Cereblon Degradation Research Articles

The cereblon-AMPK (AMP-activated protein kinase) axis in chondrocytes regulates the pathogenesis of osteoarthritis

ObjectiveAMP-activated protein kinase (AMPK) dysregulation is implicated in osteoarthritis (OA), but the mechanisms underlying this dysregulation remain unclear. We investigated the role of cereblon, a substrate-recognition protein within the E3-ligase ubiquitin complex, in AMPK dysregulation and OA pathogenesis. MethodsCereblon expression was examined in human (n=5) and mouse (n=10) OA cartilage. The role of cereblon was investigated through its adenoviral overexpression (n=10) or knockout (KO, n=15) in destabilization of the medial meniscus (DMM)-operated mice. The therapeutic potentials of the chemical cereblon degrader, TD-165, and the AMPK activator, metformin, were assessed through intra-articular (IA) injection to mice (n=15). ResultsImmunostaining revealed that cereblon is upregulated in human and mouse OA cartilage. In DMM model mice, cartilage destruction was exacerbated by overexpression of cereblon in mouse joint tissues (OARSI grade; 1.11 [95% CI: 0.50 to 2.75]), but inhibited in global (-2.50 [95% CI: -3.00 to -1.17]) and chondrocyte-specific (-2.17 [95% CI: -3.14 to -1.06]) cereblon KO mice. The inhibitory effects were more pronounced in mice fed a high-fat diet compared to a regular diet. The degradation of cereblon through IA injection of TD-165 inhibited OA cartilage destruction (-2.47 [95% CI: -3.22 to -1.56]). Mechanistically, cereblon exerts its catabolic effects by negatively modulating AMPK activity within chondrocytes. Consistently, activation of AMPK by IA injection of metformin inhibited posttraumatic OA cartilage destruction (-1.20 ([95% CI: -1.89 to -0.45]). ConclusionsThe cereblon-AMPK axis acts as a catabolic regulator of OA pathogenesis and seems to be a promising therapeutic target in animal models of OA.

Nx-2127, a Degrader of BTK and IMiD Neosubstrates, for the Treatment of B-Cell Malignancies

Bruton's tyrosine kinase (BTK) plays a key role in cell survival in B cell malignancies, such as chronic lymphocytic leukemia (CLL). Covalent inhibitors of BTK, such as ibrutinib and acalabrutinib, while effective, have been associated with the occurrence of resistance mutations. The most prevalent site of mutation, C481, renders covalent BTK inhibitors unable to form a covalent bond with BTK leading to diminished efficacy and disease progression. Small molecule-induced protein degradation offers a unique approach to target BTK for the treatment of B-cell malignancies. Chimeric Targeting Molecules (CTMs) catalyze ubiquitylation and proteasomal degradation of target proteins and are comprised of a ubiquitin ligase binding element ("harness"), a linker, and a target binding element ("hook"). NX-2127 is a CTM that contains a BTK hook linked to a cereblon (CRBN) harness. NX-2127 degrades 50% of cellular BTK (DC50) at < 5 nM across multiple cancer cell lines and in human PBMCs. BTK CTMs impair viability in the BTK-dependent ABC-DLBCL cell line, TMD8 (EC50: < 15 nM after 72 hours). Importantly, NX-2127 induces degradation of the mutated BTK-C481S in cells and inhibits proliferation of BTK-C481S mutant TMD8 cells more effectively than ibrutinib (NX-2127 EC50 values of < 30 nM versus > 1 μM for ibrutinib). Oral administration of NX-2127 in mice leads to dose-proportional exposure in plasma and BTK degradation to <10% of baseline levels in circulating and splenic B cells. In both WT TMD8 and C481S mutant xenograft models, daily oral administration of NX-2127 resulted in superior tumor growth inhibition (TGI) as compared to ibrutinib. NX-2127 also demonstrates potent degradation of BTK in cynomolgus monkeys with oral administration. Following 14 days of once daily, oral dosing in cynomolgus monkey, BTK levels are suppressed to <10% of baseline levels at doses as low as 1 mg/kg. In addition to potent BTK degradation, NX-2127 possesses IMiD-like properties through the design of the CRBN binding harness that catalyzes the degradation of CRBN neosubstrates Aiolos (IKZF3) and Ikaros (IKZF1). This activity is associated with increased T cell activation and anti-tumor effects of the IMiD drugs lenalidomide and pomalidomide. In primary human T cells, NX-2127 catalyzes the degradation of Aiolos and Ikaros with of 25 nM and 54 nM, respectively, potencies which are similar to those of lenalidomide (20 nM and 343 nM, respectively). Corresponding with such degradation, NX-2127 stimulates T cell activation as measured by increased IL-2 production in primary human T Cells in a manner similar to lenalidomide and pomalidomide. The dual activity of BTK degradation combined with immunomodulation of NX-2127 supports its development for the treatment of B-cell malignancies. Disclosures Robbins: Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Kelly:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tan:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. McIntosh:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Wu:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Konst:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Kato:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Peng:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mihalic:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Weiss:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Perez:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tung:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Kolobova:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Borodovsky:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Rountree:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tenn-McClellan:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Noviski:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ye:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Basham:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ingallinera:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. McKinnell:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Karr:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Powers:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Guiducci:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sands:Nurix Therapeutics: Current Employment, Current equity holder in publicly-traded company.

