Overcoming antigen tolerance to develop GB22–45-2, an anti-DKK1 antibody for gastric cancer

<h3>Purpose/Objective(s)</h3> Head and neck squamous cell carcinoma (HNSCC) is associated with high immune cell infiltration, and immunotherapy is currently one of the most promising treatments for HNSCC. Dickkopf-1 (DKK1), a Wnt-signaling inhibitor and mediator of immune activation, is highly associated with tumor progression and the tumor microenvironment and, thus, disease prognosis. However, the associations between DKK1 expression, HNSCC prognosis, and tumor-infiltrating lymphocytes remain unknown. <h3>Materials/Methods</h3> We investigated these relationships in this study by exploring DKK1-expression differences in multiple tumor tissues and normal tissues, using the TIMER, Oncomine, and UALCAN databases. We searched the Kaplan–Meier database to assess correlations between DKK1 mRNA-expression levels and clinical outcomes. Subsequently, we evaluate the correlation between DKK1 expression and tumor immune-cell infiltration using the TIMER platform and TISIDB website. In addition, we assessed DKK1, CD3, and CD4 expression in 27 head and neck tumor samples and DKK1 expression in 15 normal head and neck tissue samples by immunohistochemistry. Moreover, we used the cBioPortal and UCSC Xena databases to explore aberrant DKK1 expression during tumorigenesis. <h3>Results</h3> DKK1 significantly upregulated in HNSCC tissues versus normal tissues. DKK1 expression was related to the HNSCC tumor stage, tumor grade, histology, lymph node metastasis, and a poor clinical prognosis. DKK1 expression correlated negatively with CD3+ (P < 0.0001) and CD4+ (P < 0.0001) immune cell infiltration in HNSCC tumor tissues. Moreover, DKK1 closely correlated with genetic markers of various immune cells. The prognosis of HNSCC correlated negatively with DKK1 expression in immune cells. Copy-number variation and DNA methylation may promote abnormal DKK1 upregulation in HNSCC. These results help understand the role of DKK1 in HNSCC immunotherapy and provide a rationale for clinical research on treating HNSCC by targeting DKK1. <h3>Conclusion</h3> DKK1 may therefore serve as a prognostic indicator and a biomarker for immune infiltration in HNSCC, and potentially as a central target for improving the diagnosis and immunotherapy efficacy of HNSCC.

Objective: To clear the expression of transcription factor Dickkopf-1 (DKK1) in Oral Squamous Cell Carcinoma (OSCC) using the method of bioinformatics analysis. And to clarify the relationship between the expression of DKK1 and the clinicopathological characteristics of OSCC using the method of molecular biology and cytobiology, in order to determine the early diagnosis and significance of OSCC according to the marker of DKK1. Methods: In this study, the expression level of DKK1 in OSCC tissues was analyzed using GEPIA and TCGA databases, and then verified in vitro by qRTPCR and Western-blot analysis. The correlation between DKK1 gene expression and the clinical pathological parameters of OSCC, and also the impact of DKK1 on prognosis were determined using the LinkedOmics database. In addition, DKK1 was knocked down by RNA interference in SCC-4 and SCC-25 OSCC cell lines and the proliferation ability of OSCC cells was assessed by MTT assay. Results: High expression of DKK1 in OSCC is positively correlated with the pathological grade and T stage of OSCC. According to the TCGA results, DKK1 mRNA was highly expressed and it is related to the overall survival rate of OSCC. In addition, the expression level of both DKK1mRNA and protein were significantly raised in the cell line SCC-25 and SCC-4. Furthermore, MTT analysis showed that DKK1 knockdown resulted in reduced proliferation of OSCC cells. Conclusions: TCGA database analysis showed that DKK1 was highly expressed in OSCC, and it is closely correlated to the pathological parameters of OSCC, which will provide important theoretical guidance for the subsequent study of oral squamous cell carcinoma.

