Potentiates Wnt Signaling Research Articles

Secreted ADAMTS-like 2 promotes myoblast differentiation by potentiating WNT signaling

Myogenesis is the process that generates multinucleated contractile myofibers from muscle stem cells during skeletal muscle development and regeneration. Myogenesis is governed by myogenic regulatory transcription factors, including MYOD1. Here, we identified the secreted matricellular protein ADAMTS-like 2 (ADAMTSL2) as part of a Wnt-dependent positive feedback loop, which augmented or sustained MYOD1 expression and thus promoted myoblast differentiation. ADAMTSL2 depletion resulted in severe retardation of myoblast differentiation in vitro and its ablation in myogenic precursor cells resulted in aberrant skeletal muscle architecture. Mechanistically, ADAMTSL2 potentiated WNT signaling by binding to WNT ligands and WNT receptors. We identified the WNT-binding ADAMTSL2 peptide, which was sufficient to promote myogenesis in vitro. Since ADAMTSL2 was previously described as a negative regulator of TGFβ signaling in fibroblasts, ADAMTSL2 now emerges as a signaling hub that could integrate WNT, TGFβ and potentially other signaling pathways within the dynamic microenvironment of differentiating myoblasts during skeletal muscle development and regeneration.

R-SPONDIN2+ mesenchymal cells form the bud tip progenitor niche during human lung development.

The human respiratory epithelium is derived from a progenitor cell in the distal buds of the developing lung. These "bud tip progenitors" are regulated by reciprocal signaling with surrounding mesenchyme; however, mesenchymal heterogeneity and function in the developing human lung are poorly understood. We interrogated single-cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of the WNT agonist RSPO2, and we found that the adjacent bud tip progenitors are enriched for the RSPO2 receptor LGR5. Functional experiments using organoid models, explant cultures, and FACS-isolated RSPO2+ mesenchyme show that RSPO2 is a critical niche cue that potentiates WNT signaling in bud tip progenitors to support their maintenance and multipotency.

Abstract 3164: Detection and CAR T-cell directed targeting of inflammation-responsive Wnt pathway receptors on multiple myeloma cancer stem-like cells

Abstract Multiple myeloma (MM) is the second most common hematological malignancy characterized by a malignant expansion of abnormal antibody-producing cells. Despite available treatments, relapsed disease is a common occurrence with patients receiving survival predictions of less than one year. Relapsed MM is often refractory to multiple lines of therapy and occurs due to the uncontrolled regeneration of a population of malignant cells displaying progenitor-cell-like phenotypes. Selectively targeting this cancer stem cell (CSC) population provides an effective way to limit incidence of relapsed disease, improving the prognosis for high-risk patients. In MM, the defining characteristics of CSCs remain poorly understood, though signaling pathways promoting ‘stemness’ that are often deregulated in CSCs, such as interleukin-6 (IL-6) signaling, as well as canonical and non-canonical Wnt signaling are also aberrantly activated in MM. In this context, the central objective of this project was to explore the functionality of two proteins which have been shown to be downstream targets of IL-6/JAK2/STAT3 signaling and potentiate Wnt signaling, the embryonic G-protein coupled receptor LGR4 and the fetal-oncogene tyrosine kinase like orphan receptor-1 (ROR1), as CSC biomarker candidates, and explore the feasibility of CSC targeting therapeutics for MM. Receptor expression was assessed by quantitative PCR and flow cytometry in primary plasma cell dyscrasia patient samples across the spectrum of disease, as well as in human myeloma cell lines (HMCLs). Whole transcriptome sequencing analyses were carried out in HMCLs induced to overexpress LGR4, which enriched genes associated with ion transport and embryonic development. Cell adhesion and extracellular matrix genes were downregulated, along with genes associated with metabolic and mitochondrial pathways. Results of ROR1 gene expression and flow cytometry studies suggest that a subset of MM patient samples harbor a low-frequency ROR1-positive cell population, and expression is enriched in an in vitro model of acquired drug resistance. Overexpression of interferon regulatory factor-4 (IRF4) - a myeloma cell-enriched transcription factor that we recently showed promotes myeloma regeneration - also contributed to increased human ROR1 protein levels. HMCLs with high endogenous ROR1 expression were sensitive to in vitro killing by anti-ROR1 CAR T-cell treatment, which employs an scFv generated from the fully humanized ROR1-specific monoclonal antibody cirmtuzumab, at effector:target cell ratios (ET) as low as 0.33:1. Together, these results suggest a central role for Wnt signaling in MM stem cell phenotypes and activation of embryonic transcriptional pathways, while also paving the way towards development of clinical therapies that target a CSC population in MM. Citation Format: Lara C. Avsharian, Christopher S. Oh, Ashni A. Vora, Jayde Fernandez, Caitlin Shin, Navyaa Sinha, Charles Prussak, Leslie Crews. Detection and CAR T-cell directed targeting of inflammation-responsive Wnt pathway receptors on multiple myeloma cancer stem-like cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3164.

