A WNT mimetic with broad spectrum FZD-specificity decreases fibrosis and improves function in a pulmonary damage model.

BACKGROUND: Wnt/ß-catenin signaling is critical for lung development and AT2 stem cell maintenance in adults, but excessive pathway activation has been associated with pulmonary fibrosis, both in animal models and human diseases such as idiopathic pulmonary fibrosis (IPF). IPF is a detrimental interstitial lung disease, and although two approved drugs limit functional decline, transplantation is the only treatment that extends survival, highlighting the need for regenerative therapies. METHODS: Using our antibody-based platform of Wnt/ß-catenin modulators, we investigated the ability of a pathway antagonist and pathway activators to reduce pulmonary fibrosis in the acute bleomycin model, and we tested the ability of a WNT mimetic to affect alveolar organoid cultures. RESULTS: A WNT mimetic agonist with broad FZD-binding specificity (FZD1,2,5,7,8) potently expanded alveolar organoids. Upon therapeutic dosing, a broad FZD-binding specific Wnt mimetic decreased pulmonary inflammation and fibrosis and increased lung function in the bleomycin model, and it impacted multiple lung cell types in vivo. CONCLUSIONS: Our results highlight the unexpected capacity of a WNT mimetic to effect tissue repair after lung damage and support the continued development of Wnt/ß-catenin pathway modulation for the treatment of pulmonary fibrosis.

Noncanonical NF-kappaB activation requires coordinated assembly of a regulatory complex of the adaptors cIAP1, cIAP2, TRAF2 and TRAF3 and the kinase NIK.

Recent studies suggest that nuclear factor kappaB-inducing kinase (NIK) is suppressed through constitutive proteasome-mediated degradation regulated by TRAF2, TRAF3 and cIAP1 or cIAP2. Here we demonstrated that the degradation of NIK occurs upon assembly of a regulatory complex through TRAF3 recruitment of NIK and TRAF2 recruitment of cIAP1 and cIAP2. In contrast to TRAF2 and TRAF3, cIAP1 and cIAP2 seem to play redundant roles in the degradation of NIK, as inhibition of both cIAPs was required for noncanonical NF-kappaB activation and increased survival and proliferation of primary B lymphocytes. Furthermore, the lethality of TRAF3 deficiency in mice could be rescued by a single NIK gene, highlighting the importance of tightly regulated NIK.

cFLIP regulation of lymphocyte activation and development.

Cellular caspase-8 (FLICE)-like inhibitory protein (cFLIP) was originally identified as an inhibitor of death-receptor signalling through competition with caspase-8 for recruitment to FAS-associated via death domain (FADD). More recently, it has been determined that both cFLIP and caspase-8 are required for the survival and proliferation of T cells following T-cell-receptor stimulation. This paradoxical finding launched new investigations of how these molecules might connect with signalling pathways that link to cell survival and growth following antigen-receptor activation. As discussed in this Review, insight gained from these studies indicates that cFLIP and caspase-8 form a heterodimer that ultimately links T-cell-receptor signalling to activation of nuclear factor-kappaB through a complex that includes B-cell lymphoma 10 (BCL-10), mucosa-associated-lymphoid-tissue lymphoma-translocation gene 1 (MALT1) and receptor-interacting protein 1 (RIP1).

PCSK9 inhibition fails to alter hepatic LDLR, circulating cholesterol, and atherosclerosis in the absence of ApoE.

