Cullin-RING Ubiquitin Ligases Research Articles

DCAF13 promotes ovarian cancer progression by activating FRAS1-mediated FAK signaling pathway

Cullin-RING ubiquitin ligase 4 (CRL4) is closely correlated with the incidence and progression of ovarian cancer. DDB1- and CUL4-associated factor 13 (DCAF13), a substrate-recognition protein in the CRL4 E3 ubiquitin ligase complex, is involved in the occurrence and development of ovarian cancer. However, its precise function and the underlying molecular mechanism in this disease remain unclear. In this study, we confirmed that DCAF13 is highly expressed in human ovarian cancer and its expression is negatively correlated with the overall survival rate of patients with ovarian cancer. We then used CRISPR/Cas9 to knockout DCAF13 and found that its deletion significantly inhibited the proliferation, colony formation, and migration of human ovarian cancer cells. In addition, DCAF13 deficiency inhibited tumor proliferation in nude mice. Mechanistically, CRL4-DCAF13 targeted Fraser extracellular matrix complex subunit 1 (FRAS1) for polyubiquitination and proteasomal degradation. FRAS1 influenced the proliferation and migration of ovarian cancer cell through induction of the focal adhesion kinase (FAK) signaling pathway. These findings collectively show that DCAF13 is an important oncogene that promotes tumorigenesis in ovarian cancer cells by mediating FRAS1/FAK signaling. Our findings provide a foundation for the development of targeted therapeutics for ovarian cancer.

Drosophila Cul3 contributes to Diap2-mediated innate immune signaling for antimicrobial defense

The host antimicrobial immune response relies on a complex interplay of molecular mechanisms to effectively combat microbial infections. Herein, we investigate the functional role of Cullin-3 (Cul3), one critical constituent of cullin-RING ubiquitin ligases, in the Drosophila melanogaster (fruit fly) antimicrobial immune defense. We show that silencing of Cul3 leads to a decreased induction of antimicrobial peptides and high mortality in adult flies after bacterial infection. Through biochemical approaches, we demonstrate that Cul3 predominantly relies on its BTB-binding domain and neddylation domain to physically associate with death-associated inhibitor of apoptosis 2 (Diap2). Importantly, Cul3 ameliorates the Diap2-mediated ubiquitination of death-related ced-3/Nedd2-like caspase (Dredd), a process essential for robust immune deficiency signaling upon bacterial infection. Taken together, our findings highlight a previously unrecognized regulatory axis of Cul3/Diap2/Dredd in the fly antimicrobial immune defense, providing potential insights into therapeutic strategies for combating bacterial infections in humans.

The small CRL4CSA ubiquitin ligase component DDA1 regulates transcription-coupled repair dynamics

Transcription-blocking DNA lesions are specifically targeted by transcription-coupled nucleotide excision repair (TC-NER), which removes a broad spectrum of DNA lesions to preserve transcriptional output and thereby cellular homeostasis to counteract aging. TC-NER is initiated by the stalling of RNA polymerase II at DNA lesions, which triggers the assembly of the TC-NER-specific proteins CSA, CSB and UVSSA. CSA, a WD40-repeat containing protein, is the substrate receptor subunit of a cullin-RING ubiquitin ligase complex composed of DDB1, CUL4A/B and RBX1 (CRL4CSA). Although ubiquitination of several TC-NER proteins by CRL4CSA has been reported, it is still unknown how this complex is regulated. To unravel the dynamic molecular interactions and the regulation of this complex, we apply a single-step protein-complex isolation coupled to mass spectrometry analysis and identified DDA1 as a CSA interacting protein. Cryo-EM analysis shows that DDA1 is an integral component of the CRL4CSA complex. Functional analysis reveals that DDA1 coordinates ubiquitination dynamics during TC-NER and is required for efficient turnover and progression of this process.

