Human Murine Models Research Articles

Anti-IL-8 antibody activates myeloid cells and potentiates the anti-tumor activity of anti-PD-1 antibody in the humanized pancreatic cancer murine model

Pancreatic ductal adenocarcinoma(PDAC) does not respond to single-agent immune checkpoint inhibitor therapy, including anti-PD-1 antibody(aPD-1) therapy. Higher plasma levels of IL-8 are associated with poorer outcomes in patients who receive aPD-1 therapies, providing a rationale for combination immunotherapy with an anti-IL-8 antibody(aIL-8) and aPD-1. We thus investigated whether human aIL-8 therapy can potentiate the antitumor activity of aPD-1 and further investigated how the combination affects the immune response by regulating myeloid cells in the tumor microenvironment in a humanized murine model of PDAC with a reconstituted immune system consisting of human T cells and a combination of CD14+ and CD16+ myeloid cells. The results show that the combination of aIL-8 and aPD-1 treatment significantly enhanced antitumor activity following the infusion of myeloid cells. Our results further showed that the target of IL-8 is mainly present in CD16+ myeloid cells and is likely to be granulocytes. FACS analysis showed that aIL-8 treatment increased granulocytic myeloid cells in tumors. Consistently, single-nuclear RNA-sequencing analysis of tumor tissue showed that the innate immune response and cytokine response pathways in the myeloid cell cluster were activated by aIL-8 treatment. This is the first preclinical study using a humanized mouse model for new combination immunotherapeutic development and supports the further clinical testing of aIL-8 in combination with aPD-1 for PDAC treatment. This study also suggests that peripherally derived myeloid cells can potentiate the antitumor response of T cells, likely through the innate immune response, and aIL-8 re-educates tumor-infiltrating myeloid cells by activating the innate immune response.

Labile iron accumulation augments T follicular helper cell differentiation.

T follicular helper (Tfh) cells are a subset of CD4+ T cells that are essential in the pathogenesis of systemic lupus erythematosus (SLE). Notably, iron is required for activated CD4+ T lymphocytes to sustain high proliferation and metabolism. In this issue of the JCI, Gao et al. showed that CD4+ T cells from patients with SLE accumulated iron, augmenting their differentiation into Tfh cells and correlating with disease activity. Using human cells and murine models, the authors demonstrated that miR-21 was overexpressed in lupus T cells and inhibited 3-hydroxybutyrate dehydrogenase-2 (BDH2). The subsequent loss of BDH2 drove labile iron to accumulate in the cytoplasm and promoted TET enzyme activity, BCL6 gene demethylation, and Tfh cell differentiation. This work identifies a role for iron in CD4+ T cell biology and the development of pathogenic effectors in SLE. We await future investigations that could determine whether modulating iron levels could regulate Tfh cells in human health and disease.

Regulation of metabolism by protein modifications in autoimmunity

Abstract In type 1 diabetes (T1D), inflammation and oxidative stress in pancreatic islets amplifies various post-translational modifications (PTMs) of self-proteins. The loss of immune tolerance to islet PTMs triggers an autoreactive T cell response and contributes to the destruction of insulin-producing beta cells in T1D. The major function of beta cells is to secret insulin in response to glucose uptake in order to maintain glucose homeostasis. In this study, we identify autoantibodies against to beta subunit of prolyl-4-hydroxylase (P4Hb; native and carbonylated form) and glucokinase (GK; native and citrullinated form) in both human T1D and murine models. Glucokinase serves as a glucose sensor to initiate glycolysis and insulin signaling in beta cells. P4Hb is required for the accurate folding of insulin. By mass spectrometry, six carbonyl residues and eleven citrulline residues were mapped in oxidative rhP4Hb and PAD-treated rhGK, respectively. Of interest, autoreactive CD4+ T cells to citrullinated GK epitopes are present in the circulation of T1D patients. In regards to glucose metabolism, the carbonylated-P4Hb is amplified in stressed human islets coincident with decreased glucose-stimulated insulin secretion and disturb (increase) proinsulin to insulin ratios. The citrullination alters GK biologic activity (Km) and suppresses glucose-stimulated insulin secretion. Moreover, PAD2/4 inhibitors can partially restore IFNg +IL-1b suppressed glucose stimulated insulin secretion in INS-1E beta cells. Our studies implicate the crucial enzymes, glucokinase and P4Hb, as the biomarkers, providing new insights into creating autoantigens and define the impact of metabolic PTMs on the aberrant beta cell functions of T1D. This research was supported by the Juvenile Diabetes Research Foundation (Innovative Grant to M.J.M., 2-SRA-2018-551-S-B to E.A.J.), and National Institutes of Health (DK104205-01 to K.H. and M.J.M.).

