Functional characterization of the CRBN E3 ubiquitin ligase, the target of lenalidomide

• Applicant: Professor Dr. Jan Krönke
• Subject Area: Hematology, Oncology
• Term: from 2015 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 263416443

• Final Report Year: 2024

Final Report Abstract

The immunomodulatory drug (IMiD) thalidomide, that became notorious in the 1950s for causing teratogenicity, and its more potent analogs lenalidomide and pomalidomide are highly active and approved treatments for multiple myeloma. In 2014, we revealed the molecular mechanism of IMiDs: we found that IMiDs modulate the substrate specificity of cereblon (CRBN) which is the substrate adaptor of the CRBN CRL4 E3 ubiquitin ligase. Thereby, thalidomide and its analogs induce ubiquitination and degradation of the lymphoid transcription factors ikaros (IKZF1) and aiolos (IKZF3) what results in growth arrest of mutlilple myeloma cells and enhances T-cell activity by inducing interleukin-2 and other cytokines. In the first funding period of the Emmy Noether-Program we could uncover the mechanism of lenalidomide in myelodysplastic syndrome (MDS) with del(5q): Lenalidomide, but not the other IMiDs, induce CRBN-mediated degradation of casein kinase 1α (CK1α), which is haploisufficient in MDS with del(5q) via CRBNCRL4 providing a therapeutic window. We further found that non-conserved amino acids in CRBN are responsible for the primary IMiD-insensitivity of mice providing a basis for the generation of IMiD-sensitive mouse models. Since our findings were published the concept of targeted protein degradation evolved to a new key technology in drug development. In collaboration with chemical biologists we succesfully generated and validated a large series of new IMiD analogs and proteolysis targeting chimera (PROTAC) targeting cancerassociated proteins. We further explored the the function of CRBN including newly identified binding proteins like FAM46C. A major aim of the Emmy Noether funding was to elucidate resistance mechanisms and predictive markers for IMiDs in multiple myeloma, which are both of high clinical relevance. Applying genetics, transcriptomics, and proteomics on primary patient samples and integration with functional studies revealed genetic and non-genetic resistance mechanisms for IMiDs, identify new therapeutic targets and prognostic and predictive markers that allow treatment guidance. In conclusion, the results of this project revealed new mechnisms of post-translational regulation implicated in the biology and therapy of cancer that may translate to new and more effective treatments for cancer and other diseases in the future.

Publications

• Frequency and prognostic impact of casein kinase 1A1 mutations in MDS patients with deletion of chromosome 5q. Leukemia, 29(9), 1942-1945.
  Heuser, M.; Meggendorfer, M.; Cruz, M. M. A.; Fabisch, J.; Klesse, S.; Köhler, L.; Göhring, G.; Ganster, C.; Shirneshan, K.; Gutermuth, A.; Cerny-Reiterer, S.; Krönke, J.; Panagiota, V.; Haferlach, C.; Koenecke, C.; Platzbecker, U.; Thiede, C.; Schroeder, T.; Kobbe, G. ... & Thol, F.
  
• Lenalidomide induces ubiquitination and degradation of CK1α in del(5q) MDS. Nature, 523(7559), 183-188.
  Krönke, Jan; Fink, Emma C.; Hollenbach, Paul W.; MacBeth, Kyle J.; Hurst, Slater N.; Udeshi, Namrata D.; Chamberlain, Philip P.; Mani, D. R.; Man, Hon Wah; Gandhi, Anita K.; Svinkina, Tanya; Schneider, Rebekka K.; McConkey, Marie; Järås, Marcus; Griffiths, Elizabeth; Wetzler, Meir; Bullinger, Lars; Cathers, Brian E.; Carr, Steven A. ... & Ebert, Benjamin L.
  
