LGR4 and LGR5 form distinct homodimers that only LGR4 complexes with RNF43/ZNRF3 to provide high affinity binding of R-spondin ligands.

LGR4 and LGR5 are two homologous receptors that potentiate Wnt/ß-catenin signaling in response to R-spondin (RSPO) ligands. The RSPO and LGR4 complex binds to and inhibits activities of two related E3 ubiquitin ligases, RNF43 and ZNRF3, and thus protects Wnt receptors from the E3 ligase-mediated degradation. The RSPO and LGR5 complex, however, does not interact with the E3 ligases, and the structural basis of this difference remained unknown. Here we examined the affinities of monovalent and bivalent RSPO ligands in binding to LGR4, RNF43/ZNRF3, and LGR5 in whole cells and found unique features among the receptors and E3 ligases. Monovalent RSPO2 furin domain had much lower affinity in binding to LGR4 or RNF43/ZNRF3 than the bivalent form. In contrast, monovalent and bivalent forms had nearly identical affinity in binding to LGR5. Co-expression of ZNRF3 with LGR4 led to much higher binding affinity of the monovalent form whereas co-expression of ZNRF3 with LGR5 had no effect on the affinity. These results suggest that LGR4 and RNF43/ZNRF3 form a 2:2 dimer that accommodates bivalent binding of RSPO whereas LGR5 forms a homodimer that does not. Structural models are proposed to illustrate how RSPOs bind to LGR4, RNF43/ZNRF3, and LGR5 in whole cells.

G protein-coupled receptor-targeting antibody-drug conjugates: Current status and future directions.

In recent years, antibody-drug conjugates (ADCs) have emerged as promising anti-cancer therapeutic agents with several having already received market approval for the treatment of solid tumor and hematological malignancies. As ADC technology continues to improve and the range of indications treatable by ADCs increases, the repertoire of target antigens has expanded and will undoubtedly continue to grow. G protein-coupled receptors (GPCRs) are well-characterized therapeutic targets implicated in many human pathologies, including cancer, and represent a promising emerging target of ADCs. In this review, we will discuss the past and present therapeutic targeting of GPCRs and describe ADCs as therapeutic modalities. Moreover, we will summarize the status of existing preclinical and clinical GPCR-targeted ADCs and address the potential of GPCRs as novel targets for future ADC development.

Loss of LGR5 through Therapy-induced Downregulation or Gene Ablation Is Associated with Resistance and Enhanced MET-STAT3 Signaling in Colorectal Cancer Cells.

Leucine-rich repeat-containing, G protein-coupled receptor 5 (LGR5) is highly expressed in colorectal cancer and cancer stem cells (CSCs) that play important roles in tumor initiation, progression, and metastasis. Loss of LGR5 has been shown to enhance therapy resistance. However, the molecular mechanisms that mediate this resistance remain elusive. In this study, we demonstrate conversion of LGR5+ colorectal cancer cells to an LGR5- state in response to chemotherapy, LGR5- targeted antibody-drug conjugates (ADCs), or LGR5 gene ablation led to activation of STAT3. Further investigation revealed increased STAT3 activation occurred as a result of increased mesenchymal epithelial transition (MET) factor receptor activity. LGR5 overexpression decreased MET-STAT3 activity and sensitized colorectal cancer cells to therapy. STAT3 inhibition suppressed MET phosphorylation, while constitutively active STAT3 reduced LGR5 levels and increased MET activity, suggesting a potential feedback mechanism. Combination treatment of MET-STAT3 inhibitors with irinotecan or antibody-drug conjugates (ADCs) substantiated synergistic effects in colorectal cancer cells and tumor organoids. In colorectal cancer xenografts, STAT3 inhibition combined with irinotecan enhanced tumor growth suppression and prolonged survival. These findings suggest a mechanism by which drug-resistant LGR5- colorectal cancer cells acquire a survival advantage through activation of MET-STAT3 and provide rationale for new treatment strategies to target colorectal cancer.

An antibody-drug conjugate targeting GPR56 demonstrates efficacy in preclinical models of colorectal cancer.

