Introduction

Casp8 is involved in pathogenesis of different human diseases including lymphatic disorders and recent research efforts have successfully evaluated its therapeutic potential (Emricasan in phase II-III clinical trials).

By using novel transgenic mice mimicking the therapeutic inhibition of Casp8 enzymatic activity this project will provide substantial knowledge about the role of Casp8 in lymphocyte homeostasis and its therapeutic potential in B cell lymphoma.

Extrinsic apoptosis is a central regulatory mechanism of the B cell compartment and its dysfunction causes B cell diseases such as autoimmunity and lymphoma. Caspase-8 (Casp8) is the initiator caspase of the extrinsic apoptotic pathway and was initially described to induce this non-lytic programmed cell death (apoptosis). Casp8 was further characterized as the key inhibitor of necroptosis (lytic regulated cell death) and was shown to be indispensible for embryonic development.

Recently, we discovered an additional function of Casp8 demonstrating that enzymatic inactive Casp8 acts as a scaffold and induces pyroptosis (lytic inflammatory regulated cell death) and inflammation. Our studies explored Casp8 as a central molecular switch controlling apoptosis, necroptosis and pyroptosis. Based on this striking observation, we aim to investigate the role of Casp8 scaffold function versus its enzymatic activity in B cell homeostasis.

Our Aims

  1. B cell-specific roles of Casp8. Here we will specifically delete Casp8 or express inactive Casp8C362S specifically in B cells and will study B cell development in mouse.
  2. Role of Casp8 in pathogenesis of chronic lymphocytic leukemia (CLL). Here we will use CLL mouse model and delete Casp8 or express inactive Casp8C362S specifically in B cells and will study CLL development/progression in mice.

Previous Work

In order to investigate the physiologic role of Casp8 enzymatic activity we recently generated a knock-in mouse line (using CRISPR/Cas9 gene-editing) expressing enzymatically inactive Casp8C362S Fritsch et al., Nature 2019). Similar to Casp8-/-, lack of Casp8 enzymatic activity caused lethality in Casp8C362S/C362S embryos.

Our data revealed that the inhibition of necroptosis by MLKL deletion (Mlkl-/-) rescued the embryonic development in Casp8-/- mice but unexpectedly failed to prevent embryonic lethality of Casp8C362S/C362S mice, indicating that the expression of enzymatically inactive Casp8C362S causes necroptosis-independent death during embryonic development. Further extensive analysis using tissue-specific expression of Casp8C362S discovered a novel function for Casp8 demonstrating that enzymatically inactive Casp8C362S acts as a scaffold and induces pyroptosis.

Figure 1
CMMC Research
Fig1. Schematic illustration of Casp8 functions. (Fritsch et al., Nature, 2019).

We could show that Casp8C362S induces the nucleation of the inflammasome adaptor ASC, promotes the activation of the inflammatory caspase-1 (Casp1) and results in pyroptosis as well as secretion of inflammatory cytokines IL-1β and IL-18. Accordingly, only the combined deletion of MLKL together with ASC (Casp8C362S/C362S/Mlkl-/-/Asc-/-) or Casp1 (Casp8C362S/C362S/Mlkl-/-/Casp1-/-) rescued embryonic lethality of Casp8C362S/C362S mice.

Our studies explored a novel role of Casp8 in cellular death and inflammatory cascades and represent Casp8 as a central molecular switch controlling apoptosis, necroptosis and pyroptosis (Fritsch et al., Nature 2019).

