Trichodysplasia spinulosa (TS) is a folliculocentric and clinically papular dermatological disord... more Trichodysplasia spinulosa (TS) is a folliculocentric and clinically papular dermatological disorder occurring in the setting of immunosuppression typically in association with solid organ transplantation or hematolymphoid malignancies. We report the occurrence of TS in a 7-year-old girl with Down syndrome and pre-B-acute lymphoblastic leukemia (pre-BALL) who was completing chemotherapy at onset. The patient's affected follicles were dilated by an expansion of a dystrophic follicular inner root sheath cell population displaying enlarged trichohyaline cytoplasmic granules and progressing centrally to keratotic and parakeratotic debris, and superficially demonstrating some diminutive hair shaft-like material within the keratotic spicules. Electron microscopic studies of a follicular lesion demonstrated extracellular viral particles suggestive of a polyomavirus within the central follicular keratotic debris. DNA Polymerase chain reaction (PCR) and gene sequencing studies, performed on the tissue of the microscopic slide and paraffin block, for the recently identified trichodysplasia spinulosa-associated polyomavirus (TSPyV), were resulted as positive for TSPyV. PCR for the Merkel cell polyomavirus (MCPyV) was negative. To date this case is unique in representing the first case of TS confirmed by electron microscopy in which a related viral pathogen has been molecularly identified. An additional 19 reported cases classifiable as TS are tabulated and reviewed.
Multiple human polyomaviruses (HPyV) can infect the skin, but only Merkel cell polyomavirus (MCPy... more Multiple human polyomaviruses (HPyV) can infect the skin, but only Merkel cell polyomavirus (MCPyV) has been implicated in the development of a cancer, Merkel cell carcinoma (MCC). While expression of HPyV6, HPyV7, and MCPyV small T antigens (sT), all induced a senescence-associated secretory phenotype (SASP), MCPyV sT uniquely activated noncanonical NF-κB (ncNF-κB), instead of canonical NF-κB signaling, to evade p53-mediated cellular senescence. Through its large T stabilization domain, MCPyV sT activated ncNF-κB signaling both by inducing H3K4 trimethylation-mediated increases of NFKB2 and RELB transcription and also by promoting NFKB2 stabilization and activation through FBXW7 inhibition. Noncanonical NF-κB signaling was required for SASP cytokine secretion, which promoted the proliferation of MCPyV sT–expressing cells through autocrine signaling. Virus-positive MCC cell lines and tumors showed ncNF-κB pathway activation and SASP gene expression, and the inhibition of ncNF-κB sig...
Bischoff SC (2007) Role of mast cells in allergic and non-allergic immune responses: comparison o... more Bischoff SC (2007) Role of mast cells in allergic and non-allergic immune responses: comparison of human and murine data. Nat Rev Immunol 7:93-104 Bodtger U, Poulsen LK, Malling HJ (2003) Asymptomatic skin sensitization to birch predicts later development of birch pollen allergy in adults: a 3-year follow-up study. J Allergy Clin Immunol 111:149-54 Chodaczek G, Bacsi A, Dharajiya N et al. (2009) Ragweed pollen-mediated IgE-independent release of biogenic amines from mast cells via induction of mitochondrial dysfunction. Mol Immunol 46:2505-14 Gilles S, Jacoby D, Blume C et al. (2010) Pollenderived low-molecular weight factors inhibit 6-sulfo LacNAc(+) dendritic cells' capacity to induce T-helper type 1 responses. Clin Exp Allergy 40:269-78 Gilles S, Mariani V, Bryce M et al. (2009) Pollenderived E1-phytoprostanes signal via PPARgamma and NF-kappaB-dependent mechanisms.
Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterized by cutaneo... more Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterized by cutaneous symptoms, including hyperpigmentation and nail dystrophy. Some forms of DC are caused by mutations in telomerase, the enzyme that counteracts telomere shortening, suggesting a telomere-based disease mechanism. However, mice with extensively shortened telomeres due to telomerase deficiency do not develop the characteristics of DC, raising questions about the etiology of DC and/or mouse models for human telomere dysfunction. Here we describe mice engineered to undergo telomere degradation due to the absence of the shelterin component POT1b. When combined with reduced telomerase activity, POT1b deficiency elicits several characteristics of DC, including hyperpigmentation and fatal bone marrow failure at 4–5 mo of age. These results provide experimental support for the notion that DC is caused by telomere dysfunction, and demonstrate that key aspects of a human telomere-based disease can ...
Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immu... more Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations that disrupt nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts the expression of POLA1, which encodes the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency resulted in increased production of type I interferons. This enzyme is necessary for the synthesis of RNA:DNA primers during DNA replication and, strikingly, we found that POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Together this work identifies POLA1 as a critical regulator of the type I interferon response.
