Abstract Stromal fibroblasts reside in inflammatory tissues that are characterized by either immune suppression or activation. Whether and how fibroblasts adapt to these contrasting microenvironments remains unknown. Cancer-associated fibroblasts (CAF) mediate immune quiescence by producing the chemokine CXCL12, which coats cancer cells to suppress T-cell infiltration. We examined whether CAFs can also adopt an immune-promoting chemokine profile. Single-cell RNA sequencing of CAFs from mouse pancreatic adenocarcinomas identified a subpopulation of CAFs with decreased expression of Cxcl12 and increased expression of the T cell–attracting chemokine Cxcl9 in association with T-cell infiltration. TNFα and IFNγ containing conditioned media from activated CD8+ T cells converted stromal fibroblasts from a CXCL12+/CXCL9− immune-suppressive phenotype into a CXCL12−/CXCL9+ immune-activating phenotype. Recombinant IFNγ and TNFα acted together to augment CXCL9 expression, whereas TNFα alone suppressed CXCL12 expression. This coordinated chemokine switch led to increased T-cell infiltration in an in vitro chemotaxis assay. Our study demonstrates that CAFs have a phenotypic plasticity that allows their adaptation to contrasting immune tissue microenvironments.

Cancer immunotherapy frequently fails because most carcinomas have few T cells, suggesting that cancers can suppress T cell infiltration. Here, we show that cancer cells of human pancreatic ductal adenocarcinoma (PDA), colorectal cancer, and breast cancer are coated with transglutaminase-2 (TGM2)-dependent covalent CXCL12-keratin-19 (KRT19) heterodimers that are organized as filamentous networks. Since a dimeric form of CXCL12 suppresses the motility of human T cells, we determined whether this polymeric CXCL12-KRT19 coating mediated T cell exclusion. Mouse tumors containing control PDA cells exhibited the CXCL12-KRT19 coating, excluded T cells, and did not respond to treatment with anti-PD-1 antibody. Tumors containing PDA cells not expressing either KRT19 or TGM2 lacked the CXCL12-KRT19 coating, were infiltrated with activated CD8(+) T cells, and growth was suppressed with anti-PD-1 antibody treatment. Thus, carcinomas assemble a CXCL12-KRT19 coating to evade cancer immune attack.

The coxsackievirus and adenovirus receptor (CAR) is an essential cellular protein that is involved in cell adhesion, cell signaling, and viral infection. The 8-exon encoded isoform (CAREx8) resides at the apical surface of polarized epithelia, where it is accessible as a receptor for adenovirus entering the airway lumen. Given its pivotal role in viral infection, it is a target for antiviral strategies. To understand the regulation of CAREx8 and determine the feasibility of receptor downregulation, the half-life of total and apical localized CAREx8 was determined and correlated with adenovirus transduction. Total and apical CAREx8 has a relatively short half-life of approximately 2 h. The half-life of apical CAREx8 correlates well with adenovirus transduction. These results suggest that antiviral strategies that aim to degrade the primary receptor for apical adenovirus infection will be effective within a relatively short time frame after application.

