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

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The immunomodulatory drug (IMiD) lenalidomide is highly active in multiple myeloma and myelodysplastic syndrome (MDS) with del(5q). In 2014, we found that lenalidomide modulates the substrate specificity of cereblon (CRBN) which is the substrate adaptor of the CRBNCRL4 E3 ubiquitin ligase. Lenalidomide and its analogs thalidomide and pomalidomide all induce ubiquitination and degradation of the lymphoid transcription factors ikaros (IKZF1) and aiolos (IKZF3) what results in growth arrest of mutlilple myeloma cells and induces interleukin-2 release in T cells. In the first funding period of the Emmy Noether-Program we could elucidate the mechanism of lenalidomide in del(5q) MDS: Lenalidomide, but not the other IMiDs induce degradation of casein kinase 1a (CK1a) via CRBN-CRL4. Haploinsufficiency of CK1a in del(5q) MDS cells opens a therapeutic window for lenalidomide-induced degradation of CK1a resulting in TP53 activation and apoptosis. We further found that non-conserved amino acids in CRBN are responsible for the IMiD-insensitivity of mice. Expression of human CRBN overcomes this natural resistance of mouse cells. In the next funding period we aim to establish new tumor mouse models that will allow for the first time to assess the direct and indirect effects of IMiDs and combination treatments in vivo. We further generated and validated an IMiD-based homodimeric proteolysis targeting chimera (PROTAC) as first chemical inhibitor of CRBN. We will use this compound to further study the role of CRBN and effects of IMiDs in different model systems.The CRBN-binding protein FAM46C, which is recurrently mutated in multiple myeloma, was characterized as a tumor-suppressor in multiple myeloma that is affecting lenalidomide sensitivity. In ongoing studies we aim to identify the multiple myeloma-specific role of FAM46C mutations and to analyze the effects of FAM46C inactivation in vivo.In the third aim IKZF1 RNA expression levels were identified as prognostic marker in lenalidomide-treated patients. Exome and RNA sequencing of primary multiple myeloma samples obtained pre-treatment and at progression revealed a high heterogeneity of gene mutations and aberrantly expressed genes in lenalidomide-resistant cells. In the second funding period we will complement our DNA and RNA analyses by quantitative proteomics and will functionally analyze the findings in cell lines using pooled CRISPR-Cas9-based screens.The results of these projects will deepen our understanding of CRBN-CRL4 E3 ligase and the molecular mechanism of IMiDs what may translate into better treatments for multiple myeloma and MDS patients. Given the recent developments of drugs that alter ubiquitin ligases to target other proteins like IMiD-based PROTACS, our findings have important implications for cancer therapy and medical therapeutics more broadly.