Protection from Severe Influenza Virus Infections in Mice Carrying the Mx1 Influenza Virus Resistance Gene Strongly Depends on Genetic Background.

UNLABELLED: Influenza virus infections represent a serious threat to human health. Both extrinsic and intrinsic factors determine the severity of influenza. The MX dynamin-like GTPase 1 (Mx1) gene has been shown to confer strong resistance to influenza A virus infections in mice. Most laboratory mouse strains, including C57BL/6J, carry nonsense or deletion mutations in Mx1 and thus a nonfunctional allele, whereas wild-derived mouse strains carry a wild-type Mx1 allele. Congenic C57BL/6J (B6-Mx1(r/r)) mice expressing a wild-type allele from the A2G mouse strain are highly resistant to influenza A virus infections, to both mono- and polybasic subtypes. Furthermore, in genetic mapping studies, Mx1 was identified as the major locus of resistance to influenza virus infections. Here, we investigated whether the Mx1 protective function is influenced by the genetic background. For this, we generated a congenic mouse strain carrying the A2G wild-type Mx1 resistance allele on a DBA/2J background (D2-Mx1(r/r)). Most remarkably, congenic D2-Mx1(r/r) mice expressing a functional Mx1 wild-type allele are still highly susceptible to H1N1 virus. However, pretreatment of D2-Mx1(r/r) mice with alpha interferon protected them from lethal infections. Our results showed, for the first time, that the presence of an Mx1 wild-type allele from A2G as such does not fully protect mice from lethal influenza A virus infections. These observations are also highly relevant for susceptibility to influenza virus infections in humans. IMPORTANCE: Influenza A virus represents a major health threat to humans. Seasonal influenza epidemics cause high economic loss, morbidity, and deaths each year. Genetic factors of the host strongly influence susceptibility and resistance to virus infections. The Mx1 (MX dynamin-like GTPase 1) gene has been described as a major resistance gene in mice and humans. Most inbred laboratory mouse strains are deficient in Mx1, but congenic B6-Mx1(r/r) mice that carry the wild-type Mx1 gene from the A2G mouse strain are highly resistant. Here, we show that, very unexpectedly, congenic D2-Mx1(r/r) mice carrying the wild-type Mx1 gene from the A2G strain are not fully protected against lethal influenza virus infections. These observations demonstrate that the genetic background is very important for the protective function of the Mx1 resistance gene. Our results are also highly relevant for understanding genetic susceptibility to influenza virus infections in humans.

Distinct gene loci control the host response to influenza H1N1 virus infection in a time-dependent manner.

BACKGROUND: There is strong but mostly circumstantial evidence that genetic factors modulate the severity of influenza infection in humans. Using genetically diverse but fully inbred strains of mice it has been shown that host sequence variants have a strong influence on the severity of influenza A disease progression. In particular, C57BL/6J, the most widely used mouse strain in biomedical research, is comparatively resistant. In contrast, DBA/2J is highly susceptible. RESULTS: To map regions of the genome responsible for differences in influenza susceptibility, we infected a family of 53 BXD-type lines derived from a cross between C57BL/6J and DBA/2J strains with influenza A virus (PR8, H1N1). We monitored body weight, survival, and mean time to death for 13 days after infection. Qivr5 (quantitative trait for influenza virus resistance on chromosome 5) was the largest and most significant QTL for weight loss. The effect of Qivr5 was detectable on day 2 post infection, but was most pronounced on days 5 and 6. Survival rate mapped to Qivr5, but additionally revealed a second significant locus on chromosome 19 (Qivr19). Analysis of mean time to death affirmed both Qivr5 and Qivr19. In addition, we observed several regions of the genome with suggestive linkage. There are potentially complex combinatorial interactions of the parental alleles among loci. Analysis of multiple gene expression data sets and sequence variants in these strains highlights about 30 strong candidate genes across all loci that may control influenza A susceptibility and resistance. CONCLUSIONS: We have mapped influenza susceptibility loci to chromosomes 2, 5, 16, 17, and 19. Body weight and survival loci have a time-dependent profile that presumably reflects the temporal dynamic of the response to infection. We highlight candidate genes in the respective intervals and review their possible biological function during infection.

Wild-type and Hupki (human p53 knock-in) murine embryonic fibroblasts: p53/ARF pathway disruption in spontaneous escape from senescence.