Abstract 961: TCF7 promotes a mammary cancer-stem-like phenotype leading to metastasis via potentiation of a hybrid epithelial/mesenchymal (E/M) phenotype

Abstract Cancer stem cells (CSCs) are a minor population of carcinoma cells that can self-renew and give rise to differentiated progenitors that form the bulk of the tumor. CSCs cause tumor recurrence, chemoresistance and metastasis. The epithelial-to-mesenchymal transition (EMT) promotes tumor cell migration, invasion and cancer stem cell properties. The Wnt signaling pathway is a major driver of cancer stemness and is regulated by the TCF/LEF family of transcription factors. Using loss and gain of function of TCF7 (TCF1) in mouse and human breast cancer cells, we were able to demonstrate that TCF7 promotes cancer stemness through striking regulation of the EMT process. TCF7 knockdown in PyMT mammary tumor cells induced luminal epithelial differentiation, resulting in inhibition of oncosphere formation, migration, tumor growth and lung colonization. However, in keeping with a changing view of EMT as a strictly binary process, we found that TCF7 shifts tumor cells towards a hybrid epithelial/mesenchymal state (E/M) rather than a mesenchymal (M) state. This E/M population was shown to carry stemness and metastatic properties and to be interestingly represented by a small “stem-like” subpopulation in triple negative breast cancer (TNBC) cells. Using CD44 and CD104 (integrin β4) flow cytometry to isolate E/M cells from normal MCF10A and basal-like MDA-MB-468 breast cancer cells, we show that TCF7, Snail, and GSK3β phosphorylation were all highly enriched in this population whereas Zeb-1 (a marker of mesenchymal cells), was absent from E/M cells. These data support a model whereby TCF7 potentiates Wnt signaling which in turn activates GSK3β phosphorylation, resulting in Snail upregulation and enrichment of cells in the E/M state that is conducive of stemness and metastasis. Citation Format: Huizhi Liang, Outhiriaradjou Benard, Zuen Ren, Kimita Suyama, Larry Norton, Rachel B. Hazan. TCF7 promotes a mammary cancer-stem-like phenotype leading to metastasis via potentiation of a hybrid epithelial/mesenchymal (E/M) phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 961.

Activation of Drp1 promotes fatty acids-induced metabolic reprograming to potentiate Wnt signaling in colon cancer.

Cancer cells are known for their ability to adapt variable metabolic programs depending on the availability of specific nutrients. Our previous studies have shown that uptake of fatty acids alters cellular metabolic pathways in colon cancer cells to favor fatty acid oxidation. Here, we show that fatty acids activate Drp1 to promote metabolic plasticity in cancer cells. Uptake of fatty acids (FAs) induces mitochondrial fragmentation by promoting ERK-dependent phosphorylation of Drp1 at the S616 site. This increased phosphorylation of Drp1 enhances its dimerization and interaction with Mitochondrial Fission Factor (MFF) at the mitochondria. Consequently, knockdown of Drp1 or MFF attenuates fatty acid-induced mitochondrial fission. In addition, uptake of fatty acids triggers mitophagy via a Drp1- and p62-dependent mechanism to protect mitochondrial integrity. Moreover, results from metabolic profiling analysis reveal that silencing Drp1 disrupts cellular metabolism and blocks fatty acid-induced metabolic reprograming by inhibiting fatty acid utilization. Functionally, knockdown of Drp1 decreases Wnt/β-catenin signaling by preventing fatty acid oxidation-dependent acetylation of β-catenin. As a result, Drp1 depletion inhibits the formation of tumor organoids in vitro and xenograft tumor growth in vivo. Taken together, our study identifies Drp1 as a key mediator that connects mitochondrial dynamics with fatty acid metabolism and cancer cell signaling.