Merkel cell carcinoma (MCC) is an infrequent, rapidly growing skin neoplasm that carries a greate... more Merkel cell carcinoma (MCC) is an infrequent, rapidly growing skin neoplasm that carries a greater probability of regional lymph node involvement, and a grim prognosis in advanced cases. While it is seen predominantly in old age in sun-exposed body parts, the prevalence varies among different races and geographical regions. Merkel cell polyomavirus and UV radiation-induced mutations contribute to its etiopathogenesis. The clinical presentation of MCC lacks pathognomonic features and is rarely considered highly at the time of presentation. Histopathological examination frequently reveals hyperchromatic nuclei with high mitotic activity, but immunohistochemistry is required to confirm the diagnosis. Sentinel lymph node biopsy (SLNB) and imaging are advised for effective staging of the disease. Multimodal management including surgery, radiation therapy, and/or immunotherapy are deployed. Traditional cytotoxic chemotherapies may result in an initial response, but do not result in a sign...
Circular RNAs (circRNAs) are a conserved class of RNAs with diverse functions, including serving ... more Circular RNAs (circRNAs) are a conserved class of RNAs with diverse functions, including serving as messenger RNAs that are translated into peptides. Here we describe circular RNAs generated by human polyomaviruses (HPyVs), some of which encode variants of the previously described alternative large T antigen open reading frame (ALTO) protein. Circular ALTO RNAs (circALTOs) can be detected in virus positive Merkel cell carcinoma (VP-MCC) cell lines and tumor samples. CircALTOs are stable, predominantly located in the cytoplasm, and N6-methyladenosine (m6A) modified. The translation of MCPyV circALTOs into ALTO protein is negatively regulated by MCPyV-generated miRNAs in cultured cells. MCPyV ALTO expression increases transcription from some recombinant promoters in vitro and upregulates the expression of multiple genes previously implicated in MCPyV pathogenesis. MCPyV circALTOs are enriched in exosomes derived from VP-MCC lines and circALTO-transfected 293T cells, and purified exoso...
Bioinformatics and in vitro studies have revealed that single-stranded circular RNAs (circRNAs), ... more Bioinformatics and in vitro studies have revealed that single-stranded circular RNAs (circRNAs), generated through ‘backsplicing,’ occur more extensively than initially appreciated. While the functions of most circRNAs are unknown, binding of microRNAs (miRNA), regulation of splicing and transcription, and translation into proteins have all been demonstrated for specific circRNAs. Virally-derived circRNAs have recently been described in gamma-herpesviruses. Here, we report that oncogenic human papillomaviruses (HPV) generate circRNAs, including ones which encompass the entire coding region of the E7 oncogene (circE7). HPV16 circE7 can be detected by both inverse RT-PCR and Northern blots of HPV16-transformed cell lines. CircE7 is N6-methyladenosine (m6A) modified, preferentially localized to the cytoplasm, and can be translated to produce E7 oncoprotein. Specific disruption of circE7 in CaSki cervical carcinoma cells decreased E7 protein levels, inhibited cell proliferation, and inh...
Cancer cells can inhibit effector T cells (Teff) through both immunomodulatory receptors and the ... more Cancer cells can inhibit effector T cells (Teff) through both immunomodulatory receptors and the impact of cancer metabolism on the tumor microenvironment. Indeed, Teff require high rates of glucose metabolism, and consumption of essential nutrients or generation of waste products by tumor cells may impede essential T cell metabolic pathways. Clear cell renal cell carcinoma (ccRCC) is characterized by loss of the tumor suppressor von Hippel-Lindau (VHL) and altered cancer cell metabolism. Here, we assessed how ccRCC influences the metabolism and activation of primary patient ccRCC tumor infiltrating lymphocytes (TIL). CD8 TIL were abundant in ccRCC, but they were phenotypically distinct and both functionally and metabolically impaired. ccRCC CD8 TIL were unable to efficiently uptake glucose or perform glycolysis and had small, fragmented mitochondria that were hyperpolarized and generated large amounts of ROS. Elevated ROS was associated with downregulated mitochondrial SOD2. CD8 T ...
The discovery that oxidized vitamin C, dehydroascorbate (DHA), can induce oxidative stress and ce... more The discovery that oxidized vitamin C, dehydroascorbate (DHA), can induce oxidative stress and cell death in cancer cells has rekindled interest in the use of high dose vitamin C (VC) as a cancer therapy. However, high dose VC has shown limited efficacy in clinical trials, possibly due to the decreased bioavailability of oral VC. Because human erythrocytes express high levels of Glut1, take up DHA, and reduce it to VC, we tested how erythrocytes might impact high dose VC therapies. Cancer cells are protected from VC-mediated cell death when co-cultured with physiologically relevant numbers of erythrocytes. Pharmacological doses of VC induce oxidative stress, GSH depletion, and increased glucose flux through the oxidative pentose phosphate pathway (PPP) in erythrocytes. Incubation of erythrocytes with VC induced hemolysis, which was exacerbated in erythrocytes from glucose-6-phosphate dehydrogenase (G6PD) patients and rescued by antioxidants. Thus, erythrocytes protect cancer cells f...
