Contributions
Abstract: PB2065
Type: Publication Only
Background
The deregulated activation of a Ser/Thr kinase 3-phosphoinositide-dependent protein kinase 1 (PDPK1) has been shown to promote the disease progression in various solid cancers. In hematologic malignancies, we have recently identified that the constitutive activation of PDPK1 and its downstream kinase RSK2 (PDPK1/RSK2 signaling axis) plays pivotal roles in multiple myeloma (MM) pathophysiology by promoting myeloma cell survival and proliferation (Chinen Y, Cancer Res 2014; Shimura Y, Mol Cancer Ther 2012). Mantle cell lymphoma (MCL) is cytogenetically and molecularly characterized by chromosomal translocation t(11;14)(q13;q32) for deregulated cyclin D1 (CCND1) overexpression, and has remained as one of hard-to-treat subtypes of non-Hodgkin lymphomas (NHLs).
Aims
The development of novel therapeutics for MCL has been urgently needed, therefore, this study investigated the potency of PDPK1 as a therapeutic target molecule in MCL cells.
Methods
Four MCL-derived cell lines (MINO, Jeko-1, JVM-2 and Z138 cells), three diffuse large B-cell lymphoma (DLBCL)-derived cell lines (KPUM-MS3, KPUM-UH1 and A3/KAW cells) and a Burkitt lymphoma (BL)-derived cell line (Namalwa) were utilized in this study. Patient-derived biopsied specimens were obtained with informed consent and subjected to the immunohistochemical (IHC) staining of phospho (p-) PDPK1Ser241. Cell proliferation was assessed by a modified MTT assay. Antibodies utilized for Western blotting was performed for evaluating protein expression levels of PDPK1, p-PDPK1Ser241, p-RSK2Ser227, and RSK2. BX-912, a specific inhibitor for PDPK1, was purchased from Selleckchem (USA). RNA interference of PDPK1 was performed by transfecting short hairpin RNA plasmids into MCL cell lines by means of nucleofection (Lonza, Switzerland). This study was approved by the institutional review board of our institute.
Results
By means of IHC examination, our study revealed that PDPK1 was activated through phosphorylation in tumor cells of all 7 MCL patient-derived specimens examined, and this was also the case in all 5 DLBCLs examined and in all 5 follicular lymphomas examined. These indicated that PDPK1 is generally active in various types of B-cell lymphoid neoplasms. The in vitro treatment with BX-912 for 48 hours resulted in the dose-dependent inhibition of cell proliferation in all four MCL cell lines (IC50 0.9~2.5 mM), and this inhibitory effect of BX-912 was more profound in MCL cell lines compared with three DLBCL cell lines (IC50 3.7~17.0 mM) and a BL cell line (IC50 2.9 mM). In addition, the flow cytometric analysis revealed that the growth inhibition of MCL cells by PDPK1 blockade with BX-912 was at least partly mediated through the induction of apoptosis. As the molecular sequelae, PDPK1 blockade by BX-912 resulted in dephosphorylation of RSK2-NTKD, while AKT activity or CCND1 expression was unaltered by BX-912 treatment in MCL cells. By gene knockdown of PDPK1 by RNA interference using three different short hairpin RNAs, we further validated that the reduction of PDPK1 protein caused the inactivation of RSK2 and the growth inhibition in MCL cell lines. Finally, when combined with various agents those are utilized for the treatment of MCL, such as doxorubicin, etoposide, fludaraibine, bortezomib, or ABT263, BX-192 showed additive/synergistic growth inhibitory effects in MCL cell lines.
Conclusion
Collectively, our study suggested that PDPK1/RSK2 signaling axis is the potential therapeutic target in MCL.
Session topic: 18. Non-Hodgkin & Hodgkin lymphoma - Biology
Abstract: PB2065
Type: Publication Only
Background
The deregulated activation of a Ser/Thr kinase 3-phosphoinositide-dependent protein kinase 1 (PDPK1) has been shown to promote the disease progression in various solid cancers. In hematologic malignancies, we have recently identified that the constitutive activation of PDPK1 and its downstream kinase RSK2 (PDPK1/RSK2 signaling axis) plays pivotal roles in multiple myeloma (MM) pathophysiology by promoting myeloma cell survival and proliferation (Chinen Y, Cancer Res 2014; Shimura Y, Mol Cancer Ther 2012). Mantle cell lymphoma (MCL) is cytogenetically and molecularly characterized by chromosomal translocation t(11;14)(q13;q32) for deregulated cyclin D1 (CCND1) overexpression, and has remained as one of hard-to-treat subtypes of non-Hodgkin lymphomas (NHLs).
Aims
The development of novel therapeutics for MCL has been urgently needed, therefore, this study investigated the potency of PDPK1 as a therapeutic target molecule in MCL cells.
Methods
Four MCL-derived cell lines (MINO, Jeko-1, JVM-2 and Z138 cells), three diffuse large B-cell lymphoma (DLBCL)-derived cell lines (KPUM-MS3, KPUM-UH1 and A3/KAW cells) and a Burkitt lymphoma (BL)-derived cell line (Namalwa) were utilized in this study. Patient-derived biopsied specimens were obtained with informed consent and subjected to the immunohistochemical (IHC) staining of phospho (p-) PDPK1Ser241. Cell proliferation was assessed by a modified MTT assay. Antibodies utilized for Western blotting was performed for evaluating protein expression levels of PDPK1, p-PDPK1Ser241, p-RSK2Ser227, and RSK2. BX-912, a specific inhibitor for PDPK1, was purchased from Selleckchem (USA). RNA interference of PDPK1 was performed by transfecting short hairpin RNA plasmids into MCL cell lines by means of nucleofection (Lonza, Switzerland). This study was approved by the institutional review board of our institute.
Results
By means of IHC examination, our study revealed that PDPK1 was activated through phosphorylation in tumor cells of all 7 MCL patient-derived specimens examined, and this was also the case in all 5 DLBCLs examined and in all 5 follicular lymphomas examined. These indicated that PDPK1 is generally active in various types of B-cell lymphoid neoplasms. The in vitro treatment with BX-912 for 48 hours resulted in the dose-dependent inhibition of cell proliferation in all four MCL cell lines (IC50 0.9~2.5 mM), and this inhibitory effect of BX-912 was more profound in MCL cell lines compared with three DLBCL cell lines (IC50 3.7~17.0 mM) and a BL cell line (IC50 2.9 mM). In addition, the flow cytometric analysis revealed that the growth inhibition of MCL cells by PDPK1 blockade with BX-912 was at least partly mediated through the induction of apoptosis. As the molecular sequelae, PDPK1 blockade by BX-912 resulted in dephosphorylation of RSK2-NTKD, while AKT activity or CCND1 expression was unaltered by BX-912 treatment in MCL cells. By gene knockdown of PDPK1 by RNA interference using three different short hairpin RNAs, we further validated that the reduction of PDPK1 protein caused the inactivation of RSK2 and the growth inhibition in MCL cell lines. Finally, when combined with various agents those are utilized for the treatment of MCL, such as doxorubicin, etoposide, fludaraibine, bortezomib, or ABT263, BX-192 showed additive/synergistic growth inhibitory effects in MCL cell lines.
Conclusion
Collectively, our study suggested that PDPK1/RSK2 signaling axis is the potential therapeutic target in MCL.
Session topic: 18. Non-Hodgkin & Hodgkin lymphoma - Biology