Activity Of cullin-RING E3 Ubiquitin Ligases Research Articles

Nedd8-Activating Enzyme Inhibition Enhances Anti-Tumor Immunity and PD1 Blockade in In Vivo lymphoma Models

Nedd8-Activating Enzyme Inhibition Enhances Anti-Tumor Immunity and PD1 Blockade in In Vivo lymphoma Models

Neddylation Pathway Regulates Treg Differentiation and T Cell Function in Chronic Lymphocytic Leukemia (CLL) Ex Vivo and Murine In Vivo Studies

Introduction: Inhibitors of B-cell receptor associated kinases (e.g., Bruton tyrosine kinase and phoshpoinotiside-3 kinase) have led to significant improvement in outcomes of patients with CLL. Furthermore, such therapies have immunomodulatory effects. This is particularly relevant given that T cells from patients with CLL are functionally compromised and demonstrate impaired immune synapse formation, adhesion, migration and cytotoxicity. It is critical to improve understanding of the immunomodulatory properties of the novel agents as this will help understand their effect on the immune system in CLL, mitigate toxicities as well as inform future drug development. Pevonedistat, (MLN4924) forms an irreversible covalent adduct with NEDD8, a ubiquitin-like modifier, thereby inhibiting the NEDD8-activating enzyme (NAE). This leads to decreased neddylation and reduced activity of cullin-RING E3 ubiquitin ligases (CRLs). Ultimately, a decrease in CRL activity leads to reduced ubiquitination and proteasomal degradation of CRL substrates, extending the half-life of these proteins, including inhibitor of NFκB (IκB). We have shown that pevonedistat abrogates NFκB activation in CLL cells. Importantly, NFκB activation is indispensable in T-cell activation. However, there is paucity of data regarding the immune effects of targeting neddylation. Here we address this knowledge gap and demonstrate that NAE inhibition may have favorable immunomodulatory effects in CLL. Methods: Peripheral blood mononuclear cells were isolated from patients with CLL and T cells were purified using Dynabeads. Pevonedistat was provided by Millennium Pharmaceuticals, Inc. (Cambridge, MA). For gene expression analysis, FACS-sorted naïve CD4+ T cells were pre-treated with pevonedistat for 1 hour prior to T-cell receptor (TCR; αCD3/CD28) stimulation; RNA was harvested 3 or 24 hours after stimulation and analyzed on a Clariom S microarray chip. For polarization assays, FACS-sorted naïve CD4+ T cells were TCR-activated for 7 days under Th1/2/17/Treg-differentiation conditions. BALB/c mice were administered 60 mg/kg pevonedistat SC twice weekly for 3 weeks and T cell populations were analyzed by flow cytometry. Results: In vitro treatment of T cells with pevonedistat led to rapid reduction in neddylated cullins and stabilization of pIκBα. NAE inhibition did not impede proximal TCR signaling following TCR stimulation (pZAP70, pERK). GSEA demonstrated downmodulation of NFκB and IL-2 signaling pathways in pevonedistat-treated cells by 3 h. Despite this, CD4/CD8+ T cells exhibited normal induction of early activation markers (CD40L, CD69). By contrast, we observed reduced expression of CD38, HLA-DR and PD-1 and diminished CD25 following continuous treatment with pevonedistat for 72 h. This was accompanied by dose-dependent decrease in IL-2 secretion and reduced proliferation of the CD4/CD8+ T cell subsets (CFSE), but no apoptosis. Sorted naive T cells treated with pevonedistat in Th/Treg polarizing conditions exhibited an increase in IFNγ secretion and a decrease in IL-4, suggesting a shift toward Th1 phenotype. Furthermore, we observed a robust decrease of the inducible Treg (iTreg) FoxP3+ population. Loss of iTregs was accompanied by ablated IL-2/STAT5 signaling. Concurrently, we observed a modest increase in Th17 subpopulation following NAE inhibition. We found increased expression of HIF-1α, a CRL target, in pevonedistat-treated T cells, which may have contributed to this phenomenon. To mimic the clinical pharmacokinetics of pevonedistat, we performed 2 h pulse treatment with pevonedistat prior to TCR stimulation. Under these conditions NFκB activity fully recovered by 24 h. Importantly, allogeneic (OCI-LY19 cells) and autologous (CD40L-stimulated CLL cells) T-cell cytotoxicity, perforin and granzyme B production were not disrupted by NAE inhibition. In vivo administration of pevonedistat in immunocompetent BALB/c mice resulted in a decrease of Treg population, confirming in vitro data. Conclusions: Our data suggest that targeting neddylation may help rebalance T cells towards healthy immune subsets in CLL via the reduction of the Treg/Th2 phenotypes. Combined with our earlier reports that targeting NAE kills CLL cells under lymph node-mimicking conditions, these data provide a strong rationale for continued investigation of pevonedistat in CLL and lymphoid malignancies. Disclosures Berger: Millennium Pharmaceuticals, Inc., Cambridge, MA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Danilov:TG Therapeutics: Consultancy; Takeda Oncology: Research Funding; MEI: Research Funding; Verastem Oncology: Consultancy, Other: Travel Reimbursement , Research Funding; AstraZeneca: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Seattle Genetics: Consultancy; Gilead Sciences: Consultancy, Research Funding; Bristol-Meyers Squibb: Research Funding; Aptose Biosciences: Research Funding; Bayer Oncology: Consultancy, Research Funding; Celgene: Consultancy; Curis: Consultancy; Abbvie: Consultancy; Pharmacyclics: Consultancy; Janssen: Consultancy.