• IKZF1 expression is a prognostic marker in newly diagnosed standard-risk multiple myeloma treated with lenalidomide and intensive chemotherapy: a study of the German Myeloma Study Group Leukemia, 31(6), 1363-1367.
  Krönke, J.; Kuchenbauer, F.; Kull, M.; Teleanu, V.; Bullinger, L.; Bunjes, D.; Greiner, A.; Kolmus, S.; Köpff, S.; Schreder, M.; Mügge, L.-O.; Straka, C.; Engelhardt, M.; Döhner, H.; Einsele, H.; Bassermann, F.; Bargou, R.; Knop, S. & Langer, C.
  
• Prognostic impact of Ikaros expression in lenalidomide-treated multiple myeloma. Oncotarget, 8(63), 106163-106164.
  Krönke, Jan; Knop, Stefan & Langer, Christian
  
• Homo-PROTACs for the Chemical Knockdown of Cereblon. ACS Chemical Biology, 13(9), 2771-2782.
  Steinebach, Christian; Lindner, Stefanie; Udeshi, Namrata D.; Mani, Deepak C.; Kehm, Hannes; Köpff, Simon; Carr, Steven A.; Gütschow, Michael & Krönke, Jan
  
• A MedChem toolbox for cereblon-directed PROTACs. MedChemComm, 10(6), 1037-1041.
  Steinebach, Christian; Sosič, Izidor; Lindner, Stefanie; Bricelj, Aleša; Kohl, Franziska; Ng, Yuen Lam Dora; Monschke, Marius; Wagner, Karl G.; Krönke, Jan & Gütschow, Michael
  
• Chemical Inactivation of the E3 Ubiquitin Ligase Cereblon by Pomalidomide-based Homo-PROTACs. Journal of Visualized Experiments.
  Lindner, Stefanie; Steinebach, Christian; Kehm, Hannes; Mangold, Martin; Gütschow, Michael & Krönke, Jan
  
• PROTAC-mediated crosstalk between E3 ligases. Chemical Communications, 55(12), 1821-1824.
  Steinebach, Christian; Kehm, Hannes; Lindner, Stefanie; Vu, Lan Phuong; Köpff, Simon; López, Mármol Álvaro; Weiler, Corinna; Wagner, Karl G.; Reichenzeller, Michaela; Krönke, Jan & Gütschow, Michael
  
• Systematic exploration of different E3 ubiquitin ligases: an approach towards potent and selective CDK6 degraders. Chemical Science, 11(13), 3474-3486.
  Steinebach, Christian; Ng, Yuen Lam Dora; Sosič, Izidor; Lee, Chih-Shia; Chen, Sirui; Lindner, Stefanie; Vu, Lan Phuong; Bricelj, Aleša; Haschemi, Reza; Monschke, Marius; Steinwarz, Elisabeth; Wagner, Karl G.; Bendas, Gerd; Luo, Ji; Gütschow, Michael & Krönke, Jan
  
• Cereblon enhancer methylation and IMiD resistance in multiple myeloma. Blood, 138(18), 1721-1726.
  Haertle, Larissa; Barrio, Santiago; Munawar, Umair; Han, Seungbin; Zhou, Xiang; Vogt, Cornelia; Fernández, Rafael Alonso; Bittrich, Max; Ruiz-Heredia, Yanira; Da Viá, Matteo; Zovko, Josip; Garitano-Trojaola, Andoni; Bolli, Niccolò; Ruckdeschel, Anna; Stühmer, Thorsten; Chatterjee, Manik; Kull, Miriam; Krönke, Jan; Agirre, Xabier ... & Kortüm, K. Martin
  
• Comprehensive CRISPR-Cas9 screens identify genetic determinants of drug responsiveness in multiple myeloma. Blood Advances, 5(9), 2391-2402.
  Bohl, Stephan R.; Schmalbrock, Laura K.; Bauhuf, Imke; Meyer, Tatjana; Dolnik, Anna; Szyska, Martin; Blätte, Tamara J.; Knödler, Sarah; Röhner, Linda; Miller, Denise; Kull, Miriam; Langer, Christian; Döhner, Hartmut; Letai, Anthony; Damm, Frederik; Heckl, Dirk; Bullinger, Lars & Krönke, Jan
  