Prognostic potential of CUL3 ligase with differential roles in luminal A and basal type breast cancer tumors

Breast cancer is a prevalent and significant cause of mortality in women, and manifests as six molecular subtypes. Its further histologic classification into non-invasive ductal or lobular carcinoma (DCIS) and invasive carcinoma (ILC or IDC) underscores its heterogeneity. The ubiquitin–proteasome system plays a crucial role in breast cancer, with inhibitors targeting the 26S proteasome showing promise in clinical treatment. The Cullin-RING ubiquitin ligases, including CUL3, have direct links to breast cancer. This study focuses on CUL3 as a potential biomarker, leveraging high-throughput sequencing, gene expression profiling, experimental and data analysis tools. Through comprehensive analysis using databases like GEPIA2 and UALCAN, as well as TCGA datasets, CUL3's expression and its association with prognostic values were assessed. Additionally, the impact of CUL3 overexpression was explored in MCF-7 and MDA-MB-231 breast cancer cell lines, revealing distinct differences in molecular and phenotypic characteristics. We further profiled its expression and localization in breast cancer tissues identifying prominent differences between luminal A and TNBC tumors. Conclusively, CUL3 was found to be associated with cell cycle progression, and DNA damage response, exhibiting diverse roles depending on the tumor's molecular type. It exhibits a tendency to act as an oncogene in triple-negative tumors and as a tumor suppressor in luminal A types, suggesting a potential significance in breast cancer progression and therapeutic directions.

Structure of the E3 ligase CRL2-ZYG11B with substrates reveals the molecular basis for N-degron recognition and ubiquitination.

ZYG11B is a substrate specificity factor for Cullin-RING ubiquitin ligase (CRL2) involved in many biological processes, including Gly/N-degron pathways. Yet how the binding of ZYG11B with CRL2 is coupled to substrate recognition and ubiquitination is unknown. We present the Cryo-EM structures of the CRL2-ZYG11B holoenzyme alone and in complex with a Gly/N-peptide from the inflammasome-forming pathogen sensor NLRP1. The structures indicate ZYG11B folds into a Leucine-Rich Repeat followed by two armadillo repeat domains that promote assembly with CRL2 and recognition of NLRP1 Gly/N-degron. ZYG11B promotes activation of the NLRP1 inflammasome through recognition and subsequent ubiquitination of the NLRP1 Gly/N-degron revealed by viral protease cleavage. Our structural and functional data indicate that blocking ZYG11B recognition of the NLRP1 Gly/N-degron inhibits NLRP1 inflammasome activation by a viral protease. Overall, we show how the CRL2-ZYG11B E3 ligase complex recognizes Gly/N-degron substrates, including those that are involved in viral protease-mediated activation of the NLRP1 inflammasome.

MDB-57. PROTEOMIC ANALYSIS REVEALS AMBRA1 AS A NEW CRL3REN/KCTD11 SUBSTRATE PROMOTING SONIC HEDGEHOG-DEPENDENT MEDULLOBLASTOMA

Abstract BACKGROUND Sonic Hedgehog (SHH) signaling pathway is fundamental for a proper embryonic development and its uncontrolled activation is responsible for various cancers. One the most relevant SHH-dependent tumors is medulloblastoma (MB), a highly heterogeneous brain malignancy that predominantly occurs in childhood. Understanding the complex networks that govern SHH aberrant activation represents a dramatic challenge for the development of efficient personalized therapies. Here, we identify a new role of the pro-autophagic protein AMBRA1 as interactor of REN/KCTD11 (here REN), the substrate-receptor subunit of the Cullin3-RING ubiquitin ligase complex (CRL3REN) and tumor suppressor lost in ~30% of human SHH-MBs. We show a novel mechanism of action of AMBRA1 in SHH-MB pathogenesis whereby its highly expression, due to loss of REN, drives the activation of GLI1, the final effector of SHH signaling. METHODS Proteomic analysis, co-immunoprecipitation, ubiquitylation and cycloheximide assays have been performed to demonstrate that AMBRA1 is a substrate of ubiquitylation of CRL3REN and to define AMBRA1 mechanism of action in SHH-MB tumorigenesis. Transcriptomic analysis, in vitro and in vivo proliferation assays have been carried out on murine and patient-derived xenograft (PDX) models to examine the effect of AMBRA1 modulation on SHH-MB growth. RESULTS We demonstrate that CRL3REN binds and induces polyubiquitylation and proteasome-mediated degradation of AMBRA1. Interestingly, we found that genetic depletion of AMBRA1 strongly reduces SHH-dependent MB growth both in vitro and in vivo, an effect associated with the decrease in the expression of GLI1 and other SHH target genes. We hypothesize that AMBRA1 could impact on the stability of GLI1 by counteracting the activity of E3 ubiquitin ligases regulating GLI proteins. Remarkably, transcriptomic analysis of SHH PDX upon AMBRA1 knockdown revealed an impairment of SHH activity and an upregulation of cerebellum differentiation. CONCLUSION Our findings identify AMBRA1 as a novel CRL3REN substrate and promising therapeutic target for SHH-dependent cancer.

