CRL4 E3 Ubiquitin Ligase Research Articles

METTL3/METTL14 maintain human nucleoli integrity by mediating SUV39H1/H2 degradation

Nucleoli are fundamentally essential sites for ribosome biogenesis in cells and formed by liquid-liquid phase separation (LLPS) for a multilayer condensate structure. How the nucleoli integrity is maintained remains poorly understood. Here, we reveal that METTL3/METTL14, the typical methyltransferase complex catalyzing N6-methyladnosine (m6A) on mRNAs maintain nucleoli integrity in human embryonic stem cells (hESCs). METTL3/METTL14 deficiency impairs nucleoli and leads to the complete loss of self-renewal in hESCs. We further show that SUV39H1/H2 protein, the methyltransferases catalyzing H3K9me3 were dramatically elevated in METTL3/METTL14 deficient cells, which causes an accumulation and infiltration of H3K9me3 across the whole nucleolus and impairs the LLPS. Mechanistically, METTL3/METTL14 complex serves as an essential adapter for CRL4 E3 ubiquitin ligase targeting SUV39H1/H2 for polyubiquitination and proteasomal degradation and therefore prevents H3K9me3 accumulation in nucleoli. Together, these findings uncover a previously unknown role of METTL3/METTL14 to maintain nucleoli integrity by facilitating SUV39H1/H2 degradation in human cells.

Potential of GSPT1 as a novel target for glioblastoma therapy.

Glioblastoma is the most common malignant brain tumor in adults, the survival rate of which has not significantly improved over the past three decades. Therefore, there is an urgent need to develop novel treatment modalities. We previously reported that G1 to S phase transition 1 (GSPT1) depletion induces delayed cell cycle in primary astrocytes. Herein, we examined the potential of GSPT1 as a novel target for glioblastoma therapy. CC-885, a cereblon modulator that degrades GSPT1 by bridging GSPT1 to the CRL4 E3 ubiquitin ligase complex, was administered to nude mice with transplanted brain tumors of U87 glioblastoma cells. The survival period was significantly longer in CC-885 treated mice than in control mice. Furthermore, we generated GSPT1-knockout (KO) U87 cells and GSPT1-KO U87 cells with stable overexpression of FLAG-tagged GSPT1 (Rescued GSPT1-KO). Mice with transplanted GSPT1-KO U87 cells and Rescued GSPT1-KO U87 cells showed significantly longer and similar survival periods, respectively, as those with wild-type (WT) U87 cells. GSPT1-KO U87 cells showed enhanced apoptosis, detected by cleaved PARP1, compared to WT U87 cells. Brain tumors with transplantation of GSPT1-KO U87 cells also showed enhanced apoptosis compared to those with transplantation of WT and Rescued GSPT1-KO U87 cells. GSPT1 expression was confirmed in patients with glioblastoma. However, the clinical study using 87 glioblastoma samples showed that GSPT1 mRNA levels were not associated with overall survival. Taken together, we propose that GSPT1 is an essential protein for glioblastoma growth, but not its malignant characteristics, and that GSPT1 is a potential target for developing glioblastoma therapeutics.

DCAF13 inhibits the p53 signaling pathway by promoting p53 ubiquitination modification in lung adenocarcinoma