Adenoviruses (AdVs) are etiological agents of gastrointestinal, heart, eye, and respiratory tract infections that can be lethal for immunosuppressed people. Many AdVs use the coxsackievirus and adenovirus receptor (CAR) as a primary receptor. The CAR isoform resulting from alternative splicing that includes the eighth exon, CAREx8, localizes to the apical surface of polarized epithelial cells and is responsible for the initiation of AdV infection. We have shown that the membrane level of CAREx8 is tightly regulated by two MAGI-1 PDZ domains, PDZ2 and PDZ4, resulting in increased or decreased AdV transduction, respectively. We hypothesized that targeting the interactions between the MAGI-1 PDZ2 domain and CAREx8 would decrease the apical CAREx8 expression level and prevent AdV infection. Decoy peptides that target MAGI-1 PDZ2 were synthesized (TAT-E6 and TAT-NET1). PDZ2 binding peptides decreased CAREx8 expression and reduced AdV transduction. CAREx8 degradation was triggered by the activation of the regulated intramembrane proteolysis (RIP) pathway through a disintegrin and metalloproteinase (ADAM17) and γ-secretase. Further analysis revealed that ADAM17 interacts directly with the MAGI-1 PDZ3 domain, and blocking the PDZ2 domain enhanced the accessibility of ADAM17 to the substrate (CAREx8). Finally, we validated the efficacy of TAT-PDZ2 peptides in protecting the epithelia from AdV transduction in vivo using a novel transgenic animal model. Our data suggest that TAT-PDZ2 binding peptides are novel anti-AdV molecules that act by enhanced RIP of CAREx8 and decreased AdV entry. This strategy has additional translational potential for targeting other viral receptors that have PDZ binding domains, such as the angiotensin-converting enzyme 2 receptor. IMPORTANCE Adenovirus is a common threat in immunosuppressed populations and military recruits. There are no currently approved treatments/prophylactic agents that protect from most AdV infections. Here, we developed peptide-based small molecules that can suppress AdV infection of polarized epithelia by targeting the AdV receptor, coxsackievirus and adenovirus receptor (CAREx8). The newly discovered peptides target a specific PDZ domain of the CAREx8-interacting protein MAGI-1 and decrease AdV transduction in multiple polarized epithelial models. Peptide-induced CAREx8 degradation is triggered by extracellular domain (ECD) shedding through ADAM17 followed by γ-secretase-mediated nuclear translocation of the C-terminal domain. The enhanced shedding of the CAREx8 ECD further protected the epithelium from AdV infection. Taken together, these novel molecules protect the epithelium from AdV infection. This approach may be applicable to the development of novel antiviral molecules against other viruses that use a receptor with a PDZ binding domain.

C-C chemokine receptor type 2 (CCR2) is expressed on monocytes and facilitates their recruitment to tumors. Though breast cancer cells also express CCR2, its functions in these cells are unclear. We found that Ccr2 deletion in cancer cells led to reduced tumor growth and approximately twofold longer survival in an orthotopic, isograft breast cancer mouse model. Deletion of Ccr2 in cancer cells resulted in multiple alterations associated with better immune control: increased infiltration and activation of cytotoxic T lymphocytes (CTLs) and CD103(+). cross-presenting dendritic cells (DCs), as well as up-regulation of MHC class I and down-regulation of checkpoint regulator PD-L1 on the cancer cells. Pharmacological or genetic targeting of CCR2 increased cancer cell sensitivity to CTLs and enabled the cancer cells to induce DC maturation toward the CD103(+). subtype. Consistently, Ccr2(-/-) cancer cells did not induce immune suppression in Batf(3/-) mice lacking CD103(+) DCs. Our results establish that CCR2 signaling in cancer cells can orchestrate suppression of the immune response.

Notch activation is highly prevalent among cancers, in particular T-cell acute lymphoblastic leukemia (T-ALL). However, the use of pan-Notch inhibitors to treat cancers has been hampered by adverse effects, particularly intestinal toxicities. To circumvent this barrier in T-ALL, we aimed to inhibit ETS1, a developmentally important T-cell transcription factor previously shown to cobind Notch response elements. Using complementary genetic approaches in mouse models, we show that ablation of Etsl leads to strong Notch-mediated suppressive effects on T-cell development and leukemogenesis but milder intestinal effects than pan-Notch inhibitors. Mechanistically, genome-wide chromatin profiling studies demonstrate that Etsl inactivation impairs recruitment of multiple Notch-associated factors and Notch-dependent activation of transcriptional elements controlling major Notch-driven oncogenic effector pathways. These results uncover previously unrecognized hierarchical heterogeneity of Notch-controlled genes and point to Etsl-mediated enucleation of Notch-Rbpj transcriptional complexes as a target for developing specific anti-Notch therapies in T-ALL that circumvent the barriers of pan-Notch inhibition. SIGNIFICANCE: Notch signaling controls developmentally important and tissue-specific activities, raising barriers for developing anti-Notch therapies. Pivoting away from pan-Notch inhibitors, we show antileukemic but less toxic effects of targeting ETS1, a T-cell NOTCH1 cofactor. These results demonstrate the feasibility of context-dependent suppression of NOTCH1 programs for the treatment of T-ALL.

