Cell-type-specific CAG repeat expansions and toxicity of mutant Huntingtin in human striatum and cerebellum.

Brain region-specific degeneration and somatic expansions of the mutant Huntingtin (mHTT) CAG tract are key features of Huntington's disease (HD). However, the relationships among CAG expansions, death of specific cell types and molecular events associated with these processes are not established. Here, we used fluorescence-activated nuclear sorting (FANS) and deep molecular profiling to gain insight into the properties of cell types of the human striatum and cerebellum in HD and control donors. CAG expansions arise at mHTT in striatal medium spiny neurons (MSNs), cholinergic interneurons and cerebellar Purkinje neurons, and at mutant ATXN3 in MSNs from SCA3 donors. CAG expansions in MSNs are associated with higher levels of MSH2 and MSH3 (forming MutSß), which can inhibit nucleolytic excision of CAG slip-outs by FAN1. Our data support a model in which CAG expansions are necessary but may not be sufficient for cell death and identify transcriptional changes associated with somatic CAG expansions and striatal toxicity.

Hypoxia and hypoxia-inducible factors in Kaposi sarcoma-associated herpesvirus infection and disease pathogenesis.

Kaposi sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi sarcoma and several other tumors and hyperproliferative diseases seen predominantly in human immunodeficiency virus-infected and other immunocompromised persons. There is an increasing body of evidence showing that hypoxia and hypoxia-inducible factors (HIFs) play important roles in the biology of KSHV and in the pathogenesis of KSHV-induced diseases. Hypoxia and HIFs can induce lytic activation of KSHV and KSHV can in turn lead to a hypoxic-like state in infected cells. In this review, we describe the complex interactions between KSHV biology, the cellular responses to hypoxia, and the pathogenesis of KSHV-induced diseases. We also describe how interference with HIFs can lead to decreased tumor growth and/or death of infected cells and KSHV-induced tumors. Finally, we show how these observations may lead to novel strategies for the treatment of KSHV-induced diseases.

Identifying cancer-associated leukocyte profiles using high-resolution flow cytometry screening and machine learning.

Background: Machine learning (ML) is a valuable tool with the potential to aid clinical decision making. Adoption of ML to this end requires data that reliably correlates with the clinical outcome of interest; the advantage of ML is that it can model these correlations from complex multiparameter data sets that can be difficult to interpret conventionally. While currently available clinical data can be used in ML for this purpose, there exists the potential to discover new "biomarkers" that will enhance the effectiveness of ML in clinical decision making. Since the interaction of the immune system and cancer is a hallmark of tumor establishment and progression, one potential area for cancer biomarker discovery is through the investigation of cancer-related immune cell signatures. Hence, we hypothesize that blood immune cell signatures can act as a biomarker for cancer progression. Methods: To probe this, we have developed and tested a multiparameter cell-surface marker screening pipeline, using flow cytometry to obtain high-resolution systemic leukocyte population profiles that correlate with detection and characterization of several cancers in murine syngeneic tumor models. Results: We discovered a signature of several blood leukocyte subsets, the most notable of which were monocyte subsets, that could be used to train CATboost ML models to predict the presence and type of cancer present in the animals. Conclusions: Our findings highlight the potential utility of a screening approach to identify robust leukocyte biomarkers for cancer detection and characterization. This pipeline can easily be adapted to screen for cancer specific leukocyte markers from the blood of cancer patient.

Mechanism and therapeutic implications of pomalidomide-induced immune surface marker upregulation in EBV-positive lymphomas.

Epstein-Barr virus (EBV) downregulates immune surface markers to avoid immune recognition. Pomalidomide (Pom) was previously shown to increase immune surface marker expression in EBV-infected tumor cells. We explored the mechanism by which Pom leads to these effects in EBV-infected cells. Pom increased B7-2/CD86 mRNA, protein, and surface expression in EBV-infected cells but this was virtually eliminated in EBV-infected cells made resistant to Pom-induced cytostatic effects. This indicates that Pom initiates the upregulation of these markers by interacting with its target, cereblon. Interestingly, Pom increased the proinflammatory cytokines IP-10 and MIP-1∝/ß in EBV infected cells, supporting a possible role for the phosphoinositide 3-kinase (PI3K)/AKT pathway in Pom's effects. Idelalisib, an inhibitor of the delta subunit of PI3 Kinase, blocked AKT-Ser phosphorylation and Pom-induced B7-2 surface expression. PU.1 is a downstream target for AKT that is expressed in EBV-infected cells. Pom treatment led to an increase in PU.1 binding to the B7-2 promoter based on ChIP analysis. Thus, our data indicates Pom acts through cereblon leading to degradation of Ikaros and activation of the PI3K/AKT/PU.1 pathway resulting in upregulation of B7-2 mRNA and protein expression. The increased immune recognition in addition to the increases in proinflammatory cytokines upon Pom treatment suggests Pom may be useful in the treatment of EBV-positive lymphomas.

