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M. Seibold et al.
Discussion
In this study, we demonstrate the functional importance of the small GTPase RAL for survival of MM cells. Both RAL isoforms were strongly expressed in the majority of HMCL and primary MM cells when directly compared to normal plasma cells or pre-malignant MGUS cells. Moreover, GTP-bound and thus activated RAL was pres- ent in all MM cell lines analyzed, pointing to a potential functional role of RAL in transition to and/ or maintenance of the malignant tumor clone. To test this hypothesis we performed isoform-specific RNAi-mediated RAL knock- down and found that abrogation of RAL led to fast and strong cell death induction in the majority of MM cell lines. These experiments thus identified the RAL GTPases as potent pro-survival mediators in MM.
Because activation of RAL has been described as a pre- dominantly RAS-dependent oncogenic survival pathway in various cancer entities,29,45–48 we also sought to test the putative functional link between oncogenic RAS and RAL activation in MM using pulldown assays and RNA sequencing. We found that in the MM cells tested, RAL activation could not be ascribed to the presence of onco- genic RAS (as defined by harboring activating point muta- tions in NRAS or KRAS). Neither did shRNA-mediated knockdown of oncogenic RAS alter the RAL activation status. As opposed to the well-defined activating muta- tions of NRAS and KRAS, data available from the CoMMpass trial cohort (these data were generated as part of the Multiple Myeloma Research Foundation Personalized Medicine Initiatives [https://research.themmrf.org and www.themmrf.org]) and other large next generation sequencing studies49–51 revealed no oncogenic bona fide mutations affecting RAL in MM. Activation of RAL by RAS-independent mechanisms has also been shown by other groups in solid tumors such as melanoma,52 bladder carcinoma53 and malignant peripheral nerve sheath tumors54 in which deregulated RAL guanine exchange factors, direct RAL phosphorylation by protein kinase C, or involvement of secondary GTPases, respec- tively, may lead to increased RAL-dependent tumor cell proliferation.
De Gorter et al. showed that RAL could be activated by chemokines originating from the bone marrow microenvi- ronment. In their study, treatment of MM cells with stro- mal cell-derived factor-1 resulted in increased levels of GTP-bound RAL and led to enhanced cell migration.55 These effects occurred independently of RAS, which is in line with our observation that no direct link between oncogenic RAS and activated RAL could be established.
Additionally, in our transcriptome analysis we observed distinct changes of gene expression after RAL versus RAS knockdown, underpinning the notion that RAL functions as a survival pathway in its own right and warrants fur- ther validation for potential therapeutic intervention.
Due to the high affinity of the guanine nucleotides at their binding sites, small GTPases such as RAS and RAL are hard to target pharmacologically, however. Whereas to date, no clinically suitable RAS inhibitors are available,1,4,56 a small molecule RAL inhibitor has recently been described,41 showing in vitro effects in adipose tissue57 and in chronic myelogenous leukemia.58 This allosteric com- pound has been developed to stabilize RAL in its inactive GDP-bound state and thus prevent its activation.41 In our hands, in the most sensitive MM cell line INA-6, RAL acti-
vation was indeed abrogated and apoptosis induced at drug concentrations starting from 10 μM, whereas sur- vival of primary MM cells and of other HMCL was less affected even at 20 μM, warranting development of more potent second generation RAL inhibitors. To this end, Walsh et al.42 have recently observed in a murine platelet RAL knockout model that RBC8 does indeed exert specific as well as unspecific effects within similar concentration ranges, which may explain its inconsistency when tested across different (cell line) models.
In our mass-spectrometric analysis which we performed to define RAL interaction partners serving as potential downstream mediators of the RAL pathway, we identified six members of the exocyst complex among the highest scoring hits. They included the complex members EXOC- 2/Sec5 and EXOC-8/Exo84 which are known to con- tribute to RAL-induced proliferation in tumor cells.44 Interestingly, RALBP1 which is another well-defined bind- ing partner to RAL, did not appear to play a predominant role in our MM cell line analysis.
In MM, the interconnection with signals from a per se altered bone marrow microenvironment59–61 may bypass otherwise important signaling hubs such as RAS. We have previously made this observation for the PI3K/Akt path- way in MM, which can be constitutively activated inde- pendently of oncogenic RAS, possibly by involvement of upstream receptor tyrosine kinases.12,13,51 Whereas we found no indication for RAL involvement in RAS/MAPK signaling, we did find distinctly lower levels of activated Akt after extended knockdown of RALA. In keeping with this observation, while the already strong apoptotic effects of RALA depletion could not further be enhanced by simultaneous pharmacological PI3K/Akt blockade, such treatment considerably enhanced the cytotoxic effects of RALB knockdown. These observations suggest that both RAL isoforms may at least in parts play differen- tial roles in cellular signaling, and point specifically to a role for RALA as one of the potential mediators for high intrinsic levels of active Akt in a subgroup of MM cells.
Given the heterogeneity of oncogenic pathways in MM, synergistically acting combination therapies seem to be the most promising targeted treatment strategies. To this extent, our data demonstrate that RAL abrogation may be effective in combination with inhibitors of the PI3K/Akt pathway. This is particularly important because in early clinical trials, PI3K inhibitors displayed limited effectivity and will therefore most likely play a role as combination partners in tumor therapy.18,62
Taken together, our data indicate that RAL is a promis- ing molecular target for MM therapy that is functionally independent of oncogenic RAS. However, because the one existing pharmacological inhibitor targeting RAL in our hands does not perfectly mimmick the strong effects of RAL knockdown, development of more potent second- generation inhibitors for MM treatment is mandatory for clinical translation.
Funding
This work was supported by grants from the Deutsche Forschungsgemeinschaft (KFO 216) and the Interdisziplinäres Zentrum für Klinische Forschung of the Universitätsklinikum Würzburg (B-188). TSte was supported by a fellowship of the Else Kröner Fresenius-Stiftung (2010_Kolleg.52). EL and RB were supported by a grant of the Deutsche Krebshilfe (70112693).
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haematologica | 2020; 105(9)