Improving the efficacy of immunotherapies in pancreatic cancer can increase patient survival rate. Microsatellite-stable pancreatic adenocarcinoma (PDA) exclude T cells and resist immunotherapy. This may occur by cancer cells coating themselves with CXCL12, a chemokine produced by cancer associated fibroblasts (CAFs). However, PDA with microsatellite instability (MSI-H) is infiltrated with T cells and responsive to PD-1 checkpoint inhibition. We found that human MSI-H pancreatic cancer cells display the CXCL12-coat, suggesting additional factors control pancreatic cancer immune accessibility. We hypothesized that expression of neoantigens by MSI-H PDA is an independent variable that overcomes T cell exclusion mediated by the CXCL12-coat. To test this, we established a mouse hepatic PDA metastases model in which PDA cells express doxycycline-induced ovalbumin (OVA), and adoptively transferred OVA-specific OT-I CD8+ T cells report intra-tumoral accumulation of T cells. Inducing OVA expression in established metastases increases intra-tumoral accumulation and activation of OT-I cells, despite the presence of the CXCL12-coat. We are currently investigating whether high- and low-affinity antigen-expressing cancers are subject to T cell mediated elimination in the presence and absence of the CXCL12-coat, respectively. The CXCL12-coat on PDA cells and the expression of neoantigens can independently control immune response. As the formation of the CXCL12-coat is facilitated by the Transglutaminase-2 (TGM2) and can be inhibited by TGM2 inhibitors (Z Wang et al., bioRxiv, 2019), these findings reveal potential combination therapies to overcome pancreatic cancer immunotherapy resistance.

Notch1 signaling must elevate to high levels in order to drive the proliferation of CD4-CD8- double-negative (DN) thymocytes and progression to the CD4+CD8+ double-positive (DP) stage through β-selection. During this critical phase of pre-T-cell development, which is also known as the DN-DP transition, it is unclear whether the Notch1 transcriptional complex strengthens its signal output as a discrete unit or through cofactors. We previously showed that the protein inhibitor of activated STAT-like coactivator Zmiz1 is a context-dependent cofactor of Notch1 in T-cell leukemia. We also showed that withdrawal of Zmiz1 generated an early T-lineage progenitor (ETP) defect. Here, we show that this early defect seems inconsistent with loss-of-Notch1 function. In contrast, at the later pre-T-cell stage, withdrawal of Zmiz1 impaired the DN-DP transition by inhibiting proliferation, like withdrawal of Notch. In pre-T cells, but not ETPs, Zmiz1 cooperatively regulated Notch1 target genes Hes1, Lef1, and Myc. Enforced expression of either activated Notch1 or Myc partially rescued the Zmiz1-deficient DN-DP defect. We identified residues in the tetratricopeptide repeat (TPR) domain of Zmiz1 that bind Notch1. Mutating only a single residue impaired the Zmiz1-Notch1 interaction, Myc induction, the DN-DP transition, and leukemic proliferation. Similar effects were seen using a dominant-negative TPR protein. Our studies identify stage-specific roles of Zmiz1. Zmiz1 is a context-specific cofactor for Notch1 during Notch/Myc-dependent thymocyte proliferation, whether normal or malignant. Finally, we highlight a vulnerability in leukemic cells that originated from a developmentally important Zmiz1-Notch1 interaction that is hijacked during transformation from normal pre-T cells.

Division of Hematology-Oncology, Department of Internal Medicine, University of Michigan School of Medicine, Ann Arbor, MI, 48109, USA; Watson School of Biological Sciences, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 11724; Cell and Molecular Biology Program, Department of Cell and Developmental Biology, University of Michigan School of Medicine, Ann Arbor, MI, 48109, USA. Cincinnati Children's Hospital Medical Center. Cincinnati, OH; Life Sciences Institute, Department of Biostatistics, University of Michigan School of Medicine, Ann Arbor, MI, 48109, USA; Division of Hematology-Oncology, Department of Medicine and Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA 19104, USA. Department of Pathology, University of Michigan School of Medicine, Ann Arbor, MI, 48109, USA.