PPIL4 is essential for brain angiogenesis and implicated in intracranial aneurysms in humans.

Intracranial aneurysm (IA) rupture leads to subarachnoid hemorrhage, a sudden-onset disease that often causes death or severe disability. Although genome-wide association studies have identified common genetic variants that increase IA risk moderately, the contribution of variants with large effect remains poorly defined. Using whole-exome sequencing, we identified significant enrichment of rare, deleterious mutations in PPIL4, encoding peptidyl-prolyl cis-trans isomerase-like 4, in both familial and index IA cases. Ppil4 depletion in vertebrate models causes intracerebral hemorrhage, defects in cerebrovascular morphology and impaired Wnt signaling. Wild-type, but not IA-mutant, PPIL4 potentiates Wnt signaling by binding JMJD6, a known angiogenesis regulator and Wnt activator. These findings identify a novel PPIL4-dependent Wnt signaling mechanism involved in brain-specific angiogenesis and maintenance of cerebrovascular integrity and implicate PPIL4 gene mutations in the pathogenesis of IA.

Multi-Targeting DKK1 and LRP6 Prevents Myeloma-Induced Bone Disease

Multiple myeloma (MM) is characterised by an expansion of malignant plasma cells in the bone marrow, systemic bone loss and destructive osteolytic bone lesions. These are mediated by an imbalance in bone remodeling, in which bone resorption is exacerbated and bone formation is suppressed. More than 90% of MM patients present with osteolytic lesions that can lead to pain and increased risk of fracture, significantly impacting their quality of life. Bone-targeted treatments currently used in the clinic can suppress lesion progression and reduce fracture risk, however these agents cannot replace lost bone and patients continue to fracture. Therapeutic strategies aimed at promoting bone formation are therefore required to overcome the loss of skeletal integrity and subsequent fractures in MM patients.Therapeutic agents that target the canonical Wnt signaling pathway, a potent regulator of bone formation, have the potential to address these skeletal complications, where they could rebuild lost bone and improve bone strength in affected individuals. We have demonstrated a novel anti-LRP6 agent, which potentiates Wnt signaling through binding the Wnt receptor LRP6, prevented the development of myeloma-induced bone loss primarily through preventing bone resorption. However, since MM patients present with both increased bone resorption and decreased bone formation, we hypothesised that combining anti-LRP6 with the bone anabolic anti-DKK1 (100mg/kg twice weekly intravenously) would lead to more robust improvements in bone structure than single treatment approaches.MicroCT analysis demonstrated a 74% increase in femoral trabecular bone volume per tissue volume (BV/TV) in naïve, non-tumour bearing mice given the combination treatment compared to control agents (p<0.0001). Mice injected with 5TGM1eGFP murine myeloma cells had a 34% reduction in femoral BV/TV compared to naïve controls (p<0.0001). Combination treatment drastically improved BV/TV in 5TGM1-bearing mice by 111% (p<0.0001), compared to control, and this improvement with the combination treatment strategy was 25% greater than anti-LRP6 single treatment approaches (p<0.001). MicroCT analysis in L4 lumbar vertebrae demonstrated similar bone structural changes in 5TGM1-bearing mice treated with the combination strategy. Consequently, this combination significantly improved resistance to fracture in L4 vertebrae in 5TGM1eGFP-bearing mice compared to their controls (p<0.001), and it provided greater protection against fracture compared to anti-LRP6 single agent treatment. Interestingly, these improvements in bone volume were primarily due to reduced bone resorption, with significant reductions in osteoclast numbers and osteoclast surface per bone surface demonstrated in 5TGM1eGFP-bearing mice treated with the combination strategy (p<0.001) compared to control. Importantly, tumour activity was not altered with either single or combination Wnt-promoting treatment strategies.This study defines a novel therapeutic strategy, which will reduce fractures and improve quality of life in patients with MM when used in combination with tumour-targeted treatments. [Display omitted] DisclosuresCong: Novartis Institutes for Biomedical Research: Current Employment. Daley: Novartis Institutes of Biomedical Research: Current Employment.

