Inhibition of the cGAS-STING pathway ameliorates the premature senescence hallmarks of Ataxia-Telangiectasia brain organoids
Aguado, J;Chaggar, HK;Gómez-Inclán, C;Shaker, MR;Leeson, HC;Mackay-Sim, A;Wolvetang, EJ;
PMID: 34459078 | DOI: 10.1111/acel.13468
Ataxia-telangiectasia (A-T) is a genetic disorder caused by the lack of functional ATM kinase. A-T is characterized by chronic inflammation, neurodegeneration and premature ageing features that are associated with increased genome instability, nuclear shape alterations, micronuclei accumulation, neuronal defects and premature entry into cellular senescence. The causal relationship between the detrimental inflammatory signature and the neurological deficiencies of A-T remains elusive. Here, we utilize human pluripotent stem cell-derived cortical brain organoids to study A-T neuropathology. Mechanistically, we show that the cGAS-STING pathway is required for the recognition of micronuclei and induction of a senescence-associated secretory phenotype (SASP) in A-T olfactory neurosphere-derived cells and brain organoids. We further demonstrate that cGAS and STING inhibition effectively suppresses self-DNA-triggered SASP expression in A-T brain organoids, inhibits astrocyte senescence and neurodegeneration, and ameliorates A-T brain organoid neuropathology. Our study thus reveals that increased cGAS and STING activity is an important contributor to chronic inflammation and premature senescence in the central nervous system of A-T and constitutes a novel therapeutic target for treating neuropathology in A-T patients.
Scott-Wittenborn, N;D'Souza, G;Tewari, S;Rooper, L;Troy, T;Drake, V;Bigelow, EO;Windon, MJ;Ryan, WR;Ha, PK;Kiess, AP;Miles, B;Westra, WH;Mydlarz, WK;Eisele, DW;Fakhry, C;
PMID: 35132635 | DOI: 10.1002/cncr.34124
Human papillomavirus (HPV) is responsible for a growing proportion of oropharyngeal squamous cell carcinomas (OPSCCs) among men and White individuals. Whether similar trends apply to women, non-Whites, and non-oropharyngeal squamous cell carcinomas (non-OPSCCs) is unknown.This is a cross-sectional analysis combining 2 multi-institutional case series of incident head and neck squamous cell carcinoma (HNSCC) cases. Incident HNSCCs from 1995 to 2012 were enrolled retrospectively using banked tumor samples and medical record abstraction. Incident HNSCCs from 2013 to 2019 were enrolled prospectively. The prevalence of tumor HPV biomarkers was tested over 3 time periods (1995-2003, 2004-2012, and 2013-2019). Centralized testing was done for p16 immunohistochemistry (p16) and oncogenic HPV in situ hybridization (ISH).A total of 1209 incident cases of HNSCC were included. Prevalence of p16- and ISH-positive tumors increased significantly for oropharynx cancers over time. The majority were positive after 2013 for White patients (p16, 92%; P < .001; ISH 94%; P < .001), Black patients (p16, 72%; P = .021; ISH 67%; P = .011), and Hispanic patients (p16, 100%; P = .04; ISH 100%; P = .013). For women with OPSCC, the prevalence of p16- and ISH-positive tumors increased significantly to 82% (P < .001) and 78% (P = .004), respectively. For non-OPSCCs, there was increased p16 and ISH positivity overall with 24% p16 and 16% ISH positivity in the most recent time period (P < .001 for both).The majority of OPSCCs in US tertiary care centers are now p16 and ISH positive for all sex and race groups. In some populations in the United States, 91% of OPSCCs are now caused by HPV. Few non-OPSCCs are p16 and ISH positive.
Rajendra S, Yang T, Xuan W, Sharma P, Pavey D, Soon Lee C, Le S, Collins J, Wang B.
