Raymond P. Wu

Target identification using drug affinity responsive target stability (DARTS)

Identifying the molecular targets for the beneficial or detrimental effects of small-molecule drugs is an important and currently unmet challenge. We have developed a method, drug affinity responsive target stability (DARTS), which takes advantage of a reduction in the protease susceptibility of the target protein upon drug binding. DARTS is universally applicable because it requires no modification of the drug and is independent of the mechanism of drug action. We demonstrate use of DARTS to identify known small-molecule–protein interactions and to reveal the eukaryotic translation initiation machinery as a molecular target for the longevity-enhancing plant natural product resveratrol. We envisage that DARTS will also be useful in global mapping of protein–metabolite interaction networks and in label-free screening of unlimited varieties of compounds for development as molecular imaging agents.

Nrf2 responses and the therapeutic selectivity of electrophilic compounds in chronic lymphocytic leukemia

Recent studies show that redox-active small molecules are selectively cytotoxic to chronic lymphocytic leukemia (CLL). Although elevated levels of reactive oxygen species in CLL cells have been implicated, the molecular mechanism underlying this selectivity is unclear. In other cell types, the nuclear factor erythroid 2–related factor 2 (Nrf2) signaling pathway regulates the oxidative stress response. We found elevated Nrf2 signaling in untreated CLL cells compared with normal lymphocytes. Therefore, we tested 27 known electrophilic and antioxidant compounds with drug-like properties and determined their CLL-selective cytotoxicity and effect on Nrf2 signaling. The selected compounds were from five distinct structural classes; α-β unsaturated carbonyls, isothiocyanates, sulfhydryl reactive metals, flavones, and polyphenols. Our results show that compounds containing α-β unsaturated carbonyls, sulfhydryl reactive metals, and isothiocyanates are strong activators of Nrf2 in a reporter assay system and in primary human CLL based on increased expression of the Nrf2 target heme oxygenase–1. α-β Unsaturated carbonyl–containing compounds were selectively cytotoxic to CLL, and loss of the α-β unsaturation abrogated Nrf2 activity and CLL toxicity. The α-β unsaturated carbonyl containing compounds ethacrynic acid and parthenolide activated Nrf2 in normal peripheral blood mononuclear cells, but had a less potent effect in CLL cells. Furthermore, ethacrynic acid bound directly to the Nrf2-negative regulator Kelch-like ECH-associated protein 1 (Keap1) in CLL cells. These experiments document the presence of Nrf2 signaling in human CLL and suggest that altered Nrf2 responses may contribute to the observed selective cytotoxicity of electrophilic compounds in this disease.

Osteopontin deficiency does not prevent but promotes alcoholic neutrophilic hepatitis in mice

Alcoholic hepatitis (AH) is a distinct spectrum of alcoholic liver disease (ALD) with intense neutrophilic (polymorphonuclear; PMN) inflammation and high mortality. Although a recent study implicates osteopontin (SPP1) in AH, SPP1 is also shown to have protective effects on experimental ALD. To address this unsettled question, we examined the effects of SPP1 deficiency in male mice given 40% calories derived from ad libitum consumption of the Western diet high in cholesterol and saturated fat and the rest from intragastric feeding of alcohol diet without or with weekly alcohol binge. Weekly binge in this new hybrid feeding model shifts chronic ASH with macrophage inflammation and perisinusoidal and pericellular fibrosis to AH in 57% (15 of 26) of mice, accompanied by inductions of chemokines (Spp1, Cxcl1, and interleukin [Il]‐17a), progenitor genes (Cd133, Cd24, Nanog, and epithelial cell adhesion molecule), PMN infiltration, and clinical features of AH, such as hypoalbuminemia, bilirubinemia, and splenomegaly. SPP1 deficiency does not reduce AH incidence and inductions of progenitor and fibrogenic genes, but rather enhances the Il‐17a induction and PMN infiltration in some mice. Furthermore, in the absence of SPP1, chronic ASH mice without weekly binge begin to develop AH. Conclusion: These results suggest that SPP1 has a protective, rather than causal, role for experimental AH reproduced in our model. (Hepatology 2015;61:129–140)

Alcohol directly stimulates epigenetic modifications in hepatic stellate cells.

