Sujayita Roy

New cell-signaling pathways for controlling cytomegalovirus replication

Cytomegalovirus (CMV) is increasingly recognized as an accomplished modulator of cell‐signaling pathways, both directly via interaction between viral and cellular proteins, and indirectly by activating metabolic/energy states of infected cells. Viral genes, as well as captured cellular genes, enable CMV to modify these pathways upon binding to cellular receptors, up until generation of virus progeny. Deregulation of cell‐signaling pathways appears to be a well‐developed tightly balanced virus strategy to achieve the desired consequences in each infected cell type. Importantly and perhaps surprisingly, identification of new signaling pathways in cancer cells positioned CMV as a sophisticated user and abuser of many such pathways, creating opportunities to develop novel therapeutic strategies for inhibiting CMV replication (in addition to standard of care CMV DNA polymerase inhibitors). Advances in genomics and proteomics allow the identification of CMV products interacting with the cellular machinery. Ultimately, clinical implementation of candidate drugs capable of disrupting the delicate balance between CMV and cell‐signaling will depend on the specificity and selectivity index of newly identified targets.

Inhibition of Human Cytomegalovirus Replication by Artemisinins: Effects Mediated through Cell Cycle Modulation

ABSTRACT Artemisinin-derived monomers and dimers inhibit human cytomegalovirus (CMV) replication in human foreskin fibroblasts (HFFs). The monomer artesunate (AS) inhibits CMV at micromolar concentrations, while dimers inhibit CMV replication at nanomolar concentrations, without increased toxicity in HFFs. We report on the variable anti-CMV activity of AS compared to the consistent and reproducible CMV inhibition by dimer 606 and ganciclovir (GCV). Investigation of this phenomenon revealed that the anti-CMV activity of AS correlated with HFFs synchronized to the G0/G1 stage of the cell cycle. In contact-inhibited serum-starved HFFs or cells arrested at early/late G1 with specific checkpoint regulators, AS and dimer 606 efficiently inhibited CMV replication. However, in cycling HFFs, in which CMV replication was productive, virus inhibition by AS was significantly reduced, but inhibition by dimer 606 and GCV was maintained. Cell cycle analysis in noninfected HFFs revealed that AS induced early G1 arrest, while dimer 606 partially blocked cell cycle progression. In infected HFFs, AS and dimer 606 prevented the progression of cell cycle toward the G1/S checkpoint. AS reduced the expression of cyclin-dependent kinases (CDK) 2, 4, and 6 in noninfected cycling HFFs, while the effect of dimer 606 on these CDKs was moderate. Neither compound affected CDK expression in noninfected contact-inhibited HFFs. In CMV-infected cells, AS activity correlated with reduced CDK2 levels. CMV inhibition by AS and dimer 606 also correlated with hypophosphorylation (activity) of the retinoblastoma protein (pRb). AS activity was strongly associated with pRb hypophosphorylation, while its reduced anti-CMV activity was marked by pRb phosphorylation. Roscovitine, a CDK2 inhibitor, antagonized the anti-CMV activities of AS and dimer 606. These data suggest that cell cycle modulation through CDKs and pRb might play a role in the anti-CMV activities of artemisinins. Proteins involved in this modulation may be identified and targeted for CMV inhibition.

HMGA1 overexpression correlates with relapse in childhood B-lineage acute lymphoblastic leukemia