PMID: 28722212 | DOI: 10.1002/ijc.30896
We have previously demonstrated that transcriptionally active high-risk HPV (hr-HPV) is strongly incriminated in Barrett's dysplasia (BD) and oesophageal adenocarcinoma (OAC) using mainly fresh frozen tissue. This study aimed to identify biomarkers of active HPV infection in Barrett's metaplasia, (BM)/BD/OAC by immunohistochemical staining (IHC) of formalin-fixed paraffin embedded (FFPE) tissue for aberrations of p53 and the retinoblastoma (pRb) pathway which are targets for the viral oncoproteins, E6/E7 respectively. Prospectively, BM(n=81)/BD(n=72)/OAC(n=65) FFPE specimens were subjected to IHC staining for pRb, p16INK4A , cyclin D1 , p53 and RNA in-situ hybridization (ISH) for E6/E7 transcripts. HPV DNA was determined via PCR in fresh frozen specimens. Viral load measurement (real-time PCR) and Next Generation Sequencing of TP53 was also performed. Of 218 patients, 56 were HPV DNA positive [HPV16 (n=42), 18 (n=13), 6 (n=1)]. Viral load was low. Transcriptionally active HPV (DNA+ /RNA+ ) was only found in the dysplastic and adenocarcinoma group (n=21). The majority of HPV DNA+ /RNA+ BD/OAC were characterized by p16INK4Ahigh (14/21, 66.7%), pRblow (15/21, 71.4%) and p53low (20/21, 95%) and was significantly different to controls [combination of HPV DNA- /RNA- (n=94) and HPV DNA+ /RNA- cohorts (n=22)]. p53low had the strongest association with DNA+ /RNA+ oesophageal lesions (OR=23.5, 95% CI=2.94-187.8, p=0.0029). Seventeen HPV DNA+ /RNA+BD/OAC identified as p53low, were sequenced and all but one exhibited wild-type status. pRblow /p53low provided the best balance of strength of association (OR=8.0, 95% CI=2.6-25.0, p=0.0003) and sensitivity (71.4%)/specificity (71.6%) for DNA+ /RNA+ BD/OAC. Active HPV involvement in BD/OAC is characterized by wild-type p53 and aberrations of the retinoblastoma protein pathway.
Clin Cancer Res. 2018 Sep 21.
Aggarwal C, Cohen RB, Morrow MP, Kraynak KA, Sylvester AJ, Knoblock DM, Bauml J, Weinstein GS, Lin A, Boyer J, Sakata L, Tan S, Anton A, Dickerson K, Mangrolia D, Vang R, Dallas M, Oyola S, Duff S, Esser MT, Kumar R, Weiner DB, Csiki I, Bagarazzi M.
PMID: 30242022 | DOI: 10.1158/1078-0432.CCR-18-1763
Abstract
PURPOSE:
Clinical responses with programmed death (PD-1) receptor directed antibodies occur in about 20% of patients with advanced head and neck squamous cell cancer (HNSCCa). Viral neoantigens, such as the E6/E7 proteins of HPV16/18 are attractive targets for therapeutic immunization, and offer an immune activation strategy that may be complementary to PD-1 inhibition.
EXPERIMENTAL DESIGN:
We report Phase Ib/II safety, tolerability and immunogenicity results of immunotherapy with MEDI0457 (DNA immunotherapy targeting HPV16/18 E6/E7 with IL-12 encoding plasmids) delivered by electroporation with CELLECTRA® constant current device. Twenty-two patients with locally advanced, p16+ HNSCCa received MEDI0457.
RESULTS:
MEDI0457 was associated with mild injection site reactions but no treatment related grade 3-5 adverse events (AEs). Eighteen of 21 evaluable patients showed elevated antigen specific T cell activity by IFNg ELISpot and persistent cellular responses surpassing 100 SFU/106 PBMC were noted out to one year. Induction of HPV-specific CD8+ T cells was observed. MEDI0457 shifted the CD8+/FoxP3+ ratio in 4/5 post-immunotherapy tumor samples and increased the number of perforin+ immune infiltrates in all five patients. One patient developed metastatic disease and was treated with anti-PD-1 therapy with a rapid and durable complete response. Flow cytometric analyses revealed induction of HPV16 specific PD-1+ CD8+ T cells that were not found prior to MEDI0547 (0% vs. 1.8%).