Drug Conjugates of Antagonistic R-Spondin 4 Mutant for Simultaneous Targeting of Leucine-Rich Repeat-Containing G Protein-Coupled Receptors 4/5/6 for Cancer Treatment.

LGR4-6 (leucine-rich repeat-containing G-protein-coupled receptors 4, 5, and 6) are three related receptors with an upregulated expression in gastrointestinal cancers to various extents, and LGR5 is enriched in cancer stem cells. Antibody-drug conjugates (ADCs) targeting LGR5 showed a robust antitumor effect in vivo but could not eradicate tumors due to plasticity of LGR5-positive cancer cells. As LGR5-negative cancer cells often express LGR4 or LGR6 or both, we reasoned that simultaneous targeting of all three LGRs may provide a more effective approach. R-spondins (RSPOs) bind to LGR4-6 with high affinity and potentiate Wnt signaling. We identified an RSPO4 furin domain mutant (Q65R) that retains potent LGR binding but no longer potentiates Wnt signaling. Drug conjugates of a peptibody comprising the RSPO4 mutant and IgG1-Fc showed potent cytotoxic effects on cancer cell lines expressing any LGR in vitro and suppressed tumor growth in vivo without inducing intestinal enlargement or other adverse effects.

A Wnt-Independent LGR4-EGFR Signaling Axis in Cancer Metastasis.

Leucine-rich repeat-containing G protein-coupled receptors 4, 5, and 6 (LGR4/5/6) play critical roles in development and cancer. The widely accepted mechanism is that these proteins, together with their R-spondin ligands, stabilize Wnt receptors, thus potentiating Wnt signaling. Here we show that LGR4 enhanced breast cancer cell metastasis even when Wnt signaling was deactivated pharmacologically or genetically. Furthermore, LGR4 mutants that cannot potentiate Wnt signaling nevertheless promoted breast cancer cell migration and invasion in vitro and breast cancer metastasis in vivo. Multiomic screening identified EGFR as a crucial mediator of LGR4 activity in cancer progression. Mechanistically, LGR4 interacted with EGFR and blocked EGFR ubiquitination and degradation, resulting in persistent EGFR activation. Together, these data uncover a Wnt-independent LGR4-EGFR signaling axis with broad implications for cancer progression and targeted therapy. SIGNIFICANCE: This work demonstrates a Wnt-independent mechanism by which LGR4 promotes cancer metastasis.See related commentary by Stevens and Williams, p. 4397.

Disruption of a hedgehog-foxf1-rspo2 signaling axis leads to tracheomalacia and a loss of sox9+ tracheal chondrocytes.

ABSTRACTCongenital tracheomalacia, resulting from incomplete tracheal cartilage development, is a relatively common birth defect that severely impairs breathing in neonates. Mutations in the Hedgehog (HH) pathway and downstream Gli transcription factors are associated with tracheomalacia in patients and mouse models; however, the underlying molecular mechanisms are unclear. Using multiple HH/Gli mouse mutants, including one that mimics Pallister–Hall Syndrome, we show that excessive Gli repressor activity prevents specification of tracheal chondrocytes. Lineage-tracing experiments show that Sox9+ chondrocytes arise from HH-responsive splanchnic mesoderm in the fetal foregut that expresses the transcription factor Foxf1. Disrupted HH/Gli signaling results in (1) loss of Foxf1, which in turn is required to support Sox9+ chondrocyte progenitors, and (2) a dramatic reduction in Rspo2, a secreted ligand that potentiates Wnt signaling known to be required for chondrogenesis. These results reveal an HH-Foxf1-Rspo2 signaling axis that governs tracheal cartilage development and informs the etiology of tracheomalacia.This article has an associated First Person interview with the first author of the paper.