Trichodysplasia spinulosa (TS) is a proliferative skin disease observed in severely immunocomprom... more Trichodysplasia spinulosa (TS) is a proliferative skin disease observed in severely immunocompromized patients. It is characterized by papule and trichohyalin-rich spicule formation, epidermal acanthosis and distention of dysmorphic hair follicles overpopulated by inner root sheath cells (IRS). TS probably results from active infection with the TS-associated polyomavirus (TSPyV), as indicated by high viral-load, virus protein expression and particle formation. The underlying pathogenic mechanism imposed by TSPyV infection has not been solved yet. By analogy with other polyomaviruses, such as the Merkel cell polyomavirus associated with Merkel cell carcinoma, we hypothesized that TSPyV T-antigen promotes proliferation of infected IRS cells. Therefore, we analyzed TS biopsy sections for markers of cell proliferation (Ki-67) and cell cycle regulation (p16 ink4a , p21 waf , pRB, phosphorylated pRB), and the putatively transforming TSPyV early large tumor (LT) antigen. Intense Ki-67 staining was detected especially in the margins of TS hair follicles, which colocalized with TSPyV LTantigen detection. In this area, staining was also noted for pRB and particularly phosphorylated pRB, as well as p16 ink4a and p21 waf. Healthy control hair follicles did not or hardly stained for these markers. Trichohyalin was particularly detected in the center of TS follicles that stained negative for Ki-67 and TSPyV LT-antigen. In summary, we provide evidence for clustering of TSPyV LT-antigen-expressing and proliferating cells in the follicle margins that overproduce negative cell cycle regulatory proteins. These data are compatible with a scenario of TSPyV T-antigen-mediated cell cycle progression, potentially creating a pool of proliferating cells that enable viral DNA replication and drive papule and spicule formation.
Although Foxp3+ T regulatory cells (Tregs) are well documented for their ability to suppress vari... more Although Foxp3+ T regulatory cells (Tregs) are well documented for their ability to suppress various immune cells, T-cell subsets capable of counteracting Tregs have not been demonstrated. Here, we assessed phosphoantigen-activated Vγ2Vδ2 T cells for the ability to interplay with Tregs in the context of mycobacterial infection. A short-term IL-2 treatment regimen induced marked expansion of CD4+CD25+Foxp3+ T cells and subsequent suppression of mycobacterium-driven increases in numbers of Vγ2Vδ2 T cells. Surprisingly, activation of Vγ2Vδ2 T cells by adding phosphoantigen Picostim to the IL-2 treatment regimen down-regulated IL-2–induced expansion of CD4+CD25+Foxp3+ T cells. Consistently, in vitro activation of Vγ2Vδ2 T cells by phosphoantigen plus IL-2 down-regulated IL-2–induced expansion of CD4+CD25+Foxp3+ T cells. Interestingly, anti–IFN-γ–neutralizing antibody, not anti–TGF-β or anti–IL-4, reduced the ability of activated Vγ2Vδ2 T cells to down-regulate Tregs, suggesting that aut...
ABSTRACTCircular RNAs (circRNAs) are a conserved class of RNAs with diverse functions. A subset o... more ABSTRACTCircular RNAs (circRNAs) are a conserved class of RNAs with diverse functions. A subset of circRNAs are translated into peptides. Here we describe circular RNAs encoded by human polyomaviruses (HPyVs), including circular forms of RNAs encoding variants of the previously described alternative large T antigen open reading frame (ALTO) gene. Circular ALTO RNAs (circALTOs) can be detected in virus positive Merkel cell carcinoma (VP-MCC) cell lines and tumor samples. CircALTOs are stable, predominantly located in the cytoplasm, and N6-methyladenosine (m6A) modified. MCPyV circALTOs produce ALTO protein in cultured cells. MCPyV ALTO promotes the transcription of co-transfected reporter genes. MCPyV circALTOs are enriched in exosomes derived from VP-MCC lines and circALTO-transfected 293T cells, and purified exosomes can mediate ALTO expression and transcriptional activation. The related trichodysplasia spinulosa polyomavirus (TSPyV) also expresses a circALTO that can be detected i...
Single-stranded circular RNAs (circRNAs), generated through 'backsplicing', occur more extensivel... more Single-stranded circular RNAs (circRNAs), generated through 'backsplicing', occur more extensively than initially anticipated. The possible functions of the vast majority of circRNAs remain unknown. Virus-derived circRNAs have recently been described in gammaherpesviruses. We report that oncogenic human papillomaviruses (HPVs) generate cir-cRNAs, some of which encompass the E7 oncogene (circE7). HPV16 circE7 is detectable by both inverse RT-PCR and northern blotting of HPV16-transformed cells. CircE7 is N 6 -methyladenosine (m 6 A) modified, preferentially localized to the cytoplasm, associated with polysomes, and translated to produce E7 oncoprotein. Specific disruption of circE7 in CaSki cervical carcinoma cells reduces E7 protein levels and inhibits cancer cell growth both in vitro and in tumor xenografts. CircE7 is present in TCGA RNA-Seq data from HPV-positive cancers and in cell lines with only episomal HPVs. These results provide evidence that virus-derived, protein-encoding circular RNAs are biologically functional and linked to the transforming properties of some HPV.