• Generation of a lenalidomide-sensitive syngeneic murine in vivo multiple myeloma model by expression of Crbn. Experimental Hematology, 93, 61-69.e4.
  Röhner, Linda; Ng, Yuen Lam Dora; Scheffold, Annika; Lindner, Stefanie; Köpff, Simon; Brandl, Andreas; Beilhack, Andreas & Krönke, Jan
  
• Influence of Linker Attachment Points on the Stability and Neosubstrate Degradation of Cereblon Ligands. ACS Medicinal Chemistry Letters, 12(11), 1733-1738.
  Bricelj, Aleša; Dora, Ng Yuen Lam; Ferber, Dominic; Kuchta, Robert; Müller, Sina; Monschke, Marius; Wagner, Karl G.; Krönke, Jan; Sosič, Izidor; Gütschow, Michael & Steinebach, Christian
  
• The IKZF1–IRF4/IRF5 Axis Controls Polarization of Myeloma-Associated Macrophages. Cancer Immunology Research, 9(3), 265-278.
  Mougiakakos, Dimitrios; Bach, Christian; Böttcher, Martin; Beier, Fabian; Röhner, Linda; Stoll, Andrej; Rehli, Michael; Gebhard, Claudia; Lischer, Christopher; Eberhardt, Martin; Vera, Julio; Büttner-Herold, Maike; Bitterer, Katrin; Balzer, Heidi; Leffler, Magdalena; Jitschin, Simon; Hundemer, Michael; Awwad, Mohamed H.S.; Busch, Martin ... & Bruns, Heiko
  
• Proteomic profiling reveals CDK6 upregulation as a targetable resistance mechanism for lenalidomide in multiple myeloma. Nature Communications, 13(1).
  Ng, Yuen Lam Dora; Ramberger, Evelyn; Bohl, Stephan R.; Dolnik, Anna; Steinebach, Christian; Conrad, Theresia; Müller, Sina; Popp, Oliver; Kull, Miriam; Haji, Mohamed; Gütschow, Michael; Döhner, Hartmut; Walther, Wolfgang; Keller, Ulrich; Bullinger, Lars; Mertins, Philipp & Krönke, Jan
  
• Targeting the deubiquitinase USP7 for degradation with PROTACs. Chemical Communications, 58(63), 8858-8861.
  Murgai, Arunima; Sosič, Izidor; Gobec, Martina; Lemnitzer, Patricia; Proj, Matic; Wittenburg, Sophie; Voget, Rabea; Gütschow, Michael; Krönke, Jan & Steinebach, Christian
  
• The OTUD6B‐LIN28B‐MYC axis determines the proliferative state in multiple myeloma. The EMBO Journal, 41(20).
  Paulmann, Carmen; Spallek, Ria; Karpiuk, Oleksandra; Heider, Michael; Schäffer, Isabell; Zecha, Jana; Klaeger, Susan; Walzik, Michaela; Öllinger, Rupert; Engleitner, Thomas; Wirth, Matthias; Keller, Ulrich; Krönke, Jan; Rudelius, Martina; Kossatz, Susanne; Rad, Roland; Kuster, Bernhard & Bassermann, Florian
  
• Accessing three-branched high-affinity cereblon ligands for molecular glue and protein degrader design. RSC Chemical Biology, 4(3), 229-234.
  Kuchta, Robert; Heim, Christopher; Herrmann, Alexander; Maiwald, Samuel; Ng, Yuen Lam Dora; Sosič, Izidor; Keuler, Tim; Krönke, Jan; Gütschow, Michael; Hartmann, Marcus D. & Steinebach, Christian
  
• Direct-to-biology, automated, nano-scale synthesis, and phenotypic screening-enabled E3 ligase modulator discovery. Nature Communications, 14(1).
  Wang, Zefeng; Shaabani, Shabnam; Gao, Xiang; Ng, Yuen Lam Dora; Sapozhnikova, Valeriia; Mertins, Philipp; Krönke, Jan & Dömling, Alexander
  