Objective: To clear the expression of transcription factor Dickkopf-1 (DKK1) in Oral Squamous Cell Carcinoma (OSCC) using the method of bioinformatics analysis. And to clarify the relationship between the expression of DKK1 and the clinicopathological characteristics of OSCC using the method of molecular biology and cytobiology, in order to determine the early diagnosis and significance of OSCC according to the marker of DKK1. Methods: In this study, the expression level of DKK1 in OSCC tissues was analyzed using GEPIA and TCGA databases, and then verified in vitro by qRT-PCR and Western-blot analysis. The correlation between DKK1 gene expression and the clinical pathological parameters of OSCC, and also the impact of DKK1 on prognosis were determined using the Linked Omics database. In addition, DKK1 was knocked down by RNA interference in SCC-4 and SCC-25 OSCC cell lines and the proliferation ability of OSCC cells was assessed by MTT assay. Results: High expression of DKK1 in OSCC is positively correlated with the pathological grade and T stage of OSCC. According to the TCGA results, DKK1 mRNA was highly expressed and it is related to the overall survival rate of OSCC. In addition, the expression level of both DKK1mRNA and protein were significantly raised in the cell line SCC25 and SCC-4. Furthermore, MTT analysis showed that DKK1 knockdown resulted in reduced proliferation of OSCC cells. Conclusions: TCGA database analysis showed that DKK1 was highly expressed in OSCC, and it is closely correlated to the pathological parameters of OSCC, which will provide important theoretical guidance for the subsequent study of oral squamous cell carcinoma.

Wnt signalling pathways regulate proliferation, motility and survival in a variety of human cell types. Dickkopf 1 (DKK1) gene codes for a secreted Wnt inhibitory factor. It functions as tumour suppressor gene in breast cancer and as a pro-apoptotic factor in glioma cells. In this study, we aimed to demonstrate whether the different expression of DKK1 in human glioma-derived cells is dependent on microenvironmental factors like hypoxia and regulated by the intercellular crosstalk with bone-marrow-derived mesenchymal stem cells (bmMSCs). Glioma cell line U87-MG, three cell lines from human glioblastoma grade IV (glioma-derived mesenchymal stem cells) and three bmMSCs were selected for the experiment. The expression of DKK1 in cell lines under normoxic/hypoxic environment or co-culture condition was measured using real-time PCR and enzyme-linked immunoadsorbent assay. The effect of DKK1 on cell migration and proliferation was evaluated by in vitro wound healing assays and sulphorhodamine assays, respectively. Glioma-derived cells U87-MG displayed lower DKK1 expression compared with bmMSCs. Hypoxia led to an overexpression of DKK1 in bmMSCs and U87-MG when compared to normoxic environment, whereas co-culture of U87-MG with bmMSCs induced the expression of DKK1 in both cell lines. Exogenous recombinant DKK1 inhibited cell migration on all cell lines, but did not have a significant effect on cell proliferation of bmMSCs and glioma cell lines. In this study, we showed for the first time that the expression of DKK1 was hypoxia dependent in human malignant glioma cell lines. The induction of DKK1 by intracellular crosstalk or hypoxia stimuli sheds light on the intense adaption of glial tumour cells to environmental alterations.

Bone mass is inversely proportional to Dkk1 levels in mice

Dickkopf1 (DKK1) is overexpressed in various cancers and promotes cancer cell proliferation by binding to cytoskeleton-associated protein 4 (CKAP4). However, the mechanisms underlying DKK1 expression are poorly understood. RNA sequence analysis revealed that expression of the transcription factor forkhead box M1 (FOXM1) and its target genes concordantly fluctuated with expression of DKK1 in pancreatic ductal adenocarcinoma (PDAC) cells. DKK1 knockdown decreased FOXM1 expression and vice versa in PDAC and esophageal squamous cell carcinoma (ESCC) cells. Inhibition of either the DKK1-CKAP4-AKT pathway or the ERK pathway suppressed FOXM1 expression, and simultaneous inhibition of both pathways showed synergistic effects. A FOXM1 binding site was identified in the 5ʹ-untranslated region of the DKK1 gene, and its depletion decreased DKK1 expression and cancer cell proliferation. Clinicopathological and database analysis revealed that PDAC and ESCC patients who simultaneously express DKK1 and FOXM1 have a poorer prognosis. Multivariate analysis demonstrated that expression of both DKK1 and FOXM1 is the independent prognostic factor in ESCC patients. Although it has been reported that FOXM1 enhances Wnt signaling, FOXM1 induced DKK1 expression independently of Wnt signaling in PDAC and ESCC cells. These results suggest that DKK1 and FOXM1 create a positive feedback loop to promote cancer cell proliferation.