IL-20 promotes cutaneous inflammation and peripheral itch sensation in atopic dermatitis.

Atopic dermatitis (AD) is a chronic skin disease, which is associated with intense itch, skin barrier dysfunction and eczematous lesions. Aberrant IL‐20 expression has been implicated in numerous inflammatory diseases, including psoriasis. However, the role of IL‐20 in AD remains unknown. Here, RNA‐seq, Q‐PCR, and immunocytochemistry were utilized to examine disease‐driven changes of IL‐20 and its cognate receptor subunits in skin from healthy human subjects, AD patients and murine AD‐models. Calcium imaging, knockdown and cytokine array were used to investigate IL‐20‐evoked responses in keratinocytes and sensory neurons. The murine cheek model and behavioral scoring were employed to evaluate IL‐20‐elicited sensations in vivo. We found that transcripts and protein of IL‐20 were upregulated in skin from human AD and murine AD‐like models. Topical MC903 treatment in mice ear enhanced IL‐20R1 expression in the trigeminal sensory ganglia, suggesting a lesion‐associated and epidermal‐driven mechanism for sensitization of sensory IL‐20 signaling. IL‐20 triggered calcium influx in both keratinocytes and sensory neurons, and promoted their AD‐related molecule release and transcription of itch‐related genes. In sensory neurons, IL‐20 application increased TLR2 transcripts, implicating a link between innate immune response and IL‐20. In a murine cheek model of acute itch, intradermal injection IL‐20 and IL‐13 elicited significant itch‐like behavior, though only when co‐injected. Our findings provide novel insights into IL‐20 function in peripheral (skin‐derived) itch and clinically relevant intercellular neuron‐epidermal communication, highlighting a role of IL‐20 signaling in the pathophysiology of AD, thus forming a new basis for the development of a novel antipruritic strategy via interrupting IL‐20 epidermal pathways.

Combined inhibition of BET bromodomain and mTORC1/2 provides therapeutic advantage for rhabdomyosarcoma by switching cell death mechanism.

Aberrant activation of multiple complex signaling pathways underlies the pathogenesis of rhabdomyosarcoma (RMS), which remains a cause of mortality in approximately 30% of children with RMS. Bromodomain and extraterminal (BET) domain chromatin remodeling regulates several of these pathways. Here, we targeted bromodomain 4 (BRD4) in combination with another molecular metabolic tumor driver, the Akt/mTOR signaling pathway, to provide a highly effective treatment for this neoplasm. We demonstrated that a nexus of these two molecular pathways underlies RMS pathogenesis. Our data show that the combined inhibition of the BET bromodomain and mTORC1/2 signaling abrogates aggressive RMS growth. Thus, the bromodomain inhibitor RVX-208 significantly augmented the therapeutic effects of the dual mTORC1/2 inhibitors, OSI-027 and PP242, both in vitro and in a human xenograft murine model. Drug-treated residual tumors showed a decrease in the activation of underlying signaling mechanisms characterized by a reduction in the expression of p-AKT, p-mTOR, p-p70S6K, cyclin D1, and proliferation. Our ChIP-seq data demonstrated that RVX-208 effectively blocked BRD4 occupancy on its target promoters. ChIP-qPCR assays further confirmed that RVX-208 treatment resulted in a significant decrease in H3K27ac and H4K8ac signals at their target loci. While single RVX-208 treatment induces apoptosis and a single mTORC1/2 inhibitor induces macropinocytosis, their combined treatment led to necroptosis-mediated cell death. These data suggest that combined treatment with drugs targeting BRD4 and mTORC1/2 may be an effective therapeutic intervention for drug-resistant RMS.

Chronic stress promotes an immunologic inflammatory state and head and neck cancer growth in a humanized murine model.

Despite the importance of immune response and environmental stress on head and neck cancer (HNC) outcomes, no current pre-clinical stress model includes a humanized immune system. We investigated the effects of chronic stress induced by social isolation on tumor growth and human immune response in subcutaneous HNC tumors grown in NSG-SGM3 mice engrafted with a human immune system. Tumor growth (p< 0.0001) and lung metastases (p= 0.035) were increased in socially isolated versus control animals. Chronic stress increased intra-tumoral CD4+ T-cell infiltrate (p= 0.005), plasma SDF-1 (p< 0.0001) expression, and led to tumor cell dedifferentiation toward a cancer stem cell phenotype (CD44+ /ALDHhigh , p= 0.025). Chronic stress induced immunophenotypic changes, increased tumor growth, and metastasis in HNC in a murine model with a humanized immune system. This model system may provide further insight into the immunologic and oncologic impact of chronic stress on patients with HNC.