• Heterobifunctional Ligase Recruiters Enable pan-Degradation of Inhibitor of Apoptosis Proteins. Journal of Medicinal Chemistry, 66(7), 4703-4733.
  Ng, Yuen Lam Dora; Bricelj, Aleša; Jansen, Jacqueline A.; Murgai, Arunima; Peter, Kirsten; Donovan, Katherine A.; Gütschow, Michael; Krönke, Jan; Steinebach, Christian & Sosič, Izidor
  
• Leveraging Ligand Affinity and Properties: Discovery of Novel Benzamide-Type Cereblon Binders for the Design of PROTACs. Journal of Medicinal Chemistry, 66(21), 14513-14543.
  Steinebach, Christian; Bricelj, Aleša; Murgai, Arunima; Sosič, Izidor; Bischof, Luca; Ng, Yuen Lam Dora; Heim, Christopher; Maiwald, Samuel; Proj, Matic; Voget, Rabea; Feller, Felix; Košmrlj, Janez; Sapozhnikova, Valeriia; Schmidt, Annika; Zuleeg, Maximilian Rudolf; Lemnitzer, Patricia; Mertins, Philipp; Hansen, Finn K.; Gütschow, Michael ... & Hartmann, Marcus D.
  
• SUMOylation inhibition overcomes proteasome inhibitor resistance in multiple myeloma. Blood Advances, 7(4), 469-481.
  Heynen, Guus J. J. E.; Baumgartner, Francis; Heider, Michael; Patra, Upayan; Holz, Maximilian; Braune, Jan; Kaiser, Melanie; Schäffer, Isabell; Bamopoulos, Stefanos A.; Ramberger, Evelyn; Murgai, Arunima; Ng, Yuen Lam Dora; Demel, Uta Margareta; Laue, Dominik; Liebig, Sven; Krüger, Josefine; Janz, Martin; Nogai, Axel; Schick, Markus ... & Wirth, Matthias
  
• CDK6 protein expression is associated with disease progression and treatment resistance in multiple myeloma. HemaSphere, 8(1).
  Steinhart, Johannes; Möller, Peter; Kull, Miriam; Krönke, Jan & Barth, Thomas F. E.
  
• Design, Synthesis, and Evaluation of BCL‐2 Targeting PROTACs. Chemistry – A European Journal, 30(45).
  Bricelj, Aleša; Dora, Ng Yuen Lam; Gobec, Martina; Kuchta, Robert; Hu, Wanyi; Javornik, Špela; Rožič, Miha; Gütschow, Michael; Zheng, Guangrong; Krönke, Jan; Steinebach, Christian & Sosič, Izidor
  
• Making AML fly too close to the sun. Blood, 143(15), 1438-1439.
  Bullinger, Lars & Krönke, Jan
  
• Revisiting the antiangiogenic mechanisms of fluorinated thalidomide derivatives. Bioorganic & Medicinal Chemistry Letters, 110, 129858.
  Sievers, Johannes; Voget, Rabea; Lu, Feiteng; Garchitorena, Kathleen M.; Ng, Yuen Lam Dora; Chau, Cindy H.; Steinebach, Christian; Figg, William D.; Krönke, Jan & Gütschow, Michael
  
• The proteogenomic landscape of multiple myeloma reveals insights into disease biology and therapeutic opportunities. Nature Cancer, 5(8), 1267-1284.
  Ramberger, Evelyn; Sapozhnikova, Valeriia; Ng, Yuen Lam Dora; Dolnik, Anna; Ziehm, Matthias; Popp, Oliver; Sträng, Eric; Kull, Miriam; Grünschläger, Florian; Krüger, Josefine; Benary, Manuela; Müller, Sina; Gao, Xiang; Murgai, Arunima; Haji, Mohamed; Schmidt, Annika; Lutz, Raphael; Nogai, Axel; Braune, Jan ... & Krönke, Jan