BackgroundLung cancer is a malignant tumor with the highest mortality worldwide. Abnormalities in the ubiquitin proteasome system are considered to be contributed to lung cancer progression with deleterious effects. DDB1 and CUL4 associated factor 13 (DCAF13) is a substrate receptor of the E3 ubiquitin ligase CRL4, but its role in lung cancer remains unknown. In this study, we aimed to investigate the regulatory mechanisms of DCAF13 in lung adenocarcinoma (LUAD).MethodsSo as to investigate the effect of DCAF13 on lung adenocarcinoma cell function using in vivo and in vitro. Mechanistically, we have identified the downstream targets of DCAF13 by using RNA-sequencing, as well as ubiquitination assays, co-immunoprecipitation, immunofluorescence, immunohistochemistry and chromatin immunoprecipitation - qPCR experiments.ResultsOur findings reveal that DCAF13 is a carcinogenic factor in LUAD, as it is highly expressed and negatively correlated with clinical outcomes in LUAD patients. Through RNA-sequencing, it has been shown that DCAF13 negatively regulates the p53 signaling pathway and inhibits p53 downstream targets including p21, BAX, FAS, and PIDD1. We also demonstrate that DCAF13 can bind to p53 protein, leading to K48-linked ubiquitination and degradation of p53. Functionally, we have shown that DCAF13 knockdown inhibits cell proliferation and migration. Our results highlight the significant role of DCAF13 in promoting LUAD progression by inhibiting p53 protein stabilization and the p53 signaling pathway. Furthermore, our findings suggest that high DCAF13 expression is a poor prognostic indicator in LUAD, and DCAF13 may be a potential therapeutic target for treating with this aggressive cancer.ConclusionsThe DCAF13 as a novel negative regulator of p53 to promote LUAD progression via facilitating p53 ubiquitination and degradation, suggesting that DCAF13 might be a novel biomarker and therapeutical target for LUAD.

Cisplatin-activated ERβ/DCAF8 positive feedback loop induces chemoresistance in non-small cell lung cancer via PTEN/Akt axis

High levels of the estrogen receptor β (ERβ) predict poor prognosis following platinum-containing adjuvant chemotherapies in patients with non-small cell lung cancer (NSCLC). However, the precise role of ERβ remains elusive. In this study, we demonstrated that targeting ERβ could significantly increase the cytotoxicity of cisplatin both in vitro and in vivo. Mechanically, cisplatin directly binds to ERβ, which facilitates its homodimerization and nuclear translocation. ERβ activation transcriptionally represses the expression of DCAF8, an adaptor of CRL4 E3 ubiquitin ligase, which in turn attenuates the proteasomal degradation of ERβ, leading to ERβ accumulation; this positive feedback loop results in Akt activation and eventually cisplatin resistance in NSCLC through PTEN inhibition. Moreover, low expression of DCAF8 and high expression of ERβ are associated with treatment resistance in patients receiving cisplatin-containing adjuvant chemotherapy. The present results provide insights into the underlying mechanism of ERβ-induced cisplatin resistance and offer an alternative therapeutic strategy to improve the efficacy of platinum-based chemotherapy in patients with NSCLC.

The aryl sulfonamide indisulam inhibits gastric cancer cell migration by promoting the ubiquitination and degradation of the transcription factor ZEB1

Gastric cancer is one of the cancers with high morbidity and mortality worldwide. The aryl sulfonamide indisulam inhibits the proliferation of several types of cancer cells through its function as a molecular glue to promote the ubiquitination and degradation of RNA-binding motif protein 39 (RBM39). However, it is unknown whether and how indisulam regulates the migration of cancer cells. In this work, using label-free quantitative proteomics, we discover that indisulam significantly attenuates N-cadherin, a marker for epithelial to mesenchymal transition and migration of cancer cells. Our bioinformatics analysis and biochemical experiments reveal that indisulam promotes the interaction between the zinc finger E-box-binding homeobox 1 (ZEB1), a transcription factor of N-cadherin, and DCAF15, a substrate receptor of CRL4 E3 ubiquitin ligase, and enhances ZEB1 ubiquitination and proteasomal degradation. In addition, our cell line-based experiments demonstrate that indisulam inhibits the migration of gastric cancer cells in a ZEB1-dependent manner. Analyses of patient samples and datasets in public databases reveal that tumor tissues from patients with gastric cancer express high ZEB1 mRNA and this high expression reduces patient survival rate. Finally, we show that treatment of gastric tumor samples with indisulam significantly reduces ZEB1 protein levels. Therefore, this work discloses a new mechanism by which indisulam inhibits the migration of gastric cancer cells, indicating that indisulam exhibits different biological functions through distinct signaling molecules.

