RALA AND RALB MEDIATE CELL SURVIVAL INDEPENDENTLY OF ONCOGENIC RAS AND PROVIDE POTENTIAL THERAPEUTIC TARGETS IN MULTIPLE MYELOMA
(Abstract release date: 05/18/17)
EHA Library. Seibold M. 05/18/17; 182648; PB1934
Marcel Seibold
Contributions
Contributions
Abstract
Abstract: PB1934
Type: Publication Only
Background
Genetic mutations and the bone marrow microenvironment contribute to disease progression, aggressive phenotype, and shorter survival in multiple myeloma (MM). Oncogenic RAS is one of the most common mutations in MM. Pathway activation through oncogenic RAS is associated with promotion of disease progression and shorter survival. Cell survival and proliferation in MM are mainly mediated via classical signaling pathways such as MEK/ERK and PI3K/Akt. Since there is a lack of specific RAS-inhibitors for clinical use, it is important to identify and analyze associated pathways, which may provide useful alternative targets for MM therapy. The small GTPase Ral has previously been implicated in putative downstream signaling of RAS, and may therefore promote proliferation, survival and drug resistance of MM cells.
Aims
We used shRNA-mediated knockdown of RalA and RalB isoforms to appraise their role as potential therapeutic targets and to analyze their connection to important signaling pathways, which regulate MM cell survival and proliferation. Because oncogenic RAS is a potential activator of the Ral pathways, we investigated a possible link between oncogenic RAS and Ral.
Methods
Immunohistochemical stainings of bone marrow trephines of MM patients and Western analysis of primary MM cells and MM cell lines were performed to evaluate Ral protein expression. Transient or stable knockdown of RalA or RalB was achieved by electroporation of MM cell lines and the effect on cell survival and apoptosis was measured with flow cytometry using annexin V/propidium iodide staining. Ral pulldown assays were applied to test potential dependence of Ral activation on oncogenic RAS. Furthermore, RNA sequencing was performed to compare RAS and Ral gene expression signatures after respective knockdowns.
Results
Both Ral isoforms were expressed in primary MM cells and MM cell lines, with RalA showing the most prominent and consistent protein expression levels. ShRNA-mediated knockdown of RalA strongly induced apoptosis in two thirds of the tested cell lines, whereas RalB depletion did impair MM cell survival in less than half of the cell lines. Western analysis revealed no alteration of classical MEK/ERK and PI3K/Akt pathway activation after Ral knockdown. Ral activity appears to be independent of oncogenic KRAS or NRAS. In addition, RNA sequencing revealed differing gene expression signatures for RAS and Ral.
Conclusion
Ral and its effector network constitute potential therapeutic targets in MM, which are activated independently of oncogenic K- or NRAS. Therefore, investigation of the functional network of Ral may be important to identify useful clinical targets.
Session topic: 13. Myeloma and other monoclonal gammopathies - Biology
Keyword(s): Signaling, RNA interference (RNAi), Ras, Multiple Myeloma
Abstract: PB1934
Type: Publication Only
Background
Genetic mutations and the bone marrow microenvironment contribute to disease progression, aggressive phenotype, and shorter survival in multiple myeloma (MM). Oncogenic RAS is one of the most common mutations in MM. Pathway activation through oncogenic RAS is associated with promotion of disease progression and shorter survival. Cell survival and proliferation in MM are mainly mediated via classical signaling pathways such as MEK/ERK and PI3K/Akt. Since there is a lack of specific RAS-inhibitors for clinical use, it is important to identify and analyze associated pathways, which may provide useful alternative targets for MM therapy. The small GTPase Ral has previously been implicated in putative downstream signaling of RAS, and may therefore promote proliferation, survival and drug resistance of MM cells.
Aims
We used shRNA-mediated knockdown of RalA and RalB isoforms to appraise their role as potential therapeutic targets and to analyze their connection to important signaling pathways, which regulate MM cell survival and proliferation. Because oncogenic RAS is a potential activator of the Ral pathways, we investigated a possible link between oncogenic RAS and Ral.
Methods
Immunohistochemical stainings of bone marrow trephines of MM patients and Western analysis of primary MM cells and MM cell lines were performed to evaluate Ral protein expression. Transient or stable knockdown of RalA or RalB was achieved by electroporation of MM cell lines and the effect on cell survival and apoptosis was measured with flow cytometry using annexin V/propidium iodide staining. Ral pulldown assays were applied to test potential dependence of Ral activation on oncogenic RAS. Furthermore, RNA sequencing was performed to compare RAS and Ral gene expression signatures after respective knockdowns.
Results
Both Ral isoforms were expressed in primary MM cells and MM cell lines, with RalA showing the most prominent and consistent protein expression levels. ShRNA-mediated knockdown of RalA strongly induced apoptosis in two thirds of the tested cell lines, whereas RalB depletion did impair MM cell survival in less than half of the cell lines. Western analysis revealed no alteration of classical MEK/ERK and PI3K/Akt pathway activation after Ral knockdown. Ral activity appears to be independent of oncogenic KRAS or NRAS. In addition, RNA sequencing revealed differing gene expression signatures for RAS and Ral.
Conclusion
Ral and its effector network constitute potential therapeutic targets in MM, which are activated independently of oncogenic K- or NRAS. Therefore, investigation of the functional network of Ral may be important to identify useful clinical targets.
Session topic: 13. Myeloma and other monoclonal gammopathies - Biology
Keyword(s): Signaling, RNA interference (RNAi), Ras, Multiple Myeloma
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