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 signaling pathways that mediate various programs of cell death and multiple types of inflammatory responses. Strikingly, dysregulation of cell death or inflammation can lead to severe diseases, including autoimmune or degenerative disorders and cancer. It is therefore important to characterize the molecules and pathways that modulate cell death and inflammation to identify new therapeutic means to effectively treat such diseases.
Members of the tumor necrosis factor (TNF) family, e.g. TNF itself but also FasL (CD95L) and TRAIL, have previously been shown by us and others to induce inflammation as a consequence of cell death. By binding to their respective receptors which are located on the plasma membrane, these so-called death ligands activate these receptors which cannot only elicit the induction of cell death but also of gene expression which can also induce inflammation. We identified the intracellular multi-protein complex LUBAC as a component essential for regulating the signaling output of all different receptors for these death ligands. Importantly, this especially applies to the balance between cell death and gene-activatory signaling and how a deregulation of this balance can result in inflammatory disease. Additionally, we discovered that in cancer different death receptors, especially TRAIL receptors, rather than serving as cancer suppressors, which one could have assumed to be the consequence of their cell death-inducing capacity, act in a tumor-supportive manner independent of cell death.
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 lab is particularly interested in unravelling the mechanisms on how different death receptor-ligand systems, especially the TNF–TNFR, Fas–FasL and TRAIL–TRAIL-R systems, are regulated and how they impact cancer cell survival and metastasis as well as cancer-related inflammation and tumour immunity. The overarching aim of the Walczak group is to gain a deeper understanding of the interplay between cell death and inflammation and, on the basis thereof, provide novel therapies by therapeutic targeting of the perturbed physiological balance between cell death and inflammation which underlies many of the pathologies cited above, including cancer.
3. Proposed research project
Whilst induction of cell death in cancer using death ligands seemed like a promising approach to cancer therapy, cancer cells are often positively selected during tumor development for increased resistance to cell death. Hence, it is often required to decreased the threshold for cell death in cancer using chemical compounds that, in combination with death ligands, will induce cell death and prevent disease progression.
Since its discovery, the death ligand TRAIL has been considered as a promising candidate for new anti-tumor therapies. Despite the high capacity of this cytokine to selectively induce cell death in tumor cells, initial enthusiasm diminished following the use of first-generation TRAIL receptor agonists in clinical trials as these trials showed that treatment with the different TRAIL receptor agonists alone or solely in combination with chemotherapy was not effective. While we have recently discovered an unexpected pro-tumorigenic role for TRAIL signaling, different TRAIL-sensitizing therapies to induce cell death have been proposed and already shown excellent results in vitro and in vivo. We discovered CDK9 inhibition is a highly effective means to sensitize non-small cell lung cancer (NSCLC) cells to TRAIL-induced apoptosis. Mechanistically, CDK9 inhibition (CDK9i) antagonizes the expression of the antiapoptotic proteins cFLIP and Mcl1, promoting sensitization to TRAIL. However, some tumor cells overexpress other factors which prevent cell death. It is therefore necessary to further build on the TRAIL–CDK9i combination. Therefore, the aim of the present project is to identify combination treatments which synergize with the TRAIL–CDK9i combination in killing tumor cells and to determine the mechanism for the identified synergy.
The standard-of-care for patients with lung cancer is platinum-based chemotherapy. Moreover, this therapy has shown a synergistic effect with TRAIL. Therefore, our first aim is to understand whether cisplatin synergizes with CDK9i and TRAIL. Bortezomib is a proteasomal inhibitor approved by the FDA which is currently employed in the treatment of hematological malignancies. Bortezomib, but also other drugs like Thapsigargin, Taxol or Brefeldin A, activate the unfolded protein response (UPR), triggering ATF4/CHOP-mediated TRAIL-R2 up-regulation and sensitizing tumor cells to TRAIL-mediated cell death both in a TRAIL-R2-upregualtion dependent and independent manner. We will therefore test whether these drugs synergize with TRAIL–CDK9i in killing tumor cells. The antiapoptotic proteins XIAP, cIAP1 and cIAP2 antagonize effector caspases but additionally can inhibit the activation of initiator caspases, stabilizing LUBAC complex in the death-inducing signaling complex (DISC), which is a key mechanism modulating between apoptotic and non-apoptotic TRAIL signaling. XIAP, cIAP1 and cIAP2 can be inhibited by Smac mimetic drugs which have also shown a powerful capacity to sensitize tumor cells to TRAIL-induced apoptosis and we will therefore test whether Smac mimetics synergize with TRAIL–CDK9i in killing tumor cells.
In summary, the overall goal of this project is to evaluate the combination of different drugs with the TRAIL–CDK9i combination therapy in vitro and in vivo for possible novel therapeutic approaches in cancer therapy in the future.
4. Applied Methods and model organisms
Cell culture, immunoprecipitation, western blotting, cell death and viability assays, in-vitro drug screening, in-vivo work with different mouse tumor models, bulk and single cell RNAseq, qPCR, flow cytometry, CrispR/Cas9-mediated genetic alterations
5. Desirable skills and qualifications
- Master degree in Biology, Chemistry, Cell Biology, Biochemistry or equivalent.
- Experience with ex-vivo and in-vitro techniques, preferably also with in-vivo mouse models of cancer or other diseases.
- Knowledge on cell death and inflammation 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).
- Lafont E, …., Walczak H. TBK1 and IKKε prevent TNF-induced cell death by RIPK1 phosphorylation. Nat Cell Biol. 20(12):1389-1399, (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).