Kwun Wah Wen

Kaposi Sarcoma-associated Herpesvirus (KSHV): Molecular Biology and Oncogenesis

Kaposi sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA herpesvirus belonging to the gamma-herpesvirinae subfamily. KSHV has been associated with the development of three neoplastic diseases: Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD). In this review, we discuss the three KSHV-associated malignancies, KSHV genome, latent and lytic aspects of the viral lifecycle, putative viral oncogenes, as well as therapeutic regimens used for the treatment of KS, PEL, and MCD.

The Mitochondrial Proteins NLRX1 and TUFM Form a Complex that Regulates Type I Interferon and Autophagy

The mitochondrial protein MAVS (also known as IPS-1, VISA, and CARDIF) interacts with RIG-I-like receptors (RLRs) to induce type I interferon (IFN-I). NLRX1 is a mitochondrial nucleotide-binding, leucine-rich repeats (NLR)-containing protein that attenuates MAVS-RLR signaling. Using Nlrx1(-/-) cells, we confirmed that NLRX1 attenuated IFN-I production, but additionally promoted autophagy during viral infection. This dual function of NLRX1 paralleled the previously described functions of the autophagy-related proteins Atg5-Atg12, but NLRX1 did not associate with Atg5-Atg12. High-throughput quantitative mass spectrometry and endogenous protein-protein interaction revealed an NLRX1-interacting partner, mitochondrial Tu translation elongation factor (TUFM). TUFM interacted with Atg5-Atg12 and Atg16L1 and has similar functions as NLRX1 by inhibiting RLR-induced IFN-I but promoting autophagy. In the absence of NLRX1, increased IFN-I and decreased autophagy provide an advantage for host defense against vesicular stomatitis virus. This study establishes a link between an NLR protein and the viral-induced autophagic machinery via an intermediary partner, TUFM.

Hsp90 and Hsp40/Erdj3 are required for the expression and anti-apoptotic function of KSHV K1

Kaposi sarcoma-associated herpesvirus (KSHV) is a member of the gammaherpesvirus family. It is the etiological agent of three different human cancers, Kaposi sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman disease. The far left end of the KSHV genome encodes a unique transmembrane glycoprotein called K1. K1 possesses the ability to transform rodent fibroblasts and block apoptosis. K1 has also been shown to activate the PI3K/Akt/mTOR pathway in different cells. Using tandem affinity purification, we identified heat shock protein 90β (Hsp90β) and endoplasmic reticulum-associated Hsp40 (Erdj3/DnaJB11), as cellular binding partners of K1. Interactions of K1 with Hsp90β and Hsp40 were confirmed by co-immunoprecipitation in both directions. Furthermore, K1 also interacted with the Hsp90α isoform. We report that small-interfering RNAs directed against Hsp90 and Hsp40/Erdj3, as well as pharmacological inhibitors of Hsp90, dramatically reduced K1 expression, suggesting that K1 is a client protein of these chaperones. In addition, both Hsp90 and Hsp40/Erdj3 were essential for K1s anti-apoptotic function. Finally, we report that the Hsp90 inhibitors, 17-AAG and 17-DMAG, can suppress the proliferation of KSHV-positive PEL cell lines and exhibited IC50 values of 50 nM and below.

Hsp90 inhibitors are efficacious against Kaposi Sarcoma by enhancing the degradation of the essential viral gene LANA, of the viral co-receptor EphA2 as well as other client proteins.

Heat-shock protein 90 (Hsp90) inhibitors exhibit activity against human cancers. We evaluated a series of new, oral bioavailable, chemically diverse Hsp90 inhibitors (PU-H71, AUY922, BIIB021, NVP-BEP800) against Kaposi sarcoma (KS). All Hsp90 inhibitors exhibited nanomolar EC50 in culture and AUY922 reduced tumor burden in a xenograft model of KS. KS is associated with KS-associated herpesvirus (KSHV). We identified the viral latency associated nuclear antigen (LANA) as a novel client protein of Hsp90 and demonstrate that the Hsp90 inhibitors diminish the level of LANA through proteasomal degradation. These Hsp90 inhibitors also downregulated EphA2 and ephrin-B2 protein levels. LANA is essential for viral maintenance and EphA2 has recently been shown to facilitate KSHV infection; which in turn feeds latent persistence. Further, both molecules are required for KS tumor formation and both were downregulated in response to Hsp90 inhibitors. This provides a rationale for clinical testing of Hsp90 inhibitors in KSHV-associated cancers and in the eradication of latent KSHV reservoirs.