Targeting CRL4 suppresses chemoresistant ovarian cancer growth by inducing mitophagy

Chemoresistance has long been the bottleneck of ovarian cancer (OC) prognosis. It has been shown that mitochondria play a crucial role in cell response to chemotherapy and that dysregulated mitochondrial dynamics is intricately linked with diseases like OC, but the underlying mechanisms remain equivocal. Here, we demonstrate a new mechanism where CRL4CUL4A/DDB1 manipulates OC cell chemoresistance by regulating mitochondrial dynamics and mitophagy. CRL4CUL4A/DDB1 depletion enhanced mitochondrial fission by upregulating AMPKαThr172 and MFFSer172/Ser146 phosphorylation, which in turn recruited DRP1 to mitochondria. CRL4CUL4A/DDB1 loss stimulated mitophagy through the Parkin-PINK1 pathway to degrade the dysfunctional and fragmented mitochondria. Importantly, CRL4CUL4A/DDB1 loss inhibited OC cell proliferation, whereas inhibiting autophagy partially reversed this disruption. Our findings provide novel insight into the multifaceted function of the CRL4 E3 ubiquitin ligase complex in regulating mitochondrial fission, mitophagy, and OC chemoresistance. Disruption of CRL4CUL4A/DDB1 and mitophagy may be a promising therapeutic strategy to overcome chemoresistance in OC.

CRL4CRBN E3 Ligase Complex as a Therapeutic Target in Multiple Myeloma

Simple SummaryImmunomodulatory drugs (IMiDs) are effective in the treatment of multiple myeloma (MM) and other hematological malignancies. Cereblon (CRBN), a target of IMiDs, forms the CRL4 E3 ubiquitin ligase complex (CRL4CRBN) with DDB1, CUL4A and RBX1. The insight into the molecular mechanism of IMiDs action has advanced dramatically since the identification of cereblon (CRBN) as their direct target. Targeting CRBN by IMiDs modifies CRL4CRBN substrate specificity towards non-physiological protein targets which are subsequently ubiquitinated and degraded by the proteasome. To date, IMiDs are the only known group of protein degraders used in clinical practice. This review provides the current state of knowledge about thalidomide and its derivatives’ mechanisms of action, and highlights the future perspectives for targeted protein degraders.Multiple myeloma (MM) is the second most common hematological malignancy with a recurrent clinical course. The introduction of immunomodulatory drugs (IMiDs) was one of the milestones in MM therapy leading to a significant improvement in patients’ prognosis. Currently, IMiDs are the backbone of MM therapy in newly diagnosed and relapsed/refractory settings. It is now known that IMiDs exert their anti-myeloma activity mainly by binding cereblon (CRBN), the substrate receptor protein of the CRL4 E3 ubiquitin ligase (CRL4CRBN) complex. By binding CRBN, IMiDs alter its substrate specificity, leading to ubiquitination and proteasomal degradation of proteins essential for MM cell survival. Following the success of IMiDs, it is not surprising that the possibility of using the CRL4CRBN complex’s activity to treat MM is being further explored. In this review, we summarize the current state of knowledge about novel players in the MM therapeutic landscape, namely the CRBN E3 ligase modulators (CELMoDs), the next generation of IMiDs with broader biological activity. In addition, we discuss a new strategy of tailored proteolysis called proteolysis targeting chimeras (PROTACs) using the CRL4CRBN to degrade typically undruggable proteins, which may have relevance for the treatment of MM and other malignancies in the future.

Development of Photolenalidomide for Cellular Target Identification.