A phenylphthalimide derivative, TC11, induces apoptosis by degrading MCL1 in multiple myeloma cells

To date, the prognosis of multiple myeloma (MM) in patients harboring cytogenetic abnormalities (CA) involving t (4; 14) and deletion of chromosome 17 remains poor despite recent advances in drug development that include the use of immunomodulatory drugs (IMiDs) such as lenalidomide for MM. To address this issue, we have developed a novel phenylphthalimide derivative, TC11, that is structurally related to IMiDs. It remains unclear how TC11 induces apoptosis of MM cells with high-risk CA. Here, we show that TC11 does not induce degradation of CRBN’s substrates, IKZF1/3 and CK1α, and induces apoptosis of CRBN-silenced MM; this effect was independent of the cereblon (CRBN) pathway, which is involved in the mechanism of action of IMiDs used for the treatment of MM. We also revealed that TC11, in contrast to existing IMiDs, induced degradation of MCL1 and activation of caspase-9. Furthermore, inhibition of CDK1 by CGP74514A prevented TC11-induced MCL1 degradation, caspase-9 activation, and the subsequent apoptotic cell death. We showed that ectopic MCL1 expression rescued apoptosis of MM. These observations suggest that TC11 induces apoptotic death caused by degradation of MCL1 during prolonged mitotic arrest. Therefore, our findings suggest that TC11 is a potential drug candidate for high-risk MM.

Chemical Inactivation of the E3 Ubiquitin Ligase Cereblon by Pomalidomide-based Homo-PROTACs.

The immunomodulatory drugs (IMiDs) thalidomide and its analogs, lenalidomide and pomalidomide, all FDA approved drugs for the treatment of multiple myeloma, induce ubiquitination and degradation of the lymphoid transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) via the cereblon (CRBN) E3 ubiquitin ligase for proteasomal degradation. IMiDs have recently been utilized for the generation of bifunctional proteolysis targeting chimeras (PROTACs) to target other proteins for ubiquitination and proteasomal degradation by the CRBN E3 ligase. We designed and synthesized pomalidomide-based homobifunctional PROTACs and analyzed their ability to induce self-directed ubiquitination and degradation of CRBN. Here, CRBN serves as both, the E3 ubiquitin ligase and the target at the same time. The homo-PROTAC compound 8 degrades CRBN with a high potency with only minimal remaining effects on IKZF1 and IKZF3. CRBN inactivation by compound 8 had no effect on cell viability and proliferation of different multiple myeloma cell lines. This homo-PROTAC abrogates the effects of IMiDs in multiple myeloma cells. Therefore, our homodimeric pomalidomide-based compounds may help to identify CRBN's endogenous substrates and physiological functions and investigate the molecular mechanism of IMiDs.

Cereblon versus VHL: Hijacking E3 ligases against each other using PROTACs.