A biosynthetic pathway to isovaleryl-CoA in myxobacteria: the involvement of the mevalonate pathway.

A biosynthetic shunt pathway branching from the mevalonate pathway and providing starter units for branched‐chain fatty acid and secondary metabolite biosynthesis has been identified in strains of the myxobacterium Stigmatella aurantiaca. This pathway is upregulated when the branched‐chain α‐keto acid dehydrogenase gene (bkd) is inactivated, thus impairing the normal branched‐chain amino acid degradation process. We previously proposed that, in this pathway, isovaleryl‐CoA is derived from 3,3‐dimethylacrylyl‐CoA (DMA‐CoA). Here we show that DMA‐CoA is an isomerization product of 3‐methylbut‐3‐enoyl‐CoA (3MB‐CoA). This compound is directly derived from 3‐hydroxy‐3‐methylglutaryl‐CoA (HMG‐CoA) by a decarboxylation/ dehydration reaction resembling the conversion of mevalonate 5‐diphosphate to isopentenyl diphosphate. Incubation of cell‐free extracts of a bkd mutant with HMG‐CoA gave product(s) with the molecular mass of 3MB‐CoA or DMA‐CoA. The shunt pathway most likely also operates reversibly and provides an alternative source for the monomers of isoprenoid biosynthesis in myxobacteria that utilize L‐leucine as precursor.

A Leishmania major Response Locus Identified by Interval-specific Congenic Mapping of a T Helper Type 2 Cell Bias-controlling Quantitative Trait Locus

The propensity of naive CD4 T cells to become T helper (Th) type 2 cells correlates with susceptibility to infection by the protozoal parasite Leishmania major. Using genetic linkage analysis, we earlier identified Dice1 as a Th2 cell bias-controlling quantitative trait locus on chromosome 16. Using interval-specific congenic mapping, we now resolve Dice1 into two independent genetic loci, Dice1.1 and Dice1.2, which control Il4 expression from naive Th cells and thereby indirectly control Th2 cell bias. Interestingly, only one of the two congenic intervals containing Dice1.1 and Dice1.2, respectively, also contained an L. major response locus, indicating that L. major responsiveness can be insensitive to determinants that influence Th2 cell bias by controlling naive T cell Il4 expression. These results lay the groundwork for identifying the Dice1.1 and Dice1.2 genes controlling naive T cell Il4 expression and L. major responses, and for testing whether these control other Th2 cell–dependent processes such as worm expulsion, allergic asthma, and dermatitis.

Disruption of LANA in rhesus rhadinovirus generates a highly lytic recombinant virus.

ABSTRACT Rhesus monkey rhadinovirus (RRV) is a gammaherpesvirus that is closely related to human Kaposis sarcoma-associated herpesvirus (KSHV/HHV-8). RRV is the closest relative to KSHV that has a fully sequenced genome and serves as an in vitro and an in vivo model system for KSHV. The latency-associated nuclear antigen (LANA) protein of both KSHV and RRV plays key roles in the establishment and maintenance of these herpesviruses. We have constructed a RRV recombinant virus (RRVΔLANA/GFP) in which the RRV LANA open reading frame has been disrupted with a green fluorescent protein (GFP) expression cassette generated by homologous recombination. The integrity of the recombinant virus was confirmed by diagnostic PCR, restriction digestion, Southern blot analysis, and whole-genome sequencing. We compared the single-step and multistep replication kinetics of RRVΔLANA/GFP, RRV-GFP, wild-type (WT) RRV H26-95, and a revertant virus using traditional plaque assays, as well as real-time quantitative PCR-based genome quantification assays. The RRVΔLANA/GFP recombinant virus exhibited significantly higher lytic replicative properties compared to RRV-GFP, WT RRV, or the revertant virus. This was observed upon de novo infection and in the absence of chemical inducers such as phorbol esters. In addition, by using a quantitative real-time PCR-based viral array, we are the first to report differences in global viral gene expression between WT and recombinant viruses. The RRVΔLANA/GFP virus displayed increased lytic gene transcription at all time points postinfection compared to RRV-GFP. Moreover, we also examined several cellular genes that are known to be repressed by KSHV LANA and report that these genes are derepressed during de novo lytic infection with the RRVΔLANA/GFP virus compared to RRV-GFP. Finally, we also demonstrate that the RRVΔLANA/GFP virus fails to establish latency in B cells, as measured by the loss of GFP-positive cells and intracellular viral genomes.