LDL cholesterol (LDL-C) contributes to coronary heart disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) increases LDL-C by inhibiting LDL-C clearance. The therapeutic potential for PCSK9 inhibitors is highlighted by the fact that PCSK9 loss-of-function carriers exhibit 15-30% lower circulating LDL-C and a disproportionately lower risk (47-88%) of experiencing a cardiovascular event. Here, we utilized pcsk9(-/-) mice and an anti-PCSK9 antibody to study the role of the LDL receptor (LDLR) and ApoE in PCSK9-mediated regulation of plasma cholesterol and atherosclerotic lesion development. We found that circulating cholesterol and atherosclerotic lesions were minimally modified in pcsk9(-/-) mice on either an LDLR- or ApoE-deficient background. Acute administration of an anti-PCSK9 antibody did not reduce circulating cholesterol in an ApoE-deficient background, but did reduce circulating cholesterol (-45%) and TGs (-36%) in APOE*3Leiden.cholesteryl ester transfer protein (CETP) mice, which contain mouse ApoE, human mutant APOE3*Leiden, and a functional LDLR. Chronic anti-PCSK9 antibody treatment in APOE*3Leiden.CETP mice resulted in a significant reduction in atherosclerotic lesion area (-91%) and reduced lesion complexity. Taken together, these results indicate that both LDLR and ApoE are required for PCSK9 inhibitor-mediated reductions in atherosclerosis, as both are needed to increase hepatic LDLR expression.

Inhibition of secreted frizzled-related protein 5 improves glucose metabolism.

Elucidating the role of secreted frizzled-related protein 5 (SFRP5) in metabolism and obesity has been complicated by contradictory findings when knockout mice were used to determine metabolic phenotypes. By overexpressing SFRP5 in obese, prediabetic mice we consistently observed elevated hyperglycemia and glucose intolerance, supporting SFRP5 as a negative regulator of glucose metabolism. Accordingly, Sfrp5 mRNA expression analysis of both epididymal and subcutaneous adipose depots of mice indicated a correlation with obesity. Thus, we generated a monoclonal antibody (mAb) against SFRP5 to ascertain the effect of SFRP5 inhibition in vivo. Congruent with SFRP5 overexpression worsening blood glucose levels and glucose intolerance, anti-SFRP5 mAb therapy improved these phenotypes in vivo. The results from both the overexpression and mAb inhibition studies suggest a role for SFRP5 in glucose metabolism and pancreatic ß-cell function and thus establish the use of an anti-SFRP5 mAb as a potential approach to treat type 2 diabetes.

Design principles and therapeutic applications of novel synthetic WNT signaling agonists.

Wingless-related integration site or Wingless and Int-1 or Wingless-Int (WNT) signaling is crucial for embryonic development, and adult tissue homeostasis and regeneration, through its essential roles in cell fate, patterning, and stem cell regulation. The biophysical characteristics of WNT ligands have hindered efforts to interrogate ligand activity in vivo and prevented their development as therapeutics. Recent breakthroughs have enabled the generation of synthetic WNT signaling molecules that possess characteristics of natural ligands and potently activate the pathway, while also providing distinct advantages for therapeutic development and manufacturing. This review provides a detailed discussion of the protein engineering of these molecular platforms for WNT signaling agonism. We discuss the importance of WNT signaling in several organs and share insights from the initial application of these new classes of molecules in vitro and in vivo. These molecules offer a unique opportunity to enhance our understanding of how WNT signaling agonism promotes tissue repair, enabling targeted development of tailored therapeutics.

Targeted protein degradation systems to enhance Wnt signaling.

Molecules that facilitate targeted protein degradation (TPD) offer great promise as novel therapeutics. The human hepatic lectin asialoglycoprotein receptor (ASGR) is selectively expressed on hepatocytes. We have previously engineered an anti-ASGR1 antibody-mutant RSPO2 (RSPO2RA) fusion protein (called SWEETS) to drive tissue-specific degradation of ZNRF3/RNF43 E3 ubiquitin ligases, which achieved hepatocyte-specific enhanced Wnt signaling, proliferation, and restored liver function in mouse models, and an antibody-RSPO2RA fusion molecule is currently in human clinical trials. In the current study, we identified two new ASGR1- and ASGR1/2-specific antibodies, 8M24 and 8G8. High-resolution crystal structures of ASGR1:8M24 and ASGR2:8G8 complexes revealed that these antibodies bind to distinct epitopes on opposing sides of ASGR, away from the substrate-binding site. Both antibodies enhanced Wnt activity when assembled as SWEETS molecules with RSPO2RA through specific effects sequestering E3 ligases. In addition, 8M24-RSPO2RA and 8G8-RSPO2RA efficiently downregulate ASGR1 through TPD mechanisms. These results demonstrate the possibility of combining different therapeutic effects and degradation mechanisms in a single molecule.