Research on cell senescence and immortalization of murine embryonic fibroblasts (MEFs) has revealed important clues about genetic control of senescence in humans. To investigate senescence and genetic alterations in the p53 pathway that lead to senescence bypass in culture, we compared the behavior of MEFs from wild-type mice with MEFs from Hupki mice, which harbor a humanized p53 gene. We found that humanizing the p53 gene in mice preserved major features of the MEF senescence/immortalization process. In both genotypes, a significant proportion of spontaneously arising cell lines had sustained either a p53 point mutation or p19/ARF biallelic deletion. The p53 mutations selected for during Hupki MEF immortalization have been found in human tumors and are classified in the yeast transactivation assay as transcriptionally defunct, suggesting that disabling this component of p53 activity is crucial in senescence bypass. Surprisingly, in spontaneously immortalized cell lines from both wild-type and Hupki MEFs, the predominant type of p53 mutation was a G to C transversion, rather than the G to T substitutions expected from the raised oxygen levels characteristic of standard culture conditions. Over half of the cell lines did not reveal evidence of p53 mutation or loss of p19/ARF and retained a robust wild-type p53 response to DNA damage, supporting the inference from senescence bypass screens that alternative genetic routes to immortalization occur.

Correction: Lung cancer stem cells and their aggressive progeny, controlled by EGFR/MIG6 inverse expression, dictate a novel NSCLC treatment approach.

[This corrects the article DOI: 10.18632/oncotarget.26817.].

TP53 mutation signature supports involvement of aristolochic acid in the aetiology of endemic nephropathy-associated tumours.

The proposal has been put forward that the primary cause of Balkan endemic nephropathy (BEN) is exposure to food crops contaminated with seeds of Aristolochia spp, which contain high levels of aristolochic acids (AA). Recently, tumour DNA samples from patients with BEN were found to harbour principally A to T mutations in the TP53 tumour suppressor gene (Grollman et al., Proc Natl Acad Sci USA 2007;104:12129-34). Using a novel mutation assay in which we can induce and select mutations in human TP53 sequences in vitro by exposure of cultured cells to a mutagen, we found that A to T mutations were elicited by aristolochic acid at sites in TP53 rarely mutated in human cancers in general, but which were observed in the BEN patients. This concordance of specific mutations in patient tumours and aristolochic acid I-exposed cultures supports the argument that AA has a direct role in the aetiology of BEN-associated cancer.

Lung cancer stem cells and their aggressive progeny, controlled by EGFR/MIG6 inverse expression, dictate a novel NSCLC treatment approach.

The lung cancer stem cell (LuCSC) model comprises an attractive framework to explore acquired drug resistance in non-small cell lung cancer (NSCLC) treatment. Here, we used NSCLC cell line model to translate cellular heterogeneity into tractable populations to understand the origin of lung cancers and drug resistance. The epithelial LuCSCs, presumably arising from alveolar bipotent stem/progenitor cells, were lineage naïve, noninvasive, and prone to creating aggressive progeny expressing AT2/AT1 markers. LuCSC-holoclones were able to initiate rimmed niches, where their specialization created pseudo-alveoli structures. Mechanistically, LuCSC transitioning from self-renewal (ß-catenin and Nanog signaling) to malignant lineage differentiation is regulated by EGFR activation and the inverse inhibition of tumor suppressor MIG6. We further identified the functional roles of endogenous EGFR signaling in mediating progeny invasiveness and their ligands in LuCSC differentiation. Importantly, drug screening demonstrated that EGFR driving progeny were strongly responsive to TKIs; however, the LuCSCs were exclusively resistant but sensitive to AMPK agonist Metformin, antibiotic Salinomycin and to a lesser degree Carboplatin. Our data reveals previously an unknown mechanism of NSCLC resistance to EGFR-TKIs, which is associated with LuCSCs bearing a silenced EGFR and inversely expressed MIG6 suppressor gene. Taken altogether, successful NSCLC treatment requires development of a novel combination of drugs, efficiently targeting both LuCSCs and heterogeneous progeny.

RIG-I activating immunostimulatory RNA boosts the efficacy of anticancer vaccines and synergizes with immune checkpoint blockade.

BACKGROUND: Antibody-mediated targeting of regulatory T cell receptors such as CTLA-4 enhances antitumor immune responses against several cancer entities including malignant melanoma. Yet, therapeutic success in patients remains variable underscoring the need for novel combinatorial approaches. METHODS: Here we established a vaccination strategy that combines engagement of the nucleic acid-sensing pattern recognition receptor RIG-I, antigen and CTLA-4 blockade. We used in vitro transcribed 5'-triphosphorylated RNA (3pRNA) to therapeutically target the RIG-I pathway. We performed in vitro functional analysis in bone-marrow derived dendritic cells and investigated RIG-I-enhanced vaccines in different murine melanoma models. FINDINGS: We found that protein vaccination together with RIG-I ligation via 3pRNA strongly synergizes with CTLA-4 blockade to induce expansion and activation of antigen-specific CD8+ T cells that translates into potent antitumor immunity. RIG-I-induced cross-priming of cytotoxic T cells as well as antitumor immunity were dependent on the host adapter protein MAVS and type I interferon (IFN-I) signaling and were mediated by dendritic cells. INTERPRETATION: Overall, our data demonstrate the potency of a novel combinatorial vaccination strategy combining RIG-I-driven immunization with CTLA-4 blockade to prevent and treat experimental melanoma. FUND: German Research Foundation (SFB 1335, SFB 1371), EMBO, Else Kröner-Fresenius-Foundation, German Cancer Aid, European Hematology Association, DKMS Foundation for Giving Life, Dres. Carl Maximilian and Carl Manfred Bayer-Foundation.

