Kashkar, Hamid - B 5

The ubiquitin ligase activity of XIAP in the pathogenesis of cancer

Elevated expression of inhibitor of apoptosis proteins (IAPs) is frequently observed in cancer. Although initial studies mainly considered IAPs as cellular survival factors, accumulating recent evidence suggests that IAPs interfere with cellular inflammatory signaling by acting as E3 ubiquitin ligases.

Thus, the pro-inflammatory role of IAPs may in fact represent an important function of this protein family in human cancer. This project aims to characterize the ubiquitin ligase activity of IAPs in cancer and its therapeutic value as a novel anti-cancer therapy.

Introduction

The inhibitor of apoptosis protein (IAP) gene family encodes a group of structurally related proteins that were initially identified based on their ability to inhibit cell death. Although the precise mechanism is still debated, initial evidence showed that IAPs act as direct inhibitors of pro-apoptotic caspases. Given their cytoprotective properties and elevated expression level in many types of human cancer, great effort has been made to develop small pharmacological inhibitors of IAPs.

The application of such inhibitors, however, dramatically changed our view about IAPs and their possible role in cancer. These data showed that small molecules IAP antagonists kill cancer cells primarily by inducing the ubiquitin ligase activity of cIAPs and promoting the degradation of cIAP1 and 2 rather than by de-repressing caspase activity. The rapid degradation of cIAP1 and 2 activates both the canonical and non-canonical NF-kB signaling pathways, TNF secretion and cellular inflammatory response.

IAP antagonization promotes inflammatory destruction of vascular endothelium and inhibits tumor growth

The tumor stroma is increasingly recognized as an integral part of cancer initiation, growth, and progression. Cytokines produced by malignant cells including TNF plays a critical role in this context, by acting as an amplifier of the inflammatory milieu. Thus, cytokine production/secretion upon IAP antagonization may not only induce autocrine signaling within the tumor cells but also engage paracrine inflammatory signaling in adjacent tissues. We could recently show for the first time that the therapeutic antagonization of IAPs inhibits tumor growth by disrupting tumor vasculature.

Specifically, the treatment of mice bearing melanoma with an IAP antagonist compound A (Comp A) inhibited tumor growth not by inducing direct cytotoxicity against melanoma cells but rather by a hitherto unrecognized antiangiogenic activity against tumor vessels (Figure 1). Our detailed analysis showed that Comp A treatment induces NF-kB activity in 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, we shoed that the antiangiogenic and growth-attenuating effects of Comp A treatment were completely abolished in TNF-R knockout mice. Our data identified this novel targeting approach to be of clinical value in controlling pathological neoangiogenesis under inflammatory condition while sparing blood vessels under normal condition.

Bcl2 accelerates lymphomagenesis driven by a mutation in the adaptor protein MYD8

IAPs are naturally controlled by mitochondrial proteins such as SMAC which is released upon mitochondrial outer membrane permeabilization (MOMP). MOMP and mitochondrial antagonization of IAPs are tightly controlled by Bcl2 protein family.

In order to examine the physiological role of IAP antagonization, we have recently established a 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 accelerates lymphomagenesis (ABC-DLBCL) driven by a mutation in the adaptor protein MYD88 (Knittel et al., 2016). In a novel mouse model for ABC-DLBCL conditional mutation of MYD88 expression resulted in a lymphoproliferative disease and occasional transformation into clonal lymphomas. Lymphomagenesis could be accelerated by overexpression of BCL2.

Cross-validation experiments in human DLBCL samples revealed that BCL2 amplifications frequently co-occurred with MYD88 mutations and further in silico experiments revealed that MYD88-mutant ABC-DLBCL cells in particular display an actionable addiction to BCL2.

Perspectives

This project aims to identify the factors that are ubiquitylated by IAPs and to further characterize their role in cancer pathogenesis. Unique opportunities will then arise from the study of novel conditional IAP knock-in and –out mouse models recently generated in our laboratory.

Together with the data obtained in cell-free systems, tumor cell lines and primary tumor tissues this project will significantly increase our knowledge about how IAPs impact on cancer pathogenesis and will further provide insights into a novel mode of IAP-targeting strategy for anti-cancer therapy.

Selected publications (2015-2016)

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

Franz A, Pirson PA, Pilger D, Halder S, Achuthankutty D, Kashkar H, Ramadan K, Hoppe T. (2016). Chromatin-associated degradation is defined by UBXN-3/FAF1 to safeguard DNA replication fork progression. Nat. Commun., 7, 10612

Förster A, Grotha S, Seeger JM, Rabenhorst A, Gehring M, Raap U, Létard S, Dubreuil P, Kashkar H, Walczak H, Roers A, Hartmann K. (2015). Activation of KIT modulates the function of tumor necrosis factor-related apoptosis-inducing ligand receptor (TRAIL-R) in mast cells. Allergy, 70, 764-74

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

Coutelle O, Schiffmann LM, Liwschitz M, Brunold M, Goede V, Hallek M, Kashkar H*, Hacker UT*. (2015). Dual targeting of Angiopoetin-2 and VEGF potentiates effective vascular normalization without inducing empty basement membrane sleeves in xenograft tumors. Br J Cancer, 112:495-503 *equal contribution

Former Funding Period 01/2014 - 12/2016

Information from this funding period will not be updated anymore. New research related information is available here.


Prof. Dr. rer. nat. Hamid Kashkar

Inst. for Med. Microbiology, Immunology & Hygiene

Prof. Dr. rer. nat. 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)
Marie-Christine Albert (PhD student)
Melanie Fritsch (PhD student)
Saskia Günther (PhD student)
Fabian Schorn (PhD student)
Tanja Roth (technician)
Ramona Hoppe
(technician)

Figure 1

B16 melanoma cells were mixed with matrigel and subcutaneously injected into the flank region of recipient BL6 mice.After 7 or 10 days, tumors were treated with IAP antagonist (CompA) by subcutaneous injection next to the tumors. Tumor size was measured in 2 dimensions, and calculated volume was recorded daily (upper panel). Intratumoral vasculature of tumors at day 12 was analyzed by multiphoton microscopy using FITC–dextran staining of vessels.