CONCLUSIONS:
These data demonstrate that MEDI0457 can generate durable HPV16/18 antigen-specific peripheral and tumor immune responses. This approach may be used as a complementary strategy to PD-1/PD-L1 inhibition in HPV-associated HNSCCa to improve therapeutic outcomes.
Mertz, E;Makareeva, E;Mirigian, L;Leikin, S;
| DOI: 10.1002/jbm4.10701
Relevance of mineralized nodules in two-dimensional (2D) osteoblast/osteocyte cultures to bone biology, pathology, and engineering is a decades old question, but a comprehensive answer appears to be still wanting. Bone-like cells, extracellular matrix (ECM), and mineral were all reported but so were non-bone-like ones. Many studies described seemingly bone-like cell-ECM structures based on similarity to few select bone features _in vivo_, yet no studies examined multiple bone features simultaneously and none systematically studied all types of structures coexisting in the same culture. Here, we report such comprehensive analysis of 2D cultures based on light and electron microscopies, Raman microspectroscopy, gene expression, and _in situ_ mRNA hybridization. We demonstrate that 2D cultures of primary cells from mouse calvaria do form _bona fide_ bone. Cells, ECM, and mineral within it exhibit morphology, structure, ultrastructure, composition, spatial-temporal gene expression pattern, and growth consistent with intramembranous ossification. However, this bone is just one of at least five different types of cell-ECM structures coexisting in the same 2D culture, which vary widely in their resemblance to bone and ability to mineralize. We show that the other two mineralizing structures may represent abnormal (disrupted) bone and cartilage-like formation with chondrocyte-to-osteoblast trans differentiation. The two non-mineralizing cell-ECM structures may mimic periosteal cambium and pathological, non-mineralizing osteoid. Importantly, the most commonly used culture conditions (10 mM β-glycerophosphate) induce artificial mineralization of all cell-ECM structures, which then become barely distinguishable. We therefore discuss conditions and approaches promoting formation of _bona fide_ bone and simple means for distinguishing it from the other cell-ECM structures. Our findings may improve osteoblast differentiation and function analyses based on 2D cultures and extend applications of these cultures to general bone biology and tissue engineering research.
Yerly, L;Pich-Bavastro, C;Di Domizio, J;Wyss, T;Tissot-Renaud, S;Cangkrama, M;Gilliet, M;Werner, S;Kuonen, F;
PMID: 35986012 | DOI: 10.1038/s41467-022-32670-w
Tumors invade the surrounding tissues to progress, but the heterogeneity of cell types at the tumor-stroma interface and the complexity of their potential interactions hampered mechanistic insight required for efficient therapeutic targeting. Here, combining single-cell and spatial transcriptomics on human basal cell carcinomas, we define the cellular contributors of tumor progression. In the invasive niche, tumor cells exhibit a collective migration phenotype, characterized by the expression of cell-cell junction complexes. In physical proximity, we identify cancer-associated fibroblasts with extracellular matrix-remodeling features. Tumor cells strongly express the cytokine Activin A, and increased Activin A-induced gene signature is found in adjacent cancer-associated fibroblast subpopulations. Altogether, our data identify the cell populations and their transcriptional reprogramming contributing to the spatial organization of the basal cell carcinoma invasive niche. They also demonstrate the power of integrated spatial and single-cell multi-omics to decipher cancer-specific invasive properties and develop targeted therapies.