BACKGROUND & AIMS Alcohol is a primary cause of liver disease and an important co-morbidity factor in other causes of liver disease. A common feature of progressive liver disease is fibrosis, which results from the net deposition of fibril-forming extracellular matrix (ECM). The hepatic stellate cell (HSC) is widely considered to be the major cellular source of fibrotic ECM. We determined if HSCs are responsive to direct stimulation by alcohol. METHODS HSCs undergoing transdifferentiation were incubated with ethanol and expression of fibrogenic genes and epigenetic regulators was measured. Mechanisms responsible for recorded changes were investigated using ChIP-Seq and bioinformatics analysis. Ethanol induced changes were confirmed using HSCs isolated from a mouse alcohol model and from ALD patients liver and through precision cut liver slices. RESULTS HSCs responded to ethanol exposure by increasing profibrogenic and ECM gene expression including elastin. Ethanol induced an altered expression of multiple epigenetic regulators, indicative of a potential to modulate chromatin structure during HSC transdifferentiation. MLL1, a histone 3 lysine 4 (H3K4) methyltransferase, was induced by ethanol and recruited to the elastin gene promoter where it was associated with enriched H3K4me3, a mark of active chromatin. Chromatin immunoprecipitation sequencing (ChIPseq) revealed that ethanol has broad effects on the HSC epigenome and identified 41 gene loci at which both MML1 and its H3K4me3 mark were enriched in response to ethanol. CONCLUSIONS Ethanol directly influences HSC transdifferentiation by stimulating global changes in chromatin structure, resulting in the increased expression of ECM proteins. The ability of alcohol to remodel the epigenome during HSC transdifferentiation provides mechanisms for it to act as a co-morbidity factor in liver disease.

Stearoyl-CoA Desaturase Promotes Liver Fibrosis and Tumor Development in Mice via a Wnt Positive-Signaling Loop by Stabilization of Low-Density Lipoprotein-Receptor-Related Proteins 5 and 6

BACKGROUND & AIMS Stearoyl-CoA desaturase (SCD) synthesizes monounsaturated fatty acids (MUFAs) and has been associated with the development of metabolic syndrome, tumorigenesis, and stem cell characteristics. We investigated whether and how SCD promotes liver fibrosis and tumor development in mice. METHODS Rodent primary hepatic stellate cells (HSCs), mouse liver tumor-initiating stem cell-like cells (TICs), and human hepatocellular carcinoma (HCC) cell lines were exposed to Wnt signaling inhibitors and changes in gene expression patterns were analyzed. We assessed the functions of SCD by pharmacologic and conditional genetic manipulation in mice with hepatotoxic or cholestatic induction of liver fibrosis, orthotopic transplants of TICs, or liver tumors induced by administration of diethyl nitrosamine. We performed bioinformatic analyses of SCD expression in HCC vs nontumor liver samples collected from patients, and correlated levels with HCC stage and patient mortality. We performed nano-bead pull-down assays, liquid chromatography-mass spectrometry, computational modeling, and ribonucleoprotein immunoprecipitation analyses to identify MUFA-interacting proteins. We examined the effects of SCD inhibition on Wnt signaling, including the expression and stability of low-density lipoprotein-receptor-related proteins 5 and 6 (LRP5 and LRP6), by immunoblot and quantitative polymerase chain reaction analyses. RESULTS SCD was overexpressed in activated HSC and HCC cells from patients; levels of SCD messenger RNA (mRNA) correlated with HCC stage and patient survival time. In rodent HSCs and TICs, the Wnt effector β-catenin increased sterol regulatory element binding protein 1-dependent transcription of Scd, and β-catenin in return was stabilized by MUFAs generated by SCD. This loop required MUFA inhibition of binding of Ras-related nuclear protein 1 (Ran1) to transportin 1 and reduced nuclear import of elav-like protein 1 (HuR), increasing cytosolic levels of HuR and HuR-mediated stabilization of mRNAs encoding LRP5 and LRP6. Genetic disruption of Scd and pharmacologic inhibitors of SCD reduced HSC activation and TIC self-renewal and attenuated liver fibrosis and tumorigenesis in mice. Conditional disruption of Scd2 in activated HSCs prevented growth of tumors from TICs and reduced the formation of diethyl nitrosamine-induced liver tumors in mice. CONCLUSIONS In rodent HSCs and TICs, we found SCD expression to be regulated by Wnt-β-catenin signaling, and MUFAs produced by SCD provided a forward loop to amplify Wnt signaling via stabilization of Lrp5 and Lrp6 mRNAs, contributing to liver fibrosis and tumor growth. SCD expressed by HSCs promoted liver tumor development in mice. Components of the identified loop linking HSCs and TICs might be therapeutic targets for liver fibrosis and tumors.

