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

In the crosshairs: Visualizing lytic granules by high resolution microscopy and electrophysiology

Cytotoxic T lymphocytes (CTLs) form an integral part of the adaptive immune system. Their main function is to eliminate bacteria- and virus-infected target cells by releasing perforin and granzymes (the lethal hit) contained within lytic granules, at the CTL-target cell interface (the immunological synapse; IS). The formation of the IS as well as the final events at the IS leading to target-cell death are both highly complex and dynamic processes. In this review we highlight and discuss three high-resolution techniques that have proven invaluable in the effort to decipher key features of the mechanism of CTL effector function and in particular lytic granule maturation and fusion. Correlative light and electron microscopy allows the correlation between organelle morphology and localization of particular proteins, while total internal reflection fluorescence microscopy (TIRFM) enables the study of lytic granule dynamics at the IS in real time. The combination of TIRFM with patch-clamp membrane capacitance measurements finally provides a tool to quantify the size of fusing lytic granules at the IS