The thalidomide analogue lenalidomide (Len) is a clinical therapeutic that alters the substrate engagement of cereblon (CRBN), a substrate receptor for the CRL4 E3 ubiquitin ligase. Here, we report the development of photolenalidomide (pLen), a Len probe with a photoaffinity label and enrichment handle, designed for target identification by chemical proteomics. pLen preserves the substrate degradation profile, phenotypic antiproliferative and immunomodulatory properties of Len, and enhances interactions with the thalidomide-binding domain of CRBN, as revealed by binding site mapping and molecular modeling. Using pLen, we captured the known targets IKZF1 and CRBN from multiple myeloma MM.1S cells and further identified a new target, eukaryotic translation initiation factor 3 subunit i (eIF3i), from HEK293T cells. eIF3i is directly labeled by pLen and forms a ternary complex with CRBN in the presence of Len across several epithelial cell lines but is itself not ubiquitylated or degraded. These data point to the existence of a broader array of targets induced by ligands to CRBN that may or may not be degraded, which can be identified by the highly translatable application of pLen to additional biological systems.

The CRL4VPRBP(DCAF1) E3 ubiquitin ligase directs constitutive RAG1 degradation in a non-lymphoid cell line.

The development of B and T lymphocytes critically depends on RAG1/2 endonuclease activity to mediate antigen receptor gene assembly by V(D)J recombination. Although control of RAG1/2 activity through cell cycle- and ubiquitin-dependent degradation of RAG2 has been studied in detail, relatively little is known about mechanisms regulating RAG1 stability. We recently demonstrated that VprBP/DCAF1, a substrate adaptor for the CRL4 E3 ubiquitin ligase complex, is required to maintain physiological levels of RAG1 protein in murine B cells by facilitating RAG1 turnover. Loss of VprBP/DCAF1 in vivo results in elevated RAG1 expression, excessive V(D)J recombination, and immunoglobulin light chain repertoire skewing. Here we show that RAG1 is constitutively degraded when ectopically expressed in a human fibroblast cell line. Consistent with our findings in murine B cells, RAG1 turnover under these conditions is sensitive to loss of VprBP, as well as CRL4 or proteasome inhibition. Further evidence indicates that RAG1 degradation is ubiquitin-dependent and that RAG1 association with the CRL4VPRBP/DCAF1 complex is independent of CUL4 activation status. Taken together, these findings suggest V(D)J recombination co-opts an evolutionarily conserved and constitutively active mechanism to ensure rapid RAG1 turnover to restrain excessive RAG activity.

Novel Molecular Mechanism of Lenalidomide in Myeloid Malignancies Independent of Deletion of Chromosome 5q.

Simple SummaryLenalidomide is an immunomodulatory drug (IMiD) that has achieved clinical efficacies in multiple myeloma (MM) and myelodysplastic syndromes (MDS) with a single deletion of chromosome 5q (del(5q)). However, many patients treated with lenalidomide relapse and become resistant. Recent studies have demonstrated that lenalidomide binds a protein called cereblon (CRBN), leading to reduced protein levels of IKZF1 and IKZF3 and casein kinase 1 alpha. We have identified signaling molecules downstream of IKZF1, G protein-coupled receptor 68 (GPR68) and regulator of calcineurin 1 (RCAN1) in myeloid malignancies, including MDS and acute myeloid leukemia (AML) with or without del(5q). This review summarizes how lenalidomide exerts anti-tumor activity and highlights novel therapeutic targets that could enhance the anti-tumor activity of lenalidomide with a focus on myeloid malignancies, especially without del(5q).Lenalidomide as well as other immunomodulatory drugs (IMiDs) have achieved clinical efficacies in certain sub-types of hematologic malignancies, such as multiple myeloma, lower-risk myelodysplastic syndromes (MDS) with a single deletion of chromosome 5q (del(5q)) and others. Despite superior clinical response to lenalidomide in hematologic malignancies, relapse and resistance remains a problem in IMiD-based therapy. The last ten years have witnessed the discovery of novel molecular mechanism of IMiD-based anti-tumor therapy. IMiDs bind human cereblon (CRBN), the substrate receptor of the CRL4 E3 ubiquitin ligase complex. Binding of CRBN with IMiDs leads to degradation of the Ikaros family zinc finger proteins 1 and 3 (IKZF1 and IKZF3) and casein kinase 1 alpha. We have found that lenalidomide-mediated degradation of IKZF1 leads to activation of the G protein-coupled receptor 68 (GPR68)/calcium/calpain pro-apoptotic pathway and inhibition of the regulator of calcineurin 1 (RCAN1)/calcineurin pro-survival pathway in MDS and acute myeloid leukemia (AML). Calcineurin inhibitor Cyclosporin-A potentiates the anti-leukemia activity of lenalidomide in MDS/AML with or without del(5q). These findings broaden the therapeutic potential of IMiDs. This review summarizes novel molecular mechanism of lenalidomide in myeloid malignancies, especially without del(5q), in the hope to highlight novel therapeutic targets.

