Olusegun O. Onabajo

Association of germline variants in the APOBEC3 region with cancer risk and enrichment with APOBEC-signature mutations in tumors

High rates of APOBEC-signature mutations are found in many tumors, but factors affecting this mutation pattern are not well understood. Here we explored the contribution of two common germline variants in the APOBEC3 region. SNP rs1014971 was associated with bladder cancer risk, increased APOBEC3B expression, and enrichment with APOBEC-signature mutations in bladder tumors. In contrast, a 30-kb deletion that eliminates APOBEC3B and creates an APOBEC3A–APOBEC3B chimera was not important in bladder cancer, whereas it was associated with breast cancer risk and enrichment with APOBEC-signature mutations in breast tumors. In vitro, APOBEC3B expression was predominantly induced by treatment with a DNA-damaging drug in bladder cancer cell lines, and APOBEC3A expression was induced as part of the antiviral interferon-stimulated response in breast cancer cell lines. These findings suggest a tissue-specific role of environmental oncogenic triggers, particularly in individuals with germline APOBEC3 risk variants.

Expression of Interferon Lambda 4 Is Associated with Reduced Proliferation and Increased Cell Death in Human Hepatic Cells.

Interferon lambda 4 (IFN-λ4) is a novel type-III interferon that can be generated only in individuals carrying a ΔG frame-shift allele of an exonic genetic variant (rs368234815-ΔG/TT). The rs368234815-ΔG allele is strongly associated with decreased clearance of hepatitis C virus (HCV) infection. Here, we further explored the biological function of IFN-λ4 expressed in human hepatic cells—a hepatoma cell line HepG2 and fresh primary human hepatocytes (PHHs). We performed live confocal imaging, cell death and proliferation assays, mRNA expression profiling, protein detection, and antibody blocking assays using transient and inducible stable in vitro systems. Not only did we observe significant intracellular retention of IFN-λ4 but also detected secreted IFN-λ4 in the culture media of expressing cells. Secreted IFN-λ4 induced strong activation of the interferon-stimulated genes (ISGs) in IFN-λ4-expressing and surrounding cells in transwell assays. Specifically, in PHHs, secreted IFN-λ4 induced expression of the CXCL10 transcript and a corresponding pro-inflammatory chemokine, IP-10. In IFN-λ4-expressing HepG2 cells, we also observed decreased proliferation and increased cell death. All IFN-λ4-induced phenotypes—activation of ISGs, decreased proliferation, and increased cell death—could be inhibited by an anti-IFN-λ4-specific antibody. Our study offers new insights into biology of IFN-λ4 and its possible role in HCV clearance.

Actin-binding protein 1 links B-cell antigen receptors to negative signaling pathways

Significance B cells are the major mediators of humoral immunity, producing antibody to aid in the elimination of pathogens. B cells signal through their membrane receptors, resulting in transcriptional modification for the maintenance of B-cell survival, proliferation, and antibody production. B-cell receptor (BCR) signaling is tightly regulated, and elevated or sustained BCR signals lead to loss of B-cell tolerance and subsequent autoimmunity. This study reveals that the adaptor molecule Abp1 negatively regulates development of marginal zone and spontaneous germinal center B cells, as well as autoantibody production. As a link between the actin cytoskeleton and BCR signaling, Abp1 assists BCR signal attenuation by promoting BCR central cluster formation, as well as recruitment of inhibitory signaling molecules to BCR signalosomes. Prolonged or uncontrolled B-cell receptor (BCR) signaling is associated with autoimmunity. We previously demonstrated a role for actin in BCR signal attenuation. This study reveals that actin-binding protein 1 (Abp1/HIP-55/SH3P7) is a negative regulator of BCR signaling and links actin to negative regulatory pathways of the BCR. In both Abp1−/− and bone marrow chimeric mice, in which only B cells lack Abp1 expression, the number of spontaneous germinal center and marginal zone B cells and the level of autoantibody are significantly increased. Serum levels of T-independent antibody responses and total antibody are elevated, whereas T-dependent antibody responses are markedly reduced and fail to undergo affinity maturation. Upon activation, surface BCR clustering is enhanced and B-cell contraction delayed in Abp1−/− B cells, concurrent with slow but persistent increases in F-actin at BCR signalosomes. Furthermore, BCR signaling is enhanced in Abp1−/− B cells compared with wild-type B cells, including Ca2+ flux and phosphorylation of B-cell linker protein, the mitogen-activated protein kinase kinase MEK1/2, and ERK, coinciding with reductions in recruitment of the inhibitory signaling molecules hematopoietic progenitor kinase 1 and SH2-containing inositol 5-phosphatase to BCR signalosomes. Our results indicate that Abp1 negatively regulates BCR signaling by coupling actin remodeling to B-cell contraction and activation of inhibitory signaling molecules, which contributes to the regulation of peripheral B-cell development and antibody responses.