The tumor genetics of acral melanoma: What should a dermatologist know?

Dermatologists stand at the gateway of individualization of classification, treatment, and outcomes of acral melanoma patients. The acral melanoma genetic landscape differs in vital ways from that of other cutaneous melanomas. These differences have important implications in understanding pathogenesis, treatment, and prognosis. The selection of molecularly targeted therapy must be adapted for acral melanoma. It is also critical to recognize that tumor development is far more complex than an isolated event, reliably treated by a medication acting on a single target. Tumors exhibit intratumor genetic heterogeneity, metastasis may have different genetic or epigenetic features than primary tumors, and tumor resistance may develop because of the activation of alternative genetic pathways. Microenvironmental, immune, and epigenetic events contribute and sustain tumors in complex ways. Treatment strategies with multiple targets are required to effectively disrupt the tumor ecosystem. This review attempts to translate the current molecular understanding of acral melanoma into digestible concepts relevant to the practice of dermatology. The focus is tumor genetics defining potentially treatable cancer pathways, contextualized within the relevant pathologic and molecular features.

R-spondins engage heparan sulfate proteoglycans to potentiate WNT signaling.

R-spondins (RSPOs) amplify WNT signaling during development and regenerative responses. We previously demonstrated that RSPOs 2 and 3 potentiate WNT/β-catenin signaling in cells lacking leucine-rich repeat-containing G-protein coupled receptors (LGRs) 4, 5 and 6 (Lebensohn and Rohatgi, 2018). We now show that heparan sulfate proteoglycans (HSPGs) act as alternative co-receptors for RSPO3 using a combination of ligand mutagenesis and ligand engineering. Mutations in RSPO3 residues predicted to contact HSPGs impair its signaling capacity. Conversely, the HSPG-binding domains of RSPO3 can be entirely replaced with an antibody that recognizes heparan sulfate (HS) chains attached to multiple HSPGs without diminishing WNT-potentiating activity in cultured cells and intestinal organoids. A genome-wide screen for mediators of RSPO3 signaling in cells lacking LGRs 4, 5 and 6 failed to reveal other receptors. We conclude that HSPGs are RSPO co-receptors that potentiate WNT signaling in the presence and absence of LGRs.

A18 BUILDING BETTER ENTEROIDS: A NOVEL STRATEGY FOR ENRICHING SECRETORY EPITHELIAL CELL SUBTYPES

Abstract Background Inflammatory bowel diseases (IBD) are chronic gastrointestinal disorders that affect more than 270,000 Canadians, including over 7,000 children. Inflammation arising from IBD severely damages intestinal epithelial cells (IECs), however their role in disease pathogenesis is not fully understood, in part due to a lack of in vitro systems that recapitulate the epithelium’s complex cellular composition. Enteroids are an in vitro model where primary IECs are isolated and cultured as 3D ‘mini guts.’ They offer distinct advantages over cell lines, however current protocols generate enteroids primarily composed of enterocytes that seldom contain rarer IEC subtypes (goblet, enteroendocrine, tuft and Paneth cells). These cells are responsible for the gut’s mucus, antimicrobial and hormone secretory functions, yet their role in the pathophysiology of IBD is unclear. Aims Manipulate cell differentiation pathways in mouse and human enteroids to increase the prevalence of secretory IECs, as well as determine if enteroids derived from pediatric IBD patients exhibit impaired responses to differentiation treatments. Methods Mouse enteroids were derived from ileal, cecal and colonic crypts of C57BL/6 mice while human enteroids were isolated from healthy or pediatric IBD patient intestinal biopsies. Enteroids were initially supplemented with Wnt signaling activators and the extracellular matrix modified to enhance enteroid culture “stemness”. Differentiation was induced by growth media modulation of the Notch and Wnt signaling pathways. Enteroids were analyzed via flow cytometry to quantify expression of secretory cell markers, while immunofluorescent and Peroidic acid Schiff/Alcian Blue staining was used to visualize goblet cells. Results “Stem” treatment potentiated Wnt signaling and enhanced enteroid “stemness” as measured by Lgr5 and CD44 expression. Comparatively, differentiation of these “stem” enteroids led to a larger relative increase in secretory markers. Differentiation treatment increased expression of goblet cell markers (Muc2 and lectin) in the “stem” treated mouse cecal and colonic enteroids, but not in ileal enteroids. Notch inhibition produced increased expression of lysozyme, a Paneth cell marker, in all enteroids. A similar increase in secretory cell numbers was observed in control human enteroids following differentiation treatment. In contrast, enteroids derived from pediatric IBD patients displayed irregular differentation responses to treatment. Conclusions We demonstrate that manipulation of cell differentiation pathways increases the number of secretory IEC subtypes within enteroids. Furthermore, these pro-secretory cell responses differ in IBD patient enteroids, indicating that an alteration of the targeted cell signaling pathways may be linked to IBD pathogenesis. Funding Agencies CCC, CIHRBCCHRI Summer Studentship