Pyroptosis by caspase11/4‐gasdermin‐D pathway in alcoholic hepatitis in mice and patients

Alcoholic hepatitis (AH) continues to be a disease with high mortality and no efficacious medical treatment. Although severe AH is presented as acute on chronic liver failure, what underlies this transition from chronic alcoholic steatohepatitis (ASH) to AH is largely unknown. To address this question, unbiased RNA sequencing and proteomic analyses were performed on livers of the recently developed AH mouse model, which exhibits the shift to AH from chronic ASH upon weekly alcohol binge, and these results are compared to gene expression profiling data from AH patients. This cross‐analysis has identified Casp11 (CASP4 in humans) as a commonly up‐regulated gene known to be involved in the noncanonical inflammasome pathway. Immunoblotting confirms CASP11/4 activation in AH mice and patients, but not in chronic ASH mice and healthy human livers. Gasdermin‐D (GSDMD), which induces pyroptosis (lytic cell death caused by bacterial infection) downstream of CASP11/4 activation, is also activated in AH livers in mice and patients. CASP11 deficiency reduces GSDMD activation, bacterial load in the liver, and severity of AH in the mouse model. Conversely, the deficiency of interleukin‐18, the key antimicrobial cytokine, aggravates hepatic bacterial load, GSDMD activation, and AH. Furthermore, hepatocyte‐specific expression of constitutively active GSDMD worsens hepatocellular lytic death and polymorphonuclear leukocyte inflammation. Conclusion: These results implicate pyroptosis induced by the CASP11/4‐GSDMD pathway in the pathogenesis of AH. (Hepatology 2018;67:1737‐1753).

High‐mobility‐group protein 2 regulated by microRNA‐127 and small heterodimer partner modulates pluripotency of mouse embryonic stem cells and liver tumor initiating cells

High‐mobility‐group protein 2 (HMGB2) expression is up‐regulated in human liver cancer; however, little is known about its regulatory function. Here, we establish HMGB2 as a new modulator of the pluripotency of mouse embryonic stem cells. Similar to octamer‐binding transcription factor 4 (OCT4) and sex‐determining region Y‐box 2 (SOX2), HMGB2 protein is highly expressed in undifferentiated CGR8 cells, whereas it undergoes rapid decline during embryonic body formation. HMGB2 interacts with OCT4, increases protein expression of OCT4 and SOX2, and enhances their transcriptional activities. We also show that microRNA (miRNA)‐127 is a translational repressor of HMGB2 protein expression by targeting its 3′ untranslated region. We further elucidate a transcriptional mechanism controlling HMGB2 messenger RNA expression by the nuclear receptor small heterodimer partner (SHP) and transcription factor E2F1. Diminishing HMGB2 expression by ectopic expression of miR‐127 or SHP or treatment with the small molecule inhibitor inflachromene decreases OCT4 and SOX2 expression and facilitates CGR8 differentiation. In addition, HMGB2 is markedly induced in liver tumor initiating cells. Diminishing HMGB2 expression by short hairpin RNA for HMGB2 (shHMGB2), miR‐127, or SHP impairs spheroid formation. Importantly, HMGB2 expression is elevated in various human cancers. Conclusion: HMGB2 acts upstream of OCT4/SOX2 signaling to control embryonic stem cell pluripotency. Diminishing HMGB2 expression by miR‐127 or SHP may provide a potential means to decrease the pluripotency of tumor initiating cells. (Hepatology Communications 2017;1:816–830)

