Research Interest

Epigenetic chromatic modifiers in carcinogenesis and inhibitor discovery toward targeted chromatin modifiers

Epigenetic regulation in part involves a dynamic, reversible post-translational modification of histones, which convey inherited information concerning chromatin structure, and dictate the gene expression pattern. Methylation of histone tails has been recently recognized as a key post-translational modification in epigenetics owing to the discovery of histone demethylases. In conjunction with histone methyltransferases (HMTs), these counterpart modifiers work in coordination to maintain a steady global level of methylated histones, mainly at the side chains of lysine (K) and arginine (R) residues of N-terminal unstructured region of histones. Misregulated methylated status of histones from mutational inactivation or abnormal expression of these modifiers has been implicated in oncogenesis.
We are recently interested to investigate lysine demethylases of histones (KDMs), focusing on the regulation of post-translational modifications of histones during the initiation and progression of cancer as well as their link to cancer metabolism. We first target a new histone lysine demethylase KDM8 required for cell cycle progression. Structure-based inhibitor discovery toward KDMs is also underway.


Figure 1. Crystal structure of the catalytic domain of histone lysine demethylase KDM4B. (A) The 2F_o-F_c electron density map of inhibitor PD2 (orange), Ni²⁺ ion (magenta), and the substrate peptide (cyan), where lysine 9 (K9) is trimethylated. (B) Structure comparison of KDM4A (PDB code: 2OQ6), KDM4B (this study; PDB code: 4LXL), KDM4C (PDB code: 2XML), and KDM4D (PDB code: 4HON). Adapted from Chu et al., J. Med. Chem., 2014.

Regulation of key metabolic enzymes lead to alteration of cellular energy flux and gene expression pattern in cancer cells

One of the hallmarks of cancer cells is their altered metabolism to sustain their high proliferation rate referred to as aerobic glycolysis, or the Warburg effect. The tumor-preferred pathway involves an increased uptake of glucose, utilization of intracellular glucose to pyruvate via glycolysis, and the conversion into lactate in the presence of sufficient oxygen. Along this metabolic flux, PKM2 that catalyzes the dephosphorylation of phosphoenolpyruvate (PEP) to pyruvate, is a pivotal enzyme and selectively expressed in tumors cells. Its abundance and activity determine the metabolic flow to lactate, TCA cycle or biosynthetic pathway. There are four isoforms of pyruvate kinase in mammals: tissue-specific PKL and PKR encoded by PKLR, and PKM1 and PKM2 that are mutually exclusive products of PKM. Of note, PKM2 is expressed in highly multiplying cells including embryonic, adult stem cells, and re-expressed in tumor cells, while PKM1 expression is predominantly found in heart, brain and muscle cells that demand high ATP, suggesting that cancer cells favor the expression of PKM2

There are two ways that PKM2 pyruvate kinase activity can be modulated: 1) by metabolites such as FBP, serine and SAICAR as activators while Phe, Cys and T3 as negative regulators; and 2) by post-translational modifications: phosphorylation of Tyr105 by growth factor signals, oxidation of Cys358 by ROS, acetylation of Lys305 and Lys433 by high glucose. In most of these cases, suppression of PKM2 pyruvate kinase activities are favored in response to oncogenic signals.

Yet, another remarkable way of suppressing cytosolic PKM2 activity is its translocation into nucleus, where it serves as transcriptional coactivator or as a protein kinase to modulate transcriptional program. Nuclear translocation of PKM2 would stop the glycolysis flow at PEP and accumulate intermediates, which are often precursors for biosynthesis to increase biomass. Further, nuclear PKM2’s protein kinase activities are found to phosphorylate nuclear proteins H3 and Stat3. As a transcriptional activator, it induces metabolic and oncogenic genes.

An additional way to modulate PKM2’s activity is through partnership with an oncoprotein KDM8 reported by our team. KDM8 is first known as a histone lysine demethylase with specificity toward H3K36me2. It is involved in embryogenesis, oncogenesis, and stem-cell renewal. Overexpression and amplification of KDM8 was found in a variety of tumor tissues. Knockdown of KDM8 compromised the growth of cancer cells. We have demonstrated that the PKM2-KDM8 partnership facilitates PKM2’s nuclear translocation and HIF1a-mediated transactivation activity. Our data uncover a mechanism whereby PKM2 can be regulated by factor-binding-induced translocation, paving the way to cell metabolic reprogram (Wang et al., PNAS, 2014).