Cell Type Specific CAG Repeat Expansions and Toxicity of Mutant Huntingtin in Human Striatum and Cerebellum.

Brain region-specific degeneration and somatic expansions of the mutant Huntingtin (mHTT) CAG tract are key features of Huntington's disease (HD). However, the relationships between CAG expansions, death of specific cell types, and molecular events associated with these processes are not established. Here we employed fluorescence-activated nuclear sorting (FANS) and deep molecular profiling to gain insight into the properties of cell types of the human striatum and cerebellum in HD and control donors. CAG expansions arise in striatal medium spiny neurons (MSNs) and cholinergic interneurons, in cerebellar Purkinje neurons, and at mATXN3 in MSNs from SCA3 donors. CAG expansions in MSNs are associated with higher levels of MSH2 and MSH3 (forming MutSß), which can inhibit nucleolytic excision of CAG slip-outs by FAN1 in a concentration-dependent manner. Our data indicate that ongoing CAG expansions are not sufficient for cell death, and identify transcriptional changes associated with somatic CAG expansions and striatal toxicity.

Daratumumab induces cell-mediated cytotoxicity of primary effusion lymphoma and is active against refractory disease.

Primary effusion lymphoma (PEL), an aggressive non-Hodgkin lymphoma caused by Kaposi sarcoma-associated herpesvirus (KSHV), lacks standard therapy and has a median survival of 10-22 months with combination chemotherapy. PEL is a tumor of plasmablast-like B cells generally expressing CD38, the target of daratumumab (Dara). Initially, we assessed PEL cells from eight patients and established that each expressed high levels of CD38 by flow cytometry. PEL cell lines were also evaluated and most had high CD38 expression. We then assessed Dara's effects on complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) of PEL cell lines as well as its clinical benefits on two patients with PEL. Despite high CD38 expression, Dara did not induce CDC of PEL cell lines, due in part to high levels of the complement-inhibitory proteins, CD55 and CD59. However, Dara induced significant and dose-dependent increases in ADCC, particularly in those lines with high CD38 levels. Two FDA-approved drugs, all trans-retinoic acid (ATRA) and pomalidomide (Pom), significantly increased surface CD38 levels in low-CD38 expressing PEL cell lines, resulting in increased Dara-induced ADCC. Two patients with refractory PEL were treated with Dara alone or in combination with Pom. One patient with leptomeningeal PEL had a complete response to Dara and Pom combination treatment. Others had improvement in performance status and resolution of malignant ascites with Dara alone. Together, these data support the use of Dara monotherapy or in combination with ATRA or Pom as a potential therapeutic option for PEL.

Regulation of Retroviral and SARS-CoV-2 Protease Dimerization and Activity through Reversible Oxidation.

Most viruses encode their own proteases to carry out viral maturation and these often require dimerization for activity. Studies on human immunodeficiency virus type 1 (HIV-1), type 2 (HIV-2) and human T-cell leukemia virus (HTLV-1) proteases have shown that the activity of these proteases can be reversibly regulated by cysteine (Cys) glutathionylation and/or methionine oxidation (for HIV-2). These modifications lead to inhibition of protease dimerization and therefore loss of activity. These changes are reversible with the cellular enzymes, glutaredoxin or methionine sulfoxide reductase. Perhaps more importantly, as a result, the maturation of retroviral particles can also be regulated through reversible oxidation and this has been demonstrated for HIV-1, HIV-2, Mason-Pfizer monkey virus (M-PMV) and murine leukemia virus (MLV). More recently, our group has learned that SARS-CoV-2 main protease (Mpro) dimerization and activity can also be regulated through reversible glutathionylation of Cys300. Overall, these studies reveal a conserved way for viruses to regulate viral polyprotein processing particularly during oxidative stress and reveal novel targets for the development of inhibitors of dimerization and activity of these important viral enzyme targets.

