Hamid Kashkar - B 4

Mitochondria represent one of the central cellular sensory systems in response to a variety of stress cues and are involved in several cellular functions including cell death. In particular, mitochondrial outer membrane permeabilization (MOMP) and the release of several mitochondrial proteins induce cell death but also interfere with cellular immune immune response. We aim to decipher how mitochondria regulate death and inflammatory signalling and impact on cancer and infectious diseases.

Introduction

Intrinsic apoptotic pathway is initiated by mitochondrial outer membrane permeabilisation (MOMP) and leads to the release of pro-apoptotic factors including cytochrome c and SMAC from the mitochondrial inter-membrane space (IMS) into the cytosol. Whereas cytosolic cytochrome c initiates the proteolytic activation of the initiator caspase-9, cytosolic SMAC binds and inhibits the inhibitors of apoptosis proteins (IAPs) thereby potentiating caspase activity or interfering with cellular inflammatory signaling initiated by TNF or intracellular infection. MOMP is tightly regulated by Bcl2 protein family which has been viewed as a promising therapeutic target and research efforts have lately been able to develop drugs activating Bcl2 proteins and inducing MOMP in cancer. The longstanding scientific goal of our group is to discover the role and the control of cell death in tissue homeostasis with particular emphasis on the mitochondrial apoptotic pathway. In our previous work, we focused on the molecular mechanisms involved in apoptosis resistance in cancer and evaluated pathophysiological role and therapeutic value of Bcl2 and IAPs in cancer. Our recent discoveries showed for the first time that the mitochondrial apoptotic pathway interferes with and impacts on cellular inflammatory signalling and immunity against microbial pathogens. 

Mitochondrial apoptosis in B cell lymphoma-genesis and inflammation 

To address the physiological role of MOMP and mitochondrial apoptosis in different tissues, we have recently established the conditional BCL2^tg mouse line for tissue-specific overexpression of anti-apoptotic Bcl2. This transgenic mouse enables us to study the role of MOMP and Bcl2 in different tissues under healthy and disease conditions. Accordingly, we could recently show that specific overexpression of Bcl2 in B cells (by crossbreeding BCL2^tg/STOP with CD19-Cre) accelerates lymphoma-genesis (ABC-DLBCL) driven by a mutation in the adaptor protein MYD88. Here we generated a novel autochthonous mouse model of ABC-DLBCL that could be used as a preclinical platform for the development and validation of novel therapeutic approaches for the treatment of ABC-DLBCL. IL-1β is a cytokine of pivotal importance to the orchestration of inflammatory responses. Synthesized as an inactive pro-cytokine, IL-1β requires proteolytic maturation by inflammatory caspase-1 to gain biological activity. Mecha-nistically, we showed that vioprolides (cyclic peptides isolated from myxobacteria) inhibit MCL-1 and BCL2, which in turn triggers MOMP. MOMP results in the release of pro-apoptotic factors which in turn causes antagonization/depletion of IAPs. IAP depletion subsequently results in activation of apoptotic caspase-8 which provides cleavage and maturation of IL-1β (Fig. 1). These results establish the caspase-8 as a pro-inflammatory checkpoint that senses the perturbation of mitochondrial integrity and activates IL-1β secretion.

The anti-apoptotic Bcl2 member, MCL-1, regulates NOXA turnover by antagonizing CHIP-mediated lysosomal degradation of NOXA

The BH3-only protein NOXA is a key regulator of mitochondrial apoptosis by counteracting the anti-apoptotic Bcl2 protein MCL-1. We could recently discovere the E3 ubiquitin ligase CHIP as a new regulatory element that is involved in controlling NOXA protein stability and function. Our findings reveal that CHIP and MCL-1 are mutually exclusive and differentially define the fate of NOXA in response to genotoxic stress. Whereas MCL-1 triggers mitochondrial association of NOXA viadirect binding, CHIP ubiquitylates NOXA and promotes its lysosomal degradation. Our data show thatthe threshold of NOXA expression and its pro-apoptotic activity aredictatedby MCL-1. NOXA levels beyond this threshold are effectively removed by lysosomal protein degradation, which is engaged after CHIP-mediated ubiquitylation of NOXA. These results shed new light on how NOXA-related cellular stress response pathways are regulated and identified CHIP-dependent ubiquitylation of NOXA as a new cellular process that prevents cytoplasmic accumulation of NOXA after DNA damage.

