The nuclear exosome targeting (NEXT) complex is responsible for specific nuclear RNA degradation in mammalian cells. However, its function in development remains unknown. Here, we find that the depletion of a central factor of the NEXT complex, Zcchc8, in mouse results in developmental defects, a shortened lifespan, and infertility. We find that Zcchc8deficient embryonic stem cells (ESCs) exhibit proliferation abnormalities and reduced developmental potencies. Importantly, the transcripts of retrotransposon element LINE1 are found to be targeted by Zcchc8 and degraded by a Zcchc8-mediated mechanism. We further find that sustained expression of higher levels of LINE1 RNA is detected in maternal Zcchc8-depleted oocytes and embryos. Zcchc8depleted oocytes show higher chromatin accessibility and developmental defects in both meiotic maturation and embryogenesis after fertilization. Collectively, our study defines Zcchc8-mediated RNA degradation as an important post-transcription regulation of LINE1 transcripts in early embryos and ESCs, which play vital roles in the pluripotency and early development.

Response to immunotherapies can be variable and unpredictable. Pathology-based phenotyping of tumors into ‘hot’ and ‘cold’ is static, relying solely on T-cell infiltration in single-time single-site biopsies, resulting in suboptimal treatment response prediction. Dynamic vascular events (tumor angiogenesis, leukocyte trafficking) within tumor immune microenvironment (TiME) also influence anti-tumor immunity and treatment response. Here, we report dynamic cellular-level TiME phenotyping in vivo that combines inflammation profiles with vascular features through non-invasive reflectance confocal microscopic imaging. In skin cancer patients, we demonstrate three main TiME phenotypes that correlate with gene and protein expression, and response to toll-like receptor agonist immune-therapy. Notably, phenotypes with high inflammation associate with immunostimulatory signatures and those with high vasculature with angiogenic and endothelial anergy signatures. Moreover, phenotypes with high inflammation and low vasculature demonstrate the best treatment response. This non-invasive in vivo phenotyping approach integrating dynamic vasculature with inflammation serves as a reliable predictor of response to topical immune-therapy in patients.

BackgroundViral-based immunotherapy can overcome resistance to immune checkpoint blockade (ICB) and fill the unmet needs of many patients with cancer. Oncolytic viruses (OVs) are defined as engineered or naturally occurring viruses that selectively replicate in and kill cancer cells. OVs also induce antitumor immunity. The purpose of this study was to compare the antitumor effects of live oncolytic vaccinia viruses versus the inactivated versions and elucidate their underlying immunological mechanisms.MethodsWe engineered a replication-competent, oncolytic vaccinia virus (OV-GM) by inserting a murine GM-CSF gene into the thymidine kinase locus of a mutant vaccinia E3L∆83N, which lacks the Z-DNA-binding domain of vaccinia virulence factor E3. We compared the antitumor effects of intratumoral (IT) delivery of live OV-GM versus heat-inactivated OV-GM (heat-iOV-GM) in a murine B16-F10 melanoma bilateral implantation model. We also generated vvDD, a well-studied oncolytic vaccinia virus, and compared the antitumor effects of live vvDD vs heat-inactivated vvDD (heat-ivvDD) in a murine A20 B-cell lymphoma bilateral tumor implantation model.ResultsHeat-iOV-GM infection of dendritic cells (DCs) and tumor cells in vitro induced type I interferon and proinflammatory cytokines and chemokines, whereas live OV-GM did not. IT live OV-GM was less effective in generating systemic antitumor immunity compared with heat-iOV-GM. Similar to heat-iOV-GM, the antitumor effects of live OV-GM also require Batf3-dependent CD103+ dendritic cells. When combined with systemic delivery of ICB, IT heat-iOV-GM was more effective in eradicating tumors, compared with live OV-GM. IT heat-ivvDD was also more effective in treating murine A20 B-cell lymphoma, compared with live vvDD.ConclusionsTumor lysis induced by the replication of oncolytic vaccinia virus has a limited effect on the generation of systemic antitumor immunity. The activation of Batf3-dependent CD103+ DCs is critical for antitumor effects induced by both live OV-GM and heat-iOV-GM, with the latter being more potent than live OV-GM in inducing innate and adaptive immunity in both locally injected and distant, non-injected tumors. We propose that evaluations of both innate and adaptive immunity, induced by IT oncolytic viral immunotherapy at injected and non-injected tumors, should be included as potential biomarkers for host responses to viral therapy.