The discovery of NOTCH1 as the most frequently mutated oncogene in T-ALL patients raised hopes for targeted therapy in this cancer. Unfortunately, in clinical trials, the pan-Notch inhibitor GSI caused excessive GI toxicity. Mice treated continuously with GSI die from intestinal stem cell loss and severe intestinal secretory cell metaplasia. Intermittent dosing of GSI is tolerable, but has weak anti-cancer effects. Thus, the challenge has been to find ways to selectively disable Notch in T-ALL. Our idea to meet this challenge stems from work by others showing that Notch cannot activate enhancers by itself. Notch requires a favorable "chromatin context" at its enhancers that is created by cooperating transcription factors. In theory, one could target cell-specific factors at these enhancers in order to avoid the intolerable effects of pan-Notch inhibition. In support of this, others showed that ubiquitous deletion of the T-cell specific Notch-dependent Myc enhancer in mice impairs T-ALL proliferation and thymopoiesis, but has no effect on other tissues. We previously showed that the transcriptional coactivator Zmiz1 is a direct cofactor of Notch1 that selectively promotes Notch activity at the T-cell Myc enhancer. However, it was unclear what other factors promote context-dependent Notch activity. Ets1 is an attractive candidate. It can bind nucleosome-occupied regions in T-cell precursors and most Notch response elements in T-ALL cells, including the T-cell MYC enhancer. To investigate its importance, we generated conditional Ets1 knockout mice. Deletion of Ets1 in hematopoietic cells using the VavCre transgene caused a 21-fold loss of thymocytes starting at the earliest stage. This was 4-fold more severe than the loss of thymocytes in Notch-deficient mice. Deletion of Ets1 using a ubiquitous tamoxifen-inducible Cre caused a Notch loss-of-function phenotype in the intestine with a 1.4 to 2.3-fold increase in goblet cells. This was milder than the effects of GSI (3.3 to 4.2-fold increase). ~64% of the Ets1-deleted mice died from unclear causes. In vivo deletion of Ets1 in Notch1-induced murine T-ALLs reduced blast counts by 30-fold and prolonged survival. In a panel of human T-ALL cell lines, on average, knockdown with two different shEts1 reduced proliferation by 2 and 9-fold respectively over ~1.5 weeks of culture. This was superior to the effects of GSI (up to 2-fold inhibition). A small molecule inhibitor of Usp9x, the deubiquitinase of Ets1, induced Ets1 protein degradation and impaired T-ALL cell proliferation with submicromolar GI50. In PDX models, shEts1 reduced circulating blasts by 44-fold and prolonged survival. To identify the mechanism by which Ets1 promotes T-ALL, we performed endogenous co-IP assays, which showed that Ets1 interacts with Notch1 and its cofactor Zmiz1. Further, Ets1 binding by ChIP correlated with Zmiz1 binding (R2=0.93). Knockdown of Ets1 reduced Zmiz1, Ets1, and Notch1 binding to enhancers of major T-ALL oncogenes, MYC and IL7R. RNA-Seq showed that Ets1 co-regulates the expression of ~30% of Notch1 target genes. Multiple MSigDB enrichment analyses of both Ets1 and Notch-regulated genes showed that the MYC and MTORC pathways were the #1 or #2 most enriched list. Enforced expression of Myc partially rescued the proliferation of human T-ALL cell lines deprived of Ets1. Based on these data, we predicted that Ets1 inhibition would sensitize enhancers to Notch inhibition. Accordingly, Ets1 withdrawal promoted the effects of GSI in repressing Myc expression and cell proliferation. Further, in our mouse model of Notch-induced T-ALL, Ets1 deletion in combination with intermittent doses of GSI reduced blast counts and prolonged survival more effectively than either treatment alone. Our data support an emerging model in which cofactors like Ets1 create a favorable chromatin context for Notch1 to activate a subset of response elements. The context dependence of Ets1 action, which promotes certain oncogenic signals of Notch1 in T cells, might be clinically relevant. Ets1 deprivation inhibited thymopoiesis and leukemic proliferation more effectively and with less intestinal toxicity than Notch deprivation. Our data suggest that inhibiting Ets1, possibly through targeted protein degradation, would combat important drivers of the Notch pathway with reduced adverse effects linked to pan-Notch inhibition. No relevant conflicts of interest to declare.

Patients who have had their primary pancreatic ductal adenocarcinoma (PDA) surgically resected often develop metastatic disease, exemplifying the problem of latent metastases. Livers from patients and mice with PDA contained single, disseminated cancer cells (DCCs) with an unusual phenotype of being cytokeratin-19 (CK19) - and MHC class I (MHCI) - . We created a mouse model to determine how DCCs develop, their relationship to metastatic latency, and the role of immunity. Intra-portal injection of immunogenic PDA cells into pre-immunized mice seeded livers only with single, non-replicating DCCs lacking MHCI and CK19; naïve recipients had macro-metastases. Transcriptomic analysis of PDA cells with the DCC phenotype demonstrated an endoplasmic reticulum (ER) stress response. Relieving ER stress with a chemical chaperone, in combination with T cell-depletion, stimulated outgrowth of macro-metastatic lesions containing PDA cells expressing MHCI and CK19. The ER stress response is the cell-autonomous reaction that enables DCCs to escape immunity and establish latent metastases. One sentence summary: Latent pancreatic cancer metastases are created when T cells select disseminated cancer cells in which immune resistance and quiescence have been imposed by endoplasmic stress.

