Hamid Kashkar - B 4

The role of mitochondrial apoptosis in macrophages and macrophage-derived tumor-promoting inflammation

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

Apoptosis represents a mode of cell death that is used by multicellular organisms to dispose irreparably damaged cells. Apoptotic death is a tightly regulated tissue-safe-guard-program and its dysregulation has been associated with tissue malfunction and the incidence of multiple human diseases including cancer, inflammatory disorders, tissue degeneration and immune deficiency. Owing to its involvement in various diseases, apoptosis has been viewed as a promising therapeutic target and research efforts have lately been able to develop drugs restoring the apoptotic machinery in human diseases. 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 the use of small compounds restoring mitochondrial apoptosis including peptidomimetics inducing MOMP (BH3 mimetics such as ABT-199 or ABT-737) or inhibiting IAPs (Smac mimetics). Furthermore, our recent discoveries showed for the first time that the mitochondrial apoptotic pathway interferes with and impacts on cellular inflammatory signaling and immunity against microbial pathogens.

IAP antagonization promotes inflammatory destruction of vascular endothelium (Witt et al., 2015)

In this study, we show for the first time that the therapeutic antagonization of inhibitor of apoptosis proteins (IAPs) inhibits B16 melanoma growth by disrupting tumor vasculature. Specifically, the treatment of mice bearing B16 melanoma with an IAP antagonist compound A (Comp A) inhibits tumor growth not by inducing direct cytotoxicity against B16 cells but rather by a hitherto unrecognized antiangiogenic activity against tumor vessels. Our detailed analysis showed that Comp A treatment induces NF-κB activity in B16 tumor cells and facilitates the production of TNF. In the presence of Comp A, endothelial cells (ECs) become highly susceptible to TNF and undergo apoptotic cell death. Accordingly, the antiangiogenic and growth-attenuating effects of Comp A treatment were completely abolished in TNF-R knockout mice. This novel targeting approach could be of clinical value in controlling pathological neoangiogenesis under inflammatory condition while sparing blood vessels under normal condition.

B cell-specific inhibition of mitochondrial apoptosis accelerates lymphomagenesis (ABC-DLBCL) driven by a mutation in the adaptor protein MYD88 (Knittel & Seeger et al., 2016)

To address the physiological role of MOMP and mitochondrial apoptosis in different tissues, we have recently established the conditional BCL2tg 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 BCL2tg/STOP with CD19-Cre) accelerates lymphomagenesis (ABC-DLBCL) driven by a mutation in the adaptor protein MYD88. Furthermore, analyses of human DLBCL genome sequencing data confirmed that BCL2 amplifications frequently co-occurred with MYD88 mutations, further validating our approach. Finally, in silico experiments revealed that MYD88-mutant ABC-DLBCL cells in particular display an actionable addiction to BCL2. Altogether, 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.

CHIP mediates K63-linked ubiquitination and lysosomal degradation of Noxa (Albert et al., unpublished)

The BH3-only protein Noxa represents one of the critical mediators of DNA-damage-induced cell death by counteracting the anti-apoptotic protein Mcl-1. However, molecular mechanisms that govern Noxa turnover during the course of cellular homeostasis and stress responses remain largely unknown. Here we discovered a cellular regulatory circuit that involves CHIP-mediated ubiquitination and lysosomal degradation of Noxa. Our results showed that upon expression, Noxa protein is primarily targeted to mitochondria via direct interaction with Mcl-1 in order to fulfill its pro-apoptotic function. Cytosolic unbound Noxa, beyond the threshold to antagonize Mcl-1, is subject to ubiquitination by CHIP. Poly-ubiquitinated Noxa in turn is targeted to lysosomal degradation to avoid cytosolic accumulation of Noxa. Our extensive biochemical studies showed that CHIP and Mcl-1 are mutually exclusive as they both require an unmasked BH3 domain. Whereas Mcl-1 inhibits Noxa ubiquitination by masking BH3 domain, prior ubiquitination of Noxa at a single lysine residue within the BH3 domain of Noxa efficiently blocks its interaction with Mcl-1. This process safeguards the balance between Noxa and Mcl-1 and represents a novel cellular mechanism that governs Noxa protein homeostasis under physiologic and stress conditions.

Perspectives 

Macrophages are the major regulators of inflammation, coordinate tissue homeostasis/repair and provide the forefront of innate immune defense. These fundamental functions can be manipulated by a number of non-autonomous insults resulting in a causal association of macrophages with diseases such as cancer. Indeed, macrophages represent the major components of tumor microenvironment and the analysis of their function has led to the dissection of tumor-promoting inflammation. It is increasingly evident that macrophage-derived inflammation can be modulated through macrophage-apoptosis either by limiting macrophage life-span or by interfering with intracellular inflammatory signaling. However, how this dynamic process is regulated and coordinates tissue inflammation is not yet completely understood. Here we aim to decipher the role of the mitochondrial apoptotic pathway in macrophage life span and macrophage-derived tissue inflammation in cancer. Unique opportunities will arise from the study of novel conditional Bcl2 knock-in mouse (inhibiting mitochondrial apoptosis) enabling us to block mitochondrial apoptosis selectively in myeloid compartment and the use of small molecules BH3 mimetic (ABT-199) and SMAC mimetic (birinapant) (promoting apoptosis) currently used in clinical trial for cancer patients. These analyses will provide the first evidence about how macrophage stress responses to systemic anti-cancer treatment may coordinate tumor associated inflammatory environment and impact on cancer progression.

Selected publications

Saita S, Nolte H, Fiedler KU, Kashkar H, Venne AS, Zahedi RP, Krüger M, Langer T. (2017).PARL mediates Smac proteolytic maturation in mitochondria to promote apoptosis. Nat Cell Biol, 19(4):318-328

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. * and § equal contribution

Witt A, Seeger JM, Coutelle O, Zigrino P, Broxtermann P, Andree M, Brinkmann K, Jüngst C, Schauss AC, Schüll S, Wohlleber D, Knolle P, Krönke M, Mauch C, Kashkar H. (2015). IAP antagonization induces inflammatory destruction of vascular endothelium EMBO Rep., 16:719-727

Schüll S, Günther SD, Brodesser S, Seeger JM, Tosetti B, Wiegmann K, Pongratz C, Diaz F, Witt A, Andree M, Brinkmann K, Krönke M, Wiesner RJ, Kashkar H. (2015). Cytochrome c oxidase deficiency accelerates mitochondrial apoptosis by activating ceramide synthase 6. Cell Death Dis., 6:e1691


Prof. Dr. Hamid Kashkar

Inst. for Med. Microbiology, Immunology and Hygiene (IMMIH)

Prof. Dr. Hamid Kashkar

Principal Investigator B 4

h.kashkar@uni-koeln.de

Work +49 221 478 84091

Institute for Medical Microbiology, Immunology and Hygiene (IMMIH)
Goldenfelsstraße 19-21
50935 Cologne

http://immih.uk-koeln.de/forschung/ag-kashkar

Publications - Hamid Kashkar

Link to PubMed

Group Members

Jens-Michael Seeger (PostDoc)
Pia Nora Broxtermann (PostDoc)
Saskia Günther (PostDoc)
Lars Schiffmann (PostDoc)
Marie-Christine Albert (PhD student)
Melanie Fritsch (PhD student)
Fabian Schorn (PhD student)
Jan Paul Werthenbach (PhD student)
Mila Daoud (PhD student)
Maureen Menning (technician)
Tanja Roth (technician)
Ramona Hoppe (technician)
Ali Manav (technician)