EZH2-driven immune evasion defines high-risk pediatric AML with t(16;21) FUS::ERG gene fusion.

Minimal improvement in outcomes for high-risk pediatric acute myeloid leukemia (pAML) patients has been made in the past decades. Nowhere is this more evident than in patients carrying a t(16;21)(p11;q22) FUS::ERG translocation; quick time to relapse and universal failure of hematopoietic stem cell transplant contribute to one of the lowest survival rates in childhood leukemia. Here, we have identified a unique, defining immune-evasion phenotype in FUS::ERG pAML driven by EZH2 and characterized by loss of MHC class I and II molecules and immune co-stimulatory receptors. This loss of immune engagement, present at diagnosis, allows pervasiveness of blasts that prove resistant to standard treatment. We demonstrate that treatment with the FDA-approved EZH2 inhibitor tazemetostat, in combination with IFN-γ, reverses the phenotype, re-expresses MHC receptor expression, and reduces blast viability. EZH2 inhibitors provide a novel therapeutic option for this high-risk population and may prove a beneficial supplemental treatment for FUS::ERG pAML.

Crystal structure of the Frizzled 4 receptor in a ligand-free state.

Frizzled receptors (FZDs) are class-F G-protein-coupled receptors (GPCRs) that function in Wnt signalling and are essential for developing and adult organisms1,2. As central mediators in this complex signalling pathway, FZDs serve as gatekeeping proteins both for drug intervention and for the development of probes in basic and in therapeutic research. Here we present an atomic-resolution structure of the human Frizzled 4 receptor (FZD4) transmembrane domain in the absence of a bound ligand. The structure reveals an unusual transmembrane architecture in which helix VI is short and tightly packed, and is distinct from all other GPCR structures reported so far. Within this unique transmembrane fold is an extremely narrow and highly hydrophilic pocket that is not amenable to the binding of traditional GPCR ligands. We show that such a pocket is conserved across all FZDs, which may explain the long-standing difficulties in the development of ligands for these receptors. Molecular dynamics simulations on the microsecond timescale and mutational analysis uncovered two coupled, dynamic kinks located at helix VII that are involved in FZD4 activation. The stability of the structure in its ligand-free form, an unfavourable pocket for ligand binding and the two unusual kinks on helix VII suggest that FZDs may have evolved a novel ligand-recognition and activation mechanism that is distinct from that of other GPCRs.

CUL5 is required for thalidomide-dependent inhibition of cellular proliferation.

Angiogenesis is essential for cancer metastasis, thus the discovery and characterization of molecules that inhibit this process is important. Thalidomide is a teratogenic drug which is known to inhibit angiogenesis and effectively inhibit cancer metastasis, yet the specific cellular targets for its effect are not well known. We discovered that CUL5 (previously identified as VACM-1), a scaffold protein in E3 ligase complexes, is involved in thalidomide-dependent inhibition of endothelial cell growth. Our results show that in human endothelial cells (HUVEC), thalidomide-dependent decrease in cell growth was associated with decreased nuclear localization of CUL5. In HUVEC transfected with anti-VACM-1 siRNA, thalidomide failed to decrease cell growth. Previously it was established that the antiproliferative effect of CUL5 is inhibited in rat endothelial cells (RAMEC) transfected with mutated CUL5 which is constitutively modified by NEDD8, a ubiquitin-like protein. In this study, the antiproliferative response to thalidomide was compromised in RAMEC expressing mutated CUL5. These results suggest that CUL5 protein is involved in the thalidomide-dependent regulation of cellular proliferation in vitro. Consequently, CUL5 may be an important part of the mechanism for thalidomide-dependent inhibition of cellular proliferation, as well as a novel biomarker for predicting a response to thalidomide for the treatment of disorders such as multiple myeloma and HIV infection.

Assembly and architecture of the Wnt/ß-catenin signalosome at the membrane.