Figure 2. The proposed model that depicts JMJD5 as a major regulator in PKM2-stimulated HIF-1α metabolic reprogramming. JMJD5 and PKM2 are corecruited to HREs of LDHA and thereby specifically enhance HIF-1α binding. 

Molecular pathogenesis and resistance of Helicobacter pylori

The PI’s lab has been working in the molecular pathogenesis of Helicobacter pylori that leads to severe gastrointestinal diseases including gastric cancers. We have focused on several virulence factors including VacA,  CagA, the blood group antigen-binding adhesion (BabA2) and more recently, cholesterol-α-glucosyltransferase involved in adhesion, invasion, as well as underlying mechanisms in hijacking host-cell signaling. We have also reported antimicrobial resistance in H. pylori isolates in Taiwan, the relationship of IL-1b and IL-10 polymorphisms and H. pylori infection with erosive reflux esophagitis and gastritis and the expression of Foxp3+ Treg that was positively associated with the severity of gastroduodenal diseases.

Figure 3. A proposed model of accumulated cholesteryl glucosides (CGs) in the membranes leads to the raft phase coalescence that triggers the formation of the TFSS pilus. CGs that are synthesized by membrane-bound cholesterol-a-glucosyltransferase (CGT) are associated with the bacterial membranes in the absence of host contact (step 1; ①).  Upon attachment with the host membranes (step 2; ②), cholesterol is extracted from the host cells for glucosylation by bacterial CGT. In parallel, the fluctuating CGs in bacteria may drift into the lipid bilayer of the plasma membrane (magnified illustration). Accumulated cholesterol and CGs facilitate the selective lateral-phase segregation and induce the membrane assemblage and raft coalescence on the host-bacterium contact sites, which may serve as a signal to trigger the formation of the TFSS pilus. The mature TFSS subsequently delivers the effectors, CagA and peptidoglycan, into the host cytoplasm (step 3; ③). After translocation, CagA is tyrosine phosphorylated by Src family kinases and triggers downstream signaling events leading to cytoskeletal rearrangement and hummingbird phenotype.  The injected peptidoglycan induces the production of pro-inflammatory cytokine interleukin-8 (IL-8). CGs include cholesterol-α-D-glucopyranoside (αCG), cholesteryl-6’-O-tetradecanoyl-α-D-glucopyranoside (αCAG), cholesteryl-6’-O-phosphatidyl-α-D-glucopyranoside (αCGP). Chol, cholesterol; GPL, glycerophospholipid; CGT, cholesterol-α-glucosyltranferase; Csk, C-terminal Src kinase; SHP-2, SH2 domain-containing tyrosine phosphatase-2. Adapted from Wang et al., Mol. Microbiol., 2012.

Target-based Drug Discovery toward the Shikimate Pathway Enzymes of Helicobacter pylori

Apart from the H. pylori pathogenesis, a combined biochemical/crystallographic strategy has been utilized in the PI’s lab to study important enzymes as well as discovery of inhibitors toward the enzymes of the shikimate pathway. We have developed the structure-based means by Core Site-moiety Maps to interrogate pharmacologically H. pylori shikimate kinase and Mycobacterium tuberculosis shikimate kinase, and identified six potent inhibitors (<8.0 μM). The structure of the inhibitor complex, E114A∙162535, was also determined, which revealed a dramatic shift in the elastic LID region and resulted in conformational locking into a distinctive form.

Figure 4. Framework of the orthSiMMap-based screening method. In Step 1, GEMDOCK was used to generate docked poses for HpSK and MtSK by screening compound libraries (Maybridge and NCI). For each target (HpSK or MtSK), the protein-compound interacting profile was derived from fusing the top ranked 2% (~6,000) compounds. In Step 3, conserved interactions of the target protein and chemical moieties of ligands are identified to deduce the anchors of HpSK and MtSK. The orthSiMMap is constructed based on the conserved features between orthologous target site-moiety maps, which will be used to select candidate compounds for the enzymatic assay. Finally, the model is refined based on the bioassay of candidate compounds.