Surrogate R-spondins for tissue-specific potentiation of Wnt Signaling.

Secreted R-spondin1-4 proteins (RSPO1-4) orchestrate stem cell renewal and tissue homeostasis by potentiating Wnt/β-catenin signaling. RSPOs induce the turnover of negative Wnt regulators RNF43 and ZNRF3 through a process that requires RSPO interactions with Leucine-rich repeat-containing G-protein coupled receptors (LGRs), or through an LGR-independent mechanism that is enhanced by RSPO binding to heparin sulfate proteoglycans (HSPGs). Here, we describe the engineering of ‘surrogate RSPOs’ that function independently of LGRs to potentiate Wnt signaling on cell types expressing a target surface marker. These bispecific proteins were generated by fusing an RNF43- or ZNRF3-specific single chain antibody variable fragment (scFv) to the immune cytokine IL-2. Surrogate RSPOs mimic the function of natural RSPOs by crosslinking the extracellular domain (ECD) of RNF43 or ZNRF3 to the ECD of the IL-2 receptor CD25, which sequesters the complex and results in highly selective amplification of Wnt signaling on CD25+ cells. Furthermore, surrogate RSPOs were able substitute for wild type RSPO in a colon organoid growth assay when intestinal stem cells were transduced to express CD25. Our results provide proof-of-concept for a technology that may be adapted for use on a broad range of cell- or tissue-types and will open new avenues for the development of Wnt-based therapeutics for regenerative medicine.

NRARP displays either pro- or anti-tumoral roles in T-cell acute lymphoblastic leukemia depending on Notch and Wnt signaling

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with a dismal prognosis in patients with resistant or relapsed disease. Although NOTCH is a known driver in T-ALL, its clinical inhibition has significant limitations. Our previous studies suggested that NRARP, a negative regulator of Notch signaling, could have a suppressive role in T-ALL. Here, we report that NRARP levels are significantly increased in primary T-ALL cells suggesting that NRARP is not sufficient to block NOTCH oncogenic signals. Interestingly, although NRARP overexpression blocks NOTCH1 signaling and delays the proliferation of T-ALL cells that display high levels of Notch1 signaling, it promotes the expansion of T-ALL cells with lower levels of Notch1 activity. We found that NRARP interacts with lymphoid enhancer-binding factor 1 (LEF1) and potentiates Wnt signaling in T-ALL cells with low levels of Notch. Together these results indicate that NRARP plays a dual role in T-ALL pathogenesis, regulating both Notch and Wnt pathways, with opposite functional effects depending on Notch activity. Consistent with this hypothesis, mice transplanted with T-cells co-expressing NOTCH1 and NRARP develop leukemia later than mice transplanted with T-NOTCH1 cells. Importantly, mice transplanted with T-cells overexpressing NRARP alone developed leukemia with similar kinetics to those transplanted with T-NOTCH1 cells. Our findings uncover a role for NRARP in T-ALL pathogenesis and indicate that Notch inhibition may be detrimental for patients with low levels of Notch signaling, which would likely benefit from the use of Wnt signaling inhibitors. Importantly, our findings may extend to other cancers where Notch and Wnt play a role.

