 32D cells to lenalidomide, demonstrating the multiple myeloma-specific effect of IMiDs. In MOPC315.BM cells, expression of hCRBN or mCrbnI391V had no effect on the sensitivity of other potent anti-multiple myeloma drugs including bortezomib and dexamethasone, confirming the specific role of CRBN as a drug target for IMiDs.
Conclusion
These results demonstrate that mouse multiple myeloma cells can be specifically sensitized to IMiDs by expression of hCRBN or mCrbnI391V and provide a basis for further development of in vivo models for IMiDs.
Session topic: E-poster
Keyword(s): Ikaros, Multiple myeloma
Abstract: E1246
Type: Eposter Presentation
Background
The immunomodulatory drugs (IMiDs) lenalidomide, thalidomide, and pomalidomide are highly active in the treatment of multiple myeloma. We and others have shown that IMiDs inhibit growth of multiple myeloma cells by inducing ubiquitination and degradation of the lymphoid transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) by the cereblon (CRBN) E3 ubiquitin ligase. Recently, we identified another lenalidomide-induced substrate of the CRBN E3 ligase, Casein Kinase 1A1 (CK1α), the degradation of which is responsible for lenalidomide’s activity in del(5q) myelodysplastic syndrome. However, in contrast to human cells, mouse cells are resistant to lenalidomide-induced degradation of CK1α due to a single amino acid change in the mouse CRBN (mCRBN) protein at position 391 (valine in human CRBN (hCRBN)), isoleucine in mCRBN) that can be overcome by expression of the human variant.
Aims
To determine whether expression of hCRBN and mCrbnI391V sensitizes mouse multiple myeloma cells to lenalidomide.
Methods
We studied the mouse multiple myeloma cell line MOPC-315.BM that has been previously shown to induce multiple myeloma in BALB/c mice after transplantation. The different CRBN forms were expressed by retroviral vectors.
Results
Western blot analyses showed that MOPC-315.BM multiple myeloma cells had high protein levels of IKZF1 and IKZF3 as compared to non-myeloma mouse cells such as Ba/F3 pro-B cells and myeloblast-like 32D cells. However, lenalidomide treatment of MOPC-315.BM neither induced degradation of IKZF1 and IKZF3 nor had an impact on proliferation. Using retroviral transduction we expressed mCrbn, hCRBN, and mCrbnI391V in MOPC-315.BM cells. After lenalidomide treatment, we observed effective degradation of IKZF1 and IKZF3 proteins by western blot analysis in hCRBN and mCrbnI391V expressing MOPC-315.BM cells but not in those expressing mCrbn or empty vector. Immunoprecipitation analysis revealed that in the presence of lenalidomide IKZF3 bound to hCRBN and mCrbnI391V but not mCRBN, indicating that the isoleucine present in mCRBN at position 391 prevents direct interaction with IKZF3. Next, we examined the effect of expressing different CRBN forms on drug sensitivity of MOPC315.BM cells. As in the parental cells, mCrbn-expressing cells were insensitive to lenalidomide. In contrast, lenalidomide inhibited proliferation of hCRBN and mCrbnI391V expressing cells in a dose-dependent fashion. Similar results were obtained for pomalidomide and thalidomide. Expression of a dominant negative IKZF3 isoform that inactivates IKZF3 and IKZF1 inhibited proliferation of MOPC315.BM cells, demonstrating that these cells depend on both transcription factors as their human counterparts. Expression of hCRBN and mCrbnI391V did not sensitize Ba/F3 or 32D cells to lenalidomide, demonstrating the multiple myeloma-specific effect of IMiDs. In MOPC315.BM cells, expression of hCRBN or mCrbnI391V had no effect on the sensitivity of other potent anti-multiple myeloma drugs including bortezomib and dexamethasone, confirming the specific role of CRBN as a drug target for IMiDs.
Conclusion
These results demonstrate that mouse multiple myeloma cells can be specifically sensitized to IMiDs by expression of hCRBN or mCrbnI391V and provide a basis for further development of in vivo models for IMiDs.
Session topic: E-poster
Keyword(s): Ikaros, Multiple myeloma
Type: Eposter Presentation
Background
The immunomodulatory drugs (IMiDs) lenalidomide, thalidomide, and pomalidomide are highly active in the treatment of multiple myeloma. We and others have shown that IMiDs inhibit growth of multiple myeloma cells by inducing ubiquitination and degradation of the lymphoid transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) by the cereblon (CRBN) E3 ubiquitin ligase. Recently, we identified another lenalidomide-induced substrate of the CRBN E3 ligase, Casein Kinase 1A1 (CK1α), the degradation of which is responsible for lenalidomide’s activity in del(5q) myelodysplastic syndrome. However, in contrast to human cells, mouse cells are resistant to lenalidomide-induced degradation of CK1α due to a single amino acid change in the mouse CRBN (mCRBN) protein at position 391 (valine in human CRBN (hCRBN)), isoleucine in mCRBN) that can be overcome by expression of the human variant.
Aims
To determine whether expression of hCRBN and mCrbnI391V sensitizes mouse multiple myeloma cells to lenalidomide.
Methods
We studied the mouse multiple myeloma cell line MOPC-315.BM that has been previously shown to induce multiple myeloma in BALB/c mice after transplantation. The different CRBN forms were expressed by retroviral vectors.
Results
Western blot analyses showed that MOPC-315.BM multiple myeloma cells had high protein levels of IKZF1 and IKZF3 as compared to non-myeloma mouse cells such as Ba/F3 pro-B cells and myeloblast-like 32D cells. However, lenalidomide treatment of MOPC-315.BM neither induced degradation of IKZF1 and IKZF3 nor had an impact on proliferation. Using retroviral transduction we expressed mCrbn, hCRBN, and mCrbnI391V in MOPC-315.BM cells. After lenalidomide treatment, we observed effective degradation of IKZF1 and IKZF3 proteins by western blot analysis in hCRBN and mCrbnI391V expressing MOPC-315.BM cells but not in those expressing mCrbn or empty vector. Immunoprecipitation analysis revealed that in the presence of lenalidomide IKZF3 bound to hCRBN and mCrbnI391V but not mCRBN, indicating that the isoleucine present in mCRBN at position 391 prevents direct interaction with IKZF3. Next, we examined the effect of expressing different CRBN forms on drug sensitivity of MOPC315.BM cells. As in the parental cells, mCrbn-expressing cells were insensitive to lenalidomide. In contrast, lenalidomide inhibited proliferation of hCRBN and mCrbnI391V expressing cells in a dose-dependent fashion. Similar results were obtained for pomalidomide and thalidomide. Expression of a dominant negative IKZF3 isoform that inactivates IKZF3 and IKZF1 inhibited proliferation of MOPC315.BM cells, demonstrating that these cells depend on both transcription factors as their human counterparts. Expression of hCRBN and mCrbnI391V did not sensitize Ba/F3 or 32D cells to lenalidomide, demonstrating the multiple myeloma-specific effect of IMiDs. In MOPC315.BM cells, expression of hCRBN or mCrbnI391V had no effect on the sensitivity of other potent anti-multiple myeloma drugs including bortezomib and dexamethasone, confirming the specific role of CRBN as a drug target for IMiDs.
Conclusion
These results demonstrate that mouse multiple myeloma cells can be specifically sensitized to IMiDs by expression of hCRBN or mCrbnI391V and provide a basis for further development of in vivo models for IMiDs.
Session topic: E-poster
Keyword(s): Ikaros, Multiple myeloma
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