Anal squamous cell carcinoma (ASCC) is a rare, potentially fatal malignancy primarily caused by h... more Anal squamous cell carcinoma (ASCC) is a rare, potentially fatal malignancy primarily caused by high-risk human papillomaviruses (HPV). The prognostic implication of programmed death-ligand 1 (PD-L1) expression remains controversial, and glucose transporter 1 (GLUT1) expression has never been examined in ASCC. Covalently closed circular RNAs have recently been shown to be widespread in cancers and are proposed to be biomarkers. We discovered HPV16 expresses a circular E7 RNA (circE7) which has not been assessed as a potential biomarker. A retrospective, translational case series at UT Southwestern was conducted to analyze PD-L1, GLUT1, HPV-ISH, and HPV circE7 in relation to the clinical features and overall survival of patients with ASCC. Twenty-two (22) subjects were included in the study. Improved overall survival was predicted by basaloid histology (p = 0.013), PD-L1 expression (p = 0.08), and HPV-ISH positivity (p < 0.001), but not GLUT1 expression. High levels of circE7 by quantitative RT-PCR predicted improved overall survival in ASCC (p = 0.023) and analysis of The Cancer Genome Atlas sequencing from HPV-positive head and neck cancer and cervical cancer suggested high circE7 marked improved survival in 875 subjects (p = 0.074). While our study suggests that circE7 levels correlate with improved survival in ASCC, larger, prospective studies are necessary to confirm the potential role of circE7 as a biomarker.
Journal of the American Academy of Dermatology, Jan 28, 2016
Human polyomavirus (HPyV)6 and HPyV7 are shed chronically from human skin. HPyV7, but not HPyV6, ... more Human polyomavirus (HPyV)6 and HPyV7 are shed chronically from human skin. HPyV7, but not HPyV6, has been linked to a pruritic skin eruption of immunosuppression. We determined whether biopsy specimens showing a characteristic pattern of dyskeratosis and parakeratosis might be associated with polyomavirus infection. We screened biopsy specimens showing "peacock plumage" histology by polymerase chain reaction for HPyVs. Cases positive for HPyV6 or HPyV7 were then analyzed by immunohistochemistry, electron microscopy, immunofluorescence, quantitative polymerase chain reaction, and complete sequencing, including unbiased, next-generation sequencing. We identified 3 additional cases of HPyV6 or HPyV7 skin infections. Expression of T antigen and viral capsid was abundant in lesional skin. Dual immunofluorescence staining experiments confirmed that HPyV7 primarily infects keratinocytes. High viral loads in lesional skin compared with normal-appearing skin and the identification ...
We report an unexpected role for Tel2 in the expression of all mammalian phosphatidylinositol 3-k... more We report an unexpected role for Tel2 in the expression of all mammalian phosphatidylinositol 3-kinase-related protein kinases (PIKKs). Although Tel2 was identified as a budding yeast gene required for the telomere length maintenance, we found no obvious telomeric function for mammalian Tel2. Targeted gene deletion showed that mouse Tel2 is essential in embryonic development, embryonic stem (ES) cells, and embryonic fibroblasts. Conditional deletion of Tel2 from embryonic fibroblasts compromised their response to IR and UV, diminishing the activation of checkpoint kinases and their downstream effectors. The effects of Tel2 deletion correlated with significantly reduced protein levels for the PI3K-related kinases ataxia telangiectasia mutated (ATM), ATM and Rad3 related (ATR), DNA-dependent protein kinase catalytic subunit ataxia (DNA-PKcs). Tel2 deletion also elicited specific depletion of the mammalian target of rapamycin (mTOR), suppressor with morphological effect on genitalia 1 (SMG1), and transformation/transcription domain-associated protein (TRRAP), and curbed mTOR signaling, indicating that Tel2 affects all six mammalian PIKKs. While Tel2 deletion did not alter PIKK mRNA levels, in vivo pulse labeling experiments showed that Tel2 controls the stability of ATM and mTOR. Each of the PIKK family members associated with Tel2 in vivo and in vitro experiments indicated that Tel2 binds to part of the HEAT repeat segments of ATM and mTOR. These data identify Tel2 as a highly conserved regulator of PIKK stability.
Cell growth is regulated by two antagonistic processes: TOR signaling and autophagy. These proces... more Cell growth is regulated by two antagonistic processes: TOR signaling and autophagy. These processes integrate signals including growth factors, amino acids, and energy status to ensure that cell growth is appropriate to environmental conditions. Autophagy responds indirectly to the cellular milieu as a downstream inhibitory target of TOR signaling and is also directly controlled by nutrient availability, cellular energy status, and cell stress. The control of cell growth by TOR signaling and autophagy are relevant to disease, as altered regulation of either pathway results in tumorigenesis. Here we give an overview of how TOR signaling and autophagy integrate nutritional status to regulate cell growth, how these pathways are coordinately regulated, and how dysfunction of this regulation might result in tumorigenesis.
