Chris Lu

Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling

The stability of the Wnt pathway transcription factor β-catenin is tightly regulated by the multi-subunit destruction complex. Deregulated Wnt pathway activity has been implicated in many cancers, making this pathway an attractive target for anticancer therapies. However, the development of targeted Wnt pathway inhibitors has been hampered by the limited number of pathway components that are amenable to small molecule inhibition. Here, we used a chemical genetic screen to identify a small molecule, XAV939, which selectively inhibits β-catenin-mediated transcription. XAV939 stimulates β-catenin degradation by stabilizing axin, the concentration-limiting component of the destruction complex. Using a quantitative chemical proteomic approach, we discovered that XAV939 stabilizes axin by inhibiting the poly-ADP-ribosylating enzymes tankyrase 1 and tankyrase 2. Both tankyrase isoforms interact with a highly conserved domain of axin and stimulate its degradation through the ubiquitin-proteasome pathway. Thus, our study provides new mechanistic insights into the regulation of axin protein homeostasis and presents new avenues for targeted Wnt pathway therapies.

Selective inhibition of Ezh2 by a small molecule inhibitor blocks tumor cells proliferation

Ezh2 (Enhancer of zeste homolog 2) protein is the enzymatic component of the Polycomb repressive complex 2 (PRC2), which represses gene expression by methylating lysine 27 of histone H3 (H3K27) and regulates cell proliferation and differentiation during embryonic development. Recently, hot-spot mutations of Ezh2 were identified in diffused large B-cell lymphomas and follicular lymphomas. To investigate if tumor growth is dependent on the enzymatic activity of Ezh2, we developed a potent and selective small molecule inhibitor, EI1, which inhibits the enzymatic activity of Ezh2 through direct binding to the enzyme and competing with the methyl group donor S-Adenosyl methionine. EI1-treated cells exhibit genome-wide loss of H3K27 methylation and activation of PRC2 target genes. Furthermore, inhibition of Ezh2 by EI1 in diffused large B-cell lymphomas cells carrying the Y641 mutations results in decreased proliferation, cell cycle arrest, and apoptosis. These results provide strong validation of Ezh2 as a potential therapeutic target for the treatment of cancer.

Rescue Screens with Secreted Proteins Reveal Compensatory Potential of Receptor Tyrosine Kinases in Driving Cancer Growth

The overall power of kinase inhibitors is substantially overshadowed by the acquisition of drug resistance. To address this issue, we systematically assessed the potential of secreted proteins to induce resistance to kinase inhibitors. To this end, we developed a high-throughput platform for screening a cDNA library encoding 3,432 secreted proteins in cellular assays. Using cancer cells originally dependent on either MET, FGFR2, or FGFR3, we observed a bypass of dependence through ligand-mediated activation of alternative receptor tyrosine kinases (RTK). Our findings indicate a broad and versatile potential for RTKs from the HER and FGFR families as well as MET to compensate for loss of each other. We further provide evidence that combined inhibition of simultaneously active RTKs can lead to an added anticancer effect.

Nuclear receptor NR4A1 promotes breast cancer invasion and metastasis by activating TGF-β signalling

In advanced cancers, the TGF-β pathway acts as an oncogenic factor and is considered to be a therapeutic target. Here using a genome-wide cDNA screen, we identify nuclear receptor NR4A1 as a strong activator of TGF-β signalling. NR4A1 promotes TGF-β/SMAD signalling by facilitating AXIN2-RNF12/ARKADIA-induced SMAD7 degradation. NR4A1 interacts with SMAD7 and AXIN2, and potently and directly induces AXIN2 expression. Whereas loss of NR4A1 inhibits TGF-β-induced epithelial-to-mesenchymal transition and metastasis, slight NR4A1 ectopic expression stimulates metastasis in a TGF-β-dependent manner. Importantly, inflammatory cytokines potently induce NR4A1 expression, and potentiate TGF-β-mediated breast cancer cell migration, invasion and metastasis in vitro and in vivo. Notably, NR4A1 expression is elevated in breast cancer patients with high immune infiltration and its expression weakly correlates with phosphorylated SMAD2 levels, and is an indicator of poor prognosis. Our results uncover inflammation-induced NR4A1 as an important determinant for hyperactivation of pro-oncogenic TGF-β signalling in breast cancer.