MTORC1/S6K1 signaling promotes sustained oncogenic translation through modulating CRL3IBTK-mediated ubiquitination of eIF4A1 in cancer cells.

Enhanced protein synthesis is a crucial molecular mechanism that allows cancer cells to survive, proliferate, metastasize, and develop resistance to anti-cancer treatments, and often arises as a consequence of increased signaling flux channeled to mRNA-bearing eukaryotic initiation factor 4F (eIF4F). However, the post-translational regulation of eIF4A1, an ATP-dependent RNA helicase and subunit of the eIF4F complex, is still poorly understood. Here, we demonstrate that IBTK, a substrate-binding adaptor of the Cullin 3-RING ubiquitin ligase (CRL3) complex, interacts with eIF4A1. The non-degradative ubiquitination of eIF4A1 catalyzed by the CRL3IBTK complex promotes cap-dependent translational initiation, nascent protein synthesis, oncogene expression, and cervical tumor cell growth both in vivo and in vitro. Moreover, we show that mTORC1 and S6K1, two key regulators of protein synthesis, directly phosphorylate IBTK to augment eIF4A1 ubiquitination and sustained oncogenic translation. This link between the CRL3IBTK complex and the mTORC1/S6K1 signaling pathway, which is frequently dysregulated in cancer, represents a promising target for anti-cancer therapies.

MTORC1/S6K1 signaling promotes sustained oncogenic translation through modulating CRL3IBTK-mediated ubiquitination of eIF4A1 in cancer cells

Enhanced protein synthesis is a crucial molecular mechanism that allows cancer cells to survive, proliferate, metastasize, and develop resistance to anti-cancer treatments, and often arises as a consequence of increased signaling flux channeled to mRNA-bearing eukaryotic initiation factor 4F (eIF4F). However, the post-translational regulation of eIF4A1, an ATP-dependent RNA helicase and subunit of the eIF4F complex, is still poorly understood. Here, we demonstrate that IBTK, a substrate-binding adaptor of the Cullin 3-RING ubiquitin ligase (CRL3) complex, interacts with eIF4A1. The non-degradative ubiquitination of eIF4A1 catalyzed by the CRL3IBTK complex promotes cap-dependent translational initiation, nascent protein synthesis, oncogene expression, and cervical tumor cell growth both in vivo and in vitro. Moreover, we show that mTORC1 and S6K1, two key regulators of protein synthesis, directly phosphorylate IBTK to augment eIF4A1 ubiquitination and sustained oncogenic translation. This link between the CRL3IBTK complex and the mTORC1/S6K1 signaling pathway, which is frequently dysregulated in cancer, represents a promising target for anti-cancer therapies.

The Shigella kinase effector OspG modulates host ubiquitin signaling to escape septin-cage entrapment

Shigella flexneri is a Gram-negative bacterium causing severe bloody dysentery. Its pathogenesis is largely dictated by a plasmid-encoded type III secretion system (T3SS) and its associated effectors. Among these, the effector OspG has been shown to bind to the ubiquitin conjugation machinery (E2~Ub) to activate its kinase activity. However, the cellular targets of OspG remain elusive despite years of extensive efforts. Here we show by unbiased phosphoproteomics that a major target of OspG is CAND1, a regulatory protein controlling the assembly of cullin-RING ubiquitin ligases (CRLs). CAND1 phosphorylation weakens its interaction with cullins, which is expected to impact a large panel of CRL E3s. Indeed, global ubiquitome profiling reveals marked changes in the ubiquitination landscape when OspG is introduced. Notably, OspG promotes ubiquitination of a class of cytoskeletal proteins called septins, thereby inhibiting formation of cage-like structures encircling cytosolic bacteria. Overall, we demonstrate that pathogens have evolved an elaborate strategy to modulate host ubiquitin signaling to evade septin-cage entrapment.