The von Hippel-Lindau (VHL) and cereblon (CRBN) proteins are substrate recognition subunits of two ubiquitously expressed and biologically important Cullin RING E3 ubiquitin ligase complexes. VHL and CRBN are also the two most popular E3 ligases being recruited by bifunctional Proteolysis-targeting chimeras (PROTACs) to induce ubiquitination and subsequent proteasomal degradation of a target protein. Using homo-PROTACs, VHL and CRBN have been independently dimerized to induce their own degradation. Here we report the design, synthesis and cellular activity of VHL-CRBN hetero-dimerizing PROTACs featuring diverse conjugation patterns. We found that the most active compound 14a induced potent, rapid and profound preferential degradation of CRBN over VHL in cancer cell lines. At lower concentrations, weaker degradation of VHL was instead observed. This work demonstrates proof of concept of designing PROTACs to hijack different E3 ligases against each other, and highlights a powerful and generalizable proximity-induced strategy to achieve E3 ligase knockdown.

Pomalidomide-Based Homo-Protacs for the Chemical Knockdown of Cereblon

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. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Homo-PROTACs for the Chemical Knockdown of Cereblon.

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.

A genome-scale CRISPR-Cas9 screening in myeloma cells identifies regulators of immunomodulatory drug sensitivity.

Immunomodulatory drugs (IMiDs) including lenalidomide and pomalidomide bind cereblon (CRBN) and activate the CRL4CRBN ubiquitin ligase to trigger proteasomal degradation of the essential transcription factors IKZF1 and IKZF3 and multiple myeloma (MM) cytotoxicity. We have shown that CRBN is also targeted for degradation by SCFFbxo7 ubiquitin ligase. In the current study, we explored the mechanisms underlying sensitivity of MM cells to IMiDs using genome-wide CRISPR-Cas9 screening. We validate that CSN9 signalosome complex, a deactivator of Cullin-RING ubiquitin ligase, inhibits SCFFbxo7 E3 ligase-mediated CRBN degradation, thereby conferring sensitivity to IMiDs; conversely, loss of function of CSN9 signalosome activates SCFFbxo7 complex, thereby enhancing degradation of CRBN and conferring IMiD resistance. Finally, we show that pretreatment with either proteasome inhibitors or NEDD8 activating enzyme (NAE) inhibitors can abrogate degradation and maintain levels of CRBN, thereby enhancing sensitivity to IMiDs. These studies therefore demonstrate that CSN9 signalosome complex regulates sensitivity to IMiDs by modulating CRBN expression.

Abstract 4631: SCFFbxo7 ubiquitin ligase targets cereblon to limit the anti-myeloma activity of IMiDs

Abstract Multiple myeloma (MM) patients have witnessed significantly improved survival after the approved treatment with the proteasome inhibitor bortezomib and immunomodulatory drugs (IMiDs) including thalidomide and its derivatives lenalidomide and pomalidomide. IMiDs directly bind cereblon (CRBN), an adaptor of the Cul4-DDB1 (CRL4) ubiquitin ligase, and activate CRL4CRBN to target B cell-specific transcription factors IKZF1 and IKZF3 for ubiquitination. IKZF1 and IKZF3 are essential for MM cell survival and their proteasomal degradation induced by IMiDs provides insights into how these drugs work. Paradoxically, IMiDs achieve better clinical response in MM patients when combined with bortezomib, which blocks IKZF1 and IKZF3 turnover and would in principle counteract IMiDs therapy. We performed a CRISPR-based genome-wide knockout screening for regulators of pomalidomide sensitivity in MM cells, and found that CRBN is inherently unstable and its level is controlled by the COP9 signalosome (CSN) to sustain IMiDs cytotoxicity. When unbound to CRL4, CRBN is recruited by the F-box protein Fbxo7 to the Cul1-Skp1 (SCF) ubiquitin ligase for ubiquitination and degradation, a process suppressed by CSN-mediated de-neddylation of Cul1. Deletion of SCFFbxo7 components, or treatment with bortezomib or neddylation inhibitor MLN4924 stabilizes CRBN, and IMiDs further promote the loading of CRBN to CRL4 by inhibiting SCFFbxo7-targeted CRBN degradation, leading to enhanced turnover of IKZF1 and IKZF3 in MM cells and synergistic inhibition of MM cell proliferation. Our findings unravel a dual role of IMiDs in activating CRL4CRBN E3 ligase in MM cells and provide a framework to select patients most susceptible to bortezomib and IMiDs combination therapy. Citation Format: Jiye Liu, Wenrong Zhou, Yan Feng, Tianyu Song, Shaobing Gao, Junfeng Wu, Zhenggang Peng, Yong Cang. SCFFbxo7 ubiquitin ligase targets cereblon to limit the anti-myeloma activity of IMiDs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4631.