Immune Dysfunction, Cytokine Disruption, and Stromal Changes in Myelodysplastic Syndrome: A Review.

Myelodysplastic syndromes (MDS) are myeloid neoplasms characterized by bone marrow dysfunction and increased risk of transformation to leukemia. MDS represent complex and diverse diseases that evolve from malignant hematopoietic stem cells and involve not only the proliferation of malignant cells but also the dysfunction of normal bone marrow. Specifically, the marrow microenvironment—both hematopoietic and stromal components—is disrupted in MDS. While microenvironmental disruption has been described in human MDS and murine models of the disease, only a few current treatments target the microenvironment, including the immune system. In this review, we will examine current evidence supporting three key interdependent pillars of microenvironmental alteration in MDS—immune dysfunction, cytokine skewing, and stromal changes. Understanding the molecular changes seen in these diseases has been, and will continue to be, foundational to developing effective novel treatments that prevent disease progression and transformation to leukemia.

IL-17 Receptor Signaling through IL-17A or IL-17F Is Sufficient to Maintain Innate Response and Control of Helicobacter pylori Immunopathogenesis.

IL-17R signaling is required for control of extracellular pathogens and is also implicated in development of chronic inflammatory processes. The response to the human pathogen Helicobacter pylori results in Th1 and Th17 cell activation and a chronic inflammatory process that can lead to adverse outcomes, such as gastric cancer. Previously, we identified IL-17RA as a requirement for the recruitment of neutrophils and control of H. pylori colonization in the gastric mucosa. Unexpectedly, H. pylori-infected Il17ra -/- mice had significantly more chronic inflammation than H. pylori-infected wild-type mice. In this study, human epithelial cell lines and murine models were used to investigate differential roles for IL-17A, IL-17F, and IL-17A/F during H. pylori infection. Moreover, the hypothesis that IL-17RA signaling, specifically in lymphocytes, provides an autocrine feedback loop that downregulates Th17 cytokine production was investigated. The data indicate that epithelial cells exhibit a stronger response to IL-17A and IL-17A/F than IL-17F, and that IL-17A and IL-17A/F can synergize with TNF and IL-22 to induce antimicrobial genes of gastric epithelial cells. In vivo deficiencies of IL-17A or IL-17F alone did not significantly change the immunopathological response to H. pylori, but if both cytokines were absent, a hyperinflammatory lymphocytic response developed. Using a cre/flox targeting approach for IL-17RA combined with infection, our findings demonstrate that increased chronic inflammation in Il17ra -/- mice was not attributed to a T cell-intrinsic defect. These data imply that IL-17A and IL-17F may have overlapping roles in maintenance of the gastric mucosal response to infection.

ApoA-I mimetics reduce systemic and gut inflammation in chronic treated HIV.

Novel therapeutic strategies are needed to attenuate increased systemic and gut inflammation that contribute to morbidity and mortality in chronic HIV infection despite potent antiretroviral therapy (ART). The goal of this study is to use preclinical models of chronic treated HIV to determine whether the antioxidant and anti-inflammatory apoA-I mimetic peptides 6F and 4F attenuate systemic and gut inflammation in chronic HIV. We used two humanized murine models of HIV infection and gut explants from 10 uninfected and 10 HIV infected persons on potent ART, to determine the in vivo and ex vivo impact of apoA-I mimetics on systemic and intestinal inflammation in HIV. When compared to HIV infected humanized mice treated with ART alone, mice on oral apoA-I mimetic peptide 6F with ART had consistently reduced plasma and gut tissue cytokines (TNF-α, IL-6) and chemokines (CX3CL1) that are products of ADAM17 sheddase activity. Oral 6F attenuated gut protein levels of ADAM17 that were increased in HIV-1 infected mice on potent ART compared to uninfected mice. Adding oxidized lipoproteins and endotoxin (LPS) ex vivo to gut explants from HIV infected persons increased levels of ADAM17 in myeloid and intestinal cells, which increased TNF-α and CX3CL1. Both 4F and 6F attenuated these changes. Our preclinical data suggest that apoA-I mimetic peptides provide a novel therapeutic strategy that can target increased protein levels of ADAM17 and its sheddase activity that contribute to intestinal and systemic inflammation in treated HIV. The large repertoire of inflammatory mediators involved in ADAM17 sheddase activity places it as a pivotal orchestrator of several inflammatory pathways associated with morbidity in chronic treated HIV that make it an attractive therapeutic target.