Effective depletion of immune suppressive regulatory T cells (Tregs) in the tumor microenvironment without triggering systemic autoimmunity is an important strategy for cancer immunotherapy. Modified vaccinia virus Ankara (MVA) is a highly attenuated, non-replicative vaccinia virus with a long history of human use. Here, we report rational engineering of an immune-activating recombinant MVA (rMVA, MVA∆E5R-Flt3L-OX40L) with deletion of the vaccinia E5R gene (encoding an inhibitor of the DNA sensor cyclic GMP-AMP synthase, cGAS) and expression of two membrane-anchored transgenes, Flt3L and OX40L. Intratumoral (IT) delivery of rMVA (MVA∆E5R-Flt3L-OX40L) generates potent antitumor immunity, dependent on CD8+ T cells, the cGAS/STING-mediated cytosolic DNA-sensing pathway, and type I IFN signaling. Remarkably, IT rMVA (MVA∆E5R-Flt3L-OX40L) depletes OX40hi regulatory T cells via OX40L/OX40 interaction and IFNAR signaling. Single-cell RNA-seq analyses of tumors treated with rMVA showed the depletion of OX40hiCCR8hi Tregs and expansion of IFN-responsive Tregs. Taken together, our study provides a proof-of-concept for depleting and reprogramming intratumoral Tregs via an immune-activating rMVA.

Spammer detection is essentially a process of judging the authenticity of users, and thus can be regarded as a classification problem. In order to improve the classification performance, multi-classifier information fusion is usually used to realize the automatic detection of spammers by utilizing the information from multiple classifiers. However, the existing fusion strategies do not reasonably take the uncertainty from the results of different classifiers (views) into account, and the relative importance and reliability of each classifier are not strictly distinguished. Therefore, in order to detect spammers effectively, this paper develops a novel multi-classifier information fusion model based on the evidential reasoning (ER) rule. Firstly, according to the user's characterization strategy, the base classifiers are constructed through the profile-based, content-based and behavior-based. Then, the idea of multi-classifier fusion is combined with the ER rule, and the results of base classifiers are aggregated by considering their weights and reliabilities. Extensive experimental results on the real-world dataset verify the effectiveness of the proposed model.

Multiple treatment modalities for Kaposi sarcoma (KS) have been reported, including chemotherapy, radiation therapy, surgical excision, electrochemotherapy, and cryotherapy. Common topical treatments include timolol, imiquimod, and alitretinoin. We searched our institutional database for patients with ICD‐9 or 10 codes for KS seen by a dermatologist with experience in KS management from July 1, 2004 to January 1, 2022. We screened patient charts to include patients who received combination therapy of cryotherapy followed by topical imiquimod three times a week for 2 months (n = 9). Patients were followed in the clinic every 3 months. Time to resolution was assessed by photographic evidence of resolution as determined by a dermatologist and corroborated with clinical documentation in patient charts. Median age (IQR) at KS diagnosis was 58 (27.5) years. All patients were male (n = 9, 100%). Majority were white (n = 7, 78%) and non‐Hispanic (n = 8, 89%). Five (56%) had classic KS, one (11%) had HIV‐associated KS, and three (33%) were HIV‐negative men who have sex with men. Median time to resolution was 30.5 weeks, with a median of two treatments. In our study, 93% (n = 42/45) of lesions and 89% (n = 8/9) of patients experienced complete resolution during a median (range) duration of follow‐up of 58 (13–209) weeks. Side effects were limited to pain during cryotherapy, occasional blister formation after cryotherapy, and mild inflammation due to imiquimod. No infections were observed. Combination therapy of cryotherapy and topical imiquimod may be an efficacious and comparatively low‐risk treatment for limited, cutaneous KS.