BACKGROUND: Long-term prognosis remains poor for colorectal cancer (CRC) patients with advanced disease due to treatment resistance. The identification of novel targets is essential for the development of new therapeutic approaches. GPR56, an adhesion GPCR, is highly expressed in CRC tumours and correlates with poor survival. Here, we describe the generation and preclinical evaluation of a novel ADC consisting of an anti-GPR56 antibody (10C7) conjugated with the DNA-damaging payload duocarmycin. METHODS: RNA-seq dataset analysis was performed to determine GPR56 expression in CRC subtypes. The specificity of binding, epitope mapping, and internalisation of 10C7 was examined. 10C7 was conjugated to payload and ADC cytotoxicity was assessed against a panel of CRC cell lines and tumour organoids. Antitumour efficacy was evaluated in xenograft models of CRC cell lines and patient-derived tumours. RESULTS: High GPR56 was shown to be associated with the microsatellite stable (MSS) subtype that accounts for 80-85% of CRC. GPR56 ADC selectively induced cytotoxicity in CRC cells and tumour organoids at low nanomolar potency in a GPR56-dependent manner and showed significant antitumour efficacy against GPR56-expressing xenograft models. CONCLUSIONS: This study provides the rationale for the future development of a GPR56-targeted ADC approach to potentially treat a large fraction of MSS CRC patients.

Chromatin state dynamics confers specific therapeutic strategies in enhancer subtypes of colorectal cancer.

OBJECTIVE: Enhancer aberrations are beginning to emerge as a key epigenetic feature of colorectal cancers (CRC), however, a comprehensive knowledge of chromatin state patterns in tumour progression, heterogeneity of these patterns and imparted therapeutic opportunities remain poorly described. DESIGN: We performed comprehensive epigenomic characterisation by mapping 222 chromatin profiles from 69 samples (33 colorectal adenocarcinomas, 4 adenomas, 21 matched normal tissues and 11 colon cancer cell lines) for six histone modification marks: H3K4me3 for Pol II-bound and CpG-rich promoters, H3K4me1 for poised enhancers, H3K27ac for enhancers and transcriptionally active promoters, H3K79me2 for transcribed regions, H3K27me3 for polycomb repressed regions and H3K9me3 for heterochromatin. RESULTS: We demonstrate that H3K27ac-marked active enhancer state could distinguish between different stages of CRC progression. By epigenomic editing, we present evidence that gains of tumour-specific enhancers for crucial oncogenes, such as ASCL2 and FZD10, was required for excessive proliferation. Consistently, combination of MEK plus bromodomain inhibition was found to have synergistic effects in CRC patient-derived xenograft models. Probing intertumour heterogeneity, we identified four distinct enhancer subtypes (EPIgenome-based Classification, EpiC), three of which correlate well with previously defined transcriptomic subtypes (consensus molecular subtypes, CMSs). Importantly, CMS2 can be divided into two EpiC subgroups with significant survival differences. Leveraging such correlation, we devised a combinatorial therapeutic strategy of enhancer-blocking bromodomain inhibitors with pathway-specific inhibitors (PARPi, EGFRi, TGFßi, mTORi and SRCi) for EpiC groups. CONCLUSION: Our data suggest that the dynamics of active enhancer underlies CRC progression and the patient-specific enhancer patterns can be leveraged for precision combination therapy.

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.

Anti-GPR56 monoclonal antibody potentiates GPR56-mediated Src-Fak signaling to modulate cell adhesion.