BackgroundVitamin D is a fat-soluble vitamin that has a protective role in colorectal cancer. Several studies have identified the association between vitamin D and changes in DNA methylation in different types of tumours. Dickkopf-1 (DKK1) inhibits the Wnt/β-catenin signalling pathway, and 1,25(OH)2D3 can induce DKK1 expression in colorectal cancer. However, whether 1,25(OH)2D3 can affect DKK1 expression by regulating DNA methylation in colorectal cancer is not known.MethodsFifty-seven colorectal cancer (CRC) patients and fifty-five healthy controls were included in this study. Serum DKK1 and 25(OH)D levels were measured via ELISA and liquid chromatography‒tandem mass spectrometry, respectively, and the associations of DKK1 with clinicopathological characteristics and 25(OH)D were analysed. A DKK1 expression plasmid was transfected into cells to assess the functional significance of DKK1 in CRC progression via CCK8, wound healing and migration assays. BiSulphite Amplicon Sequencing (BSAS) and methylation-specific PCR were used to detect the DKK1 methylation status of colorectal cancer cells and tissues. The effect of 1,25(OH)2D3 on DKK1 methylation was investigated by pyrosequencing. A dual-luciferase reporter assay was performed to investigate the influence of CpG island methylation on DKK1 transcriptional activity.ResultsA decreased serum DKK1 level was closely associated with nerve infiltration and 25(OH)D status in patients with colorectal cancer. Overexpression of DKK1 reduced the proliferative and migratory capabilities of colorectal cancer cells. The methylation patterns of DKK1 (− 195 to + 231), including 31 CpG sites, were assayed via BSAS in CRC cells and tissues. Compared with those in adjacent normal tissues, the methylation levels of multiple CpG sites located in the promoter, 5’UTR and exon 1 were increased in tumour tissues. DKK1 hypermethylation was associated with decreased DKK1 expression in colorectal cancer cells and tissues. 1,25(OH)2D3 induced DKK1 expression in colorectal cancer cells, and pyrosequencing revealed that 1,25(OH)2D3 treatment induced demethylation of CpG sites located in the promoter (− 97 to − 32) and 5’UTR (+ 39 to + 97). The dual-luciferase reporter assay further confirmed that CpG island methylation (-120 to + 225) directly represses DKK1 transcription.ConclusionDKK1 functions as a tumour suppressor in colorectal cancer, and 1,25(OH)2D3 upregulates DKK1 expression by inducing demethylation of the DKK1 promoter and 5’UTR in specific colorectal cancer cell lines.

<h3>Background</h3> Dickkopf 1 (DKK1) is associated with tumor progression. However, whether DKK1 influences the tumor response to programmed cell death protein 1 (PD-1) blockade in colorectal cancers (CRCs) with deficient...