Despite dramatic improvements in treatment and survival, acute lymphoblastic leukemic (ALL) remains the leading cause of death in children with cancer [1]. ALL is the most common pediatric cancer, accounting for 25% of all cancers occurring in children under the age of 15 years [1]. The majority of cases are a B-lineage cell subtype of ALL (B-ALL). Although most children with ALL are cured with current chemotherapeutic regimens, approximately 15% will relapse and 8–9% will ultimately succumb to their disease [1]. Risk assessment is a critical component in the selection of appropriate therapy for individual patients. Unfortunately, current criteria used to stratify patients into groups at increasing risk for treatment failure or relapse are limited, and rely primarily on clinical characteristics (age, white blood cell count, immunophenotype, presence/absence of central nervous system disease or testicular disease) and underlying genetic lesions. Good risk genetic lesions include ETV6–RUNX1 fusion or hyperdiploidy (50 or more chromosomes) with favorable chromosomal trisomies (4 + 10), while poor risk genetic lesions include MLL rearrangements (MLL-R), hypodiploidy, intrachromosome 21 (iAMP21) or Philadelphia chromosome positive (Ph+) ALL [1]. In addition, early treatment response is included in risk stratification and determined by measurement of minimal residual disease (MRD) at specific time points early in therapy. The 5-year overall survival in ALL has increased from only 10% in the 1960s to about 80% in the 1980s, and very recent studies indicate that 92% of children with ALL will be cured with current therapies [1,2]. Nonetheless, many patients are over-treated, whereas a significant number of children who were initially classified as favorable risk suffer a relapse. Thus, elucidating the molecular underpinnings of refractory disease in ALL is needed to identify patients who are likely to relapse and require more intensive chemotherapeutic regimens. Moreover, identifying key molecular pathways could also uncover novel therapeutic targets.

Inactivation of the Cdkn2a locus cooperates with HMGA1 to drive T-cell leukemogenesis

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive leukemia with high relapse rates compared to B-lineage ALL. We previously showed that HMGA1a transgenic mice develop aggressive T-ALL, indicating that HMGA1 causes leukemic transformation in vivo. HMGA1 is also highly expressed in embryonic stem cells, hematopoietic stem cells and diverse, refractory human cancers. Disruption of the CDKN2A tumor suppressor locus occurs in most cases of T-ALL and is thought to contribute to leukemic transformation. To determine whether loss of function of CDKN2A cooperates with HMGA1 in T-ALL, we crossed HMGA1a transgenics onto a Cdkn2a null background. We discovered that T-ALL is markedly accelerated in HMGA1a transgenic Cdkn2a null mice. In addition, these mice recapitulate salient clinical and pathologic features of human T-ALL. HMGA1 is also highly overexpressed in human T-ALL. These findings suggest that HMGA1 plays a causative role in T-ALL and could represent a rational therapeutic target.

Role of nucleotide-binding oligomerization domain 1 (NOD1) and its variants in human cytomegalovirus control in vitro and in vivo

Significance Infection with human cytomegalovirus (HCMV) is a growing health problem, creating diagnostic and therapeutic challenges. Biomarkers for risk of infection are lacking, and the limited drugs that inhibit HCMV have major side effects. New strategies for virus control are needed. We report on the role of nucleotide-binding oligomerization domain 1 (NOD1), a cytoplasmic pattern recognition receptor, in HCMV suppression. NOD1 activation (through IKKα and IRF3) resulted in IFN response and HCMV inhibition. Specific mutations in NOD1 showed differential effects on HCMV replication in vitro. In a nested study of HCMV vaccine, specific polymorphisms in NOD1 were detected in HCMV-infected women compared with noninfected women. Our work provides direction for studies of innate immune response to HCMV and genetic susceptibility through NOD1. Induction of nucleotide-binding oligomerization domain 2 (NOD2) and downstream receptor-interacting serine/threonine-protein kinase 2 (RIPK2) by human cytomegalovirus (HCMV) is known to up-regulate antiviral responses and suppress virus replication. We investigated the role of nucleotide-binding oligomerization domain 1 (NOD1), which also signals through RIPK2, in HCMV control. NOD1 activation by Tri-DAP (NOD1 agonist) suppressed HCMV and induced IFN-β. Mouse CMV was also inhibited through NOD1 activation. NOD1 knockdown (KD) or inhibition of its activity with small molecule ML130 enhanced HCMV replication in vitro. NOD1 mutations displayed differential effects on HCMV replication and antiviral responses. In cells overexpressing the E56K mutation in the caspase activation and recruitment domain, virus replication was enhanced, but in cells overexpressing the E266K mutation in the nucleotide-binding domain or the wild-type NOD1, HCMV was inhibited, changes that correlated with IFN-β expression. The interaction of NOD1 and RIPK2 determined the outcome of virus replication, as evidenced by enhanced virus growth in NOD1 E56K mutant cells (which failed to interact with RIPK2). NOD1 activities were executed through IFN-β, given that IFN-β KD reduced the inhibitory effect of Tri-DAP on HCMV. Signaling through NOD1 resulting in HCMV suppression was IKKα-dependent and correlated with nuclear translocation and phosphorylation of IRF3. Finally, NOD1 polymorphisms were significantly associated with the risk of HCMV infection in women who were infected with HCMV during participation in a glycoprotein B vaccine trial. Collectively, our data indicate a role for NOD1 in HCMV control via RIPK2- IKKα-IRF3 and suggest that its polymorphisms predict the risk of infection.