Coupled monoubiquitylation of the co-E3 ligase DCNL1 by Ariadne-RBR E3 ubiquitin ligases promotes cullin-RING ligase complex remodeling

Cullin-RING E3 ubiquitin ligases (CRLs) are large and diverse multisubunit protein complexes that contribute to about one-fifth of ubiquitin-dependent protein turnover in cells. CRLs are activated by the attachment of the ubiquitin-like protein neural precursor cell expressed, developmentally down-regulated 8 (NEDD8) to the cullin subunits. This cullin neddylation is essential for a plethora of CRL-regulated cellular processes and is vital for life. In mammals, neddylation is promoted by the five co-E3 ligases, defective in cullin neddylation 1 domain-containing 1–5 (DCNL1–5); however, their functional regulation within the CRL complex remains elusive. We found here that the ubiquitin-associated (UBA) domain–containing DCNL1 is monoubiquitylated when bound to CRLs and that this monoubiquitylation depends on the CRL-associated Ariadne RBR ligases TRIAD1 (ARIH2) and HHARI (ARIH1) and strictly requires the DCNL1's UBA domain. Reconstitution of DCNL1 monoubiquitylation in vitro revealed that autoubiquitylated TRIAD1 mediates binding to the UBA domain and subsequently promotes a single ubiquitin attachment to DCNL1 in a mechanism previously dubbed coupled monoubiquitylation. Moreover, we provide evidence that DCNL1 monoubiquitylation is required for efficient CRL activity, most likely by remodeling CRLs and their substrate receptors. Collectively, this work identifies DCNL1 as a critical target of Ariadne RBR ligases and coupled monoubiquitylation of DCNL1 as an integrated mechanism that affects CRL activity and client–substrate ubiquitylation at multiple levels.

Targeted inhibition of the COP9 signalosome for treatment of cancer.

The COP9 signalosome (CSN) is a central component of the activation and remodelling cycle of cullin-RING E3 ubiquitin ligases (CRLs), the largest enzyme family of the ubiquitin–proteasome system in humans. CRLs are implicated in the regulation of numerous cellular processes, including cell cycle progression and apoptosis, and aberrant CRL activity is frequently associated with cancer. Remodelling of CRLs is initiated by CSN-catalysed cleavage of the ubiquitin-like activator NEDD8 from CRLs. Here we describe CSN5i-3, a potent, selective and orally available inhibitor of CSN5, the proteolytic subunit of CSN. The compound traps CRLs in the neddylated state, which leads to inactivation of a subset of CRLs by inducing degradation of their substrate recognition module. CSN5i-3 differentially affects the viability of tumour cell lines and suppresses growth of a human xenograft in mice. Our results provide insights into how CSN regulates CRLs and suggest that CSN5 inhibition has potential for anti-tumour therapy.