Irradiation immunity interactions.

The immune system can influence cancer development by both impeding and/or facilitating tumour growth and spread. A better understanding of this complex relationship is fundamental to optimise current and future cancer therapeutic strategies. Although typically regarded as a localised and immunosuppressive anti-cancer treatment modality, radiation therapy has been associated with generating profound systemic effects beyond the intended target volume. These systemic effects are immune-driven suggesting radiation therapy can enhance anti-tumour immunosurveillance in some instances. In this review, we summarise how radiation therapy can positively and negatively affect local and systemic anti-tumour immune responses, how co-administration of immunotherapy with radiation therapy may help promote anti-tumour immunity, and how the use of immune biomarkers may help steer radiation therapy-immunotherapy personalisation to optimise clinical outcomes.

Machine learning predicts cancer subtypes and progression from blood immune signatures.

Clinical adoption of immune checkpoint inhibitors in cancer management has highlighted the interconnection between carcinogenesis and the immune system. Immune cells are integral to the tumour microenvironment and can influence the outcome of therapies. Better understanding of an individual's immune landscape may play an important role in treatment personalisation. Peripheral blood is a readily accessible source of information to study an individual's immune landscape compared to more complex and invasive tumour bioipsies, and may hold immense diagnostic and prognostic potential. Identifying the critical components of these immune signatures in peripheral blood presents an attractive alternative to tumour biopsy-based immune phenotyping strategies. We used two syngeneic solid tumour models, a 4T1 breast cancer model and a CT26 colorectal cancer model, in a longitudinal study of the peripheral blood immune landscape. Our strategy combined two highly accessible approaches, blood leukocyte immune phenotyping and plasma soluble immune factor characterisation, to identify distinguishing immune signatures of the CT26 and 4T1 tumour models using machine learning. Myeloid cells, specifically neutrophils and PD-L1-expressing myeloid cells, were found to correlate with tumour size in both the models. Elevated levels of G-CSF, IL-6 and CXCL13, and B cell counts were associated with 4T1 growth, whereas CCL17, CXCL10, total myeloid cells, CCL2, IL-10, CXCL1, and Ly6Cintermediate monocytes were associated with CT26 tumour development. Peripheral blood appears to be an accessible means to interrogate tumour-dependent changes to the host immune landscape, and to identify blood immune phenotypes for future treatment stratification.

Regulation of the Dimerization and Activity of SARS-CoV-2 Main Protease through Reversible Glutathionylation of Cysteine 300.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for coronavirus disease 2019 (COVID-19), encodes two proteases required for replication. The main protease (Mpro), encoded as part of two polyproteins, pp1a and pp1ab, is responsible for 11 different cleavages of these viral polyproteins to produce mature proteins required for viral replication. Mpro is therefore an attractive target for therapeutic interventions. Certain proteins in cells under oxidative stress undergo modification of reactive cysteines. We show Mpro is susceptible to glutathionylation, leading to inhibition of dimerization and activity. Activity of glutathionylated Mpro could be restored with reducing agents or glutaredoxin. Analytical studies demonstrated that glutathionylated Mpro primarily exists as a monomer and that modification of a single cysteine with glutathione is sufficient to block dimerization and inhibit its activity. Gel filtration studies as well as analytical ultracentrifugation confirmed that glutathionylated Mpro exists as a monomer. Tryptic and chymotryptic digestions of Mpro as well as experiments using a C300S Mpro mutant revealed that Cys300, which is located at the dimer interface, is a primary target of glutathionylation. Moreover, Cys300 is required for inhibition of activity upon Mpro glutathionylation. These findings indicate that Mpro dimerization and activity can be regulated through reversible glutathionylation of a non-active site cysteine, Cys300, which itself is not required for Mpro activity, and provides a novel target for the development of agents to block Mpro dimerization and activity. This feature of Mpro may have relevance to the pathophysiology of SARS-CoV-2 and related bat coronaviruses. IMPORTANCE SARS-CoV-2 is responsible for the devastating COVID-19 pandemic. Therefore, it is imperative that we learn as much as we can about the biochemistry of the coronavirus proteins to inform development of therapy. One attractive target is the main protease (Mpro), a dimeric enzyme necessary for viral replication. Most work thus far developing Mpro inhibitors has focused on the active site. Our work has revealed a regulatory mechanism for Mpro activity through glutathionylation of a cysteine (Cys300) at the dimer interface, which can occur in cells under oxidative stress. Cys300 glutathionylation inhibits Mpro activity by blocking its dimerization. This provides a novel accessible and reactive target for drug development. Moreover, this process may have implications for disease pathophysiology in humans and bats. It may be a mechanism by which SARS-CoV-2 has evolved to limit replication and avoid killing host bats when they are under oxidative stress during flight.