Targeting intrinsic RIG-I signaling turns melanoma cells into type I interferon-releasing cellular antitumor vaccines.

Resistance to cell death and evasion of immunosurveillance are major causes of cancer persistence and progression. Tumor cell-intrinsic activation of the RNA receptor retinoic acid-inducible gene-I (RIG-I) can trigger an immunogenic form of programmed tumor cell death, but its impact on antitumor responses remains largely unexplored. We show that activation of intrinsic RIG-I signaling induces melanoma cell death that enforces cross-presentation of tumor-associated antigens by bystander dendritic cells. This results in systemic expansion and activation of tumor-antigen specific T cells in vivo with subsequent regression of pre-established melanoma. These processes were dependent on the signaling hub MAVS and type I interferon (IFN-I) signaling in the host cell. Using melanoma cells deficient for the transcription factors IRF3 and IRF7, we demonstrate that RIG-I-activated tumor cells used as a vaccine are a relevant source of IFN-I during T cell cross-priming in vivo. Thus, our findings may facilitate translational development of personalized anticancer vaccines.

RIG-I activation is critical for responsiveness to checkpoint blockade.

Achieving durable clinical responses to immune checkpoint inhibitors remains a challenge. Here, we demonstrate that immunotherapy with anti-CTLA-4 and its combination with anti-PD-1 rely on tumor cell-intrinsic activation of the cytosolic RNA receptor RIG-I. Mechanistically, tumor cell-intrinsic RIG-I signaling induced caspase-3-mediated tumor cell death, cross-presentation of tumor-associated antigen by CD103+ dendritic cells, subsequent expansion of tumor antigen-specific CD8+ T cells, and their accumulation within the tumor tissue. Consistently, therapeutic targeting of RIG-I with 5'- triphosphorylated RNA in both tumor and nonmalignant host cells potently augmented the efficacy of CTLA-4 checkpoint blockade in several preclinical cancer models. In humans, transcriptome analysis of primary melanoma samples revealed a strong association between high expression of DDX58 (the gene encoding RIG-I), T cell receptor and antigen presentation pathway activity, and prolonged overall survival. Moreover, in patients with melanoma treated with anti-CTLA-4 checkpoint blockade, high DDX58 RIG-I transcriptional activity significantly associated with durable clinical responses. Our data thus identify activation of RIG-I signaling in tumors and their microenvironment as a crucial component for checkpoint inhibitor-mediated immunotherapy of cancer.

Modelling mutational landscapes of human cancers in vitro.

Experimental models that recapitulate mutational landscapes of human cancers are needed to decipher the rapidly expanding data on human somatic mutations. We demonstrate that mutation patterns in immortalised cell lines derived from primary murine embryonic fibroblasts (MEFs) exposed in vitro to carcinogens recapitulate key features of mutational signatures observed in human cancers. In experiments with several cancer-causing agents we obtained high genome-wide concordance between human tumour mutation data and in vitro data with respect to predominant substitution types, strand bias and sequence context. Moreover, we found signature mutations in well-studied human cancer driver genes. To explore endogenous mutagenesis, we used MEFs ectopically expressing activation-induced cytidine deaminase (AID) and observed an excess of AID signature mutations in immortalised cell lines compared to their non-transgenic counterparts. MEF immortalisation is thus a simple and powerful strategy for modelling cancer mutation landscapes that facilitates the interpretation of human tumour genome-wide sequencing data.

The codon 72 polymorphism of p53 regulates interaction with NF-{kappa}B and transactivation of genes involved in immunity and inflammation.

A common polymorphism at codon 72 in the p53 tumor suppressor gene encodes either proline (P72) or arginine (R72). Several groups have reported that in cultured cells, this polymorphism influences p53's transcriptional, senescence, and apoptotic functions. However, the impact of this polymorphism within the context of a living organism is poorly understood. We generated knock-in mice with the P72 and R72 variants and analyzed the tissues of these mice for apoptosis and transcription. In the thymus, we find that the P72 variant induces increased apoptosis following ionizing radiation, along with increased transactivation of a subset of p53 target genes, which includes murine Caspase 4 (also called Caspase 11), which we show is a direct p53 target gene. Interestingly, the majority of genes in this subset have roles in inflammation, and their promoters contain NF-κB binding sites. We show that caspase 4/11 requires both p53 and NF-κB for full induction after DNA damage and that the P72 variant shows increased interaction with p65 RelA, a subunit of NF-κB. Consistent with this, we show that P72 mice have a markedly enhanced response to inflammatory challenge compared to that of R72 mice. Our data indicate that the codon 72 polymorphism impacts p53's role in inflammation.