357 Background: Dickkopf-1 (DKK1) modulates Wnt signaling and contributes to an immune suppressive tumor microenvironment. DKN-01 (D), a neutralizing DKK1 antibody + pembrolizumab (P) has demonstrated safety and clinical activity in advanced GEA. We report response and survival outcomes in anti-PD-1/anti-PD-L1 naïve GEA patients by high/low tumoral DKK1 expression. Methods: We enrolled advanced anti-PD-1/PD-L1 naïve GEA patients (pts) in a Phase 1b/2a study of D + P (NCT02013154). Tumoral DKK1 mRNA expression was assessed by an in situ hybridization RNAscope assay. Objective response rate (ORR), disease control rate (DCR), progression free survival (PFS) and overall survival (OS) were compared between DKK1 high and low groups. Kaplan-Meier method and Cox-PH model was used for survival analysis and logistic regression was used for clinical benefit/response outcome. Results: 34 GEA pts were enrolled to receive 300 mg D + P. 31 GEA pts had DKK1 expression available (25 response-evaluable/RE) and 27 had both DKK1 and PD-L1 expression available (22 RE). In RE pts, DKK1 high (H-score ≥ upper-tertile [≥35]) had an ORR of 50% (5 PR/10), DCR of 80% (8/10) while DKK1 low ( &lt; upper-tertile) had an ORR of 0% (0/15) and DCR of 20% (3/15); DKK1 high (vs. low) had an OR of 16 (95%CI: 2.2, 118.3; n = 25) and adjusted (for PD-L1 CPS ≥10 vs. &lt; 10) OR of 17.6 (95%CI:1.6, 194.4; n = 22) for clinical benefit/response (PR/SD vs. PD). Median PFS was 22.1 weeks in DKK1 high (n = 11) vs. 5.9 weeks in DKK1 low (n = 20); HR of 0.23 (95%CI: 0.082, 0.66; n = 31). Adjusted (for PD-L1 expression) HR for DKK1 high was 0.20 (95%CI: 0.061,0.68; n = 27) for PFS. DKK1 high (n = 11) had a median OS of 31.6 weeks vs. 17.4 weeks in DKK1 low (n = 20); HR of 0.45 (95%CI: 0.16, 1.3; n = 31) and adjusted HR of 0.62 (95%CI: 0.2,1.9; n = 27). Conclusions: High levels of tumoral DKK1 expression identify advanced anti-PD-1/PD-L1 naïve GEA pts with the greatest benefit from D + P. Improvements in response/clinical benefit and PFS were observed independent of PD-L1 expression. Tumoral DKK1 as a potential predictive biomarker for DKN-01 treated GEA pts will be evaluated in future studies. Overall survival follow-up is ongoing. Clinical trial information: NCT02013154.

e15047 Introduction: Inactivation of the vitamin D receptor (VDR) and up-regulation of its down-stream molecules, Dickkopf-1 (DKK-1) and DKK-4 are implicated in the aggressive progression of colorectal adenocarcinomas (CRC). In this study, the expression of these molecules was evaluated in sporadic CRCs, and their expression levels were assessed for correlation with patient prognosis. Methods: By the qRT-PCR, 98 CRCs and matching normal tissues were analyzed for expression of VDR, DKK-1, and DKK-4 at the mRNA level. These levels were normalized to b-actin expression and calculated as ratios of copy numbers. The expression levels were assessed for correlation with clinicopathological features and for their prognostic importance. The survival probabilities were estimated by the Kaplan-Meier method. Results: VDR expression was 1.5 times lower in CRCs (53 of 98, 54%) than in their corresponding normal tissues. Expression of DKK-1 was found in 51 of 98 (52%) of the CRCs and in 29 of 98 (29%) of the normal tissues. Expression of DKK-4 was found in only 14 of 98 (14%) of the CRCs and in none of the normal tissues. VDR expression was 2.0 fold lower in tumors that exhibited metastasis, and poor differentiation and in large tumors ( &gt; 5) cm as compared to tumors without metastasis, well or moderately differentiated tumors and tumors of small size (&lt; 5 cm), respectively. In contrast, increased (3-fold) expression of DKK-1 and DKK-4 was observed in tumors with distant and/or nodal metastases as compared to tumors without such metastases. Expression of DKK-1 was higher in tumors with poor differentiation and in large tumors. There was an inverse relationship between VDR and DKK-4 expression. Survival analyses suggested that CRCs expressing DKK-1 (log rank, P = 0.046) and those with decreased or lack of expression of VDR (log rank, P = 0.043) were associated with poor patient survival. There was, however, no prognostic value for DKK-4 expression (log rank P=0.624). Conclusions: These findings suggest that, for CRCs, increased expression of DKK-1 and reduced or lack of expression of VDR, are associated with aggressiveness and with a poor prognosis for patients. These results provide evidence augmenting the VDR-DKK- pathway may represent a rational therapeutic strategy in colorectal carcinoma. No significant financial relationships to disclose.