Abstract 4891: Hepatitis B virus remodels host protein interaction networks to generate distinct cellular dependencies

Abstract Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide. Advanced HCC has proven particularly difficult to treat because of a scarcity of clear genetic drivers of cancer progression; thus, there are currently no predictive markers that guide HCC therapy. HCC arises in the context of co-morbid hepatitis due to hepatitis B virus (HBV), hepatitis C (HCV) or fatty liver disease. We hypothesize that protein-protein interactions (PPIs) between viral proteins and HCC genes may contribute to tumor initiation and maintenance. In order to characterize these PPIs, we performed affinity purification - mass spectrometry (APMS), defining 145 HBV/host PPIs including known and novel interacting partners. We next used a network propagation algorithm to identify host genes and protein complexes that were preferentially mutated in the absence of HBV infection. HBV is a small DNA virus, with 4 genes of which only one has enzymatic activity, raising a question as to how HBV interaction modifies host behavior. Using AP-MS of host proteins, we found that the HBV X protein (HBx) remodels multiple host protein complexes through direct interaction. These physical effects on complex components result in distinct biochemical behavior from the CRL4 E3 ubiquitin ligase complex as well as the phosphatase PP2A, as determined through global phosphoproteomics and ubiquitin analysis. We show that this remodeling driven by HBx substantially changes cellular protein turnover and downstream signaling dynamics. We followed this up with assessments of cellular viability and proliferation in response to pharmacological inhibition or CRISPRi-based knockdown of HBx effectors. Our data support a model where HBV proteins alter the components and behavior of key regulatory protein complexes in the cell, altering tumor behavior and raising the possibility of precision therapeutics for HCC. Citation Format: John D. Gordan, Adriana Pitea, Manon Eckhardt, Gwendolyn Jang, Rigney E. Turnham, Alex L M. Choi, John Von Dollen, Huat C. Lim, Elizabeth F. Thayer, R. Katie Kelley, Danielle L. Swaney, Wei Zhang, Fabian J. Theis, Trey Ideker, Nevan J. Krogan. Hepatitis B virus remodels host protein interaction networks to generate distinct cellular dependencies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4891.

P63 is a cereblon substrate involved in thalidomide teratogenicity.

Cereblon (CRBN) is a primary target of thalidomide and mediates its multiple pharmacological activities, including teratogenic and antimyeloma activities. CRBN functions as a substrate receptor of the E3 ubiquitin ligase CRL4, whose substrate specificity is modulated by thalidomide and its analogs. Although a number of CRL4CRBN substrates have recently been identified, the substrate involved in thalidomide teratogenicity is unclear. Here we show that p63 isoforms are thalidomide-dependent CRL4CRBN neosubstrates that are responsible, at least in part, for its teratogenic effects. The p53 family member p63 is associated with multiple developmental processes. ∆Np63α is essential for limb development, while TAp63α is important for cochlea development and hearing. Using a zebrafish model, we demonstrate that thalidomide exerts its teratogenic effects on pectoral fins and otic vesicles by inducing the degradation of ∆Np63α and TAp63α, respectively. These results may contribute to the invention of new thalidomide analogs lacking teratogenic activity.