RNF12 Controls Embryonic Stem Cell Fate and Morphogenesis in Zebrafish Embryos by Targeting Smad7 for Degradation

TGF-β members are of key importance during embryogenesis and tissue homeostasis. Smad7 is a potent antagonist of TGF-β family/Smad-mediated responses, but the regulation of Smad7 activity is not well understood. We identified the RING domain-containing E3 ligase RNF12 as a critical component of TGF-β signaling. Depletion of RNF12 dramatically reduced TGF-β/Smad-induced effects in mammalian cells, whereas ectopic expression of RNF12 strongly enhanced these responses. RNF12 specifically binds to Smad7 and induces its polyubiquitination and degradation. Smad7 levels were increased in RNF12-deficient mouse embryonic stem cells, resulting in mitigation of both BMP-mediated repression of neural induction and activin-induced anterior mesoderm formation. RNF12 also antagonized Smad7 during Nodal-dependent and BMP-dependent signaling and morphogenic events in early zebrafish embryos. The gastrulation defects induced by ectopic and depleted Smad7 were rescued in part by RNF12 gain and loss of function, respectively. These findings demonstrate that RNF12 plays a critical role in TGF-β family signaling.

Structure of Human SMYD2 Protein Reveals the Basis of p53 Tumor Suppressor Methylation

Background: SMYD2 is a lysine methyltransferase that mediates functions of target protein by specific site methylation. Results: SMYD2 prefers to monomethylate Lys-370 of p53, and the specificity is explained by high resolution structure of the enzyme bound to p53. Conclusion: CTD domain and a unique EDEE motif play critical roles in p53 Lys-370 methylation by SMYD2. Significance: The findings provide molecular insights into the mechanism of p53 recognition by SMYD2. SMYD2 belongs to a subfamily of histone lysine methyltransferase and was recently identified to methylate tumor suppressor p53 and Rb. Here we report that SMYD2 prefers to methylate p53 Lys-370 over histone substrates in vitro. Consistently, the level of endogenous p53 Lys-370 monomethylation is significantly elevated when SMYD2 is overexpressed in vivo. We have solved the high resolution crystal structures of the full-length SMYD2 protein in binary complex with its cofactor S-adenosylmethionine and in ternary complex with cofactor product S-adenosylhomocysteine and p53 substrate peptide (residues 368–375), respectively. p53 peptide binds to a deep pocket of the interface between catalytic SET(1–282) and C-terminal domain (CTD) with an unprecedented U-shaped conformation. Subtle conformational change exists around the p53 binding site between the binary and ternary structures, in particular the tetratricopeptide repeat motif of the CTD. In addition, a unique EDEE motif between the loop of anti-parallel β7 and β8 sheets of the SET core not only interacts with p53 substrate but also forms a hydrogen bond network with residues from CTD. These observations suggest that the tetratricopeptide repeat and EDEE motif may play an important role in determining p53 substrate binding specificity. This is further verified by the findings that deletion of the CTD domain drastically reduces the methylation activity of SMYD2 to p53 protein. Meanwhile, mutation of EDEE residues impairs both the binding and the enzymatic activity of SMYD2 to p53 Lys-370. These data together reveal the molecular basis of SMYD2 in specifically recognizing and regulating functions of p53 tumor suppressor through Lys-370 monomethylation.

Fine-tuning BMP7 signalling in adipogenesis by UBE2O/E2-230K-mediated monoubiquitination of SMAD6