Suramin inhibits cullin-RING E3 ubiquitin ligases

Cullin-RING E3 ubiquitin ligases (CRL) control a myriad of biological processes by directing numerous protein substrates for proteasomal degradation. Key to CRL activity is the recruitment of the E2 ubiquitin-conjugating enzyme Cdc34 through electrostatic interactions between E3's cullin conserved basic canyon and the acidic C terminus of the E2 enzyme. This report demonstrates that a small-molecule compound, suramin, can inhibit CRL activity by disrupting its ability to recruit Cdc34. Suramin, an antitrypansomal drug that also possesses antitumor activity, was identified here through a fluorescence-based high-throughput screen as an inhibitor of ubiquitination. Suramin was shown to target cullin 1's conserved basic canyon and to block its binding to Cdc34. Suramin inhibits the activity of a variety of CRL complexes containing cullin 2, 3, and 4A. When introduced into cells, suramin induced accumulation of CRL substrates. These observations help develop a strategy of regulating ubiquitination by targeting an E2-E3 interface through small-molecule modulators.

The cullin-4 complex DCDC does not require E3 ubiquitin ligase elements to control heterochromatin in Neurospora crassa.

The cullin-4 (CUL4) complex DCDC (DIM-5/-7/-9/CUL4/DDB1 complex) is essential for DNA methylation and heterochromatin formation in Neurospora crassa. Cullins form the scaffold of cullin-RING E3 ubiquitin ligases (CRLs) and are modified by the covalent attachment of NEDD8, a ubiquitin-like protein that regulates the stability and activity of CRLs. We report that neddylation is not required for CUL4-dependent DNA methylation or heterochromatin formation but is required for the DNA repair functions. Moreover, the RING domain protein RBX1 and a segment of the CUL4 C terminus that normally interacts with RBX1, the E2 ligase, CAND1, and CSN are dispensable for DNA methylation and heterochromatin formation by DCDC. Our study provides evidence for the noncanonical functions of core CRL components.

Inhibition of Cullin RING Ligases by Cycle Inhibiting Factor: Evidence for Interference with Nedd8-Induced Conformational Control

Cycle inhibiting factor (Cif) is produced by pathogenic intracellular bacteria and injected into the host cells via a type III secretion system. Cif is known to interfere with the eukaryotic cell cycle by inhibiting the function of cullin RING E3 ubiquitin ligases (CRLs). Cullin proteins form the scaffold protein of CRLs and are modified with the ubiquitin-like protein Nedd8, which exerts important conformational control required for CRL activity. Cif has recently been shown to catalyze the deamidation of Gln40 in Nedd8 to Glu. Here, we addressed how Nedd8 deamidation inhibits CRL activity. Our results indicate that Burkholderia pseudomallei Cif (also known as CHBP) inhibits the deconjugation of Nedd8 in vivo by inhibiting binding of the deneddylating COP9 signalosome (CSN) complex. We provide evidence that the reduced binding of CSN and the inhibition of CRL activity by Cif are due to interference with Nedd8-induced conformational control, which is dependent on the interaction between the Nedd8 hydrophobic patch and the cullin winged-helix B subdomain. Of note, mutation of Gln40 to Glu in ubiquitin, an additional target of Cif, inhibits the interaction between the hydrophobic surface of ubiquitin and the ubiquitin-binding protein p62/SQSTM1, showing conceptually that Cif activity can impair ubiquitin/ubiquitin-like protein non-covalent interactions. Our results also suggest that Cif may exert additional cellular effects by interfering with the association between ubiquitin and ubiquitin-binding proteins.