Abstract P075: NTX-1088, a potent first-in-class, anti-PVR mAb, restores expression and function of DNAM1 for optimal DNAM1-mediated antitumor immunity

Abstract The poliovirus receptor (PVR, CD155) represents a resistance mechanism to approved immune checkpoint inhibitors (ICIs). It is a key regulator of immune activation, that modifies immune function through multiple mechanisms. Increased levels of PVR expression on tumor cells have been associated with resistance to anti-PD-(L)1 therapy in clinical settings, while loss of PVR led to reduced tumor growth in multiple pre-clinical models. Targeting PVR using blocking mAbs offers an attractive therapeutic approach for patients with advanced cancer. NTX-1088 is a first-in-class, potent, anti-PVR mAb being developed for the treatment of solid tumors. The antibody binds to PVR with high affinity, blocks its interactions with TIGIT and CD96, and thus interrupt their immunosuppressive signaling. However, NTX-1088 forte is manifested through its ability to block the critical interaction between PVR and the costimulatory receptor DNAM1 (CD226). This blockade prevents internalization of DNAM1, restores its expression on the surface of immune cells and results in a robust antitumor activation. NTX-1088 was tested using several tumor and immune cell co-culture systems. Various cancer cell lines were co-incubated with relevant immune effector cells from healthy human donors, in the presence of NTX-1088, as a single agent and in combination with anti-PD-1 mAb (pembrolizumab). NTX-1088 significantly increased immune cell activation, as measured by IFNg release from activated polyclonal CD8+ T cells, induction of CD137 and killing of tumor cells. When tested in combination with pembrolizumab, NTX-1088 further increased all measured activation parameters, suggesting a potential synergistic effect. When compared to anti-TIGIT mAb (tiragolumab), NTX-1088 demonstrated clear superiority in its ability to activate T and NK cells. Furthermore, NTX-1088 in combination with pembrolizumab was significantly superior to the combination of pembrolizumab with anti-TIGIT mAb. Interestingly, NTX-1088 as a single agent showed a comparable effect to that of the combined blockade of TIGIT and CD112R, and further synergized with anti-CD112R for maximal activity. NTX-1088 was the only intervention that significantly restored DNAM1 levels, whereas blockade of DNAM1 reduced the activity of NTX-1088 to levels comparable to that of anti-TIGIT mAb. Humanized murine models confirmed the above observations; NTX-1088 exhibited strong efficacy, inducing a robust tumor growth inhibition, accompanied by significantly higher prevalence of CD137+, DNAM1+, CD8+ tumor infiltrating cells, compared to control treated mice. This is the first report of drug-induced DNAM1 restoration and immune activation. NTX-1088 shows, for the first time, exclusive triple mechanism of action, whereby simultaneous and effective blockade of TIGIT and CD96 is complemented by the efficient restoration of DNAM1. This is a step change in antitumor immune activation, which will soon be tested in the clinic. Citation Format: Pini Tsukerman, Anas Atieh, Akram Obeidat, Keren Paz, Guy Cinamon, Tihana Lenac Rovis, Paola Kucan Brlic, Lea Hirsl, Stipan Jonjić, Ofer Mandelboim. NTX-1088, a potent first-in-class, anti-PVR mAb, restores expression and function of DNAM1 for optimal DNAM1-mediated antitumor immunity [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P075.

Daratumumab Prevents Experimental Xenogeneic Graft-Versus-Host Disease by Skewing Proportions of T Cell Functional Subsets and Inhibiting T Cell Activation and Migration.

Graft-versus-host disease (GVHD) remains the major cause of mortality and morbidity in non-relapse patients after allogeneic hematopoietic cell transplantation (allo-HCT). As the number of patients undergoing allo-HCT increases, it will become imperative to determine safe and effective treatment options for patients with GVHD, especially those who become refractory to systemic steroid therapy. Daratumumab (Dara), a humanized IgG1 (ĸ subclass) monoclonal antibody targeting the CD38 epitope, is used for the treatment of multiple myeloma. CD38 is a multifunctional ectoenzyme that behaves either as an enzyme, a cell adhesion molecule or a cell surface receptor involved in cell signaling. CD38 is also expressed on various immune effector and suppressor cells. However, the role of CD38 in the immune response remains elusive. We questioned whether CD38 is a potential therapeutic target against alloreactive T cells in the GVHD pathological process. Here, we investigated the impact of Dara on xenogeneic GVHD (xeno-GVHD) and graft-versus-leukemia (GVL) effects in a humanized murine model of transplantation, where human peripheral blood mononuclear cells were adoptively transplanted into immunocompromised NOD.SCID.gc-null (NSG) mice. Mice receiving Dara treatment experienced less weight loss, longer survival and lower GVHD scores compared with those in the control group. Histological evaluations, flow cytometry, RNA-sequencing and RT-qPCR analysis revealed that Dara efficaciously mitigated GVHD through multiple mechanisms including inhibition of the proliferation, activation and differentiation of CD8+ cytotoxic T cells, reduced expression of cytotoxic effector molecules, pro-inflammatory cytokines, chemokines and chemoattractant receptors by T cells and promotion of immunosuppressive T cells. More importantly, Dara preserved the GVL effect in a humanized mouse model of leukemia by metabolic reprograming of T cells to promote the induction of Th17, Th1/17and Tc1/17 cells. Our findings indicate that Dara may be an attractive therapeutic option to separate GVHD from GVL effects in patients with hematopoietic malignancies receiving allo-HCT.