Regulation of the Dimerization and Activity of SARS-CoV-2 Main Protease through Reversible Glutathionylation of Cysteine 300.

SARS-CoV-2 encodes main protease (Mpro), an attractive target for therapeutic interventions. We show Mpro is susceptible to glutathionylation leading to inhibition of dimerization and activity. Activity of glutathionylated Mpro could be restored with reducing agents or glutaredoxin. Analytical studies demonstrated that glutathionylated Mpro primarily exists as a monomer and that a single modification with glutathione is sufficient to block dimerization and loss of activity. Proteolytic digestions of Mpro revealed Cys300 as a primary target of glutathionylation, and experiments using a C300S Mpro mutant revealed that Cys300 is required for inhibition of activity upon Mpro glutathionylation. These findings indicate that Mpro dimerization and activity can be regulated through reversible glutathionylation of Cys300 and provides a novel target for the development of agents to block Mpro dimerization and activity. This feature of Mpro may have relevance to human disease and the pathophysiology of SARS-CoV-2 in bats, which develop oxidative stress during flight.

Pomalidomide restores immune recognition of primary effusion lymphoma through upregulation of ICAM-1 and B7-2.

Pomalidomide (Pom) is an immunomodulatory drug that has efficacy against Kaposi's sarcoma, a tumor caused by Kaposi's sarcoma-associated herpesvirus (KSHV). Pom also induces direct cytotoxicity in primary effusion lymphoma (PEL), a B-cell malignancy caused by KSHV, in part through downregulation of IRF4, cMyc, and CK1α as a result of its interaction with cereblon, a cellular E3 ubiquitin ligase. Additionally, Pom can reverse KSHV-induced downregulation of MHCI and co-stimulatory immune surface molecules ICAM-1 and B7-2 on PELs. Here, we show for the first time that Pom-induced increases in ICAM-1 and B7-2 on PEL cells lead to an increase in both T-cell activation and NK-mediated cytotoxicity against PEL. The increase in T-cell activation can be prevented by blocking ICAM-1 and/or B7-2 on the PEL cell surface, suggesting that both ICAM-1 and B7-2 are important for T-cell co-stimulation by PELs. To gain mechanistic insights into Pom's effects on surface markers, we generated Pom-resistant (PomR) PEL cells, which showed about 90% reduction in cereblon protein level and only minimal changes in IRF4 and cMyc upon Pom treatment. Pom no longer upregulated ICAM-1 and B7-2 on the surface of PomR cells, nor did it increase T-cell and NK-cell activation. Cereblon-knockout cells behaved similarly to the pomR cells upon Pom-treatment, suggesting that Pom's interaction with cereblon is necessary for these effects. Further mechanistic studies revealed PI3K signaling pathway as being important for Pom-induced increases in these molecules. These observations provide a rationale for the study of Pom as therapy in treating PEL and other KSHV-associated tumors.

A small molecule compound with an indole moiety inhibits the main protease of SARS-CoV-2 and blocks virus replication.

Except remdesivir, no specific antivirals for SARS-CoV-2 infection are currently available. Here, we characterize two small-molecule-compounds, named GRL-1720 and 5h, containing an indoline and indole moiety, respectively, which target the SARS-CoV-2 main protease (Mpro). We use VeroE6 cell-based assays with RNA-qPCR, cytopathic assays, and immunocytochemistry and show both compounds to block the infectivity of SARS-CoV-2 with EC50 values of 15 ± 4 and 4.2 ± 0.7 µM for GRL-1720 and 5h, respectively. Remdesivir permitted viral breakthrough at high concentrations; however, compound 5h completely blocks SARS-CoV-2 infection in vitro without viral breakthrough or detectable cytotoxicity. Combination of 5h and remdesivir exhibits synergism against SARS-CoV-2. Additional X-ray structural analysis show that 5h forms a covalent bond with Mpro and makes polar interactions with multiple active site amino acid residues. The present data suggest that 5h might serve as a lead Mpro inhibitor for the development of therapeutics for SARS-CoV-2 infection.