Increased aldehyde dehydrogenase activity in high-risk chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL), the most common type of leukemia in the Western world, has a clinically variable course, with differences in the pace of malignant B-lymphocyte accumulation and in the time until cytoreductive treatment is needed. In various solid tumors, the expression of stem cell associated markers, such as aldehyde dehydrogenase (ALDH), has been associated with aggressiveness and a poor prognosis. However, the level of ALDH has not been assessed in CLL B cells. Despite their mature appearance, the B cells from patients with CLL possess immature characteristics both functionally and biochemically. CLL B cells often display biochemical markers of cells early in the blood lineage, including ROR1 [1], Wntl6 [2] and LEF1. In addition, CLL B cells have higher levels of reactive oxygen species (ROS) than normal B cells [3] The ALDH enzyme family is thought to protect cells from accumulated aldehydes formed during lipid peroxidation and poorly regulated oxidative metabolism in a low oxygen environment [4]. Cells with a high level of ALDH activity are considered to have improved self-renewal capacity [5]. The goal of this study was to ascertain whether increased ALDH activity is associated with more aggressive disease in CLL. To this end, we compared ALDH activity with two CLL biomarkers, immunoglobulin heavy chain variable gene (IgHV) mutation status and ZAP-70 expression. These biomarkers are regularly used to classify patients with CLL as high-risk: IgHV unmutated (≥98% homology to IgHVgermline gene) or ZAP-70 positive (≥ 20% of CD19 CLL B cells expressing ZAP-70) and low-risk: IgHV mutated and ZAP-70 negative [6]. Blood samples had been previously collected and viably stored from 40 consenting subjects diagnosed with CLL and followed at the University of California San Diego (UCSD) Moores Cancer Center, with the approval of the UCSD Institutional Review Board. Twenty cases were selected with unmutated IgHV or that were ZAP-70 positive (high risk), and an additional 18 cases were selected which had both mutated IgHV and were ZAP-70 negative (low risk) [6]. Two samples were excluded because of insufficient clinical data. All blood samples were collected from patients prior to treatment. In addition, peripheral blood mononuclear cells (PBMCs) were prepared from samples collected from healthy volunteers registered at the San Diego Blood Bank, as previously described [6]. Cells (2.5 × 106) were stained for ALDH activity with an Aldefluor assay kit (StemCell Technologies, Vancouver, Canada) according to the manufacturer’s instructions. The cells were then stained for the B cell surface marker CD19, and analyzed using a FACSCalibur instrument (BD Biosciences, San Diego, CA). A total of 50,000 events were acquired. Data analysis was performed using Flowjo software (Ashland, OR) and Prism (GraphPad Software, La Jolla, CA). The cells were gated for viable CD19+ cells and mean fluorescent activity was determined. Receiver operating characteristic (ROC) curves were computed in R v2.13.0 [7] using the package ROCR, with standard errors and corresponding 95% confidence intervals (CIs) computed based on the standard errors of Somers’ Dxy rank correlation using the function rcorr.cens from the Hmisc package [8]. Significance was attained at the 5% level if the CI for the AUC was entirely above 0.50, demonstrating greater predictive ability than by chance alone. We assayed mean ALDH levels in CD19− B cells in 18 previously characterized low-risk (ZAP-70 negative and IgHV mutated) and 20 high-risk (IgHV unmutated or ZAP-70 positive) patients with CLL, as well as PBMCs [6] from six healthy individuals. The characteristics of the patients are summarized in Table I. Table I Characteristics of patients with CLL. The mean ALDH activity was elevated in the high-risk CLL samples, compared to the low-risk CLL group (p = 0.002, one-sided Wilcoxon test) [Figure 1(A); combined high-risk groups, n = 20, vs. low risk group, n = 18]. Normal CD19− B cells exhibited low levels of ALDH activity. The area under the curve (AUC) for classifying high-risk versus low-risk samples using the ALDH level was 0.77 (95% CI: 0.62, 0.92) [Figure 1(B)], indicating that ALDH activity had a moderate but statistically significant ability to discriminate between these two groups. Considering each biomarker separately, ALDH had a similar ability to classify IgHV mutational status alone [AUC of 0.77; 95% CI: 0.62, 0.92; Figure 1(C)] and ZAP-70 status alone [AUC of 0.76; 95% CI: 0.59, 0.93; Figure 1(D)]. Moreover, for the 16 samples with unmutated IgHV and an ALDH value above 400, ZAP-70 expression correlated with ALDH activity (r = 0.90 with p 400 ALDH]. These data collectively show: (i) that ALDH activity is increased in CLL B cells, (ii) that ALDH has a similar moderate ability to classify patients with CLL according to IgHV mutational status and ZAP-70 expression status alone, and jointly, and (iii) that increased ALDH activity is associated with high-risk CLL. Figure 1 ALDH activity in chronic lymphocytic leukemia. (A) Mean ALDH activities were determined in the gated CD19+ population. Mean ALDH activities of different groups of CLL and CD19+ B cells from healthy volunteers are shown. Bars indicate mean values. (B–D) ... The observation that ALDH activity is increased in CLL samples obtained from patients with high-risk characteristics has implications related to disease pathogenesis and treatment. Recently, elevated ALDH activity in CD34+ CD38− stem-like cells from acute myelogenous leukemia has been correlated with subsequent clinical relapse [9]. Increased ALDH activity in CLL cells might similarly reflect an immature, aggressive phenotype. ALDH enzymes enable cells to remove toxic products formed in a hypoxic environment as byproducts of oxidative metabolism. Together with other factors, the elevated ALDH in high-risk CLL may promote cell survival in stressful tissue compartments. ALDH may also aid in the detoxification of oxidation products resulting from overexpression of c-Myc and activated phosphatidylinositol-3 kinase (PI3K), as commonly observed in CLL [10,11]. In summary, ALDH activity, which can be measured relatively easily, warrants further investigation for use as a screening biomarker to classify patients with CLL according to a high-risk versus low-risk disease course. Future studies are needed to determine the precise role of ALDH in the pathogenesis of CLL.