SMAD6 is a crucial feedback inhibitory regulator of bone morphogenetic protein (BMP)/SMAD signalling. Although little is known regarding the post‐transcriptional modification of inhibitory SMADs and the mechanism by which their function is regulated. In this study, using a whole proteomic interaction screen for SMAD6, we identified a large putative E2 ubiquitin‐conjugating enzyme UBE2O (E2‐230K) as a novel interacting protein of SMAD6. We showed that UBE2O functions as an E2‐E3 hybrid to monoubiquitinate SMAD6 at lysine 174 and that the cysteine 885 residue of human UBE2O is necessary for SMAD6 monoubiquitination. Inactivation of the SMAD6 monoubiquitination site specially potentiates the inhibitory ability of SMAD6 against BMP7‐induced SMAD1 phosphorylation and transcriptional responses. We also found that UBE2O potentiated BMP7 signalling in a SMAD6‐dependent manner. Addressing the molecular mechanism by which UBE2O and monoubiquitinated SMAD6 potentiate BMP7 signalling, we demonstrated that monoubiquitinated SMAD6 impairs the binding affinity of non‐modified SMAD6 to the BMP type I receptor. Moreover, UBE2O and SMAD6 cooperated in the regulation of BMP7‐induced adipogenesis.

Histone methyltransferase SETDB1 regulates liver cancer cell growth through methylation of p53

SETDB1 is a histone H3K9 methyltransferase that has a critical role in early development. It is located within a melanoma susceptibility locus and facilitates melanoma formation. However, the mechanism by which SETDB1 regulates tumorigenesis remains unknown. Here we report the molecular interplay between SETDB1 and the well-known hotspot gain-of-function (GOF) TP53 R249S mutation. We show that in hepatocellular carcinoma (HCC) SETDB1 is overexpressed with moderate copy number gain, and GOF TP53 mutations including R249S associate with this overexpression. Inactivation of SETDB1 in HCC cell lines bearing the R249S mutation suppresses cell growth. The TP53 mutation status renders cancer cells dependent on SETDB1. Moreover, SETDB1 forms a complex with p53 and catalyses p53K370 di-methylation. SETDB1 attenuation reduces the p53K370me2 level, which subsequently leads to increased recognition and degradation of p53 by MDM2. Together, we provide both genetic and biochemical evidence for a mechanism by which SETDB1 regulates cancer cell growth via methylation of p53.

NSD2 is recruited through its PHD domain to oncogenic gene loci to drive multiple myeloma.

Histone lysine methyltransferase NSD2 (WHSC1/MMSET) is overexpressed frequently in multiple myeloma due to the t(4;14) translocation associated with 15% to 20% of cases of this disease. NSD2 has been found to be involved in myelomagenesis, suggesting it may offer a novel therapeutic target. Here we show that NSD2 methyltransferase activity is crucial for clonogenicity, adherence, and proliferation of multiple myeloma cells on bone marrow stroma in vitro and that NSD2 is required for tumorigenesis of t(4;14)+ but not t(4;14)- multiple myeloma cells in vivo. The PHD domains in NSD2 were important for its cellular activity and biological function through recruiting NSD2 to its oncogenic target genes and driving their transcriptional activation. By strengthening its disease linkage and deepening insights into its mechanism of action, this study provides a strategy to therapeutically target NSD2 in multiple myeloma patients with a t(4;14) translocation.

Absolute Quantification of Histone PTM Marks by MRM-Based LC-MS/MS

The N-terminal tails of core histones harbor the sites of numerous post-translational modifications (PTMs) with important roles in the regulation of chromatin structure and function. Profiling histone PTM marks provides data that help understand the epigenetics events in cells and their connections with cancer and other diseases. Our previous study demonstrated that specific derivatization of histone peptides by NHS propionate significantly improved their chromatographic performance on reversed phase columns for LC/MS analysis. As a step forward, we recently developed a multiple reaction monitoring (MRM) based LC-MS/MS method to analyze 42 targeted histone peptides. By using stable isotopic labeled peptides as internal standards that are spiked into the reconstituted solutions, this method allows to measure absolute concentration of the tryptic peptides of H3 histone proteins extracted from cancer cell lines. The method was thoroughly validated for the accuracy and reproducibility through analyzing recombinant histone proteins and cellular samples. The linear dynamic range of the MRM assays was achieved in 3 orders of magnitude from 1 nM to 1 μM for all targeted peptides. Excellent intrabatch and interbatch reproducibility (<15% CV) was obtained. This method has been used to study translocated NSD2 (a histone lysine methyltransferase that catalyzes the histone lysine 36 methylation) function with its overexpression in KMS11 multiple myeloma cells. From the results we have successfully quantitated both individual and combinatorial histone marks in parental and NSD2 selective knockout KMS11 cells.