GPR56 is a member of the adhesion G-protein-coupled receptor family shown to play important roles in cell adhesion, brain development, immune function, and tumorigenesis. GPR56 is highly upregulated in colorectal cancer and correlates with poor prognosis. Several studies have shown GPR56 couples to the Gα12/13 class of heterotrimeric G-proteins to promote RhoA activation. However, due to its structural complexity and lack of a high-affinity receptor-specific ligand, the complete GPR56 signaling mechanism remains largely unknown. To delineate the activation mechanism and intracellular signaling functions of GPR56, we generated a monoclonal antibody (mAb) that binds with high affinity and specificity to the extracellular domain (ECD). Using deletion mutants, we mapped the mAb binding site to the GAIN domain, which mediates membrane-proximal autoproteolytic cleavage of the ECD. We showed that GPR56 overexpression in 293T cells leads to increased phosphorylation of Src, Fak, and paxillin adhesion proteins and activation of the Gα12/13-RhoA-mediated serum response factor (SRF) pathway. Treatment with the mAb potentiated Src-Fak phosphorylation, RhoA-SRF signaling, and cell adhesion. Consistently, GPR56 knockdown in colorectal cancer cells decreased Src-Fak pathway phosphorylation and cell adhesion. Interestingly, GPR56-mediated activation of Src-Fak phosphorylation occurred independent of RhoA, yet mAb-induced potentiation of RhoA-SRF signaling was Src-dependent. Furthermore, we show that the C-terminal portion of the Serine-Threonine-Proline-rich (STP) region, adjacent to the GAIN domain, was required for Src-Fak activation. However, autoproteolytic cleavage of the ECD was dispensable. These data support a new ECD-dependent mechanism by which GPR56 functions to regulate adhesion through activation of Src-Fak signaling.

Unlike LGR4, LGR5 potentiates Wnt-ß-catenin signaling without sequestering E3 ligases.

LGR4 and LGR5 encode two homologous receptors with critical, yet distinct, roles in organ development and adult stem cell survival. Both receptors are coexpressed in intestinal crypt stem cells, bind to R-spondins (RSPOs) with high affinity, and potentiate Wnt-ß-catenin signaling, presumably by the same mechanism: forming RSPO-bridged complexes with the E3 ligases RNF43 and ZNRF3 to inhibit ubiquitylation of Wnt receptors. However, direct evidence for RSPO-bound, full-length LGR5 interacting with these E3 ligases in whole cells has not been reported, and only LGR4 is essential for the self-renewal of intestinal stem cells. Here, we examined the mechanisms of action of LGR4 and LGR5 in parallel using coimmunoprecipitation, proximity ligation, competition binding, and time-resolved FRET assays in whole cells. Full-length LGR4 formed a tight complex with ZNRF3 and RNF43 even without RSPO, whereas LGR5 did not interact with either E3 ligase with or without RSPO. Domain-swapping experiments with LGR4 and LGR5 revealed that the seven-transmembrane domain of LGR4 conferred interaction with the E3 ligases. Native LGR4 and LGR5 existed as dimers on the cell surface, and LGR5 interacted with both FZD and LRP6 of the Wnt signalosome to enhance LRP6 phosphorylation and potentiate Wnt-ß-catenin signaling. These findings provide a molecular basis for the weaker activity of LGR5 in the potentiation of Wnt signaling that may underlie the distinct roles of LGR4 and LGR5 in organ development, as well as the self-renewal and fitness of adult stem cells.

The most common RNF43 mutant G659Vfs*41 is fully functional in inhibiting Wnt signaling and unlikely to play a role in tumorigenesis.

RNF43 is an E3 ligase that inhibits Wnt signaling by ubiquitinating Wnt receptors for degradation. It is mutated in various cancer types with the most recurrent mutation being the frameshift G659Vfs*41 with frequencies of ~5-8% in colon, stomach and endometrial cancers. This mutation, a deletion of G in a 7-G repeat, has been assumed to encode an inactive enzyme that would lead to increased Wnt signaling and drive tumorigenesis, yet no functional characterization has been reported. We analyzed the distribution of G659Vfs*41 and its association with other cancer gene mutations, and found that the mutation occurred nearly exclusively in tumors with low expression of the DNA mismatch repair gene MLH1. Mutant RNF43-G659Vfs*41 was no different from wild type RNF43 in expression, stability, localization, R-spondin binding, and inhibition of Wnt signaling. No dominant negative activity of the mutant was observed. Colon tumors with RNF43-G659Vfs*41 had low Wnt/ß-catenin signaling and were frequently mutated in BRAF. A colon cancer cell line with RNF43-G659Vfs*41 and BRAF-V600E mutations was sensitive to activation of Wnt/ß-catenin signaling. These findings suggest that the frequent occurrence of RNF43-G659Vfs*41 may result from error-prone replication of the 7-G repeat in MLH1-deficient tumors and that the mutation itself does not inactivate enzyme.