Baicalein Targets GTPase‐Mediated Autophagy to Eliminate Liver Tumor–Initiating Stem Cell–Like Cells Resistant to mTORC1 Inhibition

Drug resistance is a major problem in the treatment of liver cancer. Mammalian Target of Rapamycin 1 (mTORC1) inhibitors have been tested for the treatment of liver cancer based on hyperactive mTOR in this malignancy. However, their clinical trials showed poor outcome, most likely due to their ability to upregulate CD133 and promote chemoresistance. The CD133+ tumor–initiating stem cell–like cells (TICs) isolated from mouse and human liver tumors are chemoresistant, and identification of an approach to abrogate this resistance is desired. In search of a compound that rescinds resistance of TICs to mTORC1 inhibition and improves chemotherapy, we identified baicalein (BC), which selectively chemosensitizes TICs and the human hepatocellular carcinoma (HCC) cell line Huh7 cells but not mouse and human primary hepatocytes. Nanobead pull‐down and mass‐spectrometric analysis, biochemical binding assay, and three‐dimensional computational modeling studies reveal BCs ability to competitively inhibit guanosine triphosphate binding of SAR1B guanosine triphosphatase, which is essential for autophagy. Indeed, BC suppresses autophagy induced by an mTORC1 inhibitor and synergizes cell death caused by mTORC1 inhibition in TIC and Huh7 spheroid formation and in the patient‐derived xenograft model of HCC. The BC‐induced chemosensitization is rescued by SAR1B expression and phenocopied by SAR1B knockdown in cancer cells treated with a mTORC1 inhibitor. Conclusion: These results identify SAR1B as a target in liver TICs and HCC cells resistant to mTORC1 inhibition.

Reply: To PMID 25132354.

1. Lazaro R, Wu R, Lee S, Zhu NL, Chen CL, French SW, et al. Osteopontin deficiency does not prevent but promotes alcoholic neutrophilic hepatitis in mice. HEPATOLOGY 2015;61:129-140. 2. Lorena D, Darby IA, Gadeau AP, Leen LL, Rittling S, Porto LC, et al. Osteopontin expression in normal and fibrotic liver. Altered liver healing in osteopontin-deficient mice. J Hepatol 2006;44:383-390. 3. Fickert P, Stoger U, Fuchsbichler A, Moustafa T, Marschall HU, Weiglein AH, et al. A new xenobiotic-induced mouse model of sclerosing cholangitis and biliary fibrosis. Am J Pathol 2007;171:525-536. 4. Xanthou G, Alissafi T, Semitekolou M, Simoes DC, Economidou E, Gaga M, et al. Osteopontin has a crucial role in allergic airway disease through regulation of dendritic cell subsets. Nat Med 2007:570-578. 5. Weber GF, Cantor H. Differential roles of osteopontin/Eta-1 in early and late lpr disease. Clin Exp Immunol 2001;126:578-583. 6. Inoue M, Shinohara ML. Intracellular osteopontin (iOPN) and immunity. Immunol Res 2011;49:160-172. 7. Coombes JD, Swiderska-Syn M, Doll e L, Reid D, Eksteen B, Claridge L, et al. Osteopontin neutralisation abrogates the liver progenitor cell response and fibrogenesis in mice. Gut 2014 [Epub ahead of print].