When stem cells first enter the thymus and become early T-cell precursor (ETP) cells, they are exposed to high levels of Notch1 ligand. Notch1 signal strength must be tightly regulated because on one hand, excessive Notch1 signals drive premature T-cell commitment, resulting in loss of ETP cells and alternative cell fates. On the other hand, complete loss of Notch1 signals impairs ETP proliferation, also resulting in loss of ETP cells. Thus, keeping Notch signals finely balanced in ETP cells preserves "stemness". However, after ETP cells commit to the T-cell lineage by the DN3 cell stage, Notch1 signals ramp up dramatically to drive proliferation. It is unclear how Notch1 signals are initially restrained and then amplified. We previously showed that the PIAS-like coregulator Zmiz1 is a direct, context-dependent cofactor of Notch1 in T-cell leukemia. In contrast to drosophila Zmiz1, mammalian Zmiz1 evolved a tetratricopeptide repeat (TPR) domain that binds directly to Notch1 and selectively induces oncogenic target genes such as Myc through its transactivation domain (TAD).

The Coxsackievirus and adenovirus receptor (CAR) is an essential cellular protein that is involved in cell–cell adhesion, protein trafficking, and viral infection. The major isoform of CAR is selectively sorted to the basolateral membrane of polarized epithelial cells where it co-localizes with the cellular scaffolding protein membrane-associated guanylate kinase with inverted domain structure-1 (MAGI-1). Previously, we demonstrated CAR interacts with MAGI-1 through a PDZ–domain dependent interaction. Here, we show that the PDZ3 domain of MAGI-1 is exclusively responsible for the high affinity interaction between the seven exon isoform of CAR and MAGI-1 using yeast-two-hybrid analysis and confirming this interaction biochemically and in cellular lysates by in vitro pull down assay and co-immunoprecipitation. The high affinity interaction between the PDZ3 domain and CAR C-terminus was measured by fluorescence resonance energy transfer. Further, we investigated the biological relevance of this high affinity interaction between CAR and the PDZ3 domain of MAGI-1 and found that it does not alter CAR-mediated adenovirus infection. By contrast, interruption of this high affinity interaction altered the localization of MAGI-1 indicating that CAR is able to traffic MAGI-1 to cell junctions. These data deepen the molecular understanding of the interaction between CAR and MAGI-1 and indicate that although CAR plays a role in trafficking PDZ-based scaffolding proteins to cellular junctions, association with a high affinity intracellular binding partner does not significantly alter adenovirus binding and entry via CAR.

Adenovirus-mediated gene therapy has been limited by the fact that the primary receptor for most adenovirus serotypes, the Coxsackievirus and adenovirus receptor (CAR), is inaccessible or not expressed on many cell types of interest. Few mechanisms have been discovered that regulate CAR expression and tissue specific localization. In the airway, CAR is mostly considered a cell-cell adhesion protein localized at the basolateral surface of polarized epithelia. Recently, an alternate isoform of CAR, CAREx8, has been identified at the apical surface of polarized airway epithelia and is implicated in viral infection from the apical surface. We hypothesized that upregulation of cellular mechanisms that facilitate endogenous CAREx8 protein expression at the apical surface would enhance adenovirus gene transfer. Using polarized model epithelial cell lines and primary human airway epithelia, we found that IL-8, a proinflammatory cytokine and a neutrophil chemoattractant, stimulates the protein expression and apical localization of CAREx8 via activation of AKT/S6K and inhibition of GSK3β. IL-8- mediated upregulation of CAREx8 increased AdV5-β-Gal entry and transduction by approximately 5-fold. Moreover, we found that infiltrating neutrophils bind CAREx8 at the apical surface of a polarized epithelium and, surprisingly, neutrophils enhance AdV5-β-Gal entry into the epithelium by 2-3 fold. The effect of IL-8 and neutrophils on AdV infection could be blocked by fiber-knob from AdV5 but not AdV3, a non-CAR binding serotype, indicating the importance of CAR. These findings suggest that acute inflammation may enhance adenovirus infection. Moreover, therapeutics that stimulate the AKT/S6K pathway or inhibit GSK3β may be able to augment adenovirus-mediated gene therapy.

