1. Research background

Cell death is a vital process occurring every day in the human body. Many processes such as maintenance of tissue homeostasis, support in recovery from acute injury as well as dealing with infections and regulation of the immune system rely on proper cell death function. In addition, cell death can also provoke inflammatory responses, and lytic forms of cell death can incite inflammation by promoting the production of cytokines and chemokines that modulate the innate immune response. Cell death can be both a consequence and a cause of inflammation, which can be difficult to distinguish. In the past decade, emerging knowledge on the interplay between cell death and inflammation has enriched the molecular understanding of the signalling pathways that mediate various programmes of cell death and multiple types of inflammatory responses. Strikingly, dysregulation of cell death or inflammation can lead to severe diseases, including cancer as well as autoimmune and degenerative disorders. It is therefore important to characterise the molecules and pathways that modulate cell death and inflammation to identify new therapeutic means to effectively treat such diseases.

Death ligand members of the tumour necrosis factor (TNF) family, including TNF itself but also Fas ligand (FasL) and the TNF-related apoptosis-inducing ligand (TRAIL), have previously been shown by us and others to induce inflammation as a consequence of cell death. By binding to their respective receptors, the so-called death receptors which are located on the plasma membrane, these death ligands activate their respective receptors which cannot only elicit the induction of cell death but also of gene expression which can also induce inflammation. Hence, death receptor stimulation by death ligands can result in inflammation through two different aetiologies, one cell death-driven and one gene-activation-driven. We identified the linear ubiquitin chain assembly complex (LUBAC) as an essential regulator of the signalling output of all death receptors. LUBAC is an intracellular multi-protein complex consisting of HOIL-1, SHARPIN and HOIP. LUBAC is the only currently known ubiquitin E3 capable of de novo synthesis of linear (also known as Met1/M1) ubiquitin chains. Our previous work has shown that the LUBAC-mediated formation of linear ubiquitin chains is crucial for maintaining the balance between gene activatory signals and cell death, whose deregulation results in developmental defects and inflammatory diseases including autoimmune disorders and cancer.

2. Research questions addressed by the group

Research in the Walczak Laboratory is focused on cell death and ubiquitin in inflammation, cancer and immunity. The overarching aim of the lab is to gain a deeper understanding of the interplay between cell death, inflammation and immunity and, on the basis thereof, provide novel therapies by targeting the perturbed physiological balance between cell death, inflammation and immunity which underlies many of the pathologies cited above, including cancer. With respect to linear ubiquitin, the Walczak lab is particularly interested in unravelling the mechanisms on how linear ubiquitin impacts (auto-)immunity as well as cancer cell survival and metastasis and cancer-related inflammation and tumour immunity.

3. Proposed research project

More than twenty percent of all human cancers bear oncogenic mutations in KRAS including lung and pancreatic ductal adeno carcinomas (LUAD and PDAC). Cancer driven by most forms of KRAS remain difficult to treat. Many effector pathways downstream of KRAS have been implicated in KRAS-driven tumour development and maintenance and, consequently, small molecule inhibitors targeting these pathways have been developed. Yet, clinical application of these drugs has revealed resistance-causing feedback mechanisms for virtually all of them. It is therefore essential to identify novel mechanisms that contribute and support KRAS-driven tumourigenesis to identify vulnerabilities that can be utilised for successful therapeutic intervention.

LUBAC is essential for optimal gene activation via NF-kB and prevention of cell death upon activation of different immune receptors such as TNFR1. We and others showed that deficiency in the LUBAC components SHARPIN or HOIL-1/HOIP in mice leads to severe inflammation in adulthood or embryonic lethality, respectively, due to deregulated TNFR1-mediated cell death. Similarly, upon TRAIL stimulation linear ubiquitination regulates the formation and activity of apoptosis- and necroptosis-mediating TRAIL-R signalling complexes by promoting TRAIL-induced gene activation (NF-kB) and preventing TRAIL-induced cell death. Moreover, we have recently discovered that endogenous TRAIL-R signalling drives tumourigenesis via two different routes: (i) by mediating KRAS-driven cancer progression and metastasis and (ii) by inducing NF-κB-dependent gene activation in cancer cells (most probably via LUBAC), resulting in cancer-cell mediated cytokine production, with consequent development of a tumour-supportive tumour immune microenvironment (TIME). Since depletion of any LUBAC components results in decreased NF-kB activation and since aberrant activation of NF-kB signalling has been associated with tumourigenesis of malignancies that are KRAS/p53-driven, we aim to understand how modulation of the expression of LUBAC components impacts PDAC development. Although LUBAC primarily acts through its activity as a ubiquitin E3 capable of forming M1-ubiquitin chains de novo, it also exerts functions as a scaffold, independently of its enzymatic activity. To investigate the role of LUBAC in PDAC tumour initiation and progression, we will assess its role differentiating between presence and activity. PDAC is known to be highly aggressive and refractory to conventional therapy as well as immune checkpoint inhibition. The immuno-suppressive environment is often the result of tumour-associated macrophages and myeloid-derived suppressor cells recruited to the tumours as a consequence of cancer-cell mediated cytokine production. Since LUBAC regulates different receptor-induced gene activation processes and, thereby, cytokine production, we will also study to which extent tumour-supportive and immuno-suppressive remodelling of the TIME in PDAC depends on LUBAC.