Docking Protein p130Cas Regulates Acinar to Ductal Metaplasia During Pancreatic Adenocarcinoma Development and Pancreatitis

Acinar to ductal metaplasia is the prerequisite for the initiation of Kras-driven pancreatic ductal adenocarcinoma (PDAC), and candidate genes regulating this process are emerging from genome-wide association studies. The adaptor protein p130Cas emerged as a potential PDAC susceptibility gene and a Kras-synthetic lethal interactor in pancreatic cell lines; however, its role in PDAC development has remained largely unknown. Human PDAC samples and murine KrasG12D-dependent pancreatic cancer models of increasing aggressiveness were used. p130Cas was conditionally ablated in pancreatic cancer models to investigate its role during Kras-induced tumorigenesis. We found that high expression of p130Cas is frequently detected in PDAC and correlates with higher histologic grade and poor prognosis. In a model of Kras-driven PDAC, loss of p130Cas inhibits tumor development and potently extends median survival. Deletion of p130Cas suppresses acinar-derived tumorigenesis and progression by means of repressing PI3K-AKT signaling, even in the presence of a worsening condition like pancreatitis. Our observations finally demonstrated that p130Cas acts downstream of Kras to boost the PI3K activity required for acinar to ductal metaplasia and subsequent tumor initiation. This demonstrates an unexpected driving role of p130Cas downstream of Kras through PI3K/AKT, thus indicating a rational therapeutic strategy of targeting the PI3K pathway in tumors with high expression of p130Cas.

Myeloid Ikaros–SIRT1 signaling axis regulates hepatic inflammation and pyroptosis in ischemia-stressed mouse and human liver

Although Ikaros (IKZF1) is a well-established transcriptional regulator in leukocyte lymphopoiesis and differentiation, its role in myeloid innate immune responses remains unclear. Sirtuin 1 (SIRT1) is a histone/protein deacetylase involved in cellular senescence, inflammation, and stress resistance. Whether SIRT1 signaling is essential in myeloid cell activation remains uncertain, while the molecular communication between Ikaros and SIRT1, two major transcriptional regulators, has not been studied. We undertook molecular and functional studies to interrogate the significance of the myeloid Ikaros-SIRT1 axis in innate immune activation and whether it may serve as a homeostatic sentinel in human liver transplant recipients (hepatic biopsies) and murine models of sterile hepatic inflammation (liver warm ischemia-reperfusion injury in wild-type, myeloid-specific Sirt1-knockout, and CD11b-DTR mice) as well as primary bone marrow-derived macrophage (BMM) cultures (Ikaros silencing vs. overexpression). In our clinical study, we identified increased post-reperfusion hepatic Ikaros levels, accompanied by augmented inflammasome signaling yet depressed SIRT1, as a mechanism of hepatocellular damage in liver transplant recipients. In our experimental studies, we identified infiltrating macrophages as the major source of Ikaros in IR-stressed mouse livers. Then, we demonstrated that Ikaros-regulated pyroptosis - induced by canonical inflammasome signaling in BMM cultures - was SIRT1 dependent. Consistent with the latter, myeloid-specific Ikaros signaling augmented hepatic pyroptosis to aggravate pro-inflammatory responses invivo by negatively regulating SIRT1 in an AMPK-dependent manner. Finally, myeloid-specific SIRT1 was required to suppress pyroptosis, pro-inflammatory phenotype, and ultimately mitigate hepatocellular injury in ischemia-stressed murine livers. These findings identify the Ikaros-SIRT1 axis as a novel mechanistic biomarker of pyroptosis and a putative checkpoint regulator of homeostasis in response to acute hepatic stress/injury in mouse and human livers. This report describes how crosstalk between Ikaros and SIRT1, two major transcriptional regulators, influence acute hepatic inflammation in murine models of liver ischemia-reperfusion injury and liver transplant recipients. Weshow that the myeloid Ikaros-SIRT1 axis regulates inflammasome-pyroptotic cell death and hepatocellular damage in stressed livers. Thus, the Ikaros-SIRT1 axis may serve as a novel checkpoint regulator that is required for homeostasis in response to acute liver injury in mice and humans.