Prevention of viral-induced respiratory disease begins with an understanding of the factors that increase or decrease susceptibility to viral infection. The primary receptor for most adenoviruses is the coxsackievirus and adenovirus receptor (CAR), a cell-cell adhesion protein normally localized at the basolateral surface of polarized epithelia and involved in neutrophil transepithelial migration. Recently, an alternate isoform of CAR, CAREx8, has been identified at the apical surface of polarized airway epithelia and is implicated in viral infection from the apical surface. We hypothesized that the endogenous role of CAREx8 may be to facilitate host innate immunity. We show that IL-8, a proinflammatory cytokine and a neutrophil chemoattractant, stimulates the protein expression and apical localization of CAREx8 via activation of AKT/S6K and inhibition of GSK3β. Apical CAREx8 tethers infiltrating neutrophils at the apical surface of a polarized epithelium. Moreover, neutrophils present on the apical-epithelial surface enhance adenovirus entry into the epithelium. These findings suggest that adenovirus evolved to co-opt an innate immune response pathway that stimulates the expression of its primary receptor, apical CAREx8, to allow the initial infection the intact epithelium. In addition, CAREx8 is a new target for the development of novel therapeutics for both respiratory inflammatory disease and adenoviral infection.

Pan-NOTCH inhibitors are poorly tolerated in clinical trials because NOTCH signals are crucial for intestinal homeostasis. These inhibitors might also promote cancer because NOTCH can act as a tumor suppressor. We previously reported that the PIAS-like coactivator ZMIZ1 is frequently co-expressed with activated NOTCH1 in T cell acute lymphoblastic leukemia (T-ALL). Here, we show that similar to Notch1, Zmiz1 was important for T cell development and controlled the expression of certain Notch target genes, such as Myc. However, unlike Notch, Zmiz1 had no major role in intestinal homeostasis or myeloid suppression. Deletion of Zmiz1 impaired the initiation and maintenance of Notch-induced T-ALL. Zmiz1 directly interacted with Notch1 via a tetratricopeptide repeat domain at a special class of Notch-regulatory sites. In contrast to the Notch cofactor Maml, which is nonselective, Zmiz1 was selective. Thus, targeting the NOTCH1-ZMIZ1 interaction might combat leukemic growth while avoiding the intolerable toxicities of NOTCH inhibitors.

The Notch1 receptor is required throughout normal T-cell development. NOTCH1 is also the most recurrently mutated oncogene in T-ALL, occurring in ~60% of human samples. However, Notch inhibitors cannot be used at full-strength because of intolerable on-target side effects, such as GI toxicity. Another concern is that these inhibitors would reverse the tumor suppressor functions of Notch. Lower doses of Notch inhibitors are better tolerated, but lead to residual Notch signaling. Moreover, collaborating pathways can reinforce these weak signals and drive resistance. Thus, it is important to identify the collaborative factors that selectively amplify the oncogenic functions of Notch1 as opposed to the physiological and tumor suppressor functions. We previously reported that the PIAS coactivator Zmiz1 and Notch1 alleles collaborated to induce T-ALL in mice. ZMIZ1 and activated NOTCH1 were co-expressed in 20-30% of patient samples and cell lines.

ABSTRACT Epithelial integrity is essential for homeostasis and poses a formidable barrier to pathogen entry. Major factors for viral entry into epithelial cells are the localization and abundance of the primary receptor. The coxsackievirus and adenovirus receptor (CAR) is a primary receptor for these two pathogenic groups of viruses. In polarized epithelia, a low-abundance, alternatively spliced eight-exon isoform of CAR, CAREx8, is localized apically where it can support viral infection from the air-exposed surface. Using biochemical, cell biology, genetic, and spectroscopic approaches, we show that the levels of apical CAREx8 are negatively regulated by the PDZ domain-containing protein MAGI-1 (membrane-associated guanylate kinase with inverted orientation protein-1) and that two MAGI-1 PDZ domains, PDZ1 and PDZ3, regulate CAREx8 levels in opposing ways. Similar to full-length MAGI-1, expression of the isolated PDZ3 domain significantly reduces cell surface CAREx8 abundance and adenovirus infection. In contrast, the PDZ1 domain is able to rescue CAREx8 and adenovirus infection from MAGI-1-mediated suppression. These data suggest a novel cell-based strategy to either suppress viral infection or augment adenovirus-based gene therapy.