RBX1-CRLs Promote Perinatal Heart Development by Regulating Hippo-YAP Signaling

Background: During development, the heart is the first organ to form, ensuring the pumping of blood through the body. Defects in cardiac morphogenesis can lead to congenital heart diseases (CHD) or predispose the heart to cardiomyopathies in adult life. The molecular mechanisms underlying this process remain incompletely understood. Cullin-RING E3 ubiquitin ligases (CRLs) are multi-subunit complexes critical to maintaining protein homeostasis by targeting diverse protein substrates for ubiquitination and subsequent degradation. In CRLs, RBX1 serves as the catalytic subunit that promotes the transfer of ubiquitin to the target protein. Germline ablation of RBX1 in worms, flies, and mice caused embryonic lethality. Deletion of RBX1 in zebrafish resulted in cardiac malformation and dysfunction, which was rescued by endothelial, but not myocardial, RBX1 overexpression. However, the cardiomyocyte-autonomous role of RBX1 in the heart in vertebrates is unknown. Therefore, we hypothesize that cardiomyocyte RBX1 is indispensable for cardiac development. Methods and Results: We generated a cardiomyocyte-specific RBX1 knockout (RBX1CKO) mouse model via αMHCCre. RBX1-deficient mouse embryos were initially morphologically indistinguishable from the littermate controls at E14.5, but began to show peripheral edema and hemorrhage at E16.5, and eventually died around E18.5. Histological analysis of RBX1CKO hearts revealed severe myocardial hypoplasia and bi-ventricular non-compaction by E14.5. Bulk RNA sequencing and proteomic analyses of RBX1CKO hearts identified perturbations in glycolytic to oxidative transition and dysregulation of sarcomeric proteins. EdU incorporation assay showed a significant reduction in cardiomyocyte proliferation in E14.5 RBX1-deficient hearts, accompanied by the impaired nuclear translocation of YAP, a co-transcriptional factor essential for cardiomyocyte proliferation and maturation. Mechanistically, RBX1 deficiency destabilized multiple CRLs and resulted in the accumulation of Hippo kinases and adaptor proteins such as SAV1, LATS1/2, and MOB1, leading to the elevated phosphorylation and inactivation of YAP. We further demonstrated that loss of RBX1 inhibited the degradation of the Hippo adaptor MOB1, likely due to suppressed ubiquitination. Conclusion: Our study identifies RBX1 as an essential regulator of ventricular maturation and found that RBX1-mediated proteolysis of Hippo kinases serves as a critical checkpoint to ensure YAP activation and, hence, cardiomyocyte proliferation and maturation during cardiac development. AHA Predoctoral Fellowship, American Heart Association, 23PRE1012641. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

CRL4DCAF4 E3 ligase-mediated degradation of MEN1 transcriptionally reactivates hTERT to sustain immortalization in colorectal cancer cells.

