Figure S1. (A) Protein expression of p53 in MM cell lines.; Figure S2. (A) Melphalan resistant MM cell line RPMI-LR5 (panel left) and dexamethasone resistant MM cell line MM1R (panel right) were treated with the indicated doses of amiloride for 24, 48 and 72 h, and cell viability was analyzed by CellTiter-Glo luminescent assays; respectively. Figure S3. (A) MM1S-luc cells were treated for 48 hours with the indicated concentrations of amiloride in the presence or absence of MSCs derived from newly-diagnosed (ND) and relapsed/refractory (RR) MM patients, and proliferation was analyzed by bioluminescence (photons/sec). Figure S4. Amiloride induced apoptosis in MM cells. Figure S5. Amiloride did not induce changes in the cell cycle profile of MM cells. Figure S6. Amiloride deregulated mitochondrial potential in MM cells. Figure S7. Activity of amiloride through caspase-dependent and independent mechanisms. Figure S8. The triple and double combination of dexamethasone and melphalan with amiloride displayed superior anti-MM activity and improved median survival compared with single agents and double combinations in a subcutaneous plasmacytoma model. Figure S9. Experimental design for RNA-Seq assay. Figure S10. Pathway enrichment analysis at gene level and differential transcript isoforms expression. Figure S12. Validation of gene expression. Figure S13. Validation of p53 pathway activation in patient cells.
Funding
Instituto de Salud Carlos III
AECC
Junta de Castilla y León
International Myeloma Foundation
Fundación Española de Hematología y Hemoterapia
ARTICLE ABSTRACT
Purpose: The search for new drugs that control the continuous relapses of multiple myeloma is still required. Here, we report for the first time the potent antimyeloma activity of amiloride, an old potassium-sparing diuretic approved for the treatment of hypertension and edema due to heart failure.Experimental Design: Myeloma cell lines and primary samples were used to evaluate cytotoxicity of amiloride. In vivo studies were carried out in a xenograft mouse model. The mechanisms of action were investigated using RNA-Seq experiments, qRT-PCR, immunoblotting, and immunofluorescence assays.Results: Amiloride-induced apoptosis was observed in a broad panel of multiple myeloma cell lines and in a xenograft mouse model. Moreover, amiloride also had a synergistic effect when combined with dexamethasone, melphalan, lenalidomide, and pomalidomide. RNA-Seq experiments showed that amiloride not only significantly altered the level of transcript isoforms and alternative splicing events, but also deregulated the spliceosomal machinery. In addition, disruption of the splicing machinery in immunofluorescence studies was associated with the inhibition of myeloma cell viability after amiloride exposure. Although amiloride was able to induce apoptosis in myeloma cells lacking p53 expression, activation of p53 signaling was observed in wild-type and mutated TP53 cells after amiloride exposure. On the other hand, we did not find a significant systemic toxicity in mice treated with amiloride.Conclusions: Overall, our results demonstrate the antimyeloma activity of amiloride and provide a mechanistic rationale for its use as an alternative treatment option for relapsed multiple myeloma patients, especially those with 17p deletion or TP53 mutations that are resistant to current therapies. Clin Cancer Res; 23(21); 6602–15. ©2017 AACR.