C5L2 is critical for the biological activities of the anaphylatoxins C5a and C3a.

Complement-derived anaphylatoxins regulate immune and inflammatory responses through G-protein-coupled receptor (GPCR)-mediated signalling. C5L2 (also known as GPR77) is a relatively new GPCR thought to be a non-signalling receptor binding to C5a, on the basis of sequence information and experimental evidence. Here we show, using gene targeting, that C5L2 is required to facilitate C5a signalling in neutrophils, macrophages and fibroblasts in vitro. Deficiency of C5L2 results in reduced inflammatory cell infiltration, suggesting that C5L2 is critical for optimal C5a-mediated cell infiltration in certain in vivo settings. C5L2 is also involved in optimizing C3a-induced signals. Furthermore, like mice incapable of C3a/complement 3a receptor (C3aR) signalling, C5L2-deficient mice are hypersensitive to lipopolysaccharide (LPS)-induced septic shock, show reduced ovalbumin (OVA)-induced airway hyper-responsiveness and inflammation, and are mildly delayed in haematopoietic cell regeneration after gamma-irradiation. Our data indicate that C5L2 can function as a positive modulator for both C5a- and C3a-anaphylatoxin-induced responses.

Abnormal TNF activity in Timp3-/- mice leads to chronic hepatic inflammation and failure of liver regeneration.

Tumor-necrosis factor (TNF), a pleiotropic cytokine, triggers physiological and pathological responses in several organs. Here we show that deletion of the mouse gene Timp3 resulted in an increase in TNF-alpha converting enzyme activity, constitutive release of TNF and activation of TNF signaling in the liver. The increase in TNF in Timp3(-/-) mice culminated in hepatic lymphocyte infiltration and necrosis, features that are also seen in chronic active hepatitis in humans. This pathology was prevented when deletion of Timp3 was combined with Tnfrsf1a deficiency. In a liver regeneration model that requires TNF signaling, Timp3(-/-) mice succumbed to liver failure. Hepatocytes from Timp3(-/-) mice completed the cell cycle but then underwent cell death owing to sustained activation of TNF. This hepatocyte cell death was completely rescued by a neutralizing antibody to TNF. Dysregulation of TNF occurred specifically in Timp3(-/-), and not Timp1(-/-) mice. These data indicate that TIMP3 is a crucial innate negative regulator of TNF in both tissue homeostasis and tissue response to injury.

Therapeutic blood-brain barrier modulation and stroke treatment by a bioengineered FZD4-selective WNT surrogate in mice.

Derangements of the blood-brain barrier (BBB) or blood-retinal barrier (BRB) occur in disorders ranging from stroke, cancer, diabetic retinopathy, and Alzheimer's disease. The Norrin/FZD4/TSPAN12 pathway activates WNT/ß-catenin signaling, which is essential for BBB and BRB function. However, systemic pharmacologic FZD4 stimulation is hindered by obligate palmitoylation and insolubility of native WNTs and suboptimal properties of the FZD4-selective ligand Norrin. Here, we develop L6-F4-2, a non-lipidated, FZD4-specific surrogate which significantly improves subpicomolar affinity versus native Norrin. In Norrin knockout (NdpKO) mice, L6-F4-2 not only potently reverses neonatal retinal angiogenesis deficits, but also restores BRB and BBB function. In adult C57Bl/6J mice, post-stroke systemic delivery of L6-F4-2 strongly reduces BBB permeability, infarction, and edema, while improving neurologic score and capillary pericyte coverage. Our findings reveal systemic efficacy of a bioengineered FZD4-selective WNT surrogate during ischemic BBB dysfunction, with potential applicability to adult CNS disorders characterized by an aberrant blood-brain barrier.

Novel Frizzled-specific antibody-based Wnt mimetics and Wnt superagonists selectively activate WNT/ß-catenin signaling in target tissues.