Telomerase reactivation is implicated in approximately 85% of human cancers, yet its underlying mechanism remains elusive. In this study, we elucidate that the cullin-RING ubiquitin ligase 4 (CRL4) complex drives the reactivation of human telomerase reverse transcriptase (hTERT) in colorectal cancer (CRC) by degrading the tumor suppressor, menin 1 (MEN1). Our data show that, in noncancerous intestinal epithelial cells, the transcription factor specificity protein 1 (Sp1) recruits both the histone acetyltransferase p300 and MEN1 to suppress hTERT expression, thus maintaining telomere shortness post-cell division. Inflammation-induced microenvironments trigger an activation of the CRL4DCAF4 E3 ligase, leading to MEN1 ubiquitination and degradation in CRC cells. This process nullifies MEN1's inhibitory action, reactivates hTERT expression at the transcriptional level, interrupts telomere shortening and spurs uncontrolled cellular proliferation. Notably, MEN1 overexpression in CRC cells partially counteracts these oncogenic phenotypes. NSC1517, an inhibitor of the CRL4DCAF4 complex identified through high-throughput screening from a plant-derived chemical pool, hinders MEN1 degradation, attenuates hTERT expression and suppresses tumor growth in mouse xenograft models. Collectively, our research elucidates the transcriptional mechanism driving hTERT reactivation in CRC. Targeting the CRL4DCAF4 E3 ligase emerges as a promising strategy to counteract cancer cell immortalization and curb tumor progression.

Regulation of the water channel aquaporin-2 by cullin E3 ubiquitin ligases.

Aquaporin 2 (AQP2) is a vasopressin (VP)-regulated water channel in the renal collecting duct. Phosphorylation and ubiquitylation of AQP2 play an essential role in controlling the cellular abundance of AQP2 and its accumulation on the plasma membrane in response to VP. Cullin-RING ubiquitin ligases (CRLs) are multisubunit E3 ligases involved in ubiquitylation and degradation of their target proteins, eight of which are expressed in the collecting duct. Here, we used an established cell model of the collecting duct (mpkCCD14 cells) to study the role of cullins in modulating AQP2. Western blotting identified Cul-1 to Cul-5 in mpkCCD14 cells. Treatment of cells for 4 h with a pan-cullin inhibitor (MLN4924) decreased AQP2 abundance, prevented a VP-induced reduction in AQP2 Ser261 phosphorylation, and attenuated VP-induced plasma membrane accumulation of AQP2 relative to the vehicle. AQP2 ubiquitylation levels were significantly higher after MLN4924 treatment compared with controls, and they remained higher despite VP treatment. Cullin inhibition increased ERK1/2 activity, a kinase that regulates AQP2 Ser261 phosphorylation, and VP-induced reductions in ERK1/2 phosphorylation were absent during MLN4924 treatment. Furthermore, the greater Ser261 phosphorylation and reduction in AQP2 abundance during MLN4924 treatment were attenuated during ERK1/2 inhibition. MLN4924 increased intracellular calcium levels via calcium release-activated calcium channels, inhibition of which abolished MLN4924 effects on Ser261 phosphorylation and AQP2 abundance. In conclusion, CRLs play a vital role in mediating some of the effects of VP to increase AQP2 plasma membrane accumulation and AQP2 abundance. Whether modulation of cullin activity can contribute to body water homeostasis requires further studies.NEW & NOTEWORTHY Aquaporin 2 (AQP2) is essential for body water homeostasis and is regulated by the antidiuretic hormone vasopressin. The posttranslational modification ubiquitylation is a key regulator of AQP2 abundance and plasma membrane localization. Here we demonstrate that cullin-RING E3 ligases play a vital role in mediating some of the effects of vasopressin to increase AQP2 abundance and plasma membrane accumulation. The results suggest that manipulating cullin activity could be a novel strategy to alter kidney water handling.

Inhibitory effect of a neddylation blockade on HTLV-1-infected T cells via modulation of NF-κB, AP-1, and Akt signaling

Adult T-cell leukemia (ATL), caused by HTLV-1, is the most lethal hematological malignancy. NEDD8-activating enzyme (NAE) is a component of the NEDD8 conjunction pathway that regulates cullin-RING ubiquitin ligase (CRL) activity. HTLV-1-infected T cells expressed higher levels of NAE catalytic subunit UBA3 than normal peripheral blood mononuclear cells. NAE1 knockdown inhibited proliferation of HTLV-1-infected T cells. The NAE1 inhibitor MLN4924 suppressed neddylation of cullin and inhibited the CRL-mediated turnover of tumor suppressor proteins. MLN4924 inhibited proliferation of HTLV-1-infected T cells by inducing DNA damage, leading to S phase arrest and caspase-dependent apoptosis. S phase arrest was associated with CDK2 and cyclin A downregulation. MLN4924-induced apoptosis was mediated by the upregulation of pro-apoptotic and downregulation of anti-apoptotic proteins. Furthermore, MLN4924 inhibited NF-κB, AP-1, and Akt signaling pathways and activated JNK. Therefore, neddylation inhibition is an attractive strategy for ATL therapy. Our findings support the use of MLN4924 in ATL clinical trials.

