Oncogenic RAS mutants confer resistance of rhabdomyosarcoma cells to oxidative stress-induced ferroptotic cell death

Recent genomic studies revealed a high rate of recurrent mutations in the RAS pathway in primary rhabdomyosarcoma (RMS) samples. In the present study, we therefore investigated how oncogenic RAS mutants impinge on the regulation of cell death of RMS cells. Here, we report that ectopic expression of NRAS12V, KRAS12V or HRAS12V protects RMS cells from oxidative stress-induced cell death. RMS cells engineered to express NRAS12V, KRAS12V or HRAS12V were significantly less susceptible to loss of cell viability upon treatment with several oxidative stress inducers including the thioredoxin reductase inhibitor Auranofin, the glutathione (GSH) peroxidase 4 (GPX4) inhibitor RSL3 or Erastin, an inhibitor of the cysteine/glutamate amino acid transporter system xc¬- that blocks GSH synthesis. Notably, addition of the iron-chelating compound ferrostatin-1 confers protection against Erastin- or RSL3-induced cytotoxicity, indicating that these compounds trigger ferroptosis, an iron-dependent form of programmed cell death. Furthermore, RMS cells overexpressing oncogenic RAS mutants are significantly protected against the dual PI3K/mTOR inhibitor PI103, whereas they are similarly sensitive to DNA-damaging drugs such as Doxorubicin or Etoposide. This suggests that oncogenic RAS selectively modulates cell death pathways triggered by cytotoxic stimuli in RMS cells. In conclusion, our discovery of an increased resistance to oxidative stress imposed by oncogenic RAS mutants in RMS cells has important implications for the development of targeted therapies for RMS

Hedgehog inhibitors in rhabdomyosarcoma: a comparison of 4 compounds and responsiveness of 4 cell lines

Rhabdomyosarcoma (RMS) are the most common soft tissue sarcoma in children and are divided into two major histological subgroups, i.e. embryonal (ERMS) and alveolar RMS (ARMS). RMS can show HEDGEHOG/SMOOTHENED (HH/SMO) signaling activity and several clinical trials using HH inhibitors for therapy of RMS have been launched. We here compared the antitumoral effects of the SMO inhibitors GDC-0449, LDE225, HhA and cyclopamine in two ERMS (RD, RUCH-2) and two ARMS (RMS-13, Rh41) cell lines. Our data show that the antitumoral effects of these SMO inhibitors are highly divers and do not necessarily correlate with inhibition of HH signaling. In addition, the responsiveness of the RMS cell lines to the drugs is highly heterogeneous. Whereas some SMO inhibitors (i.e. LDE225 and HhA) induce strong proapoptotic and antiproliferative effects in some RMS cell lines, others paradoxically induce cellular proliferation at certain concentrations (e.g. 10 µM GDC-0449 or 5 µM cyclopamine in RUCH-2 and Rh41 cells) or can increase HH signaling activity as judged by GLI1 expression (i.e. LDE225, HhA and cyclopamine). Similarly, some drugs (e.g. HhA) inhibit PI3K/AKT signaling or induce autophagy (e.g. LDE225) in some cell lines, whereas others cannot (e.g. GDC-0449). In addition, the effects of SMO inhibitors are concentration-dependent (e.g. 1 µM and 10 µM GDC-0449 decrease GLI1 expression in RD cells whereas 30 µM GDC-0449 does not). Together these data show that some SMO inhibitors can induce strong antitumoral effects in some, but not all, RMS cell lines. Due to the highly heterogeneous response we propose to conduct thorough pretesting of SMO inhibitors in patient-derived short term RMS cultures or patient-derived xenograft mouse models before applying these drugs to RMS patients