[PubMed] [Google Scholar] 29. immune stimulants in cell viability assays. In contrast, we report that this combination of LCL161 and VSV51-GFP reduces tumour volume and prolongs survival in a 76-9 syngeneic murine model. Our results support further exploration of the combined use of IAP antagonists and innate immune stimuli as a therapeutic approach for RMS cancers. the 5-untranslated region (UTR) internal ribosome access site (IRES). We further exhibited that reducing the levels of cIAP1 either by IGF2BP1 knockdown or by treatment with LCL161 sensitized RMS cell lines to TNF-mediated cell death. Finally, we tested this approach in a xenograft mouse model using the human ERMS cell collection Kym-1, which has autocrine TNF production and is therefore sensitive to SMC treatment as a single agent . Indeed, SMC treatment inhibited the establishment and growth of Kym-1 xenograft tumours and extended survival in mice. However, most RMS do not produce endogenous TNF and initial testing has suggested that the human RMS cell lines RD, RH41, RH30, and RH18 are resistant to treatment with LCL161 . Furthermore, LCL161 treatment did not inhibit tumour growth in six RMS xenograft tumours when used as a single agent . SMCs have proven to be safe and well tolerated in phase 1 and phase 2 clinical trials, but have limited efficacy in highly refractory and relapsed malignancy patient populations . This evidence suggests that SMCs will require other pro-death cytokine ligands to effectively treat most RMS cancers. Rostafuroxin (PST-2238) We recently exhibited that SMCs synergize with innate immune stimuli, including oncolytic viruses and recombinant interferon, to induce an effective and safe cytokine storm that promotes tumour death in murine models of breast and colon carcinomas . We hypothesize that this combined treatment paradigm will also promote cell death in RMS cancers. Here, we statement that the human RMS cell collection Kym-1 is sensitive to LCL161 as Rostafuroxin (PST-2238) a single agent, while other human RMS cell lines (RH36, RH41, RD, RH18, RH28, and RH30) and the murine RMS cell collection 76-9 are resistant to LCL161 as a single agent. The resistance of these cell lines does not appear to be related to alterations in apoptosis pathway effectors or in immune modulator receptors. Importantly, innate immune stimuli (e.g. oncolytic computer virus (VSV51-GFP), interferon (IFN), and tumour necrosis factor-like poor inducer of apoptosis (TWEAK)) synergize with LCL161 to promote TNF signaling and reduce cell viability in Kym-1 RMS malignancy cells. In contrast, cell viability assays showed that all other RMS cell lines tested were also resistant to combined treatment with LCL161 and immune stimulants. Importantly, targeting the IAPs and stimulating cytokine signaling in an 76-9 syngeneic model using LCL161 and VSV51-GFP resulted in reduced tumour volume and durable cures in mice. Our results advocate for the combined use of IAP antagonists and innate immune stimuli as a potential therapeutic approach for RMS cancers. RESULTS Kym-1 cells, but not other RMS cell lines, are sensitive to LCL161 as a single agent The human ERMS cell collection Kym-1 was highly sensitive to exposure to increasing concentrations of LCL161 for 24 h (Physique ?(Physique1A,1A, open circles). Viability of Kym-1 cells was assessed by Alamar Blue assay and was significantly reduced to 40.48%, 30.28%, and 3.80% following 24 h incubation with media containing 5 nM, 10 nM, and 25 Rostafuroxin (PST-2238) nM of LCL161, respectively. When Kym-1 cells were treated with concentrations of 100 nM of LCL161 for 24 h, cell viability was FLJ44612 reduced to levels that were indistinguishable from blanks (i.e. samples containing media and Alamar Blue reagent, but no cells). In contrast, concentrations of LCL161 up to 10 M experienced no effect on viability in all other human RMS cell lines (RH36, RH41, RD, RH30, RH28, and RH18) and in the mouse cell collection 76-9 (Physique ?(Figure1A).1A). To determine whether sensitivity of RMS cells to LCL161 was related to cIAP1 protein expression, western blotting was used to assess cIAP1 expression in cells treated with vehicle (DMSO) or LCL161 for 24 h (Physique ?(Figure1B).1B). Treatment with 5 M LCL161 (10 nM LCL161 in Kym-1 cells) for 24 h resulted in comparable reductions in cIAP1 protein expression in all human RMS cell lines tested, and therefore was not associated with LCL161 sensitivity. Similarly, treatment with 5 M LCL161 resulted in reduced cIAP1 protein expression in mouse C2C12 myoblast, 76-9 RMS, and 1863 sarcoma cell lines. Of notice, the expression of XIAP was unaffected by LCL161 treatment (Supplementary Physique S1) while cIAP2 is not.