Porphyromonas gingivalis outer membrane vesicles as the major drivers of and source for the toxic insult and iron accumulation/deposition in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neuroinflammatory and neurodegenerative disease of the brain, defined by the accumulation and deposition of beta-amyloid, which has recently been identified as an antimicrobial peptide suggesting an infectious etiology. The NIH's Institute on Aging recently announced new initiative to look for alternate etiologies for AD that included a call for the better understanding of the role of peripheral verses local infections, as well as sources of both local and systemic inflammation. Proving if in the case of an oral bacteria that it is migrating from the oral cavity to the brain verses whether the peripheral oral source of the bacteria are instead mostly secreting toxic gingipains/LPS/complexes in soluble or as OMVs is central to understanding the pathogenesis for this disease including their role in iron toxicity in neurovascular disease and AD. A hypothetical model of Porphyromonas gingivalis (Pg) is put forth to address if: 1. either translocation to the brain from the oral cavity via blood lymphatics, intracellularly or via neuronal axoplasmic flow and establishing a productive infection of the brain leading to the local production and the accumulation of Pg exo-/endo-toxins (e.g. gingipains/LPS etc.) verses; 2. the oral peripheral infection/colonization in the oral cavity shedding exo-/endo toxins and other virulence factors as either soluble forms or associated with complex actively secreted outer membrane vesicles (OMVs) into the blood and/or lymphatics and trans-migrating the neuro-vascular endothelium with entry into the neural parenchyma and its association with iron are important and notable differences in both pathogenesis of disease and the focus of treatment. As follows. Overviewing all published papers on natural cases of human AD or murine experimental models of Pg suggest strongly that despite in the experimental case of very large and prolonged oral admisntrastion, little direct evidence is presented to support that metabolically active bacteria reside in the brain and are responsible for the production of the toxic by-products therein. A review of this literature showing the incomplete study designs along with convincing foundational new data and publications supporting Pg OMVs as the major delivery mechanism will be provided.

Abstract P102: NTX-1088, A potent first-in-class, anti-PVR mAb, restores expression and function of DNAM1 for optimal DNAM1-mediated antitumor immunity

Abstract The poliovirus receptor (PVR, CD155) represents a resistance mechanism to approved immune checkpoint inhibitors (ICIs). It is a key regulator of immune activation, that modifies immune function through multiple mechanisms. Increased levels of PVR expression on tumor cells have been associated with resistance to anti-PD-(L)1 therapy in clinical settings, while loss of PVR led to reduced tumor growth in multiple pre-clinical models. Targeting PVR using blocking mAbs offers an attractive therapeutic approach for patients with advanced cancer. NTX-1088 is a first-in-class, potent, anti-PVR mAb being developed for the treatment of solid tumors. The antibody binds to PVR with high affinity, blocks its interactions with TIGIT and CD96, and thus interrupt their immunosuppressive signaling. However, NTX-1088 forte is manifested through its ability to block the critical interaction between PVR and the costimulatory receptor DNAM1 (CD226). This blockade prevents internalization of DNAM1, restores its expression on the surface of immune cells and results in a robust antitumor activation. NTX-1088 was tested using several tumor and immune cell co-culture systems. Various cancer cell lines were co-incubated with relevant immune effector cells from healthy human donors, in the presence of NTX-1088, as a single agent and in combination with anti-PD-1 mAb (pembrolizumab). NTX-1088 significantly increased immune cell activation, as measured by IFNg release from activated polyclonal CD8+ T cells, induction of CD137 and killing of tumor cells. When tested in combination with pembrolizumab, NTX-1088 further increased all measured activation parameters, suggesting a potential synergistic effect. When compared to anti-TIGIT mAb (tiragolumab), NTX-1088 demonstrated clear superiority in its ability to activate T and NK cells. Furthermore, NTX-1088 in combination with pembrolizumab was significantly superior to the combination of pembrolizumab with anti-TIGIT mAb. Interestingly, NTX-1088 as a single agent showed a comparable effect to that of the combined blockade of TIGIT and CD112R, and further synergized with anti-CD112R for maximal activity. NTX-1088 was the only intervention that significantly restored DNAM1 levels, whereas blockade of DNAM1 reduced the activity of NTX-1088 to levels comparable to that of anti-TIGIT mAb. Humanized murine models confirmed the above observations; NTX-1088 exhibited strong efficacy, inducing a robust tumor growth inhibition, accompanied by significantly higher prevalence of CD137+, DNAM1+, CD8+ tumor infiltrating cells, compared to control treated mice. This is the first report of drug-induced DNAM1 restoration and immune activation. NTX-1088 shows, for the first time, exclusive triple mechanism of action, whereby simultaneous and effective blockade of TIGIT and CD96 is complemented by the efficient restoration of DNAM1. This is a step change in antitumor immune activation, which will soon be tested in the clinic. Citation Format: Pini Tsukerman, Anas Atieh, Akram Obeidat, Keren Paz, Guy Cinamon, Tihana Lenac Roviš, Lea Hirsl, Paola Kucan Brilc, Stipan Jonjic, Ofer Mandelboim. NTX-1088, A potent first-in-class, anti-PVR mAb, restores expression and function of DNAM1 for optimal DNAM1-mediated antitumor immunity [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 P102.