Trichodysplasia spinulosa (TS) is a folliculocentric and clinically papular dermatological disord... more Trichodysplasia spinulosa (TS) is a folliculocentric and clinically papular dermatological disorder occurring in the setting of immunosuppression typically in association with solid organ transplantation or hematolymphoid malignancies. We report the occurrence of TS in a 7-year-old girl with Down syndrome and pre-B-acute lymphoblastic leukemia (pre-BALL) who was completing chemotherapy at onset. The patient's affected follicles were dilated by an expansion of a dystrophic follicular inner root sheath cell population displaying enlarged trichohyaline cytoplasmic granules and progressing centrally to keratotic and parakeratotic debris, and superficially demonstrating some diminutive hair shaft-like material within the keratotic spicules. Electron microscopic studies of a follicular lesion demonstrated extracellular viral particles suggestive of a polyomavirus within the central follicular keratotic debris. DNA Polymerase chain reaction (PCR) and gene sequencing studies, performed on the tissue of the microscopic slide and paraffin block, for the recently identified trichodysplasia spinulosa-associated polyomavirus (TSPyV), were resulted as positive for TSPyV. PCR for the Merkel cell polyomavirus (MCPyV) was negative. To date this case is unique in representing the first case of TS confirmed by electron microscopy in which a related viral pathogen has been molecularly identified. An additional 19 reported cases classifiable as TS are tabulated and reviewed.
Multiple human polyomaviruses (HPyV) can infect the skin, but only Merkel cell polyomavirus (MCPy... more Multiple human polyomaviruses (HPyV) can infect the skin, but only Merkel cell polyomavirus (MCPyV) has been implicated in the development of a cancer, Merkel cell carcinoma (MCC). While expression of HPyV6, HPyV7, and MCPyV small T antigens (sT), all induced a senescence-associated secretory phenotype (SASP), MCPyV sT uniquely activated noncanonical NF-κB (ncNF-κB), instead of canonical NF-κB signaling, to evade p53-mediated cellular senescence. Through its large T stabilization domain, MCPyV sT activated ncNF-κB signaling both by inducing H3K4 trimethylation-mediated increases of NFKB2 and RELB transcription and also by promoting NFKB2 stabilization and activation through FBXW7 inhibition. Noncanonical NF-κB signaling was required for SASP cytokine secretion, which promoted the proliferation of MCPyV sT–expressing cells through autocrine signaling. Virus-positive MCC cell lines and tumors showed ncNF-κB pathway activation and SASP gene expression, and the inhibition of ncNF-κB sig...
Bischoff SC (2007) Role of mast cells in allergic and non-allergic immune responses: comparison o... more Bischoff SC (2007) Role of mast cells in allergic and non-allergic immune responses: comparison of human and murine data. Nat Rev Immunol 7:93-104 Bodtger U, Poulsen LK, Malling HJ (2003) Asymptomatic skin sensitization to birch predicts later development of birch pollen allergy in adults: a 3-year follow-up study. J Allergy Clin Immunol 111:149-54 Chodaczek G, Bacsi A, Dharajiya N et al. (2009) Ragweed pollen-mediated IgE-independent release of biogenic amines from mast cells via induction of mitochondrial dysfunction. Mol Immunol 46:2505-14 Gilles S, Jacoby D, Blume C et al. (2010) Pollenderived low-molecular weight factors inhibit 6-sulfo LacNAc(+) dendritic cells' capacity to induce T-helper type 1 responses. Clin Exp Allergy 40:269-78 Gilles S, Mariani V, Bryce M et al. (2009) Pollenderived E1-phytoprostanes signal via PPARgamma and NF-kappaB-dependent mechanisms.
Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterized by cutaneo... more Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterized by cutaneous symptoms, including hyperpigmentation and nail dystrophy. Some forms of DC are caused by mutations in telomerase, the enzyme that counteracts telomere shortening, suggesting a telomere-based disease mechanism. However, mice with extensively shortened telomeres due to telomerase deficiency do not develop the characteristics of DC, raising questions about the etiology of DC and/or mouse models for human telomere dysfunction. Here we describe mice engineered to undergo telomere degradation due to the absence of the shelterin component POT1b. When combined with reduced telomerase activity, POT1b deficiency elicits several characteristics of DC, including hyperpigmentation and fatal bone marrow failure at 4–5 mo of age. These results provide experimental support for the notion that DC is caused by telomere dysfunction, and demonstrate that key aspects of a human telomere-based disease can ...
Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immu... more Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations that disrupt nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts the expression of POLA1, which encodes the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency resulted in increased production of type I interferons. This enzyme is necessary for the synthesis of RNA:DNA primers during DNA replication and, strikingly, we found that POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Together this work identifies POLA1 as a critical regulator of the type I interferon response.