Cereblon Control of Zebrafish Brain Size by Regulation of Neural Stem Cell Proliferation

SummaryThalidomide is a teratogen that causes multiple malformations in the developing baby through its interaction with cereblon (CRBN), a substrate receptor subunit of the CRL4 E3 ubiquitin ligase complex. CRBN was originally reported as a gene associated with autosomal recessive non-syndromic mild mental retardation. However, the function of CRBN during brain development remains largely unknown. Here we demonstrate that CRBN promotes brain development by facilitating the proliferation of neural stem cells (NSCs). Knockdown of CRBN in zebrafish embryos impaired brain development and led to small brains, as did treatment with thalidomide. By contrast, overexpression of CRBN resulted in enlarged brains, leading to the expansion of NSC regions and increased cell proliferation in the early brain field and an expanded expression of brain region-specific genes and neural and glial marker genes. These results demonstrate that CRBN functions in the determination of brain size by regulating the proliferation of NSCs during development.

Proliferating cell nuclear antigen interacts with the CRL4 ubiquitin ligase subunit CDT2 in DNA synthesis–induced degradation of CDT1

During DNA replication or repair, the DNA polymerase cofactor, proliferating cell nuclear antigen (PCNA), homotrimerizes and encircles the replicating DNA, thereby acting as a DNA clamp that promotes DNA polymerase processivity. The formation of the PCNA trimer is also essential for targeting the replication-licensing protein, chromatin-licensing, and DNA replication factor 1 (CDT1), for ubiquitin-dependent proteolysis to prevent chromosomal DNA re-replication. CDT1 uses its PCNA-interacting peptide box (PIP box) to interact with PCNA, and the CRL4 E3 ubiquitin ligase subunit CDT2 is recruited through the formation of PCNA-CDT1 complexes. However, it remains unclear how CDT1 and many other PIP box-containing proteins are marked for degradation by the CRL4CDT2 ubiquitin ligase during DNA replication or damage. Here, using recombinant protein expression coupled with site-directed mutagenesis, we report that CDT2 and PCNA directly interact and this interaction depends on the presence of a highly conserved, C-terminal PIP box-like region in CDT2. Deletion or mutation of this region abolished the CDT2-PCNA interaction between CDT2 and PCNA both in vitro and in vivo Moreover, PCNA-dependent CDT1 degradation in response to DNA damage and replication during the cell cycle requires an intact PIP box in CDT2. The requirement of the PIP boxes in both CDT2 and its substrate CDT1 suggests that the formation of the PCNA trimeric clamp around DNA during DNA replication and repair may bring together CDT1 and CRL4CDT2 ubiquitin E3 ligase to target CDT1 for proteolysis in a DNA synthesis-dependent manner.

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.

DCAF13 promotes pluripotency by negatively regulating SUV39H1 stability during early embryonic development.

Mammalian oocytes and zygotes have the unique ability to reprogram a somatic cell nucleus into a totipotent state. SUV39H1/2-mediated histone H3 lysine-9 trimethylation (H3K9me3) is a major barrier to efficient reprogramming. How SUV39H1/2 activities are regulated in early embryos and during generation of induced pluripotent stem cells (iPSCs) remains unclear. Since expression of the CRL4 E3 ubiquitin ligase in oocytes is crucial for female fertility, we analyzed putative CRL4 adaptors (DCAFs) and identified DCAF13 as a novel CRL4 adaptor that is essential for preimplantation embryonic development. Dcaf13 is expressed from eight-cell to morula stages in both murine and human embryos, and Dcaf13 knockout in mice causes preimplantation-stage mortality. Dcaf13 knockout embryos are arrested at the eight- to sixteen-cell stage before compaction, and this arrest is accompanied by high levels of H3K9me3. Mechanistically, CRL4-DCAF13 targets SUV39H1 for polyubiquitination and proteasomal degradation and therefore facilitates H3K9me3 removal and zygotic gene expression. Taken together, CRL4-DCAF13-mediated SUV39H1 degradation is an essential step for progressive genome reprogramming during preimplantation embryonic development.