GRL-0920, an Indole Chloropyridinyl Ester, Completely Blocks SARS-CoV-2 Infection.

We assessed various newly generated compounds that target the main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and various previously known compounds reportedly active against SARS-CoV-2, employing RNA quantitative PCR (RNA-qPCR), cytopathicity assays, and immunocytochemistry. Here, we show that two indole-chloropyridinyl-ester derivatives, GRL-0820 and GRL-0920, exerted potent activity against SARS-CoV-2 in cell-based assays performed using VeroE6 cells and TMPRSS2-overexpressing VeroE6 cells. While GRL-0820 and the nucleotide analog remdesivir blocked SARS-CoV-2 infection, viral breakthrough occurred. No significant anti-SARS-CoV-2 activity was found for several compounds reportedly active against SARS-CoV-2 such as lopinavir, nelfinavir, nitazoxanide, favipiravir, and hydroxychroloquine. In contrast, GRL-0920 exerted potent activity against SARS-CoV-2 (50% effective concentration [EC50] = 2.8 µM) and dramatically reduced the infectivity, replication, and cytopathic effect of SARS-CoV-2 without significant toxicity as examined with immunocytochemistry. Structural modeling shows that indole and chloropyridinyl of the derivatives interact with two catalytic dyad residues of Mpro, Cys145 and His41, resulting in covalent bonding, which was verified using high-performance liquid chromatography-mass spectrometry (HPLC/MS), suggesting that the indole moiety is critical for the anti-SARS-CoV-2 activity of the derivatives. GRL-0920 might serve as a potential therapeutic for coronavirus disease 2019 (COVID-19) and might be optimized to generate more-potent anti-SARS-CoV-2 compounds.IMPORTANCE Targeting the main protease (Mpro) of SARS-CoV-2, we identified two indole-chloropyridinyl-ester derivatives, GRL-0820 and GRL-0920, active against SARS-CoV-2, employing RNA-qPCR and immunocytochemistry and show that the two compounds exerted potent activity against SARS-CoV-2. While GRL-0820 and remdesivir blocked SARS-CoV-2 infection, viral breakthrough occurred as examined with immunocytochemistry. In contrast, GRL-0920 completely blocked the infectivity and cytopathic effect of SARS-CoV-2 without significant toxicity. Structural modeling showed that indole and chloropyridinyl of the derivatives interacted with two catalytic dyad residues of Mpro, Cys145 and His41, resulting in covalent bonding, which was verified using HPLC/MS. The present data should shed light on the development of therapeutics for COVID-19, and optimization of GRL-0920 based on the present data is essential to develop more-potent anti-SARS-CoV-2 compounds for treating COVID-19.

Induction of Kaposi's Sarcoma-Associated Herpesvirus-Encoded Thymidine Kinase (ORF21) by X-Box Binding Protein 1.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent for Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD). Like other herpesviruses, it has latent and lytic repertoires. However, there is evidence that some lytic genes can be directly activated by certain cellular factors. Cells undergoing endoplasmic reticulum stress express spliced X-box binding protein 1 (XBP-1s). XBP-1s is also present in large amounts in germinal center B cells. XBP-1s can activate the KSHV replication and transcription activator (RTA) and lytic replication. It can also directly activate KSHV-encoded viral interleukin-6 (vIL-6) and, thus, contribute to the pathogenesis of KSHV MCD. KSHV thymidine kinase (TK), the ORF21 gene product, can enhance the production of dTTP and is important for lytic replication. It can also phosphorylate zidovudine and ganciclovir to toxic moieties, enabling treatment of KSHV-MCD with these drugs. We show here that XBP-1s can directly activate ORF21 and that this activation is mediated primarily through two XBP-response elements (XRE) on the ORF21 promoter region. Deletion or mutation of these elements eliminated XBP-1s-induced upregulation of the promoter, and chromatin immunoprecipitation studies provide evidence that XBP-1s can bind to both XREs. Exposure of PEL cells to a chemical inducer of XBP-1s can induce ORF21 within 4 hours, and ORF21 expression in the lymph nodes of patients with KSHV-MCD is predominantly found in cells with XBP-1. Thus, XBP-1s may directly upregulate KSHV ORF21 and, thus, contribute to the pathogenesis of KSHV-MCD and the activity of zidovudine and valganciclovir in this disease.IMPORTANCE Spliced X-box binding protein 1 (XBP-1s), part of the unfolded protein response and expressed in developing germinal center B cells, can induce Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication and directly activate viral interleukin-6 (vIL-6). We show here that XBP-1s can also directly activate KSHV ORF21, a lytic gene. ORF21 encodes KSHV thymidine kinase (TK), which increases the pool of dTTP for viral replication and enhances lytic replication. Direct activation of ORF21 by XBP-1s can enhance viral replication in germinal center B cells and contribute to the pathogenesis of KSHV multicentric Castleman disease (MCD). KSHV-MCD is characterized by systemic inflammation caused, in part, by lytic replication and overproduction of KSHV vIL-6 in XBP-1s-expressing lymph node plasmablasts. KSHV thymidine kinase can phosphorylate zidovudine and ganciclovir to toxic moieties, and direct activation of ORF21 by XBP-1s may also help explain the effectiveness of zidovudine and valganciclovir in the treatment of KSHV-MCD.

