Induction Of Interferon Signaling Research Articles

Type-I interferon signaling is essential for robust metronomic chemo-immunogenic tumor regression in murine breast cancer.

Many patients with breast cancer have a poor prognosis with limited therapeutic options. Here, we investigated the potential of chemo-immunogenic therapy as an avenue of treatment. We utilized two syngeneic mouse mammary tumor models, 4T1 and E0771, to examine the chemo-immunogenic potential of cyclophosphamide and the mechanistic contributions of cyclophosphamide-activated type-I interferon (IFN) signaling to therapeutic activity. Chemically-activated cyclophosphamide induced robust IFNα/β receptor-1-dependent signaling linked to hundreds of IFN-stimulated gene responses in both cell lines. Further, in 4T1 tumors, cyclophosphamide given on a medium-dose, 6-day intermittent metronomic schedule induced strong IFN signaling but comparatively weak immune cell infiltration associated with long-term tumor growth stasis. Induction of IFN signaling was somewhat weaker in E0771 tumors but was followed by widespread downstream gene responses, robust immune cell infiltration and extensive, prolonged tumor regression. The immune dependence of these effective anti-tumor responses was established by CD8 T-cell immunodepletion, which blocked cyclophosphamide-induced E0771 tumor regression and led to tumor stasis followed by regrowth. Strikingly, IFNα/β receptor-1 antibody blockade was even more effective in preventing E0771 immune cell infiltration and blocked the major tumor regression induced by cyclophosphamide treatment. Type-I IFN signaling is thus essential for the robust chemo-immunogenic response of these tumors to cyclophosphamide administered on a metronomic schedule.

Abstract LBA010: Therapeutic stimulation of nucleic acid receptor RIG-I enhances efficacy of kinase inhibitor treatment in oncogene-driven tumors

Abstract Introduction: A major challenge in the treatment of kinase driven tumors is the inevitable emergence of resistance and the limited efficacy of subsequent treatments including immunotherapy. Methods: To gain deeper insights into the adaptive processes of cells surviving kinase inhibition we studied the mechanisms enabling cell persistence despite targeted treatment across various in vitro and in vivo tumor models. This included transcriptomic profiles of EGFR-, BRAF-, HER2- or ALK-driven colon, melanoma and lung cancer cells and of melanoma patients during kinase inhibition. To investigate underlying mechanisms we employed CRISPR/Cas9 and overexpression experiments and stimulation of nucleic acid receptors (NARs) TLR3, cGAS and RIG-I with synthetic agonists to assess combination therapies. Finally, we used humanized tumor xenografts and a syngeneic EGFR-mutant lung cancer model to validate the inflammatory effects of kinase inhibition and the efficacy of adding RIG-I agonist IVT4 to kinase inhibition in vivo. Results: Analysis of dynamic transcriptomic changes during kinase inhibition by time-series RNA-seq of oncogene-dependent cells displayed rapid induction of an interferon gene signature and suppression of MYC- and E2F-target genes. This was accompanied by induction of p27 protein levels, cell cycle arrest and an increase of b-galactosidase positive cells, indicating a senescence-associated inflammatory response that occurred in a cell autonomous manner in vitro and in vivo. In vivo, EGFR inhibition also increased T-cells, but anti-PD-1 therapy following EGFR inhibitor pre-treatment did not prevent tumor recurrence. Interestingly, the kinase inhibitor induced interferon signature was independent of caspase activation and of the nucleic acid sensing machinery. Instead, functional experiments indicated that induction of interferon signaling was dependent on inhibition of MAPK signaling and subsequent IRF1 activation, providing a potential mechanism of action. Numerous innate immune pathway members including NARs MDA-5, TLR3 and RIG-I were upregulated, which we aimed to exploit therapeutically. Evaluation of NAR agonists showed that only stimulation of RIG-I with its agonist IVT4 evoked cytokine secretion and synergistically enhanced cell death in vitro. In humanized xenografts and syngenic immunocompetent mouse models adding IVT4 led to additional tumor shrinkage after EGFR-inhibition and decreased T cell exhaustion. Effects of IVT4 on tumor volumes were lower in immunodeficient xenografts and after NK- or CD8-cell depletion in the syngeneic model indicating that immunological influences in addition to tumor cell intrinsic effects contribute to the efficacy in vivo. Conclusion: Taken together, our findings show that kinase inhibition may promote tumor cell-autonomous, senescence-associated inflammatory signaling and may sensitize oncogene-driven tumors to therapeutic intervention using RIG-I agonist IVT4. Our results thus provide a novel treatment strategy and a basis to explore combining immunotherapy with kinase inhibition in the future. Citation Format: Johannes Brägelmann, Carina Lorenz, Sven Borchmann, Kazuya Nishii, Julia Wegner, Jenny Ostendorp, David Ast, Alena Heimsoeth, Philipp Lohneis, Thomas Zillinger, Roland Ullrich, Kadoaki Ohashi, Martin Schlee, Martin Sos. Therapeutic stimulation of nucleic acid receptor RIG-I enhances efficacy of kinase inhibitor treatment in oncogene-driven tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA010.