GPR56 Drives Colorectal Tumor Growth and Promotes Drug Resistance through Upregulation of MDR1 Expression via a RhoA-Mediated Mechanism.

Drug resistance continues to be a major obstacle of effective therapy for colorectal cancer, leading to tumor relapse or treatment failure. Cancer stem cells (CSC) or tumor-initiating cells are a subpopulation of tumor cells which retain the capacity for self-renewal and are suggested to be implicated in drug resistance. LGR5 is highly expressed in colorectal cancer and marks CSCs that drive tumor growth and metastasis. LGR5(+) CSCs cells were shown to interconvert with more drug-resistant LGR5(-) cancer cells, and treatment with LGR5-targeted antibody-drug conjugates (ADC) eliminated LGR5(+) tumors, yet a fraction of LGR5(-) tumors eventually recurred. Therefore, it is important to identify mechanisms associated with CSC plasticity and drug resistance in order to develop curative therapies. Here, we show that loss of LGR5 in colon cancer cells enhanced resistance to irinotecan and 5-fluorouracil and increased expression of adhesion G-protein-coupled receptor, GPR56. GPR56 expression was significantly higher in primary colon tumors versus matched normal tissues and correlated with poor survival outcome. GPR56 enhanced drug resistance through upregulation of MDR1 levels via a RhoA-mediated signaling mechanism. Loss of GPR56 led to suppression of tumor growth and increased sensitivity of cancer cells to chemotherapy and monomethyl auristatin E-linked anti-LGR5 ADCs, by reducing MDR1 levels. These findings suggest that upregulation of GPR56 may be a mechanism associated with CSC plasticity by which LGR5(-) cancer cells acquire a more drug-resistant phenotype. IMPLICATIONS: Our findings suggest that targeting GPR56 may provide a new strategy for the treatment of colorectal cancer and combatting drug resistance.

Application of Immuno-PET in Antibody-Drug Conjugate Development.

Targeted therapies hold great promise for cancer treatment and may exhibit even greater efficacy when combined with patient selection tools. The clinical impact of identifying likely responders includes reducing the number of unnecessary and ineffective therapies as well as more accurately determining drug effects. Positron emission tomography (PET) imaging using zirconium-89 radiolabeled monoclonal antibodies (mAbs), also referred to as zirconium-89 (89Zr)-immuno-PET, provides a potential biomarker to measure target expression and verify optimal delivery of targeted agents to tumors. Antibody-drug conjugates (ADCs) combine the high affinity and specificity of mAbs with the potency of cytotoxic drugs to target tumor-expressing antigen and destroy cancer cells. Thus, 89Zr-immuno-PET of whole-body biodistribution, pharmacokinetics, and tumor targeting of antibodies and ADCs to predict toxicity and efficacy could help guide individualized treatment. Here, we review how 89Zr-immuno-PET is being used as a companion diagnostic with the development of ADCs. Furthermore, we discuss how 89Zr-immuno-PET may be utilized in future clinical trials as an adjunct tool with novel ADCs to select cancer patients who have the greatest potential to benefit from treatment and improve ADC dosing regimens.

Inhibition of Rspo-Lgr4 Facilitates Checkpoint Blockade Therapy by Switching Macrophage Polarization.

Therapies targeting immune checkpoints have shown great clinical potential in a subset of patients with cancer but may be hampered by a failure to reverse the immunosuppressive tumor microenvironment (TME). As the most abundant immune cells in TME, tumor-associated macrophages (TAM) play nonredundant roles in restricting antitumor immunity. The leucine-rich repeat-containing G-protein-coupled receptor 4 (Lgr4, also known as Gpr48) has been associated with multiple physiologic and pathologic functions. Lgr4 and its ligands R-spondin 1-4 have been shown to promote the growth and metastasis of tumor cells. However, whether Lgr4 can promote tumor progression by regulating the function of immune cells in the tumor microenvironment remains largely unknown. Here, we demonstrate that Lgr4 promotes macrophage M2 polarization through Rspo/Lgr4/Erk/Stat3 signaling. Notably, urethane-induced lung carcinogenesis, Lewis lung carcinoma (LLC), and B16F10 melanoma tumors were all markedly reduced in Lgr4fl/flLyz2cre/+ mice, characterized by fewer protumoral M2 TAMs and increased CD8+ T lymphocyte infiltration in the TME. Furthermore, LLC tumor growth was greatly depressed when Rspo/Lgr4/Erk/Stat3 signaling was blocked with either the LGR4 extracellular domain or an anti-Rspo1 antibody. Importantly, blocking Rspo-Lgr4 signaling overcame LLC resistance to anti-PD-1 therapy and improved the efficacy of PD-1 immunotherapy against B16F10 melanoma, indicating vital roles of Rspo-Lgr4 in host antitumor immunity and a potential therapeutic target in cancer immunotherapy.Significance: This study identifies a novel receptor as a critical switch in TAM polarization whose inhibition sensitizes checkpoint therapy-resistant lung cancer to anti-PD-1 therapy.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/17/4929/F1.large.jpg Cancer Res; 78(17); 4929-42. ©2018 AACR.