Perspectives

One of the major achievements of this project was the identification of a molecular link between mito-chondrial apoptosis, caspase-8 and inflammasome activation in macrophages. These data clearly showed that cytotoyxic anti-cancer treatments specifically inducing mitochondrial apoptosis involve caspase-8 and provoke macrophage-derived inflammation. Whether and how this macrophage response interferes with and impacts on tumour growth is currently under investigation. 

1. Knittel G, Liedgens P, Korovkina D, Seeger JM, Al-Baldawi Y, Al-Maarri M, Fritz C, Vlantis K, Bezhanova S, Scheel AH, Wolz OO, Reimann M, Möller P, López C, Schlesner M, Lohneis P, Weber AN, Trümper L, Consortium IM, Staudt LM, Ortmann M, Pasparakis M, Siebert R, Schmitt CA, Klatt AR, Wunderlich FT, Schäfer SC, Persigehl T, Montesinos-Rongen M, Odenthal M, Büttner R, Frenzel LP^§, Kashkar H^§, Reinhardt HC^§. (2016). B cell-specific conditional expression of Myd88p.L252P leads to the development of diffuse large B cell lymphoma in mice. Blood, 127(22), 2732-41. § equal contribution           

2. Schiffmann LM, Brunold M, Liwschitz M, Goede V, Loges S, Wroblewski M, Quaas A, Alakus H, Stippel D, Bruns CJ, Hallek M, Kashkar H, Hacker UT, Coutelle O. (2017). A combination of low-dose bevacizumab and imatinib enhances vascular normalisation without inducing extracellular matrix deposition. Br J Cancer. 2017, 116(5), 600-608

3. Saita S, Nolte H, Fiedler KU, Kashkar H, Venne AS, Zahedi RP, Krueger M, Langer M. (2017). PARL mediates Smac proteolytic maturation in mitochondria to promote apoptosis. Nat. Cell Biol., 19, 318–328                                  

4. Chauhan D*, Bartok E*, Gaidt MM, Bock FJ, Herrmann J, Seeger JM, Broz P, Beckmann R, Kashkar H, Tait SWG, Müller R, Hornung V. (2018). BAX/BAK induced apoptosis results in caspase-8 dependent IL-1bmaturation in primary macrophages Cell Rep., 25(9), 2354-2368 

5. Schiffmann LM, Fritsch M, Gebauer F, Günther SD, Stair NR, Seeger JM, Thangarajah F, Dieplinger G, Bludau M, Alakus H, Göbel H, Quaas A, Zander T, Hilberg F, Bruns CJ, Kashkar H*, Coutelle O*. (2019). Tumour-infiltrating neutrophils counteract anti-VEGF therapy in metastatic colorectal cancer. Br J Cancer. 2019, 120(1), 69-78 *equal contribution

6. Habich M, Salscheider SL, Murschall LM, Hoehne MN, Fischer M, Schorn F, Petrungaro C, Ali M, Erdogan AJ, Abou-Eid S, Kashkar H, Dengjel J, Riemer J. (2019). Vectorial import via a metastable disulphide-linked complex allows for a quality control step and import by the mitochondrial disulphide relay Cell Rep., 26(3), 759-774                                                  

7. Gräb J, Suarez I, van Gumpel E, Winter S, Schreiber F, Esser A, Hölscher C, Fritsch M, Herb M, Schramm M, Wachsmuth L, Pallasch C, Pasparakis M, Kashkar H, Rybniker J. (2019). Corticosteroids inhibit Mycobacterium tuberculosis-induced necrotic host cell death by abrogating mitochondrial membrane permeability transition. Nat. Commun., 10, 688                                

8. Holzer T, Probst K, Etich J, Auler M, Georgieva V, Bluhm B, Frie C, Heilig J, Niehoff A, Nüchel J, Plomann M, Seeger JM, Kashkar H, Baris OR, Wiesner RJ, Brachvogel B. (2019). Respiratory chain inactivation links cartilage-mediated growth retardation to mitochondrial diseases. J. Cell Biol., 218(6):1853-1870