With this project we aim to answer the following questions:
What is the role of LUBAC’s scaffolding function or activity in the development of PDAC? Does LUBAC-mediated activation of gene-activatory signalling drive the establishment of a pro-tumourigenic tumour immune micro-environment? What receptor system is mostly affected by modulation of LUBAC expression or activity in PDAC? Does modulation of LUBAC drive cell death of tumour-initiating cells? Does modulation of LUBAC provide therapeutic benefit for immune-checkpoint inhibition treatment?

By unravelling the biochemical, functional and (patho-)physiological consequences of targeting LUBAC genetically and pharmacologically the proposed project aims at unveiling new therapeutic avenues for more effective future treatment of KRAS-driven cancers.

4. Applied Methods and model organisms

Flow and mass cytometry, cell culture, immunoprecipitation, western blotting, mass spectrometry, in-vivo work with different mouse tumour models, bulk and single cell RNAseq, different spatial transcriptomic and proteomic methodologies

5. Desirable skills and qualifications

• M.Sc. in Biology, Chemistry, Cell Biology, Biochemistry or equivalent.
• Experience with ex-vivo and in-vitro techniques, preferably also with in-vivo models (M. musculus).
• Knowledge on cell death, inflammation and or immunity is desirable.

6. Key publications by the research group

• Peltzer N, …., Walczak H. LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis. Nature. 557(7703):112-117, (2018).
• Taraborrelli L, …., Walczak H. LUBAC prevents lethal dermatitis by inhibiting cell death induced by TNF, TRAIL and CD95L. Nat Commun. 9(1):3910, (2018).
• Hartwig T, … Walczak H. The TRAIL-Induced Cancer Secretome Promotes a Tumour-Supportive Immune Microenvironment via CCR2. Molecular Cell. 16; 65(4), (2017).
• Lafont E, … Walczak H. The linear ubiquitin chain assembly complex regulates TRAIL-induced gene activation and cell death. EMBO J. 36, 1147-1166, (2017).
• Kupka S., … Walczak H. SPATA2-Mediated Binding of CYLD to HOIP Enables CYLD Recruitment to Signalling Complexes. Cell Reports. 16(9):2271-80, (2016).
• von Karstedt S, …., Walczak H. Cancer cell-autonomous TRAIL-R signaling promotes KRAS-driven cancer progression, invasion, and metastasis. Cancer Cell. 27: 561-573, (2015).
• Draber P …., Walczak H. LUBAC-Recruited CYLD and A20 Regulate Gene Activation and Cell Death by Exerting Opposing Effects on Linear Ubiquitin in Signaling Complexes. Cell Rep. 13, 2258-2272, (2015).
• Peltzer N, …., Walczak H. HOIP deficiency causes embryonic lethality by aberrant TNFR1-mediated endothelial cell death. Cell Rep. 9, 153-165, (2014).
• Lemke J, … Walczak H. Selective CDK9 Inhibition Overcomes TRAIL Resistance by Concomitant Suppression of cFlip and Mcl-1. Cell Death and Differentiation. 21(3):491-502, (2014).
• Gerlach B, … Walczak H. Linear ubiquitination prevents inflammation and regulates immune signalling. Nature. 471, 591-596, (2011).
• Haas TL, …., Walczak H. Recruitment of the linear ubiquitin chain assembly complex stabilizes the TNF-R1 signaling complex and is required for TNF-mediated gene induction. Mol Cell.36, 831-844, (2009).