CDK-independent role of D-type cyclins in regulating DNA mismatch repair

Although mismatch repair (MMR) is essential for correcting DNA replication errors, it can also recognize other lesions, such as oxidized bases. In G0 and G1, MMR is kept in check through unknown mechanisms as it is error-prone during these cell cycle phases. We show that in mammalian cells, D-type cyclins are recruited to sites of oxidative DNA damage in a PCNA- and p21-dependent manner. D-type cyclins inhibit the proteasomal degradation of p21, which competes with MMR proteins for binding to PCNA, thereby inhibiting MMR. The ability of D-type cyclins to limit MMR is CDK4- and CDK6-independent and is conserved in G0 and G1. At the G1/S transition, the timely, cullin-RING ubiquitin ligase (CRL)-dependent degradation of D-type cyclins and p21 enables MMR activity to efficiently repair DNA replication errors. Persistent expression of D-type cyclins during S-phase inhibits the binding of MMR proteins to PCNA, increases the mutational burden, and promotes microsatellite instability.

The CRL5–SPSB3 ubiquitin ligase targets nuclear cGAS for degradation

Cyclic GMP-AMP synthase (cGAS) senses aberrant DNA during infection, cancer and inflammatory disease, and initiates potent innate immune responses through the synthesis of 2′3′-cyclic GMP-AMP (cGAMP)1–7. The indiscriminate activity of cGAS towards DNA demands tight regulatory mechanisms that are necessary to maintain cell and tissue homeostasis under normal conditions. Inside the cell nucleus, anchoring to nucleosomes and competition with chromatin architectural proteins jointly prohibit cGAS activation by genomic DNA8–15. However, the fate of nuclear cGAS and its role in cell physiology remains unclear. Here we show that the ubiquitin proteasomal system (UPS) degrades nuclear cGAS in cycling cells. We identify SPSB3 as the cGAS-targeting substrate receptor that associates with the cullin–RING ubiquitin ligase 5 (CRL5) complex to ligate ubiquitin onto nuclear cGAS. A cryo-electron microscopy structure of nucleosome-bound cGAS in a complex with SPSB3 reveals a highly conserved Asn-Asn (NN) minimal degron motif at the C terminus of cGAS that directs SPSB3 recruitment, ubiquitylation and cGAS protein stability. Interference with SPSB3-regulated nuclear cGAS degradation primes cells for type I interferon signalling, conferring heightened protection against infection by DNA viruses. Our research defines protein degradation as a determinant of cGAS regulation in the nucleus and provides structural insights into an element of cGAS that is amenable to therapeutic exploitation.

Neddylation is essential for malaria transmission in Plasmodium berghei.

Neddylation is a type of posttranslational modification known to regulate a wide range of cellular processes by covalently conjugating the ubiquitin-like protein Nedd8 to target proteins at lysine residues. However, the role of neddylation in malaria parasites has not been determined. Here, for the first time, we showed that neddylation plays an essential role in malaria transmission in Plasmodium berghei. We found that disruption of Nedd8 did not affect blood-stage propagation, gametocyte development, gamete formation, or zygote formation while abolishing the formation of ookinetes and further transmission of the parasites in mosquitoes. These phenotypic defects in Nedd8 knockout parasites were complemented by reintroducing the gene that restored mosquito transmission to wild-type levels. Our data establish the role of P. berghei Nedd8 in malaria parasite transmission.IMPORTANCENeddylation is a process by which Nedd8 is covalently attached to target proteins through three-step enzymatic cascades. The attachment of Nedd8 residues results in a range of diverse functions, such as cell cycle regulation, metabolism, immunity, and tumorigenesis. The potential neddylation substrates are cullin (CUL) family members, which are implicated in controlling the cell cycle. Cullin neddylation leads to the activation of cullin-RING ubiquitin ligases, which regulate a myriad of biological processes through target-specific ubiquitylation. Neddylation possibly regulates meiosis in zygotes, which subsequently develop into ookinetes. Our findings point to an essential function of this neddylation pathway and highlight its possible importance in designing novel intervention strategies.

