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RAL GTPases mediate multiple myeloma cell survival
pho-Akt (of both, the Thr308 and Ser473 phosphorylation sites) at day 3 (Figures 2E-F). This effect was quite pro- nounced in both of these cell lines which display relatively high constitutive levels of activated Akt.
Constitutive RAL activation in MM cells remains unaffected by knockdown of oncogenic RAS
To address our hypothesis that RAL activation is dependent on oncogenic RAS, we analyzed the change of the levels of activated GTP-bound RAL after knockdown of oncogenic KRAS or NRAS in HMCL harboring the respective mutated RAS isoform. Effective silencing of oncogenic RAS was verified by Western analysis 48 hours (h) after electroporation with the respective shRNA expression vectors. RAL-GTP levels were measured by performing RALA pulldown using RALBP1 protein-bind- ing domain agarose beads. Notably, the RAL activation status was not altered by knockdown of oncogenic KRAS in MM.1S cells or oncogenic NRAS in INA-6 cells (Figure 3A). Expression levels of total load of RALA proteins remained also unchanged.
RNAi-mediated knockdown of KRAS or RALA in MM.1S cells entails distinct transcriptomic effects
Because mutated RAS did not appear to be directly linked to RAL activation, we next analyzed in more detail the influence of both signaling hubs on the transcriptome of KRAS-mutated MM.1S cells using an RNA sequencing technique for 28,440 gene transcripts. Cells were trans- fected with shRNA expression vectors against either KRAS or RALA, or with the pSU empty-vector. Strongly transfected cells were purified via CD4Δ microbead selec- tion and harvested at day 2 post-transfection for prepara- tion of samples for transcriptomic analysis and Western blotting to confirm successful target knockdown (Figure 3B). As displayed in the Venn diagram (Figure 3C), KRAS knockdown led to changes in gene transcription in about double the number of genes (n=1,473) than RALA knock- down (n=771). Taken together, the number of transcripts that is altered in a mutually exclusive fashion (n=1,744) far outweighs the number affected by both, KRAS- or RALA- knockdown (n=235). Using the Molecular Signatures Database Hallmark Gene Set Collection,40 ontology gene mapping was performed for the classification of differen- tial gene expression after RAL versus RAS knockdown, highlighting the most distinct functional gene groups with relevance for MM biology (Figure 3D).
Effects of the small molecule compound RBC8 on survival and RAL activation of MM cells
The small molecule inhibitor RBC8 has recently been described as selective allosteric inhibitor of RALA and RALB, which stabilizes RAL in its inactive GDP-bound state.41 We treated MM cell lines (n=4) and primary MM cells (n=6) for 3 days with different concentrations of RBC8 and measured cell survival by flow cytometry using annexin V/PI staining. INA-6 cells were most sensitive to the drug with EC50/90 values of 12,5 and 17,5 μM, respec- tively. MM.1S cells, on the other hand, were unaffected by RCB-8 even at the highest concentration tested (20 μM) (Figure 4A). In accordance with these results, analysis of RALA activation by pulldown of RALA-GTP in the sensi- tive INA-6 cells showed a strong reduction after treatment with 20 μM of RBC8 for 3 h, whereas at 10 μM no marked effects were seen. Conversely, the levels of activated
RALA remained unaltered after 3 h-treatment with 20 μM of RBC8 in MM.1S cells (Figure 4B). Primary MM samples remained largely unaffected by RBC8 (20 μM) (Online Supplementary Figure S5A).
Data from combined RAL (RBC8) and MEK/MAPK or PI3K/Akt inhibition showed increased anti-myeloma effects in RBC8-sensitive cell lines, but no combination advantage in RBC8-insensitive cells (Figure 4C). These drug combinations showed at best mild effects on cell sur- vival in the primary MM samples tested (Online Supplementary Figure S5B).
We also performed Alamar Blue assays to test possible effects of RBC8 treatment on cell metabolism and proliferation. We found only minor impacts at concentra- tions up to 20 μM, most pronounced in INA-6 and AMO- 1 cells (Online Supplementary Figure S3C). Although higher concentrations (up to 40 μM) of RBC8 enhanced these effects, these concentrations may also exert unspecific cytotoxicity.42 Cell cycle distribution after RBC8 treatment was analyzed by BrdU/PI staining and revealed that in INA-6 cells which are most sensitive to treatment with RBC8, the G2/ M-phase significantly increased from 19% to 30 % at the expense of the S-phase, which decreased from 38% to 20% (Online Supplementary Figure S3D). No relevant changes were observed in AMO-1, L-363, or MM.1S cells. Finally, no effects on the constitutive levels of phospho-ERK and phospho-Akt were observed after RBC8 treatment of HCML (Online Supplementary Figure S4).
Combination of RALB depletion with PI3K/Akt inhibitors leads to enhanced MM cell death
Because RAL blockade had either no or differential effects on the activity of the MEK/MAPK or PI3K/Akt pathways (see above and Figures 2C-F), we tested the potential usefulness of pharmacologically targeting these pathways in combination with RAL knockdown.
After knockdown of either RALA or RALB, L-363 cells were treated with pharmacological inhibitors of MEK1/2 (PD0325901), Akt (MK2206) or PIK3CA (BYL-719). While combinations of either of these compounds with RALA abrogation did not significantly enhance apoptosis induc- tion in excess of the rather strong effects of RALA knock- down alone, combined depletion of RALB and Akt or PI3K inhibition, respectively, led to significantly higher rates of cell death (Figure 5).
Combination experiments in MM cell lines using the pharmacological RAL inhibitor RBC8 showed statistically significant (and functionally relevant) synergistic effects for the combination with PI3K/Akt primarily in the afore- mentioned cell line INA-6 (Figure 4C).
Mass spectrometric analysis identifies the exocyst complex as a predominant RAL interaction partner
To analyze potential downstream signaling partners of RAL in MM cells, we performed quantitative mass spec- trometry of MM.1S cells with stable expression of HA- tagged RALA protein. A total of 48 proteins were identi- fied as specific partners of RALA, including six members of the exocyst complex (EXOC-1, 2, 3, 4, 7, 8) (Online Supplementary Figure S6). Except for EXOC-7, all of these exocyst components are listed as highly confident interac- tion partners in the HitPredict database for protein-protein interactions.43 Moreover, EXOC-2/Sec5 and EXOC- 8/Exo84 have previously been described to play a role in RAL-mediated tumor cell proliferation.44
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