Differential activities and mechanisms of the four R-spondins in potentiating Wnt/ß-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/ß-catenin signaling and that RSPO2 can enhance this pathway without any LGR. LGR4 knockout (LGR4KO) in HEK293 cells completely abrogated the Wnt/ß-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/ß-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.

Evaluation of Anti-LGR5 Antibodies by ImmunoPET for Imaging Colorectal Tumors and Development of Antibody-Drug Conjugates.

Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is highly expressed in colorectal tumors and marks colon cancer stem cells that drive tumor growth and metastasis. Recently, we showed that LGR5 is a promising target for antibody-drug conjugate (ADC) therapy. However, it is important to identify LGR5-positive tumors that would respond to ADC treatment. Prior to drug conjugation, we evaluated two different anti-LGR5 monoclonal antibodies (mAbs), 8F2 and 9G5, using 89Zr-immunoPET to select the optimal mAb for ADC development and tumor imaging. Binding, specificity, and internalization were compared, and mAbs were prescreened as ADC candidates against colon cancer cells using secondary ADCs. Both mAbs demonstrated strong, specific binding in 293T-LGR5 cells but not 293T-vector cells. In DLD-1 colorectal cancer cells, which express high levels of LGR5, the mAbs rapidly internalized into lysosomes and promoted ADC-induced cytotoxicity, with 8F2 exhibiting slightly higher potency. No binding was detected in DLD-1-shLGR5 (LGR5 knockdown) cells. 89Zr-DFO-LGR5 mAbs were generated and shown to retain high affinity and LGR5-dependent uptake in vitro. PET/CT imaging of DLD-1 tumors was performed 5 days postinjection of 89Zr-DFO-LGR5 mAbs, and findings were consistent with biodistribution data, which showed significantly higher tumor uptake (%ID/g) for 89Zr-DFO-8F2 (17.9 ± 2.2) compared to 89Zr-DFO-9G5 (5.5 ± 1.2) and 89Zr-DFO-IgG (3.8 ± 1.0). No significant uptake was observed in DLD-1-shLGR5 tumors. This study identifies 8F2 as the optimal candidate for ADC development and provides initial evidence that 89Zr-DFO-LGR5 mAbs may be utilized to stratify tumors which would respond best to LGR5-targeted ADC therapy.

LGR5 receptor promotes cell-cell adhesion in stem cells and colon cancer cells via the IQGAP1-Rac1 pathway.

Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a bona fide marker of adult stem cells in several epithelial tissues, most notably in the intestinal crypts, and is highly up-regulated in many colorectal, hepatocellular, and ovarian cancers. LGR5 activation by R-spondin (RSPO) ligands potentiates Wnt/ß-catenin signaling in vitro; however, deletion of LGR5 in stem cells has little or no effect on Wnt/ß-catenin signaling or cell proliferation in vivo Remarkably, modulation of LGR5 expression has a major impact on the actin cytoskeletal structure and cell adhesion in the absence of RSPO stimulation, but the molecular mechanism is unclear. Here, we show that LGR5 interacts with IQ motif-containing GTPase-activating protein 1 (IQGAP1), an effector of Rac1/CDC42 GTPases, in the regulation of actin cytoskeleton dynamics and cell-cell adhesion. Specifically, LGR5 decreased levels of IQGAP1 phosphorylation at Ser-1441/1443, leading to increased binding of Rac1 to IQGAP1 and thus higher levels of cortical F-actin and enhanced cell-cell adhesion. LGR5 ablation in colon cancer cells and crypt stem cells resulted in loss of cortical F-actin, reduced cell-cell adhesion, and disrupted localization of adhesion-associated proteins. No evidence of LGR5 coupling to any of the four major subtypes of heterotrimeric G proteins was found. These findings suggest that LGR5 primarily functions via the IQGAP1-Rac1 pathway to strengthen cell-cell adhesion in normal adult crypt stem cells and colon cancer cells.