  • Pallasch CP, Schulz A, Kutsch N, Schwamb J, Hagist S, Kashkar H, Ultsch A, Wickenhauser C, Hallek M, Wendtner CM. Overexpression of TOSO in CLL is triggered by B-Cell receptor signaling and associated with progressive disease. Blood 2008, 112:4213-9
  • Frenzel LP, Patz m, Pallasch CP, Brinker R, Claasen J, Schulz A, Hallek M, Kashkar H*, and Wendtner CM*. Novel X-linked inhibitor of apoptosis inhibiting compound as sensitizer for TRAIL-mediated apoptosis in chronic lymphocytic leukaemia with poor prognosis. Br J Haematol. 2011, 152(2):191-200. *equal contribution
  • Huelsemann MF, Patz M, Beckmann L, Brinkmann K, Otto T, Fandrey J, Becker HJ, Theurich S, von Bergwelt-Baildon M, Pallasch CP, Zahedi RP, Kashkar H, Reinhardt HC, Hallek M, Wendtner CM, Frenzel LP. Hypoxia-induced p38 MAPK activation reduces Mcl-1 expression and facilitates sensitivity towards BH3 mimetics in chronic lymphocytic leukemia. Leukemia 2015, 29:981-4
  • 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 2016, 127(22):2732-41. * and§ equal contribution
  • Chauhan D*, Bartok E*, Gaidt MM, Bock FJ, Herrmann J, Seeger JM, Broz P, Beckmann R, Kashkar H, Tait SWG, Müller R, Hornung V. BAX/BAK induced apoptosis results in caspase-8 dependent IL-1b maturation in primary macrophages Cell Rep. 2018, 25(9):2354-2368
  • Fritsch M, Günther SD, Schwarzer R, Albert MC, Schorn F, Werthenbach JP, Schiffmann LM, Stair N, Stocks H, Seeger JM, Lamkanfi M, Krönke M, Pasparakis M, Kashkar H. Caspase-8 is the molecular switch for apoptosis, necroptosis and pyroptosis. Nature 575:683-687
  • Thomalla D, Beckmann L, Grimm C, Oliverio M, Meder L, Herling C, Nieper P, Merkel O, Feldmann T, Lorsy E, da Palma Guerreiro A, von Jan J, Kisis I, Wasserburger E, Claasen J, Faitschuk-Meyer E, Altmüller J, Nürnberg P, Yang TP, Lienhard M, Herwig R, Kreuzer KA, Pallasch C, Buettner R, Schäfer S, Hartley J, Abken H, Peifer M, Kashkar H, Knittel G, Eichhorst B, Ullrich R, Herling M, Reinhardt HC, Hallek M, Schweiger M, Frenzel L. (2022). Deregulation and epigenetic modification of BCL2-family genes cause resistance to venetoclax in hematologic malignancies. Blood doi: 10.1182/blood.2021014304. Online ahead of print.
  • Simpson DS, Pang J, Weir A, Kong IY, Fritsch M, Rashidi M, Cooney JP, Davidson K, Speir M, Djajawi MT, Hughes S, Anderton H, Doerflinger M, Deng Y, Huang AS, Conos SA, Tye H, Rahman A, Norton RS, Nicholson SE, Burgio G, Man SM, Herold M, Hawkins ED, Lawlor KE, Strasser A, Silke J, Pelligrini M, Kashkar H, Feltham R,*, Vince JE. (2022). IFNγ primes macrophages for pathogen ligand-induced killing via a caspase-8 and mitochondrial cell death pathway licensed by nitric oxide. Immunity 55(3):423-441.e9. doi: 10.1016/j.immuni.2022.01.003.
  • Thomalla D, Beckmann L, Grimm C, Oliverio M, Meder L, Herling C, Nieper P, Merkel O, Feldmann T, Lorsy E, da Palma Guerreiro A, von Jan J, Kisis I, Wasserburger E, Claasen J, Faitschuk-Meyer E, Altmüller J, Nürnberg P, Yang TP, Lienhard M, Herwig R, Kreuzer KA, Pallasch C, Buettner R, Schäfer S, Hartley J, Abken H, Peifer M, Kashkar H, Knittel G, Eichhorst B, Ullrich R, Herling M, Reinhardt HC, Hallek M, Schweiger M, Frenzel L. (2022). Deregulation and epigenetic modification of BCL2-family genes cause resistance to venetoclax in hematologic malignancies. Blood doi: 10.1182/blood.2021014304. Online ahead of print.
  • Daoud M, Broxtermann PN, Schorn F, Werthenbach JP, Seeger JM, Schiffmann LM, Brinkmann K, Vucic D, Tuting T, Mauch C, Kulms D, Zigrino P, and Kashkar H (2022). XIAP promotes melanoma growth by inducing tumour neutrophil infiltration. EMBO Rep, e53608. doi:10.15252/embr.202153608.