Induction of interferon signaling and allograft inflammatory factor 1 in macrophages in a mouse model of breast cancer metastases.

Background: Metastatic breast cancer cells recruit macrophages (metastasis-associated macrophages, or MAMs) to facilitate their seeding, survival and outgrowth. However, a comprehensive understanding of the gene expression program in MAMs and how this program contributes to metastasis remain elusive. Methods: We compared the transcriptomes of MAMs recruited to lung metastases and resident alveolar macrophages (RAMs) and identified a large variety of differentially expressed genes and their associated signaling pathways. Some of the changes were validated using qRT-PCR and immunofluorescence. To probe the functional relevance to metastatic growth, a gene-targeting mouse model of female mice in the C57BL6/J background was used to study allograft inflammatory factor 1 (AIF1, also known as ionized calcium-binding adapter molecule 1 or IBA1). Results: Interferon signaling is one of the most activated pathways in MAMs, with strong upregulation of multiple components of the pathway and a significant enrichment for the gene signatures of interferon-alpha-treated human macrophages. Aif1, an interferon-responsive gene that regulates multiple macrophage activities, was robustly induced in MAMs. Aif1 deficiency in MAMs, however, did not affect development of lung metastases, suggesting that AIF1 indicates MAM activation but is dispensable for regulating metastasis. Conclusions: The drastically different gene expression profile of MAMs as compared to RAMs suggests an important role in promoting metastatic growth. Dissection of the underlying mechanisms and functional validation of potential targets in the profile may provide novel therapeutic strategies for the treatment of metastatic diseases.

Induction of interferon signaling and allograft inflammatory factor 1 in macrophages in a mouse model of breast cancer metastases

Background: Metastatic breast cancer cells recruit macrophages (metastasis-associated macrophages, or MAMs) to facilitate their seeding, survival and outgrowth. However, a comprehensive understanding of the gene expression program in MAMs and how this program contributes to metastasis remain elusive. Methods: We compared the transcriptomes of MAMs recruited to lung metastases and resident alveolar macrophages (RAMs) and identified a large variety of differentially expressed genes and their associated signaling pathways. Some of the changes were validated using qRT-PCR and immunofluorescence. To probe the functional relevance to metastatic growth, a gene-targeting mouse model of female mice in the C57BL6/J background was used to study allograft inflammatory factor 1 (AIF1, also known as ionized calcium-binding adapter molecule 1 or IBA1). Results: Interferon signaling is one of the most activated pathways in MAMs, with strong upregulation of multiple components of the pathway and a significant enrichment for the gene signatures of interferon-alpha-treated human macrophages. Aif1, an interferon-responsive gene that regulates multiple macrophage activities, was robustly induced in MAMs. Aif1 deficiency in MAMs, however, did not affect development of lung metastases, suggesting that AIF1 indicates MAM activation but is dispensable for regulating metastasis. Conclusions: The drastically different gene expression profile of MAMs as compared to RAMs suggests an important role in promoting metastatic growth. Dissection of the underlying mechanisms and functional validation of potential targets in the profile may provide novel therapeutic strategies for the treatment of metastatic diseases.

Induction of interferon signaling and allograft inflammatory factor 1 in macrophages in a mouse model of breast cancer metastases.

Background: Metastatic breast cancer cells recruit macrophages (metastasis-associated macrophages, or MAMs) to facilitate their seeding, survival and outgrowth. However, a comprehensive understanding of the gene expression program in MAMs and how this program contributes to metastasis remain elusive. Methods: We compared the transcriptomes of MAMs recruited to lung metastases and resident alveolar macrophages (RAMs) and identified a large variety of differentially expressed genes and their associated signaling pathways. Some of the changes were validated using qRT-PCR and immunofluorescence. To probe the functional relevance to metastatic growth, a gene-targeting mouse model of female mice in the C57BL6/J background was used to study allograft inflammatory factor 1 (AIF1, also known as ionized calcium-binding adapter molecule 1 or IBA1). Results: Interferon signaling is one of the most activated pathways in MAMs, with strong upregulation of multiple components of the pathway and a significant enrichment for the gene signatures of interferon-alpha-treated human macrophages. Aif1, an interferon-responsive gene that regulates multiple macrophage activities, was robustly induced in MAMs. Aif1 deficiency in MAMs, however, did not affect development of lung metastases, suggesting that AIF1 indicates MAM activation but is dispensable for regulating metastasis. Conclusions: The drastically different gene expression profile of MAMs as compared to RAMs suggests an important role in promoting metastatic growth. Dissection of the underlying mechanisms and functional validation of potential targets in the profile may provide novel therapeutic strategies for the treatment of metastatic diseases.