Synthesis of a Fluorescently Labeled 68Ga-DOTA-TOC Analog for Somatostatin Receptor Targeting.

Fluorescently labeled imaging agents can identify surgical margins in real-time to help achieve complete resections and minimize the likelihood of local recurrence. However, photon attenuation limits fluorescence-based imaging to superficial lesions or lesions that are a few millimeters beneath the tissue surface. Contrast agents that are dual-labeled with a radionuclide and fluorescent dye can overcome this limitation and combine quantitative, whole-body nuclear imaging with intraoperative fluorescence imaging. Using a multimodality chelation (MMC) scaffold, IRDye 800CW was conjugated to the clinically used somatostatin analog, 68Ga-DOTA-TOC, to produce the dual-labeled analog, 68Ga-MMC(IRDye 800CW)-TOC, with high yield and specific activity. In vitro pharmacological assays demonstrated retention of receptor-targeting properties for the dual-labeled compound with robust internalization that was somatostatin receptor (SSTR) 2-mediated. Biodistribution studies in mice identified the kidneys as the primary excretion route for 68Ga-MMC(IRDye 800CW)-TOC, along with clearance via the reticuloendothelial system. Higher uptake was observed in most tissues compared to 68Ga-DOTA-TOC but decreased as a function of time. The combination of excellent specificity for SSTR2-expressing cells and suitable biodistribution indicate potential application of 68Ga-MMC(IRDye 800CW)-TOC for intraoperative detection of SSTR2-expressing tumors.

A Modular Dual-Labeling Scaffold That Retains Agonistic Properties for Somatostatin Receptor Targeting.

Fluorescence-guided surgery is an emerging imaging technique that can enhance the ability of surgeons to detect tumors when compared with visual observation. To facilitate characterization, fluorescently labeled probes have been dual-labeled with a radionuclide to enable cross-validation with nuclear imaging. In this study, we selected the somatostatin receptor imaging agent DOTATOC as the foundation for developing a dual-labeled analog. We hypothesized that a customized dual-labeling approach with a multimodality chelation (MMC) scaffold would minimize steric effects of dye conjugation and retain agonist properties. Methods: An MMC conjugate (MMC-TOC) was synthesized on solid-phase and compared with an analog prepared using conventional methods (DA-TOC). Both analogs were conjugated to IRDye 800 using copper-free click chemistry. The resulting compounds, MMC(IR800)-TOC and DA(IR800)-TOC, were labeled with Cu and 64Cu and tested in vitro in somatostatin receptor subtype 2-overexpressing HEK-293 cells to assess agonist properties, and in AR42J rat pancreatic cancer cells to determine receptor binding characteristics. Multimodality imaging was performed in AR42J xenografts. Results: Cu-MMC(IR800)-TOC demonstrated higher potency for cyclic adenosine monophosphate inhibition (half maximal effective concentration [EC50]: 0.21 ± 0.18 vs. 1.38 ± 0.54 nM) and receptor internalization (EC50: 41.9 ± 29.8 vs. 455 ± 299 nM) than Cu-DA(IR800)-TOC. Radioactive uptake studies showed that blocking with octreotide caused a dose-dependent reduction in 64Cu-MMC(IR800)-TOC uptake whereas 64Cu-DA(IR800)-TOC was not affected. In vivo studies revealed higher tumor uptake for 64Cu-MMC(IR800)-TOC than 64Cu-DA(IR800)-TOC (5.2 ± 0.2 vs. 3.6 ± 0.4 percentage injected dose per gram). In vivo blocking studies with octreotide reduced tumor uptake of 64Cu-MMC(IR800)-TOC by 66%. Excretion of 64Cu-MMC(IR800)-TOC was primarily through the liver and spleen whereas 64Cu-DA(IR800)-TOC was cleared through the kidneys. Ex vivo analysis at 24 h confirmed PET/CT data by showing near-infrared fluorescence signal in tumors and a tumor-to-muscle ratio of 5.3 ± 0.8 as determined by γ-counting. Conclusion: The findings demonstrate that drug design affected receptor pharmacology and suggest that the MMC scaffold is a useful tool for the development of dual-labeled imaging agents.