Appendiceal goblet cell carcinoid: common errors in staging and clinical interpretation with a proposal for an improved terminology

Goblet cell carcinoid (GCC) is staged and treated as adenocarcinoma (AC) and not as neuroendocrine tumor (NET) or neuroendocrine carcinoma. The term carcinoid may lead to incorrect interpretation as NET. The aim of the study was to explore pitfalls in staging and clinical interpretation of GCC and mixed GCC-AC, and propose strategies to avoid common errors. Diagnostic terminology, staging, and clinical interpretation were evaluated in 58 cases (27 GCCs, 31 mixed GCC-ACs). Opinions were collected from 23 pathologists using a survey. Clinical notes were reviewed to assess the interpretation of pathology diagnoses by oncologists. NET staging was incorrectly used for 25% of GCCs and 5% of mixed GCC-ACs. In the survey, 43% of pathologists incorrectly indicated that NET staging is applicable to GCCs, and 43% incorrectly responded that Ki-67 proliferation index is necessary for GCC grading. Two cases each of GCC and mixed GCC-AC were incorrectly interpreted as neuroendocrine neoplasms by oncologists, and platinum-based therapy was considered for 2 GCC-AC cases because of the mistaken impression of neuroendocrine carcinoma created by use of the World Health Organization 2010 term mixed adenoneuroendocrine carcinoma. The term carcinoid in GCC and use of mixed adenoneuroendocrine carcinoma for mixed GCC-AC lead to errors in staging and treatment. We propose that goblet cell carcinoid should be changed to goblet cell carcinoma, whereas GCC with AC should be referred to as mixed GCC-AC with a comment about the proportion of each component and the histologic subtype of AC. This terminology will facilitate appropriate staging and clinical management, and avoid errors in interpretation.

Signaling mechanisms that regulate ex vivo survival of human acute myeloid leukemia initiating cells

Acute myeloid leukemia (AML) is an aggressive malignancy of the hematopoietic system that arises through clonal expansion of myeloid precursor cells that have arrested at an early stage of differentiation and ultimately causes death in over 50% of patients. Despite the aggressive growth characteristics of AML in vivo, AML blasts are difficult to culture once removed from the patient, suggesting that AML cells depend on signals from the microenvironment. AML initiating cells (LICs), defined functionally as cells capable of initiating AML in immunocompromised mice, are also challenging to maintain in culture, presenting a major obstacle to developing new therapies for AML that target the LIC. Co-culture with stromal cells can maintain and expand LICs; however these culture systems use multiple cytokines that may promote differentiation and loss of LICs ex vivo. Small molecules such as SR1 (an arylhydrocarbon receptor antagonist) and UM729 support AML viability without stromal co-culture, but also require multiple cytokines. In this work, we demonstrate an alternative approach that enhances the viability of primary AML cells and supports ex vivo maintenance of LICs in cytokine-free medium. We show that inhibition of GSK-3, which activates Wnt signaling, markedly improves the viability of primary human AML cells in cytokine-free medium and further that combined inhibition of GSK-3 and the nutrient sensor mTORC1 maintains LICs capable of engrafting immunocompromised mice, identifying a signaling network that regulates the viability of LICs. As GSK-3 inhibition expands hematopoietic stem and progenitor cells and Gsk3 knockout causes a myeloproliferative neoplasm suggestive of AML in mice, we tested whether direct inhibition of GSK-3 would enhance the viability of primary AML cells ex vivo. AML cells in culture do not proliferate and typically die over a few days. Cells from AML patients were cultured in defined medium without cytokines in the presence of the GSK-3 inhibitor CHIR99021 (Chiron) or vehicle. After 5 days, cells cultured with vehicle alone were small with fragmented nuclei whereas cells cultured with Chiron were uniformly larger with intact, round nuclei (Fig. 1a). The fraction of viable cells upon GSK-3 inhibition increased in a dose-dependent manner over 10 days (Fig. 1b, Supplementary Fig. 1a), with a maximal effect at 1–3 μM Chiron. The mechanistically distinct GSK-3 inhibitor, lithium chloride, also increased the fraction of viable cells compared to control at concentrations similar to the established IC50 for lithium inhibition of GSK-3 (Figs. 1c, Supplementary Fig. 1b). To address potential off-target effects with these inhibitors, we knocked down GSK3 with short interfering RNAs (siRNAs). Knockdown (30% of control for GSK3A and GSK3B, Supplementary Fig. 1c, d) of either GSK3A or GSK3B alone had no effect on viability, but combined knockdown significantly increased the viable fraction relative to control siRNA, similar to chemical GSK-3 inhibitors (Gsk3i) and consistent with redundant functions for GSK3A and GSK3B (Supplementary Fig. 1e). Thus GSK-3 inhibition improves the viability of AML cells ex vivo. To test the generality of the response to GSK-3 inhibition, primary AML blasts with distinct genetic and