  • Simpson DS, Pang J, Weir A, Kong IY, Fritsch M, Rashidi M, Cooney JP, Davidson K, Speir M, Djajawi MT, Hughes S, Anderton H, Doerflinger M, Deng Y, Huang AS, Conos SA, Tye H, Rahman A, Norton RS, Nicholson SE, Burgio G, Man SM, Herold M, Hawkins ED, Lawlor KE, Strasser A, Silke J, Pelligrini M, Kashkar H, Feltham R,*, Vince JE. (2022). IFNγ primes macrophages for pathogen ligand-induced killing via a caspase-8 and mitochondrial cell death pathway licensed by nitric oxide. Immunity 55(3):423-441.e9. doi: 10.1016/j.immuni.2022.01.003.
  • Flores-Romero H, Hohorst L, John M, Albert MC, King LE, Beckmann L, Szabo T, Hertlein V, Luo X, Villunger A, Frenzel LP, Kashkar H, and Garcia-Saez AJ (2022). BCL-2-family protein tBID can act as a BAX-like effector of apoptosis. EMBO J41, e108690. doi:10.15252/embj.2021108690.
  • Flores-Romero H, Hohorst L, John M, Albert MC, King LE, Beckmann L, Szabo T, Hertlein V, Luo X, Villunger A, Frenzel LP, Kashkar H, and Garcia-Saez AJ (2021). BCL-2-family protein tBID can act as a BAX-like effector of apoptosis. EMBO J, e108690. doi:10.15252/embj.2021108690.
  • Flumann R, Rehkamper T, Nieper P, Pfeiffer P, Holzem A, Klein S, Bhatia S, Kochanek M, Kisis I, Pelzer BW, Ahlert H, Hauer J, da Palma Guerreiro A, Ryan JA, Reimann M, Riabinska A, Wiederstein J, Kruger M, Deckert M, Altmuller J, Klatt AR, Frenzel LP, Pasqualucci L, Beguelin W, Melnick AM, Sander S, Montesinos-Rongen M, Brunn A, Lohneis P, Buttner R, Kashkar H, Borkhardt A, Letai A, Persigehl T, Peifer M, Schmitt CA, Reinhardt HC, and Knittel G (2021). An Autochthonous Mouse Model of Myd88- and BCL2-Driven Diffuse Large B-cell Lymphoma Reveals Actionable Molecular Vulnerabilities. Blood Cancer Discov2, 70-91. doi:10.1158/2643-3230.BCD-19-0059.
  • Geueke A, Mantellato G, Kuester F, Schettina P, Nelles M, Seeger JM, Kashkar H, and Niemann C (2021). The anti-apoptotic Bcl-2 protein regulates hair follicle stem cell function. EMBO Rep22, e52301. doi:10.15252/embr.202052301.
  • Theobald SJ, Grab J, Fritsch M, Suarez I, Eisfeld HS, Winter S, Koch M, Holscher C, Pasparakis M, Kashkar H, and Rybniker J (2021). Gasdermin D mediates host cell death but not interleukin-1beta secretion in Mycobacterium tuberculosis-infected macrophages. Cell Death Discov7, 327. doi:10.1038/s41420-021-00716-5.
  • Theobald SJ, Simonis A, Georgomanolis T, Kreer C, Zehner M, Eisfeld HS, Albert MC, Chhen J, Motameny S, Erger F, Fischer J, Malin JJ, Grab J, Winter S, Pouikli A, David F, Boll B, Koehler P, Vanshylla K, Gruell H, Suarez I, Hallek M, Fatkenheuer G, Jung N, Cornely OA, Lehmann C, Tessarz P, Altmuller J, Nurnberg P, Kashkar H, Klein F, Koch M, and Rybniker J (2021). Long-lived macrophage reprogramming drives spike protein-mediated inflammasome activation in COVID-19. EMBO Mol Med13, e14150. doi:10.15252/emmm.202114150.
  • He L, Sehrawat TS, Verma VK, Navarro-Corcuera A, Luo X, Katsumi T, Chen J, Arab JP, Cao S, Shah S, Kashkar H, Gores GJ, Malhi H, Shah VJ. (2021). XIAP knockdown in alcohol-associated liver disease models exhibits divergent in vitro and in vivo phenotypes owing to a potential zonal inhibitory role of SMAC. Front Physiol. 12:664222. doi: 10.3389/fphys.2021.664222.
  • Albert MC, Brinkmann K, Pokrzywa W, Gunther SD, Kronke M, Hoppe T, and Kashkar H (2020). CHIP ubiquitylates NOXA and induces its lysosomal degradation in response to DNA damage. Cell Death Dis 11, 740.
     