Liang, T;Hu, Y;Zhang, H;Xu, Q;Smith, CE;Zhang, C;Kim, JW;Wang, SK;Saunders, TL;Lu, Y;Hu, JC;Simmer, JP;
PMID: 34667213 | DOI: 10.1038/s41598-021-00219-4
Non-syndromic inherited defects of tooth dentin are caused by two classes of dominant negative/gain-of-function mutations in dentin sialophosphoprotein (DSPP): 5' mutations affecting an N-terminal targeting sequence and 3' mutations that shift translation into the - 1 reading frame. DSPP defects cause an overlapping spectrum of phenotypes classified as dentin dysplasia type II and dentinogenesis imperfecta types II and III. Using CRISPR/Cas9, we generated a Dspp-1fs mouse model by introducing a FLAG-tag followed by a single nucleotide deletion that translated 493 extraneous amino acids before termination. Developing incisors and/or molars from this mouse and a DsppP19L mouse were characterized by morphological assessment, bSEM, nanohardness testing, histological analysis, in situ hybridization and immunohistochemistry. DsppP19L dentin contained dentinal tubules but grew slowly and was softer and less mineralized than the wild-type. DsppP19L incisor enamel was softer than normal, while molar enamel showed reduced rod/interrod definition. Dspp-1fs dentin formation was analogous to reparative dentin: it lacked dentinal tubules, contained cellular debris, and was significantly softer and thinner than Dspp+/+ and DsppP19L dentin. The Dspp-1fs incisor enamel appeared normal and was comparable to the wild-type in hardness. We conclude that 5' and 3' Dspp mutations cause dental malformations through different pathological mechanisms and can be regarded as distinct disorders.
Zwaans BMM, Wegner KA, Bartolone SN, Vezina CM, Chancellor MB, Lamb LE
PMID: 32109348 | DOI: 10.14814/phy2.14377
A subset of patients receiving radiation therapy for pelvic cancer develop radiation cystitis, a complication characterized by mucosal cell death, inflammation, hematuria, and bladder fibrosis. Radiation cystitis can reduce bladder capacity, cause incontinence, and impair voiding function so severely that patients require surgical intervention. Factors influencing onset and severity of radiation cystitis are not fully known. We tested the hypothesis that genetic background is a contributing factor. We irradiated bladders of female C57BL/6, C3H, and BALB/c mice and evaluated urinary voiding function, bladder shape, histology, collagen composition, and distribution of collagen-producing cells. We found that the genetic background profoundly affects the severity of radiation-induced bladder fibrosis and urinary voiding dysfunction. C57BL/6 mice are most susceptible and C3H mice are most resistant. Irradiated C57BL/6 mouse bladders are misshapen and express more abundant collagen I and III proteins than irradiated C3H and BALB/c bladders. We localized Col1a1 and Col3a1 mRNAs to FSP1-negative stromal cells in the bladder lamina propria and detrusor. The number of collagen I and collagen III-producing cells can predict the average voided volume of a mouse. Collectively, we show that genetic factors confer sensitivity to radiation cystitis, establish C57BL/6 mice as a sensitive preclinical model, and identify a potential role for FSP1-negative stromal cells in radiation-induced bladder fibrosis
O'Toole, A;Mohamed, F;Zhang, J;Brown, C;
| DOI: 10.2139/ssrn.4199232
To detail early tissue distribution and innate immune response to rabbit hemorrhagic disease virus 2 (RHDV2), 13 rabbits were orally ( Oryctolagus cuniculus ) inoculated with liver homogenate made from a feral rabbit that succumbed to RHDV2 during the 2020 outbreak in Oregon, USA. Rabbits were monitored regularly, with euthanasia and collection of tissues and swabs, at 12, 24, 36, 48, 96, and 144 hours post inoculation. Livers from these rabbits were positive by RT-rtPCR for presence of the virus. Using RNAscope for viral and replicative intermediates, rabbits had detectable viral genomic RNA at each time point, initially within the gastrointestinal tract, then in the liver by 36 hours post inoculation. Also using RNAscope, there were increasing amounts of mRNA coding for TNF-α, IL-6, and IL-1β within the liver and spleen through 48 hours post inoculation. The results of this study aided our understanding of the local innate immune response to RHDV2, as well as aspects of pathogenesis.
Gupta K, Levinsohn J, Linderman G, Chen D, Sun TY, Dong D, Taketo MM, Bosenberg M, Kluger Y, Choate K, Myung P.