Merkel cell carcinoma (MCC) is an infrequent, rapidly growing skin neoplasm that carries a greate... more Merkel cell carcinoma (MCC) is an infrequent, rapidly growing skin neoplasm that carries a greater probability of regional lymph node involvement, and a grim prognosis in advanced cases. While it is seen predominantly in old age in sun-exposed body parts, the prevalence varies among different races and geographical regions. Merkel cell polyomavirus and UV radiation-induced mutations contribute to its etiopathogenesis. The clinical presentation of MCC lacks pathognomonic features and is rarely considered highly at the time of presentation. Histopathological examination frequently reveals hyperchromatic nuclei with high mitotic activity, but immunohistochemistry is required to confirm the diagnosis. Sentinel lymph node biopsy (SLNB) and imaging are advised for effective staging of the disease. Multimodal management including surgery, radiation therapy, and/or immunotherapy are deployed. Traditional cytotoxic chemotherapies may result in an initial response, but do not result in a sign...
Circular RNAs (circRNAs) are a conserved class of RNAs with diverse functions, including serving ... more Circular RNAs (circRNAs) are a conserved class of RNAs with diverse functions, including serving as messenger RNAs that are translated into peptides. Here we describe circular RNAs generated by human polyomaviruses (HPyVs), some of which encode variants of the previously described alternative large T antigen open reading frame (ALTO) protein. Circular ALTO RNAs (circALTOs) can be detected in virus positive Merkel cell carcinoma (VP-MCC) cell lines and tumor samples. CircALTOs are stable, predominantly located in the cytoplasm, and N6-methyladenosine (m6A) modified. The translation of MCPyV circALTOs into ALTO protein is negatively regulated by MCPyV-generated miRNAs in cultured cells. MCPyV ALTO expression increases transcription from some recombinant promoters in vitro and upregulates the expression of multiple genes previously implicated in MCPyV pathogenesis. MCPyV circALTOs are enriched in exosomes derived from VP-MCC lines and circALTO-transfected 293T cells, and purified exoso...
Bioinformatics and in vitro studies have revealed that single-stranded circular RNAs (circRNAs), ... more Bioinformatics and in vitro studies have revealed that single-stranded circular RNAs (circRNAs), generated through ‘backsplicing,’ occur more extensively than initially appreciated. While the functions of most circRNAs are unknown, binding of microRNAs (miRNA), regulation of splicing and transcription, and translation into proteins have all been demonstrated for specific circRNAs. Virally-derived circRNAs have recently been described in gamma-herpesviruses. Here, we report that oncogenic human papillomaviruses (HPV) generate circRNAs, including ones which encompass the entire coding region of the E7 oncogene (circE7). HPV16 circE7 can be detected by both inverse RT-PCR and Northern blots of HPV16-transformed cell lines. CircE7 is N6-methyladenosine (m6A) modified, preferentially localized to the cytoplasm, and can be translated to produce E7 oncoprotein. Specific disruption of circE7 in CaSki cervical carcinoma cells decreased E7 protein levels, inhibited cell proliferation, and inh...
Cancer cells can inhibit effector T cells (Teff) through both immunomodulatory receptors and the ... more Cancer cells can inhibit effector T cells (Teff) through both immunomodulatory receptors and the impact of cancer metabolism on the tumor microenvironment. Indeed, Teff require high rates of glucose metabolism, and consumption of essential nutrients or generation of waste products by tumor cells may impede essential T cell metabolic pathways. Clear cell renal cell carcinoma (ccRCC) is characterized by loss of the tumor suppressor von Hippel-Lindau (VHL) and altered cancer cell metabolism. Here, we assessed how ccRCC influences the metabolism and activation of primary patient ccRCC tumor infiltrating lymphocytes (TIL). CD8 TIL were abundant in ccRCC, but they were phenotypically distinct and both functionally and metabolically impaired. ccRCC CD8 TIL were unable to efficiently uptake glucose or perform glycolysis and had small, fragmented mitochondria that were hyperpolarized and generated large amounts of ROS. Elevated ROS was associated with downregulated mitochondrial SOD2. CD8 T ...
The discovery that oxidized vitamin C, dehydroascorbate (DHA), can induce oxidative stress and ce... more The discovery that oxidized vitamin C, dehydroascorbate (DHA), can induce oxidative stress and cell death in cancer cells has rekindled interest in the use of high dose vitamin C (VC) as a cancer therapy. However, high dose VC has shown limited efficacy in clinical trials, possibly due to the decreased bioavailability of oral VC. Because human erythrocytes express high levels of Glut1, take up DHA, and reduce it to VC, we tested how erythrocytes might impact high dose VC therapies. Cancer cells are protected from VC-mediated cell death when co-cultured with physiologically relevant numbers of erythrocytes. Pharmacological doses of VC induce oxidative stress, GSH depletion, and increased glucose flux through the oxidative pentose phosphate pathway (PPP) in erythrocytes. Incubation of erythrocytes with VC induced hemolysis, which was exacerbated in erythrocytes from glucose-6-phosphate dehydrogenase (G6PD) patients and rescued by antioxidants. Thus, erythrocytes protect cancer cells f...
