The immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide, all approved for the treatment of multiple myeloma, induce targeted ubiquitination and degradation of Ikaros (IKZF1) and Aiolos (IKZF3) via the cereblon (CRBN) E3 ubiquitin ligase. IMiD-based proteolysis-targeting chimeras (PROTACs) can efficiently recruit CRBN to a protein of interest, leading to its ubiquitination and proteasomal degradation. By linking two pomalidomide molecules, we designed homobifunctional, so-called homo-PROTACs and investigated their ability to induce self-directed ubiquitination and degradation. The homodimerized compound 15a was characterized as a highly potent and efficient CRBN degrader with only minimal effects on IKZF1 and IKZF3. The cellular selectivity of 15a for CRBN degradation was confirmed at the proteome level by quantitative mass spectrometry. Inactivation by compound 15a did not affect proliferation of different cell lines, prevented pomalidomide-induced degradation of IKZF1 and IKZF3, and antagonized the effects of pomalidomide on multiple myeloma cells. Homobifunctional CRBN degraders will be useful tools for future biomedical investigations of CRBN-related signaling and may help to further elucidate the molecular mechanism of thalidomide analogues.

Protein ubiquitylation is involved in a plethora of cellular processes. While antibodies directed at ubiquitin remnants (K-ɛ-GG) have improved the ability to monitor ubiquitylation using mass spectrometry, methods for highly multiplexed measurement of ubiquitylation in tissues and primary cells using sub-milligram amounts of sample remains a challenge. Here, we present a highly sensitive, rapid and multiplexed protocol termed UbiFast for quantifying 10,000 ubiquitylation sites from as little as 500 μg peptide per sample from cells or tissue in a TMT10plex in ca. 5 h. High-field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) is used to improve quantitative accuracy for posttranslational modification analysis. We use the approach to rediscover substrates of the E3 ligase targeting drug lenalidomide and to identify proteins modulated by ubiquitylation in models of basal and luminal human breast cancer. The sensitivity and speed of the UbiFast method makes it suitable for largescale studies in primary tissue samples.

The effective treatment of bipolar disorder (BD) represents a significant unmet medical need. Although lithium remains a mainstay of treatment for BD, limited knowledge regarding how it modulates affective behavior has proven an obstacle to discovering more effective mood stabilizers with fewer adverse side effects. One potential mechanism of action of lithium is through inhibition of the serine/threonine protein kinase GSK3β, however, relevant substrates whose change in phosphorylation may mediate downstream changes in neuroplasticity remain poorly understood. Here, we used human induced pluripotent stem cell (hiPSC)-derived neuronal cells and stable isotope labeling by amino acids in cell culture (SILAC) along with quantitative mass spectrometry to identify global changes in the phosphoproteome upon inhibition of GSK3α/β with the highly selective, ATP-competitive inhibitor CHIR-99021. Comparison of phosphorylation changes to those induced by therapeutically relevant doses of lithium treatment led to the identification of collapsin response mediator protein 2 (CRMP2) as being highly sensitive to both treatments as well as an extended panel of structurally distinct GSK3α/β inhibitors. On this basis, a high-content image-based assay in hiPSC-derived neurons was developed to screen diverse compounds, including FDA-approved drugs, for their ability to mimic lithium's suppression of CRMP2 phosphorylation without directly inhibiting GSK3β kinase activity. Systemic administration of a subset of these CRMP2-phosphorylation suppressors were found to mimic lithium's attenuation of amphetamine-induced hyperlocomotion in mice. Taken together, these studies not only provide insights into the neural substrates regulated by lithium, but also provide novel human neuronal assays for supporting the development of mechanism-based therapeutics for BD and related neuropsychiatric disorders.

The immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide are all approved for the treatment of multiple myeloma. IMiDs bind cereblon (CRBN), a substrate adaptor for the CRL4 E3 ubiquitin ligase (CRL4CRBN). In multiple myeloma, IMiDs enhance the binding of the lymphoid transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) to CRL4CRBN, leading to their ubiquitination and degradation. Depletion of IKZF1 and IKZF3 results in growth inhibition in multiple myeloma cells. In addition, IMiDs can block the physiologic function of CRBN what has been shown to contribute to the anti-proliferative effects as well as other properties of the drug. Recently, IMiDs were exploited for the generation of Proteolysis Targeting Chimeras (PROTACs). These molecules link the IMiD structure to another small molecule that binds a protein of interest (POI). Such IMiD-based PROTACs are capable to guide the CRBN-CRL4 E3 ligase to the POI, resulting in its ubiquitination and degradation. Here, we designed pomalidomide-based homobifunctional PROTACs and investigated their ability to induce self-directed CRBN ubiquitination and degradation (Figure 1A). We evaluated different attachment strategies, modifications and linker lengths and tested the effect of a series of homo-dimeric compounds on their potency to deplete CRBN protein levels. The homodimeric compound 15a with a linker length of 8 atoms was identified as the most potent CRBN degrader with a minimal remaining effect on IKZF1 (Figure 1B). Homodimeric PROTACs with longer linkers exhibited a weaker capability for CRBN degradation and had a more potent effect on IKZF1 protein levels. The effect of compound 15a on CRBN was blocked after pre-treatment with a proteasome inhibitor or MLN4924, a neddylation activating enzyem inhibitor that blocks Cullin E3 ligases. Co-immunoprecipitation revealed that 15a induces interaction of two CRBN molecules. The homo-PROTAC 15a was active at low concentrations below 100 nM and had long-lasting effects on the intracellular CRBN level (Figure 1B). Applying global proteome analyses in the multiple myeloma cell line MM1.S demonstrated that PROTAC 15a specifically induced degradation of CRBN and had only weak effects on IKZF1 and IKZF3 and no effect on the other members of the CRL4 ligase family, including DDB1 and CUL4A which are in close proximity to CRBN in the CRL4CRBN complex. CRBN inactivation by our compounds had no effect on proliferation of different multiple myeloma cell lines. Pre-treatment with Homo-PROTAC 15a prevented pomalidomide-induced degradation of IKZF1 and IKZF3, and antagonized the effects of pomalidomide and lenalidomide on multiple myeloma cell growth. This was consistent with genetic inactivation of CRBN by CRISPR/Cas9. In conclusion, we generated the first chemical inhibitors of CRBN that can serve as a useful tool for future biomedical investigations on CRBN-related signaling. These compounds will also help to discriminate whether an IMiD effect depends on CRBN-mediated targeted degradation of neo-substrates or from blocking the physiologic function of CRBN. Furthermore, our data confirm the essential role of CRBN in IMiD activity in multiple myeloma. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.