Immunomodulatory drugs (IMiDs) including lenalidomide and pomalidomide bind cereblon (CRBN) and activate the CRL4 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 SCF 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 SCF E3 ligase-mediated CRBN degradation, thereby conferring sensitivity to IMiDs; conversely, loss of function of CSN9 signalosome activates SCF 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.

Background and Aims Protein S‐sulfhydration mediated by H2S has been shown to play important roles in several diseases. However, its precise role in liver disease and the related mechanism remain unclear. Approach and Results We showed that in streptozotocin (STZ)–treated and high‐fat diet (HFD)–treated low‐density lipoprotein receptor–negative (LDLr−/−) mice, the H2S donor GYY4137 ameliorated liver injury, decreased serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, mitigated lipid deposition, and reduced hepatocyte death. Strikingly, S‐sulfhydration of Kelch‐like ECH‐associated protein 1 (Keap1) was decreased in the livers of patients with fatty liver under diabetic conditions. In STZ+HFD‐treated LDLr−/− mice and in high glucose–treated and oxidized low‐density lipoprotein (ox‐LDL)–treated primary mouse hepatocytes, the GYY4137‐mediated increase in Keap1 S‐sulfhydration induced nuclear erythroid 2‐related factor 2 (Nrf2) dissociation from Keap1, which enhanced the nuclear translocation of Nrf2 itself and the consequent expression of antioxidant proteins. Keap1 Cys151 mutation significantly reduced Keap1 S‐sulfhydration and abolished the hepatoprotective effects of H2S both in vivo and in vitro. Nrf2 deficiency inhibited the H2S‐induced beneficial impacts in Nrf2−/− mice. Similarly, in CCl4‐stimulated mice, GYY4137 increased Keap1 S‐sulfhydration, improved liver function, alleviated liver fibrosis, decreased hepatic oxidative stress, and activated the Nrf2 signaling pathway; and these effects were abrogated after Keap1 Cys151 mutation. Moreover, H2S increased the binding of Nrf2 to the promoter region of LDLr‐related protein 1 (Lrp1) and consequently up‐regulated LRP1 expression, but these effects were disrupted by Keap1 Cys151 mutation. Conclusions H2S‐mediated Keap1 S‐sulfhydration alleviates liver damage through activation of Nrf2. Hence, administration of exogenous H2S in the form of the H2S donor GYY4137 may be of therapeutic benefit in the context of concurrent hyperlipidemia and hyperglycemia–induced or CCl4‐stimulated liver dysfunction.

Intellectual disability (ID), one of the most common human developmental disorders, can be caused by genetic mutations in Cullin 4B (Cul4B) and cereblon (CRBN). CRBN is a substrate receptor for the Cul4A/B-DDB1 ubiquitin ligase (CRL4) and can target voltage- and calcium-activated BK channel for ER retention. Here we report that ID-associated CRL4CRBN mutations abolish the interaction of the BK channel with CRL4, and redirect the BK channel to the SCFFbxo7 ubiquitin ligase for proteasomal degradation. Glioma cell lines harbouring CRBN mutations record density-dependent decrease of BK currents, which can be restored by blocking Cullin ubiquitin ligase activity. Importantly, mice with neuron-specific deletion of DDB1 or CRBN express reduced BK protein levels in the brain, and exhibit similar impairment in learning and memory, a deficit that can be partially rescued by activating the BK channel. Our results reveal a competitive targeting of the BK channel by two ubiquitin ligases to achieve exquisite control of its stability, and support changes in neuronal excitability as a common pathogenic mechanism underlying CRL4CRBN–associated ID.

Edited by George N. DeMartinoCullin-RING ligase 4 (CRL4), a complex of Cul4 and DDB1, regulates the cell cycle, DNA damage repair, and chromatin replication by targeting a variety of substrates for ubiquitination. CRL4 is also hijacked by viral proteins or thalidomide-derived compounds to degrade host restriction factors. Here we report that the c-Abl non-receptor kinase phosphorylates DDB1 at residue Tyr-316 to recruit a small regulatory protein, DDA1, leading to increased substrate ubiquitination. Pharmacological inhibition or genetic ablation of the Abl-DDB1-DDA1 axis decreases the ubiquitination of CRL4 substrates, including IKZF1 and IKZF3, in lenalidomide-treated multiple myeloma cells. Importantly, panobinostat, a recently approved anti-myeloma drug, and dexamethasone enhance lenalidomide-induced substrate degradation and cytotoxicity by activating c-Abl, therefore providing a mechanism underlying their combination with lenalidomide to treat multiple myeloma.The cullin-RING ubiquitin ligases (CRLs), 2 comprising the scaffold cullins, the E2-interacting RING finger protein ROC1/2, the adaptor proteins specific for each cullin family member, and adaptor-interacting substrate receptors, are the largest family of E3 ligases in eukaryotes, regulating diverse cellular pathways (1). CRL4 uses the damaged DNA binding protein 1 (DDB1) as the adaptor protein to assemble with a subset of WD40-containing proteins called DDB1-and Cullin4 (Cul4)-associated factors to target diverse substrates to regulate the cell cycle, DNA damage repair, and chromatin functions (1-3). CRL4 ubiquitinates cellular proteins such as CDT1 (4), P21 (5, 6), and DDB2 (7) for proteasomal degradation and teneleven translocation (TET) proteins (8) and histones H2A (9), H3 (10, 11), and H4 (10) to regulate their chromatin-mediated functions. Several viral proteins can hijack CRL4 to turn over host restriction factors to promote virus replication, including STAT1 targeted by SV5 virus V protein (12-14), SAMHD1 by simian HIV Vpr protein (15), and Smc5/6 by hepatitis B virus X protein (16).Additionally, immunomodulatory drugs (IMiDs), including thalidomide and its derivatives lenalidomide and pomalidomide, can repurpose CRL4 to target and destroy IKZF1 and IKZF3 (17, 18), two lymphoid transcription factors essential for multiple myeloma (MM) cell survival. Similarly, lenalidomide induces ubiquitination and degradation of casein kinase 1A1 (CK1␣), which accounts for the clinical efficacy of lenalidomide in myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)) (19). Crystal structure studies reveal that the glutarimide moiety of lenalidomide directly inserts into a hydrophobic pocket of cereblon (CRBN), a CRL4 substrate receptor, and that the exposed chemical moiety, together with CRBN, creates a new surface for receiving substrates (20 -22). Based on these results, novel chemicals conjugating glutarimide to other protein-interacting chemical structures are designed to control protein stability and thus enable targeting of previously...

c-Abl inhibits replication of hepatitis B virus by promoting CRL4Cdt2-mediated destruction of the viral polymerase.