Novel strategies to reprogram tumor-infiltrating myeloid cells for cancer immunotherapy are urgently needed, given that the primary and acquired resistance to immune checkpoint blockade (ICB) therapy has hindered the overall success of immunotherapy. Modified vaccinia virus Ankara (MVA) is a highly attenuated, non-replicative vaccinia virus and an approved vaccine against smallpox and monkeypox. Here we report rational engineering of recombinant MVA, MQ833, by removing three immune suppressive genes, E5R, E3L, and WR199, from the MVA genome and inserting three transgenes encoding Flt3L, OX40L, and IL-12. Intratumoral (IT) delivery of MQ833 generates potent antitumor responses dependent on CD8+ T cells, neutrophils, and M1-like macrophages, the nucleic acid-sensing pathways mediated by MDA5/STING, and interferon feedback loop. IT MQ833 promotes the recruitment and activation of neutrophils and inflammatory monocytes into the injected tumors, depletion of M2-like macrophages, and expansion of M1-like macrophages, generating potent antitumor immunity against tumors resistant to ICB.

Investigating the dynamic crosstalk between the tumor-immune microenvironment (TiME) and microvasculature in vivo in patients can lead to important insights into the underlying biology, help identify tumor phenotypes and reveal attractive druggable targets. Dynamic non-invasive label-free imaging of TiME and microvasculature in real-time directly in patients using reflectance confocal microscopy (RCM) was investigated on 60 skin cancer patients (basal cell carcinoma, BCC; squamous cell carcinoma, SCC), followed by automated and machine-learning based quantification of TiME and microvasculature features such as vascular density, leukocyte trafficking and immune cell density. Manual (two readers) and histopathological evaluation (dermatopathologist) of these features was also performed. Molecular correlation of imaging features and phenotypes was performed using anti-CD3/anti-CD20 IHC staining for tertiary lymphoid structures (TLS) and total lymphocyte density (n=33), flow cytometry for immune cells (n=3), and differential RNA expression (n=14). Correlation of RCM features and phenotypes at baseline (before treatment) with treatment response was also evaluated on 9 cancer lesions undergoing topical immunotherapy imiquimod. High agreement for feature presence on RCM and Histology, and manual and automated RCM features was observed. Unsupervised clustering on total TiME and microvasculature features on RCM using principal component analysis (PCA) indicates four distinct tumor phenotypes (PCA 1). The phenotype with high inflammation, high trafficking and higher density of vessels or the denoted ‘hot' phenotype correlated with higher activated CD8+ Granzyme B+ cells (67% of total CD8+cells). The clustering pattern on RCM was compared to TLS and lymphocyte density (PCA 2) and gene expression following CIBERSORT analysis (PCA 3). The clustering in RCM correlated better with gene expression (PCA 1 and 3, 100% agreement) than TLS and lymphocyte density (PCA 1 and 2, 86% agreement). The ‘hot' phenotype in RCM correlated with higher immune gene signatures and higher TLS/lymphocyte density. Increased plasma, CD8, activated CD4 memory and activated NK cells, M1 macrophages and monocytes, along with up-regulation of JAK-STAT, chemokine and cell adhesion signaling cascade were found in the ‘hot' RCM phenotype. Statistical modeling for correlating phenotypes with treatment outcomes was performed using principal component-linear discriminant analysis (PC-LDA). Two responders with tumor regression were predicted as ‘hot' phenotype while the non-responding patients (remaining 7) were classified as cold phenotype; suggesting that RCM 'hot' phenotype correlates with better treatment response. Thus, we demonstrate the potential utility of noninvasive RCM imaging in identifying ‘hot' and ‘cold' tumor phenotypes directly in patients. Citation Format: Aditi Sahu, Melissa Gill, Miguel Cordova, Anthony Santella, Kivanc Kose, Teguru Tembo, Anabel Alfonso, Pratik Chandrani, Christi Fox, Salvador Gonzalez, Nicholas Kurtansky, Melissa Pulitzer, William Phillips, Madison Li, Kimeil King, Stephen Dusza, Shuaitong Liu, Ning Yang, Haaris Jilani, Paras Mehta, Ashfaq Marghoob, Allan Halpern, Anthony Rossi, Liang Deng, Chih-Shan Jason Chen, Milind Rajadhyaksha. Dynamic imaging of tumor-immune microenvironment (TiME) and microvasculature identifies ‘hot' and ‘cold' tumor phenotypes in vivo in patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2814.