Cereblon Control of Zebrafish Brain Size by Regulation of Neural Stem Cell Proliferation

Thalidomide is a teratogen that causes multiple malformations when exposed to developing vertebrate embryos. This was recently shown to be due to its interaction with Cereblon (CRBN), a novel substrate receptor subunit of the CRL4 E3 ubiquitin ligase complex, whose enzymatic activity is controlled by thalidomide and its analogs. CRBN was originally reported as a gene associated with autosomal recessive non-syndromic mild mental retardation. However, the function of CRBN during brain development remains largely unknown. Here we demonstrate that CRBN promotes brain development by facilitating proliferation of neural stem cells (NSCs). Knockdown of CRBN in zebrafish embryos impaired brain development and led to small brains, as did treatment with thalidomide. By contrast, overexpression of CRBN resulted in enlarged brains, leading to the expansion of NSC regions in the telencephalon, the diencephalon and the midbrain, increased cell proliferation throughout the brain, and an expanded expression of brain region-specific genes and neural and glial marker genes. CRBN functions downstream of Six3 and Lhx2 to regulate neural stem cell proliferation. These results demonstrate a new function for CRBN in the determination of brain size by regulating proliferation of NSCs during development.

Abstract PR13: p53-related protein kinase is a novel prognostic marker and therapeutic target in multiple myeloma

Abstract Background: Multiple myeloma (MM) is a disease characterized by the accumulation of malignant plasma cells in the bone marrow. The introduction of novel drugs, such as immunomodulators (IMIDs), proteasome inhibitors, monoclonal antibodies, and HDAC inhibitors has revolutionized the approach to treat patients. Nevertheless, a better characterization of MM pathogenesis and of the mechanisms of action of current medications is needed to improve patient selection and hence outcomes, by increasing responses to therapies and reducing side effects. To this end, we have developed an approach based on genetic screening and drug characterization that allowed us to identify p53-related protein kinase (TP53RK) as a novel therapeutic target in MM. Methods: Gene expression profiling analysis was performed in primary MM cells and cell lines using Affymetrix U133 Plus 2.0 chips. Knockdown of proteins (TP53RK, c-Myc, p53, RRM1) was carried out using siRNA and/or shRNA. Expression of RNA and protein was assessed by real-time qPCR and immunoblotting, respectively. Cell proliferation was determined by live cell count and MTT assay. Electrophoretic mobility shift assay was used for evaluation of E2F1 activity. Pomalidomide-conjugating beads were generated for pull-down assay. Nuclear magnetic resonance spectroscopy, differential scanning fluorimetry, and isothermal titration calorimetry were used to confirm pomalidomide-Pom binding to TP53RK. Results: We identified a novel protein, called TP53RK, that is expressed at higher levels in malignant plasma cells compared to normal plasma cells. In newly diagnosed MM patients higher TP53RK level confers poor prognosis. Downregulation of TP53RK induces apoptosis and inhibits cellular growth in multiple MM cell lines. TP53RK knockdown inhibits phosphorylation of TP53 (Ser15), promoting downregulation of MYC and E2F1-dependent upregulation of BIM. Importantly, we demonstrated by standard affinity chromatography and nuclear magnetic resonance spectroscopy that lenalidomide and pomalidomide bind to TP53RK affecting its activity, with biologic sequelae similar to TP53RK knockdown. Lenalidomide binds to cereblon (CRBN), a substrate adaptor of the CRL4 E3 ubiquitin ligase, and induces the ubiquitination and degradation of IKZF1 and IKZF3. CRBN knockdown does not alter TP53RK, or p53 axis; conversely, TP53RK downregulation does not modulate CRBN expression, indicating that lenalidomide and pomalidomide independently bind and regulate TP53RK and CRBN. TP53RK knockdown-induced MM cell growth inhibition was observed even in TP53-mutated and TP53-null MM cell lines, suggesting additional molecules targeted by TP53RK. Indeed, we identified RRM1, p18, proteasome inhibitor subunits, and telomerases as potential downstream targets. Hence, our studies demonstrate that either genetic or pharmacologic inhibition of TP53RK triggers MM cell apoptosis via both p53-Myc axis-dependent and -independent pathways. Furthermore, TP53RK blockade may be one of the novel mechanisms of action responsible for IMIDs' effects in MM. Conclusions: Our results identify TP53RK as a marker of poor prognosis in MM, validate TP53RK as a novel therapeutic target in this patient subset, and delineate a novel mechanism whereby IMIDs bind TP53RK, inhibit its activity, and trigger apoptotic MM cell death. Ongoing efforts are directed to developing selective TP53RK inhibitors to improve patient outcome in this patient subgroup. This abstract is also being presented as Poster 04. Citation Format: Francesca Cottini, Teru Hideshima, Yoshihisa Nozawa, Hiroto Ohguchi, Teruhiro Utsugi, Kenneth C Anderson. p53-related protein kinase is a novel prognostic marker and therapeutic target in multiple myeloma [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr PR13.