Efficacy and Mechanism of Action of Low Dose Emetine against Human Cytomegalovirus

Infection with human cytomegalovirus (HCMV) is a threat for pregnant women and immunocompromised hosts. Although limited drugs are available, development of new agents against HCMV is desired. Through screening of the LOPAC library, we identified emetine as HCMV inhibitor. Additional studies confirmed its anti-HCMV activities in human foreskin fibroblasts: EC50−40±1.72 nM, CC50−8±0.56 μM, and selectivity index of 200. HCMV inhibition occurred after virus entry, but before DNA replication, and resulted in decreased expression of viral proteins. Synergistic virus inhibition was achieved when emetine was combined with ganciclovir. In a mouse CMV (MCMV) model, emetine was well-tolerated, displayed long half-life, preferential distribution to tissues over plasma, and effectively suppressed MCMV. Since the in vitro anti-HCMV activity of emetine decreased significantly in low-density cells, a mechanism involving cell cycle regulation was suspected. HCMV inhibition by emetine depended on ribosomal processing S14 (RPS14) binding to MDM2, leading to disruption of HCMV-induced MDM2-p53 and MDM2-IE2 interactions. Irrespective of cell density, emetine induced RPS14 translocation into the nucleus during infection. In infected high-density cells, MDM2 was available for interaction with RPS14, resulting in disruption of MDM2-p53 interaction. However, in low-density cells the pre-existing interaction of MDM2-p53 could not be disrupted, and RPS14 could not interact with MDM2. In high-density cells the interaction of MDM2-RPS14 resulted in ubiquitination and degradation of RPS14, which was not observed in low-density cells. In infected-only or in non-infected emetine-treated cells, RPS14 failed to translocate into the nucleus, hence could not interact with MDM2, and was not ubiquitinated. HCMV replicated similarly in RPS14 knockdown or control cells, but emetine did not inhibit virus replication in the former cell line. The interaction of MDM2-p53 was maintained in infected RPS14 knockdown cells despite emetine treatment, confirming a unique mechanism by which emetine exploits RPS14 to disrupt MDM2-p53 interaction. Summarized, emetine may represent a promising candidate for HCMV therapy alone or in combination with ganciclovir through a novel host-dependent mechanism.

The High Mobility Group A1 (HMGA1) gene is highly overexpressed in human uterine serous carcinomas and carcinosarcomas and drives Matrix Metalloproteinase-2 (MMP-2) in a subset of tumors