Pomalidomide increases immune surface marker expression and immune recognition of oncovirus-infected cells.

Most chronic viruses evade T-cell and natural killer (NK) immunity through downregulation of immune surface markers. Previously we showed that Pomalidomide (Pom) increases surface expression of major histocompatibility complex class I (MHC-I) in Kaposi sarcoma-associated herpesvirus-infected latent and lytic cells and restores ICAM-1 and B7-2 in latent cells. We explored the ability of Pom to increase immune surface marker expression in cells infected by other chronic viruses, including human T-cell leukemia virus type-1 (HTLV-1), Epstein-Barr virus (EBV), human papilloma virus (HPV), Merkel cell polyoma virus (MCV), and human immunodeficiency virus type-1 (HIV-1). Pom increased MHC-1, ICAM-1, and B7-2/CD86 in immortalized T-cell lines productively infected with HTLV-1 and also significantly increased their susceptibility to NK cell-mediated cytotoxicity. Pom enhancement of MHC-I and ICAM-1 in primary cells infected with HTLV-1 was abrogated by knockout of HTLV-1 orf-1. Pom increased expression of ICAM-1, B7-2 and MHC class I polypeptide related sequence A (MICA) surface expression in the EBV-infected Daudi cells and increased their T-cell activation and susceptibility to NK cells. Moreover, Pom increased expression of certain of these surface markers on Akata, Raji, and EBV lymphoblastic cell lines. The increased expression of immune surface markers in these virus-infected lines was generally associated with a decrease in IRF4 expression. By contrast, Pom treatment of HPV, MCV and HIV-1 infected cells did not increase these immune surface markers. Pom and related drugs may be clinically beneficial for the treatment of HTLV-1 and EBV-induced tumors by rendering infected cells more susceptible to both innate and adaptive host immune responses.

PTEN loss in the fallopian tube induces hyperplasia and ovarian tumor formation.

The signaling events involved in the onset of ovarian cancer from the fallopian tube epithelium (FTE) are crucial for early detection and treatment of the disease, but they remain poorly defined. Conditional homozygous knockout of PTEN mediated by PAX8-cre recombinase was sufficient to drive endometrioid and serous borderline ovarian carcinoma, providing the first model of FTE-derived borderline tumors. In addition, heterozygous PTEN deletion in the FTE resulted in hyperplasia, providing a model to study early events of human ovarian pathogenesis. To uncover the mechanism underlying the invasion of cancerous oviductal cells to the ovary, PTEN-deficient murine oviductal cells were developed and tagged with green fluorescent protein. Loss of PTEN increased cell migration, invasion, and upregulated WNT4, a key regulator of Müllerian duct development during embryogenesis. Further investigation revealed that WNT4 was required for increased migration and colonization of the ovary by PTEN-deficient oviductal cells in a ß-catenin independent manner. Human tumor microarrays and ovarian cancer cells lines confirmed WNT4 expression in cancer and its role in migration. Together, these findings provide a novel model to study the mechanism of fallopian tube tumor initiation and invasion to the ovary mediated by loss of PTEN, which may help to define early events of human ovarian carcinogenesis.

Restoration of immune surface molecules in Kaposi sarcoma-associated herpes virus infected cells by lenalidomide and pomalidomide.