WNTs are essential factors for stem cell biology, embryonic development, and for maintaining homeostasis and tissue repair in adults. Difficulties in purifying WNTs and their lack of receptor selectivity have hampered research and regenerative medicine development. While breakthroughs in WNT mimetic development have overcome some of these difficulties, the tools developed so far are incomplete and mimetics alone are often not sufficient. Here, we developed a complete set of WNT mimetic molecules that cover all WNT/ß-catenin-activating Frizzleds (FZDs). We show that FZD1,2,7 stimulate salivary gland expansion in vivo and salivary gland organoid expansion. We further describe the discovery of a novel WNT-modulating platform that combines WNT and RSPO mimetics' effects into one molecule. This set of molecules supports better organoid expansion in various tissues. These WNT-activating platforms can be broadly applied to organoids, pluripotent stem cells, and in vivo research, and serve as bases for future therapeutic development.

IL-21-induced Bepsilon cell apoptosis mediated by natural killer T cells suppresses IgE responses.

Epidemiological studies have suggested that the recent increase in the incidence and severity of immunoglobulin (Ig)E-mediated allergic disorders is inversely correlated with Mycobacterium bovis bacillus Calmette Guerin (BCG) vaccination; however, the underlying mechanisms remain uncertain. Here, we demonstrate that natural killer T (NKT) cells in mice and humans play a crucial role in the BCG-induced suppression of IgE responses. BCG-activated murine Valpha14 NKT cells, but not conventional CD4 T cells, selectively express high levels of interleukin (IL)-21, which preferentially induces apoptosis in Bepsilon cells. Signaling from the IL-21 receptor increases the formation of a complex between Bcl-2 and the proapoptotic molecule Bcl-2-modifying factor, resulting in Bepsilon cell apoptosis. Similarly, BCG vaccination induces IL-21 expression by human peripheral blood mononuclear cells (PBMCs) in a partially NKT cell-dependent fashion. BCG-activated PBMCs significantly reduce IgE production by human B cells. These findings provide new insight into the therapeutic effect of BCG in allergic diseases.

G-protein-coupled receptor GPR21 knockout mice display improved glucose tolerance and increased insulin response.

GPR21 is an orphan G-protein-coupled receptor. We found that mice deficient for the GPR21 gene were resistant to diet-induced obesity. Knockout mice were leaner than their wildtype counterpart, despite that no difference was observed in food intake. No differences were observed in the respiratory exchange rate and thermogenesis. However, knockout mice were more active than wildtype littermates, and this level of activity may be an underlying reason for the difference in energy balance. Mutant mice were more sensitive to insulin than their wildtype control and showed an improved glucose tolerance. Several inflammatory markers MCP-1, CRP and IP-10 were decreased in mutant animals, suggesting that GPR21 may also mediate its effect through anti-inflammatory mechanisms. We found that GPR21 is widely expressed in all tissues, with the highest levels found in the brain and in the spleen. Overall, these findings suggest that GPR21 may play an important role in regulating body weight and glucose metabolism.

A proprotein convertase subtilisin/kexin type 9 neutralizing antibody reduces serum cholesterol in mice and nonhuman primates.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates serum LDL cholesterol (LDL-C) by interacting with the LDL receptor (LDLR) and is an attractive therapeutic target for LDL-C lowering. We have generated a neutralizing anti-PCSK9 antibody, mAb1, that binds to an epitope on PCSK9 adjacent to the region required for LDLR interaction. In vitro, mAb1 inhibits PCSK9 binding to the LDLR and attenuates PCSK9-mediated reduction in LDLR protein levels, thereby increasing LDL uptake. A combination of mAb1 with a statin increases LDLR levels in HepG2 cells more than either treatment alone. In wild-type mice, mAb1 increases hepatic LDLR protein levels approximately 2-fold and lowers total serum cholesterol by up to 36%: this effect is not observed in LDLR(-/-) mice. In cynomolgus monkeys, a single injection of mAb1 reduces serum LDL-C by 80%, and a significant decrease is maintained for 10 days. We conclude that anti-PCSK9 antibodies may be effective therapeutics for treating hypercholesterolemia.

SZN-413, a FZD4 Agonist, as a Potential Novel Therapeutic for the Treatment of Diabetic Retinopathy.

