RAL Mediates Multiple Myeloma Cell Survival Independently of Oncogenic RAS

Introduction

Mutation of RAS is one of the most frequent oncogenic lesions in human cancers and is present in up to half of primary multiple myeloma (MM) cases. Despite its therapeutic relevance, pharmacological inhibition of oncogenic RAS has so far been unsuccessful. Therefore, downstream effectors and RAS-associated pathways may serve as alternative therapeutic targets. The small GTPase RAL is a putative downstream effector of RAS and promotes malignant transformation and tumor cell survival in solid neoplasias. We set out to analyze the functional role of RAL in MM using shRNA-mediated knockdown of the two RAL isoforms, RALA and RALB. Our data demonstrate that RAL promotes MM cell survival and is activated independently of oncogenic RAS.

Methods

Expression of RALA and RALB was analyzed by immunohistochemistry in bone marrow trephines from MM patients (n=24) and by Western blotting in purified primary MM cells (n=10). To abrogate RAL function in MM cell lines, two sequence-specific shRNA expression vectors targeting either RALA or RALB were generated. Cell viability after RAL knockdown was measured by flow cytometry using annexin V/propidium iodide staining. Effects on MEK/MAPK and PI3K/Akt signaling were determined by Western analysis. Additionally, RAL pulldown assays were used to analyze the functional link between RAL activation and oncogenic RAS. Moreover, we performed RNA sequencing to compare RAS- and RAL-dependent gene expression profiles. Finally, potential RAL interaction partners were identified by mass spectrometry.

Results

In comparison to MGUS or normal plasma cells RALA showed strong expression in all MM cells tested, whereas RALB was expressed more heterogeneously. RNAi-mediated knockdown of RALA or RALB significantly reduced cell survival in L-363 (25% and 40%, respectively) and MM.1S cells (32% and 52%, respectively). Phospho-Akt and phospho-ERK1/2 levels remained unaffected by RAL depletion. Interestingly, abrogation of oncogenic RAS did not alter RAL activation in pulldown assays. Moreover, RNA sequencing after RAS or RAL knockdown revealed differential expression of 1473 KRAS-regulated genes versus 771 RALA-regulated genes, with an overlap of 235 genes, underscoring the notion that both targets serve different signaling pathways. Mass spectrometry analysis revealed interaction partners of RAL which are currently evaluated.

Conclusion

Our data indicate that RAL GTPases serve as promising therapeutic targets in MM, which mediate tumor cell survival independently of oncogenic RAS. Development of pharmacological inhibitors targeting RAL and its downstream interaction partners is, therefore, warranted for potential clinical translation.