Claudins: Beyond Tight Junctions in Human IBD and Murine Models.

Claudins are transmembrane proteins constituting one of three tight junction protein families. In patients with inflammatory bowel disease (IBD), disease activity–dependent changes in expression of certain claudins have been noted, thus making certain claudin family members potential therapy targets. A study was undertaken with the aim of exploring expression of claudins in human disease and two different animal models of IBD: dextrane sulfate sodium–induced colitis and adoptive transfer model of colitis. The expression of sealing claudin-1, claudin-3, claudin-4, and claudin-8, and pore-forming claudin-2 in humans and rodents has been evaluated by immunohistochemistry and quantitative polymerase chain reaction. Claudins were expressed by epithelial and cells of mesodermal origin and were found to be situated at the membrane, within the cytoplasm, or within the nuclei. Claudin expression by human mononuclear cells isolated from lamina propria has been confirmed by Western blot and flow cytometry. The claudin expression pattern in uninflamed and inflamed colon varied between species and murine strains. In IBD and both animal models, diverse alterations in claudin expression by epithelial and inflammatory cells were recorded. Tissue mRNA levels for each studied claudin reflected changes within cell lineage and, at the same time, mirrored the ratio between various cell types. Based on the results of the study, it can be concluded that 1) claudins are not expressed exclusively by epithelial cells, but by certain types of cells of mesodermal origin as well; 2) changes in the claudin mRNA level should be interpreted in the context of overall tissue alterations; and 3) both IBD animal models that were analyzed can be used for investigating claudins as a therapy target, respecting their similarities and differences highlighted in this study.

Focal adhesion kinase inhibitor TAE226 combined with Sorafenib slows down hepatocellular carcinoma by multiple epigenetic effects

BackgroundHepatocellular carcinoma (HCC) is one of the most common and lethal malignant tumours worldwide. Sorafenib (SOR) is one of the most effective single-drug systemic therapy against advanced HCC, but the identification of novel combination regimens for a continued improvement in overall survival is a big challenge. Recent studies highlighted the crucial role of focal adhesion kinase (FAK) in HCC growth. The aim of this study was to investigate the antitumor effects of three different FAK inhibitors (FAKi), alone or in combination with SOR, using in vitro and in vivo models of HCC.MethodsThe effect of PND1186, PF431396, TAE226 on cell viability was compared to SOR. Among them TAE226, emerging as the most effective FAKi, was tested alone or in combination with SOR using 2D/3D human HCC cell line cultures and HCC xenograft murine models. The mechanisms of action were assessed by gene/protein expression and imaging approaches, combined with high-throughput methods.ResultsTAE226 was the more effective FAKi to be combined with SOR against HCC. Combined TAE226 and SOR treatment reduced HCC growth both in vitro and in vivo by affecting tumour-promoting gene expression and inducing epigenetic changes via dysregulation of FAK nuclear interactome. We characterized a novel nuclear functional interaction between FAK and the NuRD complex. TAE226-mediated FAK depletion and SOR-promoted MAPK down-modulation caused a decrease in the nuclear amount of HDAC1/2 and a consequent increase of the histone H3 lysine 27 acetylation, thus counteracting histone H3 lysine 27 trimethylation.ConclusionsAltogether, our findings provide the first evidence that TAE226 combined with SOR efficiently reduces HCC growth in vitro and in vivo. Also, our data highlight that deep analysis of FAK nuclear interactome may lead to the identification of new promising targets for HCC therapy.

Malaria in Pregnancy: From Placental Infection to Its Abnormal Development and Damage.