Purpose: There remains a high unmet need for therapies with new mechanisms of action to achieve reperfusion of ischemic retina in diabetic retinopathy. We examined whether a novel frizzled class receptor 4 (FZD4) agonist could promote regeneration of functional blood vessels in animal models of retinopathy. Methods: We developed a novel Norrin mimetic (SZN-413-p) targeting FZD4 and low-density lipoprotein receptor-related protein 5 (LRP5) and examined its effect on retinal and brain endothelial cells in vitro. SZN-413-p was subsequently humanized, resulting in the therapeutic candidate SZN-413, and was examined in animal models of retinopathy. In an oxygen-induced retinopathy mouse model, avascular and neovascularization areas were measured. Furthermore, in a vascular endothelial growth factor (VEGF)-induced retinal vascular leakage rabbit model, the impact on vascular leakage by SZN-413 was examined by measuring fluorescein leakage. Results: SZN-413-p induced Wnt/ß-catenin signaling and upregulated blood-brain barrier/blood-retina barrier gene expressions in endothelial cells. In the oxygen-induced retinopathy mouse model, SZN-413-p and SZN-413 significantly reduced the neovascularization area size (P < 0.001) to a level comparable to, or better than the positive control aflibercept. Both agonists also showed a reduction in avascular area size compared to vehicle (P < 0.001) and aflibercept groups (P < 0.05 and P < 0.01 for SZN-413-p and SZN-413, respectively). In the VEGF-induced retinal vascular leakage rabbit model, SZN-413 reduced retinal vascular leakage by ∼80%, compared to the vehicle-treated group (P < 0.01). Conclusions: Reduction of neovascular tufts and avascular areas and of VEGF-driven retinal vascular leakage suggests that SZN-413 can simultaneously address retinal non-perfusion and vascular leakage. Translational Relevance: FZD4 signaling modulation by SZN-413 is a novel mechanism of action that can offer a new therapeutic strategy for diabetic retinopathy.

Robust Colonic Epithelial Regeneration and Amelioration of Colitis via FZD-Specific Activation of Wnt Signaling.

BACKGROUND AND AIMS: Current management of inflammatory bowel disease leaves a clear unmet need to treat the severe epithelial damage. Modulation of Wnt signaling might present an opportunity to achieve histological remission and mucosal healing when treating IBD. Exogenous R-spondin, which amplifies Wnt signals by maintaining cell surface expression of Frizzled (Fzd) and low-density lipoprotein receptor-related protein receptors, not only helps repair intestine epithelial damage, but also induces hyperplasia of normal epithelium. Wnt signaling may also be modulated with the recently developed Wnt mimetics, recombinant antibody-based molecules mimicking endogenous Wnts. METHODS: We first compared the epithelial healing effects of RSPO2 and a Wnt mimetic with broad Fzd specificity in an acute dextran sulfate sodium mouse colitis model. Guided by Fzd expression patterns in the colon epithelium, we also examined the effects of Wnt mimetics with subfamily Fzd specificities. RESULTS: In the DSS model, Wnt mimetics repaired damaged colon epithelium and reduced disease activity and inflammation and had no apparent effect on uninjured tissue. We further identified that the FZD5/8 and LRP6 receptor-specific Wnt mimetic, SZN-1326-p, was associated with the robust repair effect. Through a range of approaches including single-cell transcriptome analyses, we demonstrated that SZN-1326-p directly impacted epithelial cells, driving transient expansion of stem and progenitor cells, promoting differentiation of epithelial cells, histologically restoring the damaged epithelium, and secondarily to epithelial repair, reducing inflammation. CONCLUSIONS: It is feasible to design Wnt mimetics such as SZN-1326-p that impact damaged intestine epithelium specifically and restore its physiological functions, an approach that holds promise for treating epithelial damage in inflammatory bowel disease.

Osteoclast differentiation independent of the TRANCE-RANK-TRAF6 axis.