Abstract 3445: Mechanism, physiological and therapeutic implications of LGR-independent potentiation of WNT signaling by R-spondins

Abstract The WNT signaling pathway encodes positional information in animals, orchestrates patterning and morphogenesis during embryonic development, and promotes tissue renewal and regeneration in adults. Dysregulation of WNT signaling has been implicated in many diseases, including developmental malformations, skeletal and dental abnormalities, cardiovascular and neurodegenerative disorders, diabetes, and many types of cancer. Members of the R-spondin family of secreted growth factors have emerged as key regulators of WNT signaling strength. The R-spondin system is a vertebrate adaptation that may be related to the appearance of sophisticated stem cell compartments, which require exquisite control of WNT signaling. The four members of the family (R-spondin 1-4) can strongly potentiate responses to WNT ligands during development and in stem cells, but the mechanisms by which they transduce signals are not fully understood and the reasons why they produce different physiological effects are largely unknown. Unexpectedly, we discovered that R-spondins 2 and 3 can uniquely potentiate WNT signaling in cells lacking LGRs 4, 5 and 6, the principal R-spondin receptors. We determined the protein domains on R-spondins necessary and sufficient for this new mode of signaling, and showed that it is mediated by an alternative interaction with cell-surface heparan sulfate proteoglycans. This finding is transformative because LGRs 4-6 were thought to be required to transduce all R-spondin signals and hence determine their site of action. Indeed, these LGRs are highly expressed in many stem cell compartments whose maintenance depends on potentiation of WNT signaling by R-spondins. Instead our work shows that there are two modes of signaling by R-spondins, an LGR-dependent and an LGR-independent mode, and that different R-spondins may use distinct molecular mechanisms to transduce signals in various biological contexts. Supporting our findings, recent work from another group demonstrated that during limb development, R-spondin 2 signals through an LGR-independent mechanism. Since components of the R-spondin signaling module are commonly mutated in cancer, therapeutic targeting of LGR-independent signaling deserves evaluation. I will present the results demonstrating our conclusions and discuss their implications in physiology and disease. Citation Format: Andres M. Lebensohn, Rajat Rohatgi. Mechanism, physiological and therapeutic implications of LGR-independent potentiation of WNT signaling by R-spondins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3445.

USP9X-mediated deubiquitination of B-cell CLL/lymphoma 9 potentiates Wnt signaling and promotes breast carcinogenesis

Hyperactivation of the canonical Wnt-signaling pathway is a prominent feature of a number of human malignancies. Transcriptional activation of this signaling cascade depends on the formation of the β-catenin-B-cell CLL/lymphoma 9 (BCL9)-pygopus (PYGO) family plant homeodomain finger 1 complex, yet how the assembly of this complex is regulated remains to be investigated. Here, using MCF-7, HeLa, HEK293T, MDA-MB-231, and Sf9 cells, along with immunoblotting and immunofluorescence, nano-HPLC-MS/MS, deubiquitination, immunoprecipitation, and chromatin immunoprecipitation (ChIP) assays, we report that BCL9 physically associates with a protein deubiquitinase, ubiquitin-specific peptidase 9, X-linked (USP9X), and that USP9X removes Lys-63-linked polyubiquitin on Lys-212 of BCL9. Importantly, the USP9X-mediated BCL9 deubiquitination facilitated the formation of the β-catenin-BCL9-PYGO complex, thereby potentiating the transcriptional activation of Wnt/β-catenin target genes. We also show that USP9X-mediated BCL9 deubiquitination promotes the proliferation and invasion of breast cancer cells. Together, these results uncover USP9X as a deubiquitinase of BCL9, implicating USP9X in Wnt/β-catenin signaling and breast carcinogenesis.