IFN-λ4 Attenuates Antiviral Responses by Enhancing Negative Regulation of IFN Signaling

Type III IFNs are important mediators of antiviral immunity. IFN-λ4 is a unique type III IFN because it is produced only in individuals who carry a dG allele of a genetic variant rs368234815-dG/TT. Counterintuitively, those individuals who can produce IFN-λ4, an antiviral cytokine, are also less likely to clear hepatitis C virus infection. In this study, we searched for unique functional properties of IFN-λ4 that might explain its negative effect on hepatitis C virus clearance. We used fresh primary human hepatocytes (PHHs) treated with recombinant type III IFNs or infected with Sendai virus to model acute viral infection and subsequently validated our findings in HepG2 cell line models. Endogenous IFN-λ4 protein was detectable only in Sendai virus–infected PHHs from individuals with the dG allele, where it was poorly secreted but highly functional, even at concentrations < 50 pg/ml. IFN-λ4 acted faster than other type III IFNs in inducing antiviral genes, as well as negative regulators of the IFN response, such as USP18 and SOCS1. Transient treatment of PHHs with IFN-λ4, but not IFN-λ3, caused a strong and sustained induction of SOCS1 and refractoriness to further stimulation with IFN-λ3. Our results suggest unique functional properties of IFN-λ4 that can be important in viral clearance and other clinical conditions.

Comparative Functional Analysis of 12 Mammalian IFN-λ4 Orthologs.

IFN-λ4 is a novel type-III interferon with strong clinical significance in humans. Only a subset of individuals—up to 10% of Asians, 50% of Europeans, and 90% of Africans—carry the ΔG allele of a genetic variant rs368234815-TT/ΔG and are genetically able to produce IFN-λ4 protein. Carriers of the ΔG allele have impaired ability to clear infection with hepatitis C virus (HCV). IFN-λ4 is also predicted to exist and be functionally important in several nonhuman mammals. In this study, we present the first comparative analysis of 12 mammalian IFN-λ4 orthologs in a human hepatic cell line, HepG2, which supports signaling of the human IFN-λ4. We show that despite differences in protein sequences, functional properties of the recombinant human and nonhuman IFN-λ4 proteins are comparable—they are all expressed as predominantly cytoplasmic proteins that are biologically active for induction of interferon signaling. We show that several IFN-λ4 orthologs can be detected by Western blotting, flow cytometry, and confocal imaging using a monoclonal antibody developed for the human IFN-λ4. Studies of IFN-λ4 in animals should help improve our understanding of the biology of this novel clinically important interferon in normal and disease conditions.

IFNL4-ΔG Allele Is Associated with an Interferon Signature in Tumors and Survival of African-American Men with Prostate Cancer

Purpose: Men of African ancestry experience an excessive prostate cancer mortality that could be related to an aggressive tumor biology. We previously described an immune-inflammation signature in prostate tumors of African-American (AA) patients. Here, we further deconstructed this signature and investigated its relationships with tumor biology, survival, and a common germline variant in the IFNλ4 (IFNL4) gene. Experimental Design: We analyzed gene expression in prostate tissue datasets and performed genotype and survival analyses. We also overexpressed IFNL4 in human prostate cancer cells. Results: We found that a distinct interferon (IFN) signature that is analogous to the previously described “IFN-related DNA damage resistance signature” (IRDS) occurs in prostate tumors. Evaluation of two independent patient cohorts revealed that IRDS is detected about twice as often in prostate tumors of AA than European-American men. Furthermore, analysis in TCGA showed an association of increased IRDS in prostate tumors with decreased disease-free survival. To explain these observations, we assessed whether IRDS is associated with an IFNL4 germline variant (rs368234815-ΔG) that controls production of IFNλ4, a type III IFN, and is most common in individuals of African ancestry. We show that the IFNL4 rs368234815-ΔG allele was significantly associated with IRDS in prostate tumors and overall survival of AA patients. Moreover, IFNL4 overexpression induced IRDS in three human prostate cancer cell lines. Conclusions: Our study links a germline variant that controls production of IFNλ4 to the occurrence of a clinically relevant IFN signature in prostate tumors that may predominantly affect men of African ancestry. Clin Cancer Res; 24(21); 5471–81. ©2018 AACR.

Abstract 4419: Exploration of alternative pathways mediated by IFNL4 and related to cell proliferation and death in a hepatoma cell line