Neuronal maturation reduces the type I IFN response to orthobunyavirus infection and leads to increased apoptosis of human neurons

BackgroundLa Crosse virus (LACV) is the leading cause of pediatric arboviral encephalitis in the USA. LACV encephalitis can result in learning and memory deficits, which may be due to infection and apoptosis of neurons in the brain. Despite neurons being the primary cell infected in the brain by LACV, little is known about neuronal responses to infection.MethodsHuman cerebral organoids (COs), which contain a spectrum of developing neurons, were used to examine neuronal responses to LACV. Plaque assay and quantitative reverse transcription (qRT) PCR were used to determine the susceptibility of COs to LACV infection. Immunohistochemistry, flow cytometry, and single-cell transcriptomics were used to determine specific neuronal subpopulation responses to the virus.ResultsOverall, LACV readily infected COs causing reduced cell viability and increased apoptosis. However, it was determined that neurons at different stages of development had distinct responses to LACV. Both neural progenitors and committed neurons were infected with LACV, however, committed neurons underwent apoptosis at a higher rate. Transcriptomic analysis showed that committed neurons expressed fewer interferon (IFN)-stimulated genes (ISGs) and genes involved IFN signaling in response to infection compared to neural progenitors. Furthermore, induction of interferon signaling in LACV-infected COs by application of recombinant IFN enhanced cell viability.ConclusionsThese findings indicate that neuronal maturation increases the susceptibility of neurons to LACV-induced apoptosis. This susceptibility is likely due, at least in part, to mature neurons being less responsive to virus-induced IFN as evidenced by their poor ISG response to LACV. Furthermore, exogenous administration of recombinant IFN to LACV COs rescued cellular viability suggesting that increased IFN signaling is overall protective in this complex neural tissue. Together these findings indicate that induction of IFN signaling in developing neurons is an important deciding factor in virus-induced cell death.

Ethanol induces interferon expression in neurons via TRAIL: role of astrocyte-to-neuron signaling

RationaleAlcohol use disorder (AUD) involves dysregulation of innate immune signaling in brain. Toll-like receptor 3 (TLR3), an innate immune receptor that is upregulated in post-mortem human alcoholics, leads to induction of interferon (IFN) signaling. IFNs have been linked to depressive-like symptoms and therefore may play a role in addiction pathology. Astrocyte-neuronal signaling may contribute to maladaptation of neuronal circuits.ObjectivesIn this manuscript, we examine ethanol (EtOH) induction of IFN signaling in neuronal, astrocyte, and microglial cell lines and assess astrocyte-neuronal interactions.MethodsU373 astrocytes, SH-SY5Y neurons, and BV2 microglia were treated with EtOH and analyzed for autocrine/paracrine IFN signaling.ResultsEtOH induced TLR3, IFNβ, and IFNγ in SH-SY5Y neurons and U373 astrocytes, but not in BV2 microglia. The IFN response gene TRAIL was also strongly upregulated by TLR3 agonist Poly(I:C) and EtOH in U373 astrocytes. TRAIL blockage via neutralizing antibody prevented induction of IFNs in SH-SY5Y neurons but not in U373 astrocytes. Blocking TRAIL in conditioned media from EtOH-treated astrocytes prevented induction of IFNs in SH-SY5Y neurons. Finally, an in vivo model of chronic 10-day binge EtOH exposure in C57BL6/J mice, as well as single acute treatment with Poly(I:C), showed increased TRAIL +IR cells in both orbitofrontal and entorhinal cortex.ConclusionsThis study establishes a role of astrocyte to neuron TRAIL release in EtOH-induced IFN responses. This may contribute to alcohol associated negative affect and suggest potential therapeutic benefit of TRAIL inhibition in AUD.

ID: 59: Combination of classical genetics, reverse genetics and transcriptomics to study reovirus’ interferon sensitivity and host-cell response

Mammalian reovirus is an oncolytic virus that discriminates between parental and cancer cells. This is due in part to altered interferon-induced antiviral response in cancer cells. However, understanding of viral and cellular determinants involved remains largely incomplete. In our current work, two different approaches were used to identify reovirus variants exhibiting increased interferon sensitivity. (1) Chemical mutagenesis followed by selection of small plaques mutants in the presence of interferon; (2) Selection on interferon-deficient cells in order to relieve the pressure to keep interferon resistance. Virus variants were sequenced and plasmid-based reverse genetics was used to examine the effect of each mutation. A single amino acid substitution in the 2′-O methyltransferase of viral mRNA capping enzyme was responsible for 10–100 fold increased interferon sensitivity for one variant; work is under progress for additional viruses. In parallel, we took advantage of these well genetically-defined viruses to investigate host-cell response using RNA-Seq. A large number of induced genes was observed in wild-type virus-infected cells, to an extent that was not revealed by previously-used approaches. Most induced genes encode sensors or antiviral response determinants (ISG, IFIT, IRF, Mx). They were similarly induced by the wild-type and interferon-sensitive virus. This suggests that altered RNA capping increases virus’ sensitivity to interferon-induced antiviral factors rather than induction of interferon signaling. Similar analysis with other viruses of different interferon sensitivity, in parental and transformed cells, should allow to better comprehend the antiviral response during reovirus infection and could contribute to the design of better oncolytic viruses.