Alternative polyadenylation quantitative trait methylation mapping in human cancers provides clues into the molecular mechanisms of APA.

Genetic variants are involved in the orchestration of alternative polyadenylation (APA) events, while the role of DNA methylation in regulating APA remains unclear. We generated a comprehensive atlas of APA quantitative trait methylation sites (apaQTMs) across 21 different types of cancer (1,612 to 60,219 acting in cis and 4,448 to 142,349 in trans). Potential causal apaQTMs in non-cancer samples were also identified. Mechanistically, we observed a strong enrichment of cis-apaQTMs near polyadenylation sites (PASs) and both cis- and trans-apaQTMs in proximity to transcription factor (TF) binding regions. Through the integration of ChIP-signals and RNA-seq data from cell lines, we have identified several regulators of APA events, acting either directly or indirectly, implicating novel functions of some important genes, such as TCF7L2, which is known for its involvement in type 2 diabetes and cancers. Furthermore, we have identified a vast number of QTMs that share the same putative causal CpG sites with five different cancer types, underscoring the roles of QTMs, including apaQTMs, in the process of tumorigenesis. DNA methylation is extensively involved in the regulation of APA events in human cancers. In an attempt to elucidate the potential underlying molecular mechanisms of APA by DNA methylation, our study paves the way for subsequent experimental validations into the intricate biological functions of DNA methylation in APA regulation and the pathogenesis of human cancers. To present a comprehensive catalog of apaQTM patterns, we introduce the Pancan-apaQTM database, available at https://pancan-apaqtm-zju.shinyapps.io/pancanaQTM/.

A dual-functional oncolytic adenovirus ZD55-aPD-L1 scFv armed with PD-L1 inhibitor potentiates its antitumor activity.

BACKGROUND: Clinical data indicate that a substantial portion of cancer patients, though eligible for immune checkpoint inhibitor (ICI) therapy, cannot fully benefit from ICI monotherapy due to the poor immunogenicity of tumors and lack of tumor-infiltrating lymphocytes within the 'cold' tumor microenvironment (TME). In addition to poor antibody penetrance into the TME, systemic delivery of ICIs is associated with immune-related adverse events (irAEs) among recipients, some of which are life-threatening. Oncolytic virotherapy is a potentially viable approach to improving the efficacy of ICI therapy because of their ability to selectively replicate and lyse tumor cells, release tumor-associated antigens (TAAs), induce inflammatory response and promote lymphocyte infiltration in tumors. METHODS: A recombinant oncolytic adenoviruses (OAd), denoted ZD55-aPD-L1 scFv, which carried the expression cassette for anti-PD-L1 scFv was constructed by molecular cloning. Western blot and ELISA assay were performed to detect aPD-L1 scFv expression. Flow cytometry were used to analyse PD-L1 expression and count tumor cells. Co-culture assay of human peripheral blood mononuclear cells (PBMCs) with tumor cells in vitro and triple-negative breast cancer (TNBC) MDA-MB-231 tumor-bearing model in vivo were evaluated the antitumor effects of recombinant oncolytic adenoviruses ZD55-aPD-L1 scFv. RESULTS: We found that cells infected with recombinant oncolytic adenovirus ZD55-aPD-L1 scFv can effectively express aPD-L1 scFv, which function similarly to its full-length anti-PD-L1 antibody. PBMCs have inherently very limited killing effect on tumor cells even with administration of anti-PD-L1 antibody as observed from our in vitro co-cultures. Treatment consisting of ZD55 alone or ZD55 combined with anti-PD-L1 antibody yielded mediocre antitumor efficacy in subsequent in vitro and in vivo investigations, but were all substantially surpassed by the synergistic antitumor effects observed with ZD55-aPD-L1 scFv treatment. We show that the concomitant direct oncolysis by the recombinant OAd and localized autocrine/paracrine interception of PD-1:PD-L1 checkpoint interaction mediated by ZD55-aPD-L1 scFv-infected cells is exceedingly superior to co-administration of ZD55 and anti-PD-L1 antibody in the human TNBC mice model. CONCLUSIONS: Our results provided evidence for the development of novel strategies, in this case an anti-PD-L1 scFv-armed OAd, to bolster immune responses to 'cold' tumors and to improve therapeutic responsiveness to ICIs.

Identification of potential functional variants and genes at 18q21.1 associated with the carcinogenesis of colorectal cancer.

Genome-wide association studies (GWAS) have identified more than 160 susceptibility loci for colorectal cancer (CRC). The effects of these variants, particularly their mechanisms, however, remain unclear. In this study, a comprehensive functional annotation of CRC-related GWAS signals was firstly conducted to identify the potential causal variants. We found that the SNP rs7229639 in intron 3 of SMAD7 at 18q21.1 might serve as a putative functional variant in CRC. The SNP rs7229639 is located in a region with evidence of regulatory potential. Dual-luciferase reporter assays revealed that three other SNPs (rs77544449, rs60385309 and rs72917785), in strong linkage disequilibrium (LD) with rs7229639, exhibited allele-specific enhancer activity, of which one of the target genes may conceivably be LIPG, as suggested by eQTL association data and Hi-C data. We also verified that LIPG promoted malignancy of CRC cells in vitro, with supporting clinical data indicating that LIPG is upregulated and correlated with a poor prognosis in CRC. Finally, pitavastatin was observed to exhibit an anti-CRC activity and modest inhibition of LIPG mRNA levels. Collectively, our data suggest that these functional variants at 18q21.1 are involved in the pathogenesis of CRC by modulating enhancer activity, and possibly LIPG expression, thus indicating a promising therapeutic target for CRC. The results of functional annotation in our investigation could also serve as an inventory for CRC susceptibility SNPs and offer guides for post-GWAS downstream functional studies.

Expression and functional identification of recombinant SARS-CoV-2 receptor binding domain (RBD) from E. coli system.

The receptor binding domain (RBD) of SARS-CoV-2 is located in the C-terminal of S1 subunit of the spike (S) protein which is responsible for recognizing and binding to the angiotensin-converting enzyme 2 (ACE2) receptor. The DNA encoding the SARS-CoV-2 RBD was inserted into pET-28a (+) to construct expression plasmid pET-28a (+)/RBD. The desired RBD protein was produced in E. coli Rosetta (DE) and purified by a Ni-NTA column. The recombinant RBD was analyzed by SDS-PAGE and Western blot. The flow cytometry analysis indicated that the recombinant RBD is capable of binding to human ACE2 (hACE2) in the ACE2-overexpressed HEK293A-hACE2 cells. Our results demonstrated that recombinant RBD expressed in E. coli Rosetta (DE) strain has bioactivities and can be used as an antigen for diagnosis and as a tool for the development of novel anti-viral drugs against SASR-CoV-2.

An efficient system to generate truncated human angiotensin converting enzyme 2 (hACE2) capable of binding RBD and spike protein of SARS-CoV2.

Human angiotensin converting enzyme 2 (hACE2) mediates the cell entry of both SARS-CoV and SARS-CoV2 and can be used as a drug target. The DNA encoding the truncated hACE2 (30-356aa) was cloned into pET-28a (+) and expressed in Escherichia coli Rosetta (DE3). The recombinant hACE2 (rhACE2) was purified by affinity chromatography on a Ni-NTA column and characterized with SDS-PAGE and Western blot. The binding activity of rhACE2 to Spike protein of SARS-CoV2 was evaluated in S protein-overexpressed HEK293A cells (HEK293A-SP cells) through flow cytometry. The prokaryotic expression system is able to produce approximately 75 mg protein per liter, which would be useful for infection mechanism study, and drug screening and development of SARS-CoV2.