Trichodysplasia spinulosa (TS) is a proliferative skin disease observed in severely immunocomprom... more Trichodysplasia spinulosa (TS) is a proliferative skin disease observed in severely immunocompromized patients. It is characterized by papule and trichohyalin-rich spicule formation, epidermal acanthosis and distention of dysmorphic hair follicles overpopulated by inner root sheath cells (IRS). TS probably results from active infection with the TS-associated polyomavirus (TSPyV), as indicated by high viral-load, virus protein expression and particle formation. The underlying pathogenic mechanism imposed by TSPyV infection has not been solved yet. By analogy with other polyomaviruses, such as the Merkel cell polyomavirus associated with Merkel cell carcinoma, we hypothesized that TSPyV T-antigen promotes proliferation of infected IRS cells. Therefore, we analyzed TS biopsy sections for markers of cell proliferation (Ki-67) and cell cycle regulation (p16 ink4a , p21 waf , pRB, phosphorylated pRB), and the putatively transforming TSPyV early large tumor (LT) antigen. Intense Ki-67 staining was detected especially in the margins of TS hair follicles, which colocalized with TSPyV LTantigen detection. In this area, staining was also noted for pRB and particularly phosphorylated pRB, as well as p16 ink4a and p21 waf. Healthy control hair follicles did not or hardly stained for these markers. Trichohyalin was particularly detected in the center of TS follicles that stained negative for Ki-67 and TSPyV LT-antigen. In summary, we provide evidence for clustering of TSPyV LT-antigen-expressing and proliferating cells in the follicle margins that overproduce negative cell cycle regulatory proteins. These data are compatible with a scenario of TSPyV T-antigen-mediated cell cycle progression, potentially creating a pool of proliferating cells that enable viral DNA replication and drive papule and spicule formation.
Although Foxp3+ T regulatory cells (Tregs) are well documented for their ability to suppress vari... more Although Foxp3+ T regulatory cells (Tregs) are well documented for their ability to suppress various immune cells, T-cell subsets capable of counteracting Tregs have not been demonstrated. Here, we assessed phosphoantigen-activated Vγ2Vδ2 T cells for the ability to interplay with Tregs in the context of mycobacterial infection. A short-term IL-2 treatment regimen induced marked expansion of CD4+CD25+Foxp3+ T cells and subsequent suppression of mycobacterium-driven increases in numbers of Vγ2Vδ2 T cells. Surprisingly, activation of Vγ2Vδ2 T cells by adding phosphoantigen Picostim to the IL-2 treatment regimen down-regulated IL-2–induced expansion of CD4+CD25+Foxp3+ T cells. Consistently, in vitro activation of Vγ2Vδ2 T cells by phosphoantigen plus IL-2 down-regulated IL-2–induced expansion of CD4+CD25+Foxp3+ T cells. Interestingly, anti–IFN-γ–neutralizing antibody, not anti–TGF-β or anti–IL-4, reduced the ability of activated Vγ2Vδ2 T cells to down-regulate Tregs, suggesting that aut...
ABSTRACTCircular RNAs (circRNAs) are a conserved class of RNAs with diverse functions. A subset o... more ABSTRACTCircular RNAs (circRNAs) are a conserved class of RNAs with diverse functions. A subset of circRNAs are translated into peptides. Here we describe circular RNAs encoded by human polyomaviruses (HPyVs), including circular forms of RNAs encoding variants of the previously described alternative large T antigen open reading frame (ALTO) gene. Circular ALTO RNAs (circALTOs) can be detected in virus positive Merkel cell carcinoma (VP-MCC) cell lines and tumor samples. CircALTOs are stable, predominantly located in the cytoplasm, and N6-methyladenosine (m6A) modified. MCPyV circALTOs produce ALTO protein in cultured cells. MCPyV ALTO promotes the transcription of co-transfected reporter genes. MCPyV circALTOs are enriched in exosomes derived from VP-MCC lines and circALTO-transfected 293T cells, and purified exosomes can mediate ALTO expression and transcriptional activation. The related trichodysplasia spinulosa polyomavirus (TSPyV) also expresses a circALTO that can be detected i...
Single-stranded circular RNAs (circRNAs), generated through 'backsplicing', occur more extensivel... more Single-stranded circular RNAs (circRNAs), generated through 'backsplicing', occur more extensively than initially anticipated. The possible functions of the vast majority of circRNAs remain unknown. Virus-derived circRNAs have recently been described in gammaherpesviruses. We report that oncogenic human papillomaviruses (HPVs) generate cir-cRNAs, some of which encompass the E7 oncogene (circE7). HPV16 circE7 is detectable by both inverse RT-PCR and northern blotting of HPV16-transformed cells. CircE7 is N 6 -methyladenosine (m 6 A) modified, preferentially localized to the cytoplasm, associated with polysomes, and translated to produce E7 oncoprotein. Specific disruption of circE7 in CaSki cervical carcinoma cells reduces E7 protein levels and inhibits cancer cell growth both in vitro and in tumor xenografts. CircE7 is present in TCGA RNA-Seq data from HPV-positive cancers and in cell lines with only episomal HPVs. These results provide evidence that virus-derived, protein-encoding circular RNAs are biologically functional and linked to the transforming properties of some HPV.