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA The ability to generate interferon lambda 4 (IFNL4), the most recently discovered member of the type-III interferon family, has been associated with impaired clearance of hepatitis C virus (HCV). IFNL4 signals through an IFNL receptor complex that serves all type-III interferons and consists of receptors IFNLR1 and IL10R2. Signaling through IFNL receptor complex leads to activation of the JAK-STAT pathway and transcriptional activation of interferon-stimulated genes (ISG). Intracellular expression of IFNL4 also causes reduced proliferation and increased cell death in human hepatic cells but it remains unclear if this is a result of JAK-STAT signaling through IFNL receptor complex or through some other alternative mechanisms. To address this question, we used CRISPR-CAS9 system to knock-out IFNLR1 in a hepatoma HepG2 cells. We designed and tested six guide RNAs (gRNAs) for IFNLR1 exons. CAS9 protein and gRNAs that most efficiently induced mutations in IFNLR1 were introduced into HepG2 cells to generate stable cell lines. No obvious defects were observed in IFNLR1-KO-HepG2 cells, indicating that loss of IFNLR1 had no deleterious effects on cell viability. We selected three stable clones which had sequencing-confirmed inactivating IFNLR1 mutations and Western blot-confirmed lack of IFNLR1 protein expression. Compared to the parental HepG2 cells the IFNLR1-KO-HepG2 cells were deficient in the ability to induce the luciferase-tagged interferon-stimulated response element (ISRE-Luc) reporter after treatment with recombinant IFNL4 and IFNL3 proteins, which signal through the same receptor complex. At the same time, response to IFNa treatment was not affected because IFNa signals through its own receptor complex. These results indicate that, as other type-III interferons, IFNL4 induces ISRE signaling through IFNL receptor complex. INFLR1-KO-HepG2 cells provide a highly efficient model for studies of factors involved in signaling of type-III interferons, including IFNL4. Next, we will investigate alternative pathways involved in anti-proliferative and cell death-related phenotypes induced by IFNL4 in HepG2 cell. We will perform RNA-seq on parental and IFNLR1-KO-HepG2 cells treated with or without IFNL4, IFNL3 and IFNa. These results will contribute to a better understanding of IFNL4 function, which can be relevant for viral infections and cancer. Citation Format: Fang Wang, Olusegun O. Onabajo, Nina Rao, Ludmila Prokunina-Olsson. Exploration of alternative pathways mediated by IFNL4 and related to cell proliferation and death in a hepatoma cell line. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4419.

ID: 87: Anti-proliferative and cell death-inducing effects of interferon lambda-4 (IFNL4) may contribute differentially to HCV pathogenesis and cancer

IFN-λ4 is a novel type-III interferon with strong clinical significance in humans. Only individuals who carry the Δ G allele of a genetic variant rs368234815-TT/ Δ G are genetically able to produce IFN-λ4 protein. Carriers of the Δ G allele have impaired ability to clear hepatitis C virus (HCV) infection (Prokunina-Olsson et al., 2013). At the same time, recent findings suggest that carriers of the Δ G allele are protected from mucinous type of ovarian cancer (Kelemen et al., 2015). We explored the function of IFN-λ4 in an inducible cell line HepG2- IFN-λ4 -GFP, which produces a partially secreted IFN-λ4. We observed two main phenotypes caused by IFN-λ4 expression: induction of interferon-stimulated genes (ISGs) both in the expressing and bystander cells, and increased cell death with reduced proliferation in the expressing cells. Both effects were inhibited by a blocking antibody for IFN-λ4, suggesting that they might be related and caused by secreted IFN-λ4. Our results suggest that chronic induction of IFN-λ4 in HCV-infected hepatocytes of individuals with rs368234815- Δ G allele could contribute to pre-activation of interferon signaling competing with effects of type-I interferons (IFN- α ) . IFN-λ4-induced death of hepatic cells may contribute to development of chronic hepatitis and eventual liver failure. At the same time, we suggest that the anti-proliferative effects of IFN-λ4 might be beneficial in other circumstances, and result in prevention of excessive proliferation and elimination of tumor cells. The pathogenic or beneficial effect of IFN-λ4 might depend on cell type specificity and triggers of its expression.

151: Interferon lambda 4 (IFN-λ4) induces a pro-inflammatory state in human hepatic cells which can be blocked by a monoclonal antibody

Interferon lambda 4 (IFN-λ4) is a novel human type-III interferon. An exonic genetic variant (rs368234815-ΔG), which creates the IFN-λ4 protein, is the strongest host factor predicting clearance of hepatitis C virus (HCV) infection. Individuals who carry the ΔG allele and thus can generate IFN-λ4, are less likely to clear HCV compared to individuals who do not carry this allele and are unable to generate IFN-λ4. We show that IFN-λ4 can be detected at a low level with a MesoScale ELISA assay in culture media of human hepatic cells (HepG2 and fresh primary human hepatocytes) transiently transfected with IFNL4-expressing construct. The secreted IFN-λ4 protein is able to induce strong expression of a panel of interferon stimulated genes (ISGs) in a transwell assay and this effect could be blocked by a monoclonal antibody against IFN-λ4 (α-IFN-λ4). Specifically, in primary hepatocytes secreted IFN-λ4 induced strong activation of the pro-inflammatory chemokine IP-10 (encoded by CXCL10) both on mRNA and protein level and this induction was blocked by the α-IFN-λ4 antibody. Increased expression of IP-10 is associated with hepatoinflammation and reduced response to HCV treatment. The α-IFN-λ4 antibody did not block the signalling of other interferons (IFN-α, IFN-β, IFN-γ, and IFN-λ3), suggesting a possible use of this antibody for targeted blocking of IFN-λ4 signalling. In conclusion, we propose a pathogenic mechanism of IFN-λ4 as a moderately secreted interferon stimulating ISGs and inducing a pro-inflammatory state in human hepatic cells; we also suggest a tool of blocking this induction with a monoclonal antibody.