Brahma related gene 1 (Brg1) contributes to liver regeneration by epigenetically activating the Wnt/β‐catenin pathway in mice

Liver regeneration is a complicated pathophysiologic process that is regulated by a myriad of signaling pathways and transcription factors. The interaction among these pathways and factors, either cooperatively or antagonistically, may ultimately lead to recovery and restoration of liver function or permanent loss of liver function and liver failure. In the present study, we investigated the mechanism whereby the chromatin remodeling protein brahma related gene 1 (Brg1) regulates liver regeneration in mice. The Smarca4-Flox strain of mice was crossbred with the Alb-Cre strain to generate hepatocyte-specific Brg1 knockout mice. Liver injury was induced by partial hepatectomy (PHx). We report that Brg1 deletion in hepatocyte compromised liver regeneration and dampened survival after PHx in mice. Brg1 interacted with β-catenin to potentiate Wnt signaling and promote hepatocyte proliferation. Mechanistically, Brg1 recruited lysine demethylase 4 (KDM4) to activate β-catenin target genes. Our data suggest that Brg1 might play an essential role maintaining hepatic homeostasis and contributing to liver repair.-Li, N., Kong, M., Zeng, S., Hao, C., Li, M., Li, L., Xu, Z., Zhu, M., Xu, Y. Brahma related gene 1 (Brg1) contributes to liver regeneration by epigenetically activating the Wnt/β-catenin pathway in mice.

Differential activities and mechanisms of the four R-spondins in potentiating Wnt/β-catenin signaling

The four R-spondins (RSPO1-4) strongly potentiate Wnt signaling and play critical roles in normal development, adult stem cell survival, and cancer development and aggressiveness. All four RSPOs have been suggested to potentiate Wnt signaling by binding to three related receptors, i.e. leucine-rich repeat-containing, G protein-coupled receptors 4, 5, and 6 (LGR4/5/6), and then inducing the clearance of two E3 ubiquitin ligases (RNF43 and ZNRF3) that otherwise would ubiquitinate Wnt receptors for degradation. Here, we show that RSPO1-4 have differential dependence on LGRs in potentiating Wnt/β-catenin signaling and that RSPO2 can enhance this pathway without any LGR. LGR4 knockout (LGR4KO) in HEK293 cells completely abrogated the Wnt/β-catenin signaling response to RSPO1 and RSPO4 and strongly impaired the response to RSPO3. RSPO2, however, retained robust activity albeit with decreased potency. Complete rescue of RSPO1-4 activity in LGR4KO cells required the seven-transmembrane domain of LGR4. Furthermore, an RSPO2 mutant with normal binding affinity to ZNRF3 but no or little binding to LGR4 or LGR5 still potentiated Wnt/β-catenin signaling in vitro, supported the growth of intestinal organoids ex vivo, and stimulated intestinal crypt growth in vivo Mechanistically, RSPO2 could increase Wnt receptor levels in the absence of any LGR without affecting ZNRF3 endocytosis and stability. These findings suggest that RSPO1-4 use distinct mechanisms in regulating Wnt and other signaling pathways, which have important implications for understanding the pleiotropic functions of RSPOs and LGRs in both normal and cancer development.

TMEM59 potentiates Wnt signaling by promoting signalosome formation

Wnt/β-catenin signaling controls development and adult tissue homeostasis by regulating cell proliferation and cell fate decisions. Wnt binding to its receptors Frizzled (FZD) and low-density lipoprotein-related 6 (LRP6) at the cell surface initiates a signaling cascade that leads to the transcription of Wnt target genes. Upon Wnt binding, the receptors assemble into large complexes called signalosomes that provide a platform for interactions with downstream effector proteins. The molecular basis of signalosome formation and regulation remains elusive, largely due to the lack of tools to analyze its endogenous components. Here, we use internally tagged Wnt3a proteins to isolate and characterize activated, endogenous Wnt receptor complexes by mass spectrometry-based proteomics. We identify the single-span membrane protein TMEM59 as an interactor of FZD and LRP6 and a positive regulator of Wnt signaling. Mechanistically, TMEM59 promotes the formation of multimeric Wnt-FZD assemblies via intramembrane interactions. Subsequently, these Wnt-FZD-TMEM59 clusters merge with LRP6 to form mature Wnt signalosomes. We conclude that the assembly of multiprotein Wnt signalosomes proceeds along well-ordered steps that involve regulated intramembrane interactions.