No abstract

Modified Vaccinia Ankara (MVA) is a highly attenuated poxvirus vector that is widely used to develop vaccines for infectious diseases and cancer. We demonstrate the construction of a vaccine platform based on a unique three-plasmid system to efficiently generate recombinant MVA vectors from chemically synthesized DNA. In response to the ongoing global pandemic caused by SARS coronavirus-2 (SARS-CoV-2), we use this vaccine platform to rapidly produce fully synthetic MVA (sMVA) vectors co-expressing SARS-CoV-2 spike and nucleocapsid antigens, two immunodominant antigens implicated in protective immunity. We show that mice immunized with these sMVA vectors develop robust SARS-CoV-2 antigen-specific humoral and cellular immune responses, including potent neutralizing antibodies. These results demonstrate the potential of a vaccine platform based on synthetic DNA to efficiently generate recombinant MVA vectors and to rapidly develop a multi-antigenic poxvirus-based SARS-CoV-2 vaccine candidate.

Disseminated cancer cells rely on intricate interactions among diverse cell types in the tumor-associated stroma, vasculature, and immune system for survival and growth. Ubiquitous expression of c-Jun N-terminal kinase (jnk) genes in various cell types permits their control of metastasis. In early stages of metastasis, JNKs affect tumor-associated inflammation and angiogenesis as well as tumor cell migration and intravasation. Within the tumor stroma, JNKs are essential for the release of growth factors that promote epithelial-to-mesenchymal transition (EMT) in tumor cells. JNK3, the least ubiquitous isoform, facilitates angiogenesis by increasing endothelial cell migration. Importantly, JNK expression in tumor cells integrates stromal signals to promote tumor cell invasion. However, JNK isoforms differentially regulate migration toward the endothelial barrier. Once tumor cells enter the bloodstream, JNKs increase circulating tumor cell (CTC) survival and homing to tissues. By promoting fibrosis, JNKs improve CTC attachment to the endothelium. Once anchored, JNKs stimulate EMT to facilitate tumor cell extravasation and enhance the secretion of endothelial barrier disrupters. Tumor cells attract barrier-disrupting macrophages by JNK-dependent transcription of macrophage chemoattractant molecules. In the secondary tissue, JNKs are instrumental in the premetastatic niche and stimulate tumor cell proliferation. JNK expression in cancer cells stimulates tissue-remodeling macrophages to improve tumor colonization. However, in T-cells, JNKs alter cytokine production that increases tumor surveillance and inhibits the recruitment of tissue-remodeling macrophages. Therapeutically targeting JNKs for metastatic disease is attractive considering their promotion of metastasis; however, specific JNK tools are needed to determine their definitive actions within the context of the entire metastatic cascade.

Oncogenes induce cell proliferation leading to replicative stress, DNA damage and genomic instability. A wide variety of cellular stresses activate c-Jun N-terminal kinase (JNK) proteins, but few studies have directly addressed the roles of JNK isoforms in tumor development. Herein, we show that jnk2 knockout mice expressing the Polyoma Middle T Antigen transgene developed mammary tumors earlier and experienced higher tumor multiplicity compared to jnk2 wildtype mice. Lack of jnk2 expression was associated with higher tumor aneuploidy and reduced DNA damage response, as marked by fewer pH2AX and 53BP1 nuclear foci. Comparative genomic hybridization further confirmed increased genomic instability in PyV MT/jnk2−/− tumors. In vitro, PyV MT/jnk2−/− cells underwent replicative stress and cell death as evidenced by lower BrdU incorporation, and sustained chromatin licensing and DNA replication factor 1 (CDT1) and p21Waf1 protein expression, and phosphorylation of Chk1 after serum stimulation, but this response was not associated with phosphorylation of p53 Ser15. Adenoviral overexpression of CDT1 led to similar differences between jnk2 wildtype and knockout cells. In normal mammary cells undergoing UV induced single stranded DNA breaks, JNK2 localized to RPA (Replication Protein A) coated strands indicating that JNK2 responds early to single stranded DNA damage and is critical for subsequent recruitment of DNA repair proteins. Together, these data support that JNK2 prevents replicative stress by coordinating cell cycle progression and DNA damage repair mechanisms.

Ataxia-telangiectasia mutated (ATM) protein kinase regulates the DNA damage response (DDR) and is associated with cancer suppression. Here we report a cancer-promoting role for ATM. ATM depletion in metastatic cancer cells reduced cell migration and invasion. Transcription analyses identified a gene network, including the chemokine IL-8, regulated by ATM. IL-8 expression required ATM and was regulated by oxidative stress. IL-8 was validated as an ATM target by its ability to rescue cell migration and invasion defects in ATM-depleted cells. Finally, ATM-depletion in human breast cancer cells reduced lung tumors in a mouse xenograft model and clinical data validated IL-8 in lung metastasis. These findings provide insights into how ATM activation by oxidative stress regulates IL-8 to sustain cell migration and invasion in cancer cells to promote metastatic potential. Thus, in addition to well-established roles in tumor suppression, these findings identify a role for ATM in tumor progression.DOI: http://dx.doi.org/10.7554/eLife.07270.001