Kaposi sarcoma-associated herpesvirus (KSHV) is the cause of several tumors, including Kaposi sarcoma and primary effusion lymphoma (PEL). Most viruses have evolved means of escaping immune recognition. KSHV downregulates MHC-I expression during lytic infection, and expression of ICAM-1 and B7-2 (CD86) during latent infection, allowing evasion of T cell and natural killer immunity respectively. These effects are largely mediated by two KSHV-encoded proteins, K3 and K5. We show here that lenalidomide (Len) and pomalidomide (Pom) prevent down-regulation of MHC-I during lytic activation, and restore ICAM-1 and B7-2 surface expression in latently infected PEL cells. Importantly, these changes occurred at clinically achievable concentrations and prior to any cytotoxic effects. Exploration of the mechanism revealed that Pom blocked lytic down-regulation of MHC-I induced by transfection with K3 but not K5. Although Pom alone did not significantly increase HLA mRNA expression in PEL cells, it did blunt the butyrate-induced decrease in MHC-I mRNA expression and decreased the upregulation of K3 mRNA in lytic cells. Virus-induced tumors express foreign antigens, but immunotherapy can be thwarted by viral strategies to evade immune recognition. The effects of Pom and Len described here can prevent these strategies and support the use of these drugs to treat KSHV-induced tumors.

Hypoxia-inducible factor-1 alpha as a therapeutic target for primary effusion lymphoma.

Primary effusion lymphoma (PEL) is an aggressive B-cell lymphoma with poor prognosis caused by Kaposi's sarcoma-associated herpesvirus (KSHV). Previous studies have revealed that HIF-1α, which mediates much of the cellular response to hypoxia, plays an important role in life cycle of KSHV. KSHV infection promotes HIF-1α activity, and several KSHV genes are in turn activated by HIF-1α. In this study, we investigated the effects of knocking down HIF-1α in PELs. We observed that HIF-1α knockdown in each of two PEL lines leads to a reduction in both aerobic and anaerobic glycolysis as well as lipid biogenesis, indicating that HIF-1α is necessary for maintaining a metabolic state optimal for growth of PEL. We also found that HIF-1α suppression leads to a substantial reduction in activation of lytic KSHV genes, not only in hypoxia but also in normoxia. Moreover, HIF-1α knockdown led to a decrease in the expression of various KSHV latent genes, including LANA, vCyclin, kaposin, and miRNAs, under both normoxic and hypoxic conditions. These observations provide evidence that HIF-1α plays an important role in PEL even in normoxia. Consistent with these findings, we observed a significant inhibition of growth of PEL in normoxia upon HIF-1α suppression achieved by either HIF-1α knockdown or treatment with PX-478, a small molecule inhibitor of HIF-1α. These results offer further evidence that HIF-1α plays a critical role in the pathogenesis of PEL, and that inhibition of HIF-1α can be a potential therapeutic strategy in this disease.

Identification of functional hypoxia inducible factor response elements in the human lysyl oxidase gene promoter.

Human lysyl oxidase (LOX) is a hypoxia-responsive gene whose product catalyzes collagen crosslinking and is thought to be important in cancer metastasis and osteoarthritis. We previously demonstrated that LOX was upregulated by hypoxia inducible factor 2 (HIF-2) more strongly than hypoxia inducible 1 (HIF-1). Here, we further investigated the response of the LOX gene and LOX promoter to HIFs. LOX mRNA, measured by real time reverse transcriptase-PCR, was strongly up-regulated (almost 40-fold), by transfection of HEK-293T cells with a plasmid encoding the HIF-2α subunit of HIF-2, but only three-fold by a plasmid encoding HIF-1α. LOX protein was detectable by Western blot of cells transfected with HIF-2α, but not with HIF-1α. Analysis of a 1487 bp promoter sequence upstream of the human LOX gene revealed 9 potential hypoxia response elements (HREs). Promoter truncation allowed the mapping of two previously unidentified functional HREs, called here HRE8 and HRE7; -455 to -451 and -382 to -386 bp, respectively, upstream of the start codon for LOX. Removal or mutation of these HREs led to a substantial reduction in both HIF-1α and HIF-2α responsiveness. Also, expression of LOX was significantly inhibited by a small molecule specific HIF-2 inhibitor. In conclusion, LOX is highly responsive to HIF-2α and this is largely mediated by two previously unidentified HREs. These observations enhance our understanding of the regulation of this important gene involved in cancer and osteoarthritis, and suggest that these conditions may be targeted by HIF-2 inhibitors.