Emerging Roles of Cullin-RING Ubiquitin Ligases in Cardiac Development.

Heart development is a spatiotemporally regulated process that extends from the embryonic phase to postnatal stages. Disruption of this highly orchestrated process can lead to congenital heart disease or predispose the heart to cardiomyopathy or heart failure. Consequently, gaining an in-depth understanding of the molecular mechanisms governing cardiac development holds considerable promise for the development of innovative therapies for various cardiac ailments. While significant progress in uncovering novel transcriptional and epigenetic regulators of heart development has been made, the exploration of post-translational mechanisms that influence this process has lagged. Culling-RING E3 ubiquitin ligases (CRLs), the largest family of ubiquitin ligases, control the ubiquitination and degradation of ~20% of intracellular proteins. Emerging evidence has uncovered the critical roles of CRLs in the regulation of a wide range of cellular, physiological, and pathological processes. In this review, we summarize current findings on the versatile regulation of cardiac morphogenesis and maturation by CRLs and present future perspectives to advance our comprehensive understanding of how CRLs govern cardiac developmental processes.

SALL4 is a CRL3REN/KCTD11 substrate that drives Sonic Hedgehog-dependent medulloblastoma

The Sonic Hedgehog (SHH) pathway is crucial regulator of embryonic development and stemness. Its alteration leads to medulloblastoma (MB), the most common malignant pediatric brain tumor. The SHH-MB subgroup is the best genetically characterized, however the molecular mechanisms responsible for its pathogenesis are not fully understood and therapeutic benefits are still limited. Here, we show that the pro-oncogenic stemness regulator Spalt-like transcriptional factor 4 (SALL4) is re-expressed in mouse SHH-MB models, and its high levels correlate with worse overall survival in SHH-MB patients. Proteomic analysis revealed that SALL4 interacts with REN/KCTD11 (here REN), a substrate receptor subunit of the Cullin3-RING ubiquitin ligase complex (CRL3REN) and a tumor suppressor lost in ~30% of human SHH-MBs. We demonstrate that CRL3REN induces polyubiquitylation and degradation of wild type SALL4, but not of a SALL4 mutant lacking zinc finger cluster 1 domain (ΔZFC1). Interestingly, SALL4 binds GLI1 and cooperates with HDAC1 to potentiate GLI1 deacetylation and transcriptional activity. Notably, inhibition of SALL4 suppresses SHH-MB growth both in murine and patient-derived xenograft models. Our findings identify SALL4 as a CRL3REN substrate and a promising therapeutic target in SHH-dependent cancers.

Reduction of oligomer size modulates the competition between cluster formation and phase separation of the tumor suppressor SPOP

Phase separation compartmentalizes many cellular pathways. Given that the same interactions that drive phase separation mediate the formation of soluble complexes below the saturation concentration, the contribution of condensates vs complexes to function is sometimes unclear. Here, we characterized several new cancer-associated mutations of the tumor suppressor Speckle-type POZ protein (SPOP), a substrate recognition subunit of the Cullin3-RING ubiquitin ligase (CRL3). These pointed to a strategy for generating separation-of-function mutations. SPOP self-associates into linear oligomers and interacts with multivalent substrates, and this mediates the formation of condensates. These condensates bear the hallmarks of enzymatic ubiquitination activity. We characterized the effect of mutations in the dimerization domains of SPOP on its linear oligomerization, binding to its substrate DAXX, and phase separation with DAXX. We showed that the mutations reduce SPOP oligomerization and shift the size distribution of SPOP oligomers to smaller sizes. The mutations therefore reduce the binding affinity to DAXX but unexpectedly enhance the poly-ubiquitination activity of SPOP towards DAXX. Enhanced activity may be explained by enhanced phase separation of DAXX with the SPOP mutants. Our results provide a comparative assessment of the functional role of complexes versus condensates and support a model in which phase separation is an important factor in SPOP function. Our findings also suggest that tuning of linear SPOP self-association could be used by the cell to modulate activity and provide insights into the mechanisms underlying hypermorphic SPOP mutations. The characteristics of cancer-associated SPOP mutations suggest a route for designing separation-of-function mutations in other phase-separating systems.