LGR5-Targeted Antibody-Drug Conjugate Eradicates Gastrointestinal Tumors and Prevents Recurrence.

Gastrointestinal cancer is one of the leading causes of cancer-related mortality in men and women worldwide. The adult stem cell marker LGR5 (leucine-rich repeat-containing, G protein-coupled receptor 5) is highly expressed in a significant fraction of gastrointestinal tumors of the colon, liver, pancreas, and stomach, relative to normal tissues. LGR5 is located on the cell surface and undergoes rapid, constitutive internalization independent of ligand. Furthermore, LGR5-high cancer cells have been shown to exhibit the properties of tumor-initiating cells or cancer stem cells (CSC). On the basis of these attributes, we generated two LGR5-targeting antibody-drug conjugates (ADC) by tethering the tubulin-inhibiting cytotoxic drug monomethyl auristatin E to a highly specific anti-LGR5 mAb via a protease cleavable or noncleavable chemical linker and compared them in receptor binding, cell internalization, and cytotoxic efficacy in cancer cells. Here, we show that both ADCs bind LGR5 with high specificity and equivalent nanomolar affinity and rapidly internalize to the lysosomes of LGR5-expressing gastrointestinal cancer cells. The anti-LGR5 ADCs effectively induced cytotoxicity in LGR5-high gastrointestinal cancer cells, but not in LGR5-negative or -knockdown cancer cell lines. Overall, we demonstrate that the cleavable ADC exhibited higher potency in vitro and was able to eradicate tumors and prevent recurrence in a xenograft model of colon cancer. These findings provide preclinical evidence for the potential of LGR5-targeting ADCs as effective new therapeutics for the treatment and eradication of gastrointestinal tumors and CSCs with high LGR5 expression. Mol Cancer Ther; 15(7); 1580-90. ©2016 AACR.

RSPO-LGR4 functions via IQGAP1 to potentiate Wnt signaling.

R-spondins (RSPOs) and their receptor leucine-rich repeat-containing G-protein coupled receptor 4 (LGR4) play pleiotropic roles in normal and cancer development as well as the survival of adult stem cells through potentiation of Wnt signaling. Current evidence indicates that RSPO-LGR4 functions to elevate levels of Wnt receptors through direct inhibition of two membrane-bound E3 ligases (RNF43 and ZNRF3), which otherwise ubiquitinate Wnt receptors for degradation. Whether RSPO-LGR4 is coupled to intracellular signaling proteins to regulate Wnt pathways remains unknown. We identified the intracellular scaffold protein IQ motif containing GTPase-activating protein 1 (IQGAP1) as an LGR4-interacting protein that mediates RSPO-LGR4's interaction with the Wnt signalosome. IQGAP1 binds to and modulates the activities of a plethora of signaling molecules, including MAP kinases, Rho GTPases, and components of the Wnt signaling pathways. Interaction of LGR4 with IQGAP1 brings RSPO-LGR4 to the Wnt signaling complex through enhanced IQGAP1-DVL interaction following RSPO stimulation. In this configuration, RSPO-LGR4-IQGAP1 potentiates ß-catenin-dependent signaling by promoting MEK1/2-medidated phosphorylation of LRP5/6 as well as ß-catenin-independent signaling through regulation of actin dynamics. Overall, these findings reveal that RSPO-LGR4 not only induces the clearance of RNF43/ZNRF3 to increase Wnt receptor levels but also recruits IQGAP1 into the Wnt signaling complex, leading to potent and robust potentiation of both the canonical and noncanonical pathways of Wnt signaling.