  • Gunther SD, Fritsch M, Seeger JM, Schiffmann LM, Snipas SJ, Coutelle M, Kufer TA, Higgins PG, Hornung V, Bernardini ML, Honing S, Kronke M, Salvesen GS, and Kashkar H (2020). Cytosolic Gram-negative bacteria prevent apoptosis by inhibition of effector caspases through lipopolysaccharide. Nat Microbiol 5, 354-67.
     
  • Haumann S, Boix J, Knuever J, Bieling A, Vila Sanjurjo A, Elson JL, Blakely EL, Taylor RW, Riet N, Abken H, Kashkar H, Hornig-Do HT, and Wiesner RJ (2020). Mitochondrial DNA mutations induce mitochondrial biogenesis and increase the tumorigenic potential of Hodgkin and Reed-Sternberg cells. Carcinogenesis 10.1093/carcin/bgaa032.
     
  • Schiffmann LM, Werthenbach JP, Heintges-Kleinhofer F, Seeger JM, Fritsch M, Gunther SD, Willenborg S, Brodesser S, Lucas C, Jungst C, Albert MC, Schorn F, Witt A, Moraes CT, Bruns CJ, Pasparakis M, Kronke M, Eming SA, Coutelle O, and Kashkar H (2020). Mitochondrial respiration controls neoangiogenesis during wound healing and tumour growth. Nat Commun 11.
  • Lampl S, Janas MK, Donakonda S, Brugger M, Lohr K, Schneider A, Manske K, Sperl LE, Wettmarshausen J, Müller C, Laschinger M, Hartmann D, Hüser N, Perrochi F, Schmidt-Kopplin P, Hagn F, Zender L, Hornung V, Borner C, Pichlmair A, Kashkar H, Klingenspor M, Prinz M, Schreiner S, Conrad M, Jost PJ, Zischka H, Steiger K, Krönke M, Zehn D, Protzer U, Heikenwälder M, Knolle PA, Wohlleber D. (2020). Reduced mitochondrial resilience enables non-canonical induction of apoptosis after TNFR signaling in virus-infected hepatocytes. J. Hepatol 2020, 73(6):1347-1359
Prof. Dr. Hamid Kashkar
Prof. Dr. Hamid Kashkar

Institute for Molecular Immunology | CECAD Research Center

CMMC - PI - B 06
Head - CMMC Cell Sorting Facility

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Institute for Molecular Immunology | CECAD Research Center

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50931 Cologne

Curriculum Vitae - Hamid Kashkar
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