Rejection following liver and kidney transplantation remains a major barrier to long-term graft survival. Early and reliable detection of rejection is crucial for optimizing patient outcomes and guiding personalized therapeutic approaches. Despite ongoing efforts, currently available serum-based biomarkers often fail to provide sufficient sensitivity and specificity for early diagnosis. Dickkopf-1 (DKK1) and cytoskeleton-associated protein 4 (CKAP4) are molecules involved in Wnt signaling, immune regulation, fibrosis, and tissue remodeling. Their upregulation has been associated with inflammatory and fibrotic processes in various pathological contexts. These properties make them strong candidates as novel molecular biomarkers in transplant rejection. This prospective observational study aimed to investigate the association between DKK1 and CKAP4 mRNA expression levels and the occurrence of rejection in liver and kidney transplant recipients. Peripheral blood samples from 55 transplant patients diagnosed with rejection (30 kidney, 25 liver) and 35 healthy controls were analyzed for DKK1 and CKAP4 mRNA expression using real-time polymerase chain reaction. Expression profiles were evaluated in relation to clinical and histopathological parameters. DKK1 and CKAP4 mRNA expression levels were significantly elevated in transplant recipients with rejection compared with healthy controls. In kidney transplant patients, both markers showed increased expression, although no significant histopathological correlations were detected. In liver transplant recipients, DKK1 expression was significantly associated with cellular rejection and portal inflammation. These findings suggest that DKK1 and CKAP4 may serve as promising molecular biomarkers for transplant rejection monitoring. In particular, DKK1 may provide additional diagnostic value in identifying cellular rejection and portal inflammation in liver grafts. Further multicenter studies are required to validate these results and assess their potential for clinical application.

DKK1 is a secreted protein that antagonizes Wnt signaling and plays essential roles in vertebrate embryogenesis including head induction, skeletal development, and limb patterning. DKK1 is also implicated in osteoporosis, arthritis, and cancer and represents a potential therapeutic target for the treatment of these diseases. DKK1 is a high affinity antagonistic ligand for LRP6, which is a Wnt coreceptor that acts together with the Frizzled serpentine receptor to initiate Wnt signal transduction. Two different models have been proposed to account for the mechanism by which DKK1 antagonizes LRP6 function. One model suggests that DKK1 binding to LRP6 disrupts Wnt-induced Frizzled-LRP6 complex formation, whereas the other model proposes that DKK1 interaction with LRP6 promotes LRP6 internalization and degradation, thereby reducing the cell surface LRP6 level. To clarify the molecular basis of DKK1 action, we examined how DKK1 affects the endogenous LRP6 in several mammalian cell lines including mouse embryonic fibroblasts. Here we show that DKK1 inhibits Wnt signaling but induces neither LRP6 down-regulation from the cell surface nor reduction of total LRP6 protein level and that DKK1 has no effect on the rate of continuous internalization of LRP6 and the half-life (about 4.7 h) of LRP6. We conclude that DKK1 inhibition of LRP6 is independent of LRP6 internalization and degradation.

Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, characterized by extensive heterogeneity, high rates of metastasis and recurrence, and poor prognosis. Chemotherapy with severe toxicity still remains the mainstay systemic treatment. Although various molecular therapeutic targets exist for TNBC, the low proportion regarding the expression of those targets in TNBC tissues limits the number of the patients who benefit from the molecular therapeutics. Therefore, there is an urgent need to identify novel molecular targets those are expressed in the substantial proportion of TNBC patients. Methods: Dickkopf-1 (DKK1) protein expression within clinical TNBC tissues was evaluated using immunohistochemistry (IHC) assay, and the corresponding H-score was calculated. DKK1-positive TNBC tissues were defined by an H-score greater than 50 in the IHC analysis. As genetic approaches, DKK1 knockout MDA-MB-231 (231-DKK1 KO) / 4T1 (4T1-DKK1 KO) cell lines using CRISPR/Cas9 technology was constructed, respectively. As a pharmacologic approach, a single-strand nucleic acid aptamer specifically against DKK1 was then utilized to synthesize a DKK1 aptamer-based PROTAC (Apc102) with both exceptional cellular internalization ability and intracellular DKK1 degradation activity. The cellular internalization of DKK1 antibody and Apc102 was evaluated using fluorescence microscopy. Cell proliferative ability was evaluated using colony formation assay. Cell migratory and invasive abilities were evaluated using transwell assay. Results: In our clinical TNBC biobank (n=48), it was found that DKK1, an osteocyte-derived secretory protein, was positively expressed (H-score &amp;gt; 50) in a substantial proportion (73%) of clinical TNBC tissues. Bioinformatic analysis indicated that DKK1 expression was associated with poor prognosis in TNBC patients. DKK1 knockout significantly inhibited cell proliferation, migration, and invasion in both 231-DKK1 KO and 4T1-DKK1 KO in vitro. It suggested the vital role of DKK1 in TNBC. Anti-DKK1 antibody could not be internalized by TNBC cells and demonstrated no effects on cell proliferation, migration, and invasion in TNBC cells in vitro. It suggested that the role of extracellular DKK1 could be excluded in TNBC in vitro. Pharmacologically, Apc102 led to a significant inhibition of cell proliferation, migration, and invasion in MDA-MB-231 cell line in vitro. It suggested the vital role of intracellular DKK1 in TNBC. Conclusions: DKK1 was positively expressed in a substantial proportion of TNBC patients and associated with poor prognosis. Intracellular DKK1 could play a vital role in promoting TNBC cell proliferation, migration, and invasion. Citation Format: YIHAO ZHANG, ZIQI CHEN, YUFEI PAN, MEIHENG SUN, YUMENG LIU, MENGHAO CHEN, HANG LUO, WEI KANG, AIPING LU, SIFAN YU, YUANYUAN YU, GE ZHANG, BAOTING ZHANG. Intracellular DKK1 could play a vital role in promoting triple-negative breast cancer cell proliferation, migration, and invasion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Functional and Genomic Precision Medicine in Cancer: Different Perspectives, Common Goals; 2025 Mar 11-13; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(5 Suppl):Abstract nr A048.

The aim of this study was to investigate the presence and prognostic value of Dickkopf-1 (DKK1) in epithelia ovarian carcinoma. The expression of DKK1 was determined in 56 epithelial ovarian carcinoma tissues, 35 benign ovarian cystadenoma tissues, and 12 normal ovarian tissues by quantitative real-time PCR and immunohistochemistry. The DKK1 mRNA level in the carcinoma tissues was upregulated (5.5+/-2.7-fold increase) compared with that in the normal tissues (p<0.0001). The mRNA level of DKK1 in the cystadenoma tissues (1.1+/-0.4-fold increase) was not statistically different from that in the normal tissues (p=0.486). DKK1 protein expression in the carcinoma tissues was also higher (89.29%) than in cystadenoma (65.71%) and normal tissues (58.33%) (p=0.006 and p=0.009, respectively). In epithelial ovarian carcinoma, DKK1 gene and protein overexpression was associated with advanced FIGO stage (p=0.007, p=0.004) and poor differentiation grade (p=0.027, p=0.010). Elevated DKK1 protein levels in ovarian carcinoma samples were associated with a poor outcome in univariate and multivariate analysis (p<0.0001 and p<0.0001, respectively). The study indicated that DKK1 maymbe a useful prognostic and diagnostic indicator for epithelial ovarian carcinoma.

Purpose Dickkopf 1 (DKK1) functions as a natural antagonist of the canonical Wnt/β-catenin pathway. The purpose of this study was to examine the expression of DKK1 in vitreous samples of patients with pathological myopia, in order to search for possible correlations between DKK1 and axial length. Materials and Methods The expression of DKK1 and other cytokines in vitreous samples of 44 non-myopic eyes, 42 eyes with low-to-moderate myopia, and 51 eyes with pathological myopia were examined using multiplex cytokine detection technology. Ophthalmologic characteristics, including axial length and subfoveal choroidal thickness, were clinically measured for further analysis. Results The intravitreous levels of DKK1 (P < .0001) were markedly higher in the pathological myopia group than in the control group. There were no differences of DKK1 levels in different vitreoretinal conditions. Additionally, we found that the DKK1 levels were positively correlated with HGF (β = 0.268, P = .032), and TIMP-3 (β = 0.209, P = .047) levels, as well as with axial length (β = 0.714, P < .0001) in the pathological myopia group. Conclusions Elevated levels of DKK1 were found in the eyes with elongated axial length.