Osteoclasts are derived from myeloid lineage cells, and their differentiation is supported by various osteotropic factors, including the tumor necrosis factor (TNF) family member TNF-related activation-induced cytokine (TRANCE). Genetic deletion of TRANCE or its receptor, receptor activator of nuclear factor kappaB (RANK), results in severely osteopetrotic mice with no osteoclasts in their bones. TNF receptor-associated factor (TRAF) 6 is a key signaling adaptor for RANK, and its deficiency leads to similar osteopetrosis. Hence, the current paradigm holds that TRANCE-RANK interaction and subsequent signaling via TRAF6 are essential for the generation of functional osteoclasts. Surprisingly, we show that hematopoietic precursors from TRANCE-, RANK-, or TRAF6-null mice can become osteoclasts in vitro when they are stimulated with TNF-alpha in the presence of cofactors such as TGF-beta. We provide direct evidence against the current paradigm that the TRANCE-RANK-TRAF6 pathway is essential for osteoclast differentiation and suggest the potential existence of alternative routes for osteoclast differentiation.

Cellular FLICE-inhibitory protein is required for T cell survival and cycling.

Fas-associated death domain (FADD) and caspase-8 are key signal transducers for death receptor-induced apoptosis, whereas cellular FLICE-inhibitory protein (cFLIP) antagonizes this process. Interestingly, FADD and caspase-8 also play a role in T cell development and T cell receptor (TCR)-mediated proliferative responses. To investigate the underlying mechanism, we generated cFLIP-deficient T cells by reconstituting Rag-/- blastocysts with cFLIP-deficient embryonic stem cells. These Rag chimeric mutant mice (rcFLIP-/-) had severely reduced numbers of T cells in the thymus, lymph nodes, and spleen, although mature T lymphocytes did develop. Similar to FADD- or caspase-8-deficient cells, rcFLIP-/- T cells were impaired in proliferation in response to TCR stimulation. Further investigation revealed that cFLIP is required for T cell survival, as well as T cell cycling in response to TCR stimulation. Interestingly, some signaling pathways from the TCR complex appeared competent, as CD3 plus CD28 cross-linking was capable of activating the ERK pathway in rcFLIP-/- T cells. We demonstrate an essential role for cFLIP in T cell function.

Differential role for c-Rel and C/EBPbeta/delta in TLR-mediated induction of proinflammatory cytokines.

TLR stimulation triggers a signaling pathway via MyD88 and IL-1R-associated kinase 4 that is essential for proinflammatory cytokine induction. Although NF-kappaB has been shown to be one of the key transcriptional regulators of these cytokines, evidence suggests that other factors may also be important. In this study, we showed that MyD88-deficient macrophages have defective c-Rel activation, which has been linked to IL-12p40 induction, but not IL-6 or TNF-alpha. We also investigated other transcription factors and showed that C/EBPbeta and C/EBPdelta expression was limited in MyD88- or IL-1R-associated kinase 4-deficient macrophages treated with LPS. Importantly, the absence of both C/EBPbeta and C/EBPdelta resulted in the impaired induction of proinflammatory cytokines stimulated by several TLR ligands. Our results identify c-Rel and C/EBPbeta/delta as important transcription factors in a MyD88-dependent pathway that regulate the induction of proinflammatory cytokines.

Beyond tumor necrosis factor receptor: TRADD signaling in toll-like receptors.

Tumor necrosis factor receptor 1-associated death domain protein (TRADD) is the core adaptor recruited to TNF receptor 1 (TNFR1) upon TNFalpha stimulation. In cells from TRADD-deficient mice, TNFalpha-mediated apoptosis and TNFalpha-stimulated NF-kappaB, JNK, and ERK activation are defective. TRADD is also important for germinal center formation, DR3-mediated costimulation of T cells, and TNFalpha-mediated inflammatory responses in vivo. TRADD deficiency does not enhance IFNgamma-induced signaling. Importantly, TRADD has a novel role in TLR3 and TLR4 signaling. TRADD participates in the TLR4 complex formed upon LPS stimulation, and TRADD-deficient macrophages show impaired cytokine production in response to TLR ligands in vitro. Thus, TRADD is a multifunctional protein crucial both for TNFR1 signaling and other signaling pathways relevant to immune responses.