OBJECTIVES Although uterine cancer is the fourth most common cause for cancer death in women worldwide, the molecular underpinnings of tumor progression remain poorly understood. The High Mobility Group A1 (HMGA1) gene is overexpressed in aggressive cancers and high levels portend adverse outcomes in diverse tumors. We previously reported that Hmga1a transgenic mice develop uterine tumors with complete penetrance. Because HMGA1 drives tumor progression by inducing MatrixMetalloproteinase (MMP) and other genes involved in invasion, we explored the HMGA1-MMP-2 pathway in uterine cancer. METHODS To investigate MMP-2 in uterine tumors driven by HMGA1, we used a genetic approach with mouse models. Next, we assessed HMGA1 and MMP-2 expression in primary human uterine tumors, including low-grade carcinomas (endometrial endometrioid) and more aggressive tumors (endometrial serous carcinomas, uterine carcinosarcomas/malignant mesodermal mixed tumors). RESULTS Here, we report for the first time that uterine tumor growth is impaired in Hmga1a transgenic mice crossed on to an Mmp-2 deficient background. In human tumors, we discovered that HMGA1 is highest in aggressive carcinosarcomas and serous carcinomas, with lower levels in the more indolent endometrioid carcinomas. Moreover, HMGA1 and MMP-2 were positively correlated, but only in a subset of carcinosarcomas. HMGA1 also occupies the MMP-2 promoter in human carcinosarcoma cells. CONCLUSIONS Together, our studies define a novel HMGA1-MMP-2 pathway involved in a subset of human carcinosarcomas and tumor progression in murine models. Our work also suggests that targeting HMGA1 could be effective adjuvant therapy for more aggressive uterine cancers and provides compelling data for further preclinical studies.

Human Cytomegalovirus Inhibits the PARsylation Activity of Tankyrase—A Potential Strategy for Suppression of the Wnt Pathway

Human cytomegalovirus (HCMV) was reported to downregulate the Wnt/β-catenin pathway. Induction of Axin1, the negative regulator of the Wnt pathway, has been reported as an important mechanism for inhibition of β-catenin. Since Tankyrase (TNKS) negatively regulates Axin1, we investigated the effect of HCMV on TNKS expression and poly-ADP ribose polymerase (PARsylation) activity, during virus replication. Starting at 24 h post infection, HCMV stabilized the expression of TNKS and reduced its PARsylation activity, resulting in accumulation of Axin1 and reduction in its PARsylation as well. General PARsylation was not changed in HCMV-infected cells, suggesting specific inhibition of TNKS PARsylation. Similarly, treatment with XAV939, a chemical inhibitor of TNKS’ activity, resulted in the accumulation of TNKS in both non-infected and HCMV-infected cell lines. Reduction of TNKS activity or knockdown of TNKS was beneficial for HCMV, evidenced by its improved growth in fibroblasts. Our results suggest that HCMV modulates the activity of TNKS to induce Axin1, resulting in inhibition of the β-catenin pathway. Since HCMV replication is facilitated by TNKS knockdown or inhibition of its activity, TNKS may serve as an important virus target for control of a variety of cellular processes.

IRF-5 - A New Link to Autoimmune Diseases

Transcription factors of the interferon regulatory factor (IRF) family have a critical role in the activation of interferon (IFN) genes. All cellular IRFs share a region of homology in the amino terminus encompassing a highly conserved DNA binding motif characterized by five tryptophan repeats, but show variability in the carboxy (C-) terminal part of the IRF polypeptides. While some of these IRFs like IRF-3 and IRF-7 have a critical role in the antiviral response, the others like IRF-1, IRF-4 and IRF-8 have basic roles in the development and function of lymphoid cells. Recently, the importance of IRF-5 in the antiviral and inflammatory response in vivo has been clearly established, but it was also shown that this IRF has a basic function in apoptosis and B cells and macrophage differentiation. More interestingly, the role of IRF-5 pathogenicity in autoimmune diseases has been also established, as IRF-5 has been identified as one of the primary risk factors associated with Systemic Lupus Erythematosus (SLE) and other autoimmune diseases. This chapter will review the current knowledge of the mechanisms of IRF-5 activation by the TLR7 pathway and the genetic modifications of IRF-5 that may contribute to the dysregulation of the innate and adaptive immune response associated with the autoimmune disease. Furthermore we will summarize the contribution of the SLE mouse models to our understanding of the role of IRF-5 and TLR7 in the induction of the autoimmune diseases.