Pancreatic ductal adenocarcinoma (PDAC) is amongst the deadliest cancers with a 5-year survival rate of below 5%. This poor prognosis is caused by absence of specific early symptoms and primary resistance to chemotherapy highlighting an urgent need to address these two problems. Molecularly, PDAC is characterised by the presence of activating point mutation in the small GTPase KRAS in 95% of the cases. This point mutation leads to constitutive activation of KRAS, promoting proliferation, invasion and cell death resistance. We recently contributed to a study identifying that the cystine/glutamate antiporter xCT (SLC7A11) promotes Ras-mediated transformation by protecting cells from reactive oxygen species (ROS) overload (Lim et al., 2019). Interestingly, xCT has been implicated in the protection against a recently discovered form of regulated necrosis, termed ferroptosis. Sensitivity to ferroptosis, in turn, has emerged as a selective vulnerability in cancer cells which escape targeted treatment. Based on these findings, we have experimentally explored whether the KRASG12D mutation which is prevalent in PDAC might be promoting malignancy through evading ferroptosis. The aim of this project is to establish i) whether ferroptosis evasion might determine KRASG12D fitness in PDAC development ii) and how breaking ferroptosis evasion might affect the development of PDAC precursor lesions in a genetically-engineered mouse model. We thereby anticipate to identify cell death vulnerabilities in conjunction with potential markers during early PDAC progression for the development of novel treatment strategies. 

The poor 5-year survival rate of below 5% of PDAC patients has not significantly improved since the 1970ies. This has marked PDAC as one of the cancers with unmet needs warranting the urgent requirement to explore novel therapeutic strategies. We propose to test ferroptosis-inducing therapy (FIT) in in-vitro and in-vivo PDAC model systems in order to address this need. Findings made in these model systems will be corroborated in a PDAC patient tissue bank.

1. Elucidate mechanisms of ferroptosis response in KRAS-mutated cells
   • Define proteomics of KRAS mutated and wild type cells upon induction of ferroptosis
   • Define expression changes of KRAS mutated and wild type cells upon induction of ferroptosis
   • Identify differential adaptation mechanisms in KRAS-mutated PDAC upon ferroptosis
   • Validation of role of differentially regulated factors
2. Elucidate the role of genetic ferroptosis induction in the development and progression of PDAC
   • Determine the role of ferroptosis in PDAC development in vivo
   • Test FIT in KRAS-driven genetically engineered mouse models
   • Validation of expression of ferroptosis pathway components and potential prognostic value in a patient biobank

Most tumours are known to undergo constant cycles of selection via immune effector cells. One pathway via which immune effector cells kill target cells is extrinsic apoptosis induction via ligand/receptor binding of Tumor necrosis factor (TNF)- and TNF-receptor (TNFR) superfamily (SF) members (Karstedt et al., 2017). Yet, given the fast kinetics and efficiency by which extrinsic apoptosis can eliminate targeted cells, it is not surprising that tumours frequently develop escape mechanisms against extrinsic apoptosis. In KRAS-driven PDAC, we previously found that extrinsic apoptosis is disabled (Karstedt et al., 2015). Interestingly, Ferroptosis is a recently discovered type of regulated necrosis which, unlike apoptosis, is independent of caspase. Instead, ferroptotic cells die following iron-dependent lipid peroxidation, a process which is antagonised by glutathione peroxidase 4 (GPX4), by the cystine/glutamate antiporter xCT (SLC7A11) and ferroptosis suppressor protein 1 (FSP1) (reviewed in Bebber et al. 2020). Importantly, tumour cells escaping other forms of regulated cell death maintain or acquire sensitivity to ferroptosis (Bebber et al. 2021). Moreover, ferroptosis is a lytic type of cell death during which cellular plasmamembranes are permeabilised (Pedrera et al. 2020) making it potentially immunogenic and thereby particularly interesting for cancer therapy. As part of this project, we tested whether KRAS-mutated PDAC can respond to ferroptosis-inducing therapy (FIT).

Interestingly, we identified that expression of oncogenic as compared to WT KRAS in isogenic cellular systems renders cells more resistant to ferroptosis. Mechanistically, we find that cells with mutant KRAS show a specific lack of ferroptosis-induced lipid peroxidation along with upregulated expression of ferroptosis suppressor protein 1 (FSP1). Indeed, elevated levels of FSP1 in KRAS mutant cells are responsible for mediating ferroptosis resistance. Consequently, we find that pharmacological induction of ferroptosis in pancreatic organoids derived from the LsL-KRASG12D expressing mouse model is effective at killing organoids only in combination with FSP1 inhibition. Lastly, FSP1 is upregulated in non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) as compared to the respective normal tissue of origin. Based on these data, we propose that ferroptosis-inducing therapy should be combined with FSP1 inhibitors for efficient therapy of KRAS-mutant cancers (Müller et al. under review).