HIV-1 Vpr protein directly loads helicase-like transcription factor (HLTF) onto the CRL4-DCAF1 E3 ubiquitin ligase

The viral protein R (Vpr) is an accessory virulence factor of HIV-1 that facilitates infection in immune cells. Cellular functions of Vpr are tied to its interaction with DCAF1, a substrate receptor component of the CRL4 E3 ubiquitin ligase. Recent proteomic approaches suggested that Vpr degrades helicase-like transcription factor (HLTF) DNA helicase in a proteasome-dependent manner by redirecting the CRL4-DCAF1 E3 ligase. However, the precise molecular mechanism of Vpr-dependent HLTF depletion is not known. Here, using in vitro reconstitution assays, we show that Vpr mediates polyubiquitination of HLTF, by directly loading it onto the C-terminal WD40 domain of DCAF1 in complex with the CRL4 E3 ubiquitin ligase. Mutational analyses suggest that Vpr interacts with DNA-binding residues in the N-terminal HIRAN domain of HLTF in a manner similar to the recruitment of another target, uracil DNA glycosylase (UNG2), to the CRL4-DCAF1 E3 by Vpr. Strikingly, Vpr also engages a second, adjacent region, which connects the HIRAN and ATPase/helicase domains. Thus, our findings reveal that Vpr utilizes common as well as distinctive interfaces to recruit multiple postreplication DNA repair proteins to the CRL4-DCAF1 E3 ligase for ubiquitin-dependent proteasomal degradation.

Cereblon and its downstream substrates as molecular targets of immunomodulatory drugs.

Thalidomide was first developed as a sedative around 60years ago, but exhibited teratogenicity, leading to serious defects such as limb deformities. Nevertheless, thalidomide is now recognized as a therapeutic drug for the treatment of Hansen's disease and myeloma. Immunomodulatory drugs (IMiDs), a new class of anti-cancer drug derived from thalidomide, have also been developed and exert potent anti-cancer effects. Although the molecular mechanism of thalidomide and IMiDs remained unclear for a long time, cereblon, a substrate receptor of the CRL4 E3 ubiquitin ligase was identified as a primary direct target by a new affinity technique. A growing body of evidence suggests that the effect of IMiDs on myeloma and other cancer cells is mediated by CRBN. Each IMiD binds to CRBN and alters the substrate specificity of the CRBN E3 ubiquitin ligase complex, resulting in breakdown of intrinsic downstream proteins such as Ikaros and Aiolos. Here we give an overview of the current understanding of mechanism of action of IMiDs via CRBN and prospects for the development of new drugs that degrade protein of interest.