Wnt/ß-catenin signalling is initiated by a ternary Wnt-Frizzled (FZD)-LDL receptor-related protein (LRP) 5/6 binding event. The resulting conformational changes in the FZD and LRP5/6 receptors promote the assembly of an intracellular signalosome driven by Dishevelled and Axin co-polymerization. Recent evidence suggests that the FZD receptor and LRP5/6 participate in the assembly of this signalosome by forming regulatory scaffolds for stabilizing Dishevelled and Axin adapters. In this review, we focus on the contributions of Wnts and their receptors in the assembly of the signalosome. We present an emerging model, which unifies Wnt receptor oligomerization with intracellular signalosome formation, and then discuss how FZD receptors might be targeted to either disrupt or enhance their capacity as a dynamic sensor of Wnt binding. LINKED ARTICLES: This article is part of a themed section on WNT Signalling: Mechanisms and Therapeutic Opportunities. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.24/issuetoc.

Wnt5a promotes Frizzled-4 signalosome assembly by stabilizing cysteine-rich domain dimerization.

Wnt/ß-catenin signaling is activated when extracellular Wnt ligands bind Frizzled (FZD) receptors at the cell membrane. Wnts bind FZD cysteine-rich domains (CRDs) with high affinity through a palmitoylated N-terminal "thumb" and a disulfide-stabilized C-terminal "index finger," yet how these binding events trigger receptor activation and intracellular signaling remains unclear. Here we report the crystal structure of the Frizzled-4 (FZD4) CRD in complex with palmitoleic acid, which reveals a CRD tetramer consisting of two cross-braced CRD dimers. Each dimer is stabilized by interactions of one hydrophobic palmitoleic acid tail with two CRD palmitoleoyl-binding grooves oriented end to end, suggesting that the Wnt palmitoleoyl group stimulates CRD-CRD interaction. Using bioluminescence resonance energy transfer (BRET) in live cells, we show that WNT5A stimulates dimerization of membrane-anchored FZD4 CRDs and oligomerization of full-length FZD4, which requires the integrity of CRD palmitoleoyl-binding residues. These results suggest that FZD receptors may form signalosomes in response to Wnt binding through the CRDs and that the Wnt palmitoleoyl group is important in promoting these interactions. These results complement our understanding of lipoprotein receptor-related proteins 5 and 6 (LRP5/6), Dishevelled, and Axin signalosome assembly and provide a more complete model for Wnt signalosome assembly both intracellularly and at the membrane.

Nuclear localization signal sequence is required for VACM-1/CUL5-dependent regulation of cellular growth.

VACM-1/CUL5 is a member of the cullin family of proteins involved in the E3 ligase-dependent degradation of diverse proteins that regulate cellular proliferation. The ability of VACM-1/CUL5 to inhibit cellular growth is affected by its posttranslational modifications and its localization to the nucleus. Since the mechanism of VACM-1/CUL5 translocation to the nucleus is not clear, the goal of this project was to determine the role that the putative nuclear localization signal (NLS) we identified in the VACM-1/CUL5 (640PKLKRQ646) plays in the cellular localization of VACM-1/CUL5 and its effect on cellular growth. We used site-directed mutagenesis to change Lys642 and Lys644 to Gly and the mutated cDNA constructs were transfected into COS-1 cells. Mutation of the NLS in VACM-1/CUL5 significantly reduced its localization to the nucleus and compromised its effect on cellular growth. We have shown previously that the antiproliferative effect of VACM-1/CUL5 could be reversed by mutation of PKA-specific phosphorylation sequence (S730AVACM-1/CUL5), which was associated with its increased nuclear localization and modification by NEDD8. Thus, we examined whether these properties can be controlled by the NLS. The mutation of NLS in S730AVACM-1/CUL5 cDNA compromised its proliferative effect and reduced its localization to the nucleus. The immunocytochemistry results showed that, in cells transfected with the mutant cDNAs, the nuclear NEDD8 signal was decreased. Western blot analysis of total cell lysates, however, showed that VACM-1/CUL5 neddylation was not affected. Together, these results suggest that the presence of the NLS, both in VACM-1/CUL5 and in S730AVACM-1/CUL5 sequences, is critical for their control of cell proliferation.