Malaria remains a global health burden with Plasmodium falciparum accounting for the highest mortality and morbidity. Malaria in pregnancy can lead to the development of placental malaria, where P. falciparum-infected erythrocytes adhere to placental receptors, triggering placental inflammation and subsequent damage, causing harm to both mother and her infant. Histopathological studies of P. falciparum-infected placentas revealed various placental abnormalities such as excessive perivillous fibrinoid deposits, breakdown of syncytiotrophoblast integrity, trophoblast basal lamina thickening, increased syncytial knotting, and accumulation of mononuclear immune cells within intervillous spaces. These events in turn, are likely to impair placental development and function, ultimately causing placental insufficiency, intrauterine growth restriction, preterm delivery and low birth weight. Hence, a better understanding of the mechanisms behind placental alterations and damage during placental malaria is needed for the design of effective interventions. In this review, using evidence from human studies and murine models, an integrated view on the potential mechanisms underlying placental pathologies in malaria in pregnancy is provided. The molecular, immunological and metabolic changes in infected placentas that reflect their responses to the parasitic infection and injury are discussed. Finally, potential models that can be used by researchers to improve our understanding on the pathogenesis of malaria in pregnancy and placental pathologies are presented.

HER3 Is an Actionable Target in Advanced Prostate Cancer.

It has been recognized for decades that ERBB signaling is important in prostate cancer, but targeting ERBB receptors as a therapeutic strategy for prostate cancer has been ineffective clinically. However, we show here that membranous HER3 protein is commonly highly expressed in lethal prostate cancer, associating with reduced time to castration resistance (CR) and survival. Multiplex immunofluorescence indicated that the HER3 ligand NRG1 is detectable primarily in tumor-infiltrating myelomonocytic cells in human prostate cancer; this observation was confirmed using single-cell RNA sequencing of human prostate cancer biopsies and murine transgenic prostate cancer models. In castration-resistant prostate cancer (CRPC) patient-derived xenograft organoids with high HER3 expression as well as mouse prostate cancer organoids, recombinant NRG1 enhanced proliferation and survival. Supernatant from murine bone marrow-derived macrophages and myeloid-derived suppressor cells promoted murine prostate cancer organoid growth in vitro, which could be reversed by a neutralizing anti-NRG1 antibody and ERBB inhibition. Targeting HER3, especially with the HER3-directed antibody-drug conjugate U3-1402, exhibited antitumor activity against HER3-expressing prostate cancer. Overall, these data indicate that HER3 is commonly overexpressed in lethal prostate cancer and can be activated by NRG1 secreted by myelomonocytic cells in the tumor microenvironment, supporting HER3-targeted therapeutic strategies for treating HER3-expressing advanced CRPC. SIGNIFICANCE: HER3 is an actionable target in prostate cancer, especially with anti-HER3 immunoconjugates, and targeting HER3 warrants clinical evaluation in prospective trials.

Distinctive roles of tumor necrosis factor receptor type 1 and type 2 in a mouse disc degeneration model.

BackgroundElevated tumor necrosis factor alpha (TNF-α) expression is correlated with the progression of intervertebral disc degeneration (IVDD). Progranulin binding to tumor necrosis factor receptor (TNFR) and its derivative Atsttrin are effective for treating inflammatory arthritis. We hypothesize that Atsttrin has a protective effect in IVDD through different roles of TNFR receptor type 1 (TNFR1) and TNFR receptor type 2 (TNFR2) in degenerated discs.MethodsIVDD models were established in TNFR1−/−, TNFR2−/− mice and their control littermates. Nucleus Pulpous (NP) samples from human patients and IVDD murine models were evaluated by X-ray, micro-MRI, μCT, histological staining and immunofluorescence staining. NP cells isolated from wild-type (WT), TNFR1−/− and TNFR2−/− mice were treated with TNF-α or Atsttrin and then assayed by Western blotting, qRT–PCR, and ELISA.ResultsTNFR1 and TNFR2 expression was significantly elevated in the disc tissues of both human patients and IVDD murine models. TNFR1 knockout contributed to reduced disc degeneration. In contrast, TNFR2 knockout was associated with enhanced IVDD severity, including degraded cellular composition, increased cell apoptosis and elevated vertebral destruction. Atsttrin protected against IVDD in WT and TNFR1−/− mouse models but had no effect in TNFR2−/− IVDD models. Additionally, in vitro NP cell-based assays demonstrated that TNF-α-stimulated catabolism and Atsttrin-activated anabolism depended on TNFR1 and TNFR2, respectively.ConclusionTNFR1 is associated with the degenerative progression of IVDD, while TNFR2 contributes to the protective effect on the discs. Atsttrin protects against IVDD at least partially by inhibiting the TNFα/TNFR1 inflammatory/catabolic pathway and activating the TNFR2 protective/anabolic pathway.The translational potential of this articleThis study demonstrates that TNFR1 and TNFR2 have disparate roles in disc degeneration and hlights the potential use of Atsttrin as a therapeutic agent against IVDD in mice.