The Spop/BRD4/c-Myc Axis Modulates IMiDs Sensitivity in Multiple Myeloma

The development of novel agents including immunomodulatory drugs (IMiDs) lenalidomide (Len) and pomalidomide (Pom) has led to improved patient outcomes in multiple myeloma (MM); however, MM patients inevitably experience relapse and drug resistance. The expression level of CRBN, CRBN-binding proteins, and CRL4 CRBN ubiquitin ligase is considered to be associated with IMiDs resistance. Clinical studies have shown that MM patients can exhibit IMiDs resistance even without CRBN abnormalities, suggesting the molecular mechanisms of intrinsic resistance to IMiDs are not fully understood. To delineate the molecular mechanisms underlying IMiDs resistance, we performed genome-wide knockout (KO) screening in IMiDs-sensitive MM cells. For in vitro work, we used the MM cell lines MM1S, and H929. We used sgRNA to KO SPOP and BRD4 in MM cells. After performing a CRISPR KO screen in MM cells, we discovered that in addition to CRBN and its associated genes, sgRNAs targeting SPOP (speckle-type POZ protein, a speckled zinc finger protein) were highly enriched after treatment with Poma. Importantly, SPOP KO MM cell lines acquired significant resistance to Pom and Len treatment. To examine whether SPOP KO-induced IMiDs resistance was CRBN-pathway dependent, we assessed CRBN and its downstream interacting protein levels. SPOP KO showed no effect on CRBN expression; moreover, IMiDs can still trigger IKZF1 and IKZF3 degradation, associated with the downregulation of IRF4, suggesting that SPOP mediates sensitivity to IMiDs in a mechanism independent of CRBN-IKZF1/3 axis.Compared to plasma cells from normal donors, we found that the expression levels of SPOP were significantly decreased in MGUS patients, smoldering myeloma patients, newly diagnosed MM patients, and refractory relapsed MM patients (p-values: 2.4e-2, 2.0e-4, 9.9e-3, 3.17e-6, respectively). SPOP is the adaptor protein of the Cullin3-RING ubiquitin ligase complex. Therefore, the function of SPOP is determined by the characteristics of the substrates it recruits. We performed immunoprecipitation with overexpressed SPOP in MM cells, followed by mass spectrometry analysis, and identified BRD4 as a candidate protein binding to SPOP in MM, which was confirmed by Co-IP. KO of SPOP led to an upregulation of BRD4 protein levels in MM cells. As a downstream regulator of BRD4, the oncogene c-Myc showed a significant increase in protein expression in IMiDs-resistant MM patients. Furthermore, the KO of SPOP notably enhanced c-Myc expression in MM cells, while the KO of BRD4 in SPOP KO MM cells decreaed c-Myc expression and restored MM cell sensitivity to IMiD treatment. These findings suggest that SPOP modulates c-Myc expression through BRD4 in a CRBN-IKZF1/3 independent manner, thereby mediating MM cell sensitivity to IMiDs. BRD4 plays a significant role in the development and progression of various tumors. The BRD4 inhibitor JQ1 has been shown to exhibit anti-tumor effects in multiple types of cancer. We found that combination treatment with JQ-1 and IMiDs reversed IMiDs resistance of SPOP KO MM cell lines and primary MM cells from relapsed patients. Our data show that SPOP is a CRBN-independent modulator of IMiDs sensitivity by regulating the BRD4/c-Myc axis and provides the preclinical rationale for combining IMiDs with BRD4 inhibitors to overcome IMiDs resistance and improve patient outcomes.