Targeting RBM39-MEK5 Axis Synergizes with Bortezomib to Inhibit Multiple Myeloma Malignancy

Multiple myeloma (MM) is a type of haematological malignancy characterised by the accumulation and expansion of malignant plasma cells in the bone marrow (BM). Despite recent advances in chemotherapy, MM remains an incurable disease for most patients and therefore requires novel therapies. Abnormal alternative splicing is a hallmark of cancer and is associated with poor survival and an ultra-high-risk population in MM (Bauer et. al. Haematologica 2021). Targeting splicing is a promising therapeutic strategy in MM. Our previous study showed that RBM39 is highly expressed in primary MM cells and cell lines and correlates with poor overall survival (Tong et. al. Haematologica 2020). To investigate the underlying role of RBM39 in MM, we knocked down RBM39 in MM cell lines. We found that knockdown of RBM39 inhibited MM cell proliferation, induced apoptosis and cell cycle arrest at the G2/M phase, and inhibited subcutaneous tumour growth. Indisulam has been reported to induce RBM39 degradation via the proteasomal pathway by acting as a molecular glue to link RBM39 to the E3 ubiquitin ligase DCAF15 (Han et. al. Science 2017). In our study, Indisulam treatment in MM cells was shown to degrade RBM39 via DCAF15. Indisulam could induce apoptosis and G2/M phase arrest in MM cell lines and inhibite tumour growth in vivo. It also significantly decreased MM cell viability in a co-culture system with MM BM-derived mesenchymal stromal cells. Indisulam was also able to inhibit primary CD138+ cells isolated from newly diagnosed MM patients.

To explore the mechanistic role of RBM39 in promoting MM malignancy, we performed RNA immunoprecipitation (RIP)-Seq and RNA sequencing in RBM39 knockdown MM cells to investigate the alternative splicing targets of RBM39. KEGG pathway analysis of differentially expressed and alternatively spliced genes revealed the enrichment of the MAPK pathway. We then identified MEK5, a non-redundant member of the MAPK family, as a potential target of RBM39, which is highly expressed in MM. Full-length MEK5 contributes to increased proliferation and survival of MM cells. We further demonstrated that MEK5 is bound and alternatively spliced by RBM39 and that splicing of full-length MEK5 is dependent on RBM39. Given the importance of the RBM39-MEK5 axis in MM, we also found that inhibition of RBM39 with Indisulam or MEK5 with the inhibitor BIX02189 increased the cytotoxicity of bortezomib in vivo and in vitro.

In conclusion, we have demonstrated that the RBM39-MEK5 axis, in which RBM39 maintains splicing of full-length MEK5, is essential for MM cell survival and proliferation. Mechanistically, shRNA-mediated knockdown or Indisulam-mediated degradation of RBM39 caused extensive altered splicing, including mis-splicing of MEK5. Targeting RBM39 or MEK5 could not only inhibit MM malignancy, but also enhance the cytotoxicity of bortezomib for MM.