Anal squamous cell carcinoma (ASCC) is a rare, potentially fatal malignancy primarily caused by h... more Anal squamous cell carcinoma (ASCC) is a rare, potentially fatal malignancy primarily caused by high-risk human papillomaviruses (HPV). The prognostic implication of programmed death-ligand 1 (PD-L1) expression remains controversial, and glucose transporter 1 (GLUT1) expression has never been examined in ASCC. Covalently closed circular RNAs have recently been shown to be widespread in cancers and are proposed to be biomarkers. We discovered HPV16 expresses a circular E7 RNA (circE7) which has not been assessed as a potential biomarker. A retrospective, translational case series at UT Southwestern was conducted to analyze PD-L1, GLUT1, HPV-ISH, and HPV circE7 in relation to the clinical features and overall survival of patients with ASCC. Twenty-two (22) subjects were included in the study. Improved overall survival was predicted by basaloid histology (p = 0.013), PD-L1 expression (p = 0.08), and HPV-ISH positivity (p < 0.001), but not GLUT1 expression. High levels of circE7 by quantitative RT-PCR predicted improved overall survival in ASCC (p = 0.023) and analysis of The Cancer Genome Atlas sequencing from HPV-positive head and neck cancer and cervical cancer suggested high circE7 marked improved survival in 875 subjects (p = 0.074). While our study suggests that circE7 levels correlate with improved survival in ASCC, larger, prospective studies are necessary to confirm the potential role of circE7 as a biomarker.
Journal of the American Academy of Dermatology, Jan 28, 2016
Human polyomavirus (HPyV)6 and HPyV7 are shed chronically from human skin. HPyV7, but not HPyV6, ... more Human polyomavirus (HPyV)6 and HPyV7 are shed chronically from human skin. HPyV7, but not HPyV6, has been linked to a pruritic skin eruption of immunosuppression. We determined whether biopsy specimens showing a characteristic pattern of dyskeratosis and parakeratosis might be associated with polyomavirus infection. We screened biopsy specimens showing "peacock plumage" histology by polymerase chain reaction for HPyVs. Cases positive for HPyV6 or HPyV7 were then analyzed by immunohistochemistry, electron microscopy, immunofluorescence, quantitative polymerase chain reaction, and complete sequencing, including unbiased, next-generation sequencing. We identified 3 additional cases of HPyV6 or HPyV7 skin infections. Expression of T antigen and viral capsid was abundant in lesional skin. Dual immunofluorescence staining experiments confirmed that HPyV7 primarily infects keratinocytes. High viral loads in lesional skin compared with normal-appearing skin and the identification ...
We report an unexpected role for Tel2 in the expression of all mammalian phosphatidylinositol 3-k... more We report an unexpected role for Tel2 in the expression of all mammalian phosphatidylinositol 3-kinase-related protein kinases (PIKKs). Although Tel2 was identified as a budding yeast gene required for the telomere length maintenance, we found no obvious telomeric function for mammalian Tel2. Targeted gene deletion showed that mouse Tel2 is essential in embryonic development, embryonic stem (ES) cells, and embryonic fibroblasts. Conditional deletion of Tel2 from embryonic fibroblasts compromised their response to IR and UV, diminishing the activation of checkpoint kinases and their downstream effectors. The effects of Tel2 deletion correlated with significantly reduced protein levels for the PI3K-related kinases ataxia telangiectasia mutated (ATM), ATM and Rad3 related (ATR), DNA-dependent protein kinase catalytic subunit ataxia (DNA-PKcs). Tel2 deletion also elicited specific depletion of the mammalian target of rapamycin (mTOR), suppressor with morphological effect on genitalia 1 (SMG1), and transformation/transcription domain-associated protein (TRRAP), and curbed mTOR signaling, indicating that Tel2 affects all six mammalian PIKKs. While Tel2 deletion did not alter PIKK mRNA levels, in vivo pulse labeling experiments showed that Tel2 controls the stability of ATM and mTOR. Each of the PIKK family members associated with Tel2 in vivo and in vitro experiments indicated that Tel2 binds to part of the HEAT repeat segments of ATM and mTOR. These data identify Tel2 as a highly conserved regulator of PIKK stability.
Cell growth is regulated by two antagonistic processes: TOR signaling and autophagy. These proces... more Cell growth is regulated by two antagonistic processes: TOR signaling and autophagy. These processes integrate signals including growth factors, amino acids, and energy status to ensure that cell growth is appropriate to environmental conditions. Autophagy responds indirectly to the cellular milieu as a downstream inhibitory target of TOR signaling and is also directly controlled by nutrient availability, cellular energy status, and cell stress. The control of cell growth by TOR signaling and autophagy are relevant to disease, as altered regulation of either pathway results in tumorigenesis. Here we give an overview of how TOR signaling and autophagy integrate nutritional status to regulate cell growth, how these pathways are coordinately regulated, and how dysfunction of this regulation might result in tumorigenesis.
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Papers by Richard Wang