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RAL GTPases mediate multiple myeloma cell survival
whereas combined blockade in patients with solid tumors resulted in high levels of toxicity.23–26 The identification of alternative RAS-driven pathways to target MM cells is therefore highly warranted.
Here, we investigated the functional role of RAS-like protein (RAL) in MM, which has sometimes been branded “the third pathway” in the context of RAS-dependent oncogenic signaling.27–29 RAL belongs to the RAS super- family of small GTPases that – like RAS itself – are characterized by cycling between a GTP-bound active and a GDP-bound inactive state. The two isoforms of RAL: RALA and RALB have both been shown to be involved in malignant transformation, tumor cell survival, and tumor cell growth and metastasis, although their functional role(s) may depend to some extent on the tumor entity and/or model tested.30–32
In our study, we sought to analyze the functional impor- tance of RAL in MM as bona fide downstream effector of oncogenic RAS by using RNAi-mediated knockdown approaches. We found that RAL is important for MM cell survival, but that its constitutive activation is not directly linked to oncogenic RAS. Furthermore, knockdown of RAL entails very different transcriptomic changes than RAS depletion. Therefore, we infer that the RAL pathway constitutes a potential clinical target in its own right.
Methods
Culture of MM cell lines and preparation of primary MM cells
Cell culture conditions of human myeloma cell line (HMCL) and isolation of CD138-positive primary MM cells were previous- ly described.33 Bone marrow aspirates of MM patients were obtained after informed consent according to the Declaration of Helsinki, and with permission of the Ethics Committee of the University of Würzburg (reference no. 76/13). See the Online Supplementary Materials and Methods for details.
Immunohistochemical stainings of bone marrow biopsies
To evaluate protein expression of the RAL isoforms in plasma cells we performed immunohistochemical analysis in formalin- fixed, paraffin-embedded bone marrow biopsies from 26 patients with MM as previously described.8,14 For comparison, we ana- lyzed patients with monoclonal gammopathy of undetermined significance (MGUS) (n=10) and bone marrow trephines contain- ing reactive, polyclonal plasma cells (n=5). Slides were evaluated by experienced hematopathologists. See the Online Supplementary Materials and Methods for details.
Cell death assay
Fractions of unaffected and (pre-)apoptotic cells were measured by flow cytometry after staining with propidium iodide (PI) and annexin V labeled with either PromoFluor 647, allophycocyanin (APC) or fluorescein isothiocyanate (FITC) as previously described.34 Cell death measurements were conducted at days 3 and 4 after transfection.
Cell metabolism, proliferation and cell cycle assays
Alamar Blue and bromodeoxyuridine (BrdU)/PI assays were performed to analyze cell metabolism, proliferation and cell cycle distribution after RAL knockdown or pharmacological inhibition with RBC8. See the Online Supplementary Materials and Methods for details.
Construction of shRNA expression vectors
Construction of pSUPER-based small hairpin RNA (shRNA) expression vectors was performed as previously described.35 See the Online Supplementary Materials and Methods for sequences.36,37
Transfection of MM cells by electroporation
Transient transfection of HMCL was previously described in detail.34 HMCL were electroporated with pSUPER-based shRNA expression vectors. ShRNA expression plasmid concentrations in the final electroporation mix were 20 μg/mL (15 μg/mL for trans- fections with subsequent drug treatment). Strongly transfected cells were purified by microbead selection for co-expressed CD4 or, in the case of AMO-1, by fluorescence-activated cell sorting for co-expressed enhanced green-fluorescent protein (EGFP).
RALA activity assay
INA-6 and MM.1S cells were transfected with shRNA expression plasmids and harvested two days after electroporation. The activa- tion status of RALA was measured using the RAL Activation Assay from Cell Biolabs (no. STA-408, San Diego, CA, USA) according to the manufacturer's instructions. Subsequent Western blotting was performed to analyze RAL-GTP levels and total RAL protein loads. Antibodies against RALA were diluted 1:500 or 1:1,000.
Western analysis
Western blotting of cell lysates was performed according to standard protocols as previously described.12,34 See the Online Supplementary Materials and Methods for details.
RNA sequencing analysis
For transcriptome analyses, MM.1S cells were transfected with pSUPER-based shRNA expression vectors against either KRAS or RALA. Control cells were transfected with empty pSUPER plas- mids. RNA sequencing data are deposited in Gene Expression Omnibus in entry GSE126794. See the Online Supplementary Materials and Methods for details.
Mass spectrometry-based interactome analysis
To identify RAL interaction partners we performed quantitative mass spectrometric analysis of MM.1S cells with stable expression of HA-tagged RALA protein. Detailed description of sample preparation and analysis is provided in the Online Materials and Methods and by Cox et al.38,39
Statistical analysis
Statistical significance (P<0.05) was determined by a two-tailed Student’s t-test. Three independent experiments were performed.
Results
RAL expression in multiple myeloma cells
RAL protein expression in a panel of MM cell lines (n=7) and primary MM samples (n=10) was analyzed for each isoform using Western blotting. Both proteins were detected at fairly equal levels in all (RALA), and in 6 of 7 (RALB) HMCL, respectively (Figure 1A, top). Cell line U- 266 was notable for its complete lack of RALB expression. Interestingly, all cell lines showed constitutive RALA acti- vation through the presence of GTP-bound RALA as detected by a pulldown assay (Figure 1A, bottom). In CD138-positive primary MM cells isolated from bone marrow aspirates of MM patients, both RAL proteins were always present. Accounting for differences in the amounts of sample loading, RALA and RALB expression
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