Use of DNA flow cytometry in the diagnosis, risk stratification, and management of gastric epithelial dysplasia

The natural history of gastric epithelial dysplasia and the consequential surveillance strategies are not well defined in the West. To date, the diagnosis relies on morphology, and no reliable adjunct methods, either immunohistochemical or molecular, have reproducibly been able to confirm the diagnosis and/or risk stratify gastric epithelial dysplasia. Yet, such a tool would be useful in confirming the diagnosis, and developing objective and rational surveillance guidelines. DNA flow cytometry was performed using formalin-fixed paraffin-embedded gastric tissue from 23 cases of high-grade dysplasia and 38 cases of low-grade dysplasia. Twenty-four benign background mucosal samples from the same cohort (20 biopsies and 4 surgical resections from 16 low- and 8 high-grade dysplasia cases) were utilized as controls. The presence of DNA content abnormality (aneuploidy or elevated 4N fraction) correlated with increasing levels of dysplasia, as DNA content abnormality was detected in 18 (78%) of 23 high-grade dysplasia, 5 (13%) of 38 low-grade dysplasia, and none of 24 non-dysplastic samples. 1 and 4-year detection rates of high-grade dysplasia or gastric adenocarcinoma in low-grade dysplasia patients with DNA content abnormality were 80% (p = 0.003) and 100% (p = 0.005), respectively, whereas patients with low-grade dysplasia but with normal DNA content had 1, 4, and 12-year detection rates of 23, 32, and 54%, respectively. The univariate hazard ratio (HR) for subsequent detection of high-grade dysplasia or gastric adenocarcinoma in low-grade dysplasia patients with DNA content abnormality was 6.9 (p = 0.001). Older patients (HR = 1.1, p = 0.005) and those with familial adenomatous polyposis (HR = 9.7, p = 0.029) also had an increased risk for developing high-grade dysplasia or gastric adenocarcinoma in the univariate analysis, but only DNA content abnormality demonstrated a significantly elevated HR of 5.9 in the multivariate analysis (p = 0.005). While older age showed a minimally elevated risk (HR = 1.1, p = 0.013), no other potential risk factors, including male gender, ethnicity, polypoid endoscopic appearance, Helicobacter pylori infection, and intestinal metaplasia, were significantly associated with subsequent detection of high-grade dysplasia or gastric adenocarcinoma in the multivariate analysis. Among the 18 high-grade dysplasia cases with DNA content abnormality, 13 cases (72%) developed gastric adenocarcinoma within a mean follow-up time of 9 months, conferring a HR of 2.5; however, this did not reach statistical significance. In conclusion, the presence of DNA content abnormality can identify a subset of low-grade dysplasia patients who are at increased risk for subsequent detection of high-grade dysplasia or gastric adenocarcinoma. It can also provide confirmatory evidence to a morphologic impression or suspicion of high-grade dysplasia. The majority of gastric epithelial dysplasia patients with DNA content abnormality developed high-grade dysplasia or gastric adenocarcinoma within a year and thus may benefit from more thorough and rigorous endoscopic surveillance.