PMID: 30595533 | DOI: 10.1016/j.devcel.2018.11.032
Delineating molecular and cellular events that precede appendage morphogenesis has been challenging due to the inability to distinguish quantitative molecular differences between cells that lack histological distinction. The hair follicle (HF) dermal condensate (DC) is a cluster of cells critical for HF development and regeneration. Events that presage emergence of this distinctive population are poorly understood. Using unbiased single-cell RNA sequencing and in vivo methods, we infer a sequence of transcriptional states through which DC cells pass that begins prior to HF morphogenesis. Our data indicate that Wnt/β-catenin signaling is required to progress into an intermediate stage that precedes quiescence and differentiation. Further, we provide evidence that quiescent DC cells are recent progeny of selectively proliferating cells present prior to morphogenesis and that are later identified in the peri-DC zone during DC expansion. Together, these findings provide an inferred path of molecular states that lead to DC cell differentiation.
Nature biomedical engineering
You, Y;Tian, Y;Yang, Z;Shi, J;Kwak, KJ;Tong, Y;Estania, AP;Cao, J;Hsu, WH;Liu, Y;Chiang, CL;Schrank, BR;Huntoon, K;Lee, D;Li, Z;Zhao, Y;Zhang, H;Gallup, TD;Ha, J;Dong, S;Li, X;Wang, Y;Lu, WJ;Bahrani, E;Lee, LJ;Teng, L;Jiang, W;Lan, F;Kim, BYS;Lee, AS;
PMID: 36635419 | DOI: 10.1038/s41551-022-00989-w
The success of messenger RNA therapeutics largely depends on the availability of delivery systems that enable the safe, effective and stable translation of genetic material into functional proteins. Here we show that extracellular vesicles (EVs) produced via cellular nanoporation from human dermal fibroblasts, and encapsulating mRNA encoding for extracellular-matrix α1 type-I collagen (COL1A1) induced the formation of collagen-protein grafts and reduced wrinkle formation in the collagen-depleted dermal tissue of mice with photoaged skin. We also show that the intradermal delivery of the mRNA-loaded EVs via a microneedle array led to the prolonged and more uniform synthesis and replacement of collagen in the dermis of the animals. The intradermal delivery of EV-based COL1A1 mRNA may make for an effective protein-replacement therapy for the treatment of photoaged skin.
Gajewski, T;Rouhani, S;Trujillo, J;Pyzer, A;Yu, J;Fessler, J;Cabanov, A;Higgs, E;Cron, K;Zha, Y;Lu, Y;Bloodworth, J;Abasiyanik, M;Okrah, S;Flood, B;Hatogai, K;Leung, M;Pezeshk, A;Kozloff, L;Reschke, R;Strohbehn, G;Chervin, CS;Kumar, M;Schrantz, S;Madariaga, ML;Beavis, K;Yeo, KT;Sweis, R;Segal, J;Tay, S;Izumchenko, E;Mueller, J;Chen, L;
PMID: 34845442 | DOI: 10.21203/rs.3.rs-1083825/v1
The mechanisms explaining progression to severe COVID-19 remain poorly understood. It has been proposed that immune system dysregulation/over-stimulation may be implicated, but it is not clear how such processes would lead to respiratory failure. We performed comprehensive multiparameter immune monitoring in a tightly controlled cohort of 128 COVID-19 patients, and used the ratio of oxygen saturation to fraction of inspired oxygen (SpO2 / FiO2) as a physiologic measure of disease severity. Machine learning algorithms integrating 139 parameters identified IL-6 and CCL2 as two factors predictive of severe disease, consistent with the therapeutic benefit observed with anti-IL6-R antibody treatment. However, transcripts encoding these cytokines were not detected among circulating immune cells. Rather, in situ analysis of lung specimens using RNAscope and immunofluorescent staining revealed that elevated IL-6 and CCL2 were dominantly produced by infected lung type II pneumocytes. Severe disease was not associated with higher viral load, deficient antibody responses, or dysfunctional T cell responses. These results refine our understanding of severe COVID-19 pathophysiology, indicating that aberrant cytokine production by infected lung epithelial cells is a major driver of immunopathology. We propose that these factors cause local immune regulation towards the benefit of the virus.
