DKK-1 cell screening model

The Wnt signaling pathway has always been one of the important focuses of drug discovery and immunotherapy. It has been found to be involved in embryonic development, including cardiovascular development and neural development, as well as various cell behaviors, such as cell proliferation, migration, adhesion and Polarity etc. As the first Wnt pathway discovered, the classic Wnt signaling pathway has been widely studied. Although modern medicine has made many advances in treating various diseases, many patients still suffer from diseases related to dysfunction of canonical Wnt signaling. The Wnt signaling pathway is mediated by Wnt protein, a secreted cysteine-rich glycoprotein that regulates specific downstream target genes. Over the past few decades, many studies in this area have emphasized the importance of developing drugs that stimulate the Wnt pathway and exploring possible therapeutic strategies. Given the difficulty and cost of obtaining biologically active Wnt proteins for the development of agonists, a viable alternative to activating the Wnt pathway is to block naturally occurring inhibitors. It is well known that DKK1 is an endogenous inhibitor of the classic Wnt signaling pathway;

In addition, it participates in other signaling pathways, including the c-Jun NH2-terminal kinase (JNK) pathway and the DKK1/CKAP4 pathway. High levels of DKK1 expression have been detected in a variety of diseases, and DKK1-mediated inhibition has been confirmed to be one of the causes of many abnormal body functions. Due to the importance of DKK1 in Wnt signaling, effective strategies to treat diseases related to this signaling pathway could include the development of highly effective inhibitors, such as antibodies and small molecules, to target the function of DKK1 or at the protein and gene levels. inhibit the expression of DKK1. In recent years, many studies have been conducted on DKK1 inhibitors, and several DKK1 antibody drugs have been developed and tested in clinical trials.

The Dickkopf family encodes four major secreted glycoproteins of 255–350 amino acids: DKK1, DKK2, DKK3, and DKK4. DKK1, DKK2 and DKK4 are highly similar in sequence, especially DKK2 has up to 70% homology with DKK1 in the C-terminal region; however, this is not the case for DKK3, which may be due to its inability to bind to the same receptor. to regulate Wnt signaling. Although DKK1/2/4 are mainly reported to have inhibitory effects on Wnt signaling, DKK2 has also been found to show the opposite effect in some studies, which mainly depends on the cellular microenvironment.

Structurally, DKK1 is a 266-amino-acid protein composed of two conserved cysteine-rich domains separated by a sequence of unknown function and variable length. , which was originally discovered in Xenopus laevis and identified as an important regulator involved in inducing head formation in amphibian embryos. DKK1 has a potential N-linked glycosylation site at Asn225 and two O-linked glycans at Ser30, resulting in a discrepancy between its apparent molecular size and theoretical molecular weight. The function may be related to enhanced invasive activity and anti-apoptotic signaling pathways; and the C-terminal domain of DKK1 (DKK1c) has been identified as an essential domain for Wnt pathway inhibition and is responsible for binding to LRP6.

As a key molecule in the classic Wnt pathway, DKK1 has been confirmed to be associated with a variety of cancers. Research evidence shows that DKK1 may inhibit the pro-apoptotic effect of β-catenin on tumor cells by downregulating β-catenin. In addition, in a variety of tumors, the high expression level of DKK1 is also an important prospective biomarker for diagnosis and prognosis. For example, a study on tumor tissue samples from 205 cancer patients showed that the expression of DKK1 was positively correlated with angiogenic mimicry, which led to enhanced invasiveness of cancer cells; another study found that serum DKK1 levels in 470 cases were higher than those in the control group This is further supported by the observation of more severe bone metastases in patients with non-small cell lung cancer (NSCLC); similarly, high serum DKK1 levels make DKK1 an important factor in esophageal squamous cell carcinoma, pancreatic cancer, prostate cancer, Promising biomarkers in cholangiocarcinoma, laryngeal squamous cell carcinoma, and liver cancer. In addition, statistics show that increased DKK1 expression is associated with poor prognosis in patients with liver cancer, but has nothing to do with OS and recurrence-free survival rate. In addition to the above-mentioned cancers, DKK1 is also involved in a variety of gynecological cancers. Higher DKK1 levels in cervical cancer patients can lead to lymph node metastasis and are positively correlated with tumor diameter; in ovarian cancer, DKK1 overexpression provides a way to suppress anti-tumor immunity. The tumor microenvironment of the cell population; a study of serum DKK1 expression levels in 89 cancer patients showed that it was significantly higher than that of healthy controls. The difference was more obvious in patients with bone metastasis, and a small molecule drug called dorsomorphin has been was shown to significantly reduce DKK1 mRNA and protein levels in cancer breast cells, suggesting that DKK1 may be an effective target in cancer.

DKK1 has immune-modulating properties, and its inhibitory effect on the immune system makes it a promising target for immunotherapy. Previously, Leap Therapeutics developed anti-DKK1 neutralizing antibody DKN-01 and was evaluated in Phase I or Phase II trials in advanced gastroesophageal junction and gastric cancer (GEJ/GC) and gynecological cancers in regimens that included combination with paclitaxel or Comparison of pembrolizumab monotherapy and combination therapy. Notably, gynecological cancer patients with Wnt signaling mutations may benefit from DKN-01 treatment. Another ongoing clinical study from GEJ/GC also shows improved survival and objective response outcomes in patients whose tumors express high levels of DKK1 (DKK1-high); very encouraging results showing that DKN-01 Adding Keytruda treatment can increase the median progression-free survival (PFS) to more than 22 weeks and the median overall survival (OS) to nearly 32 weeks in patients with DKK1 high GEJ/GC who have not received anti-PD-1/PD-L1 treatment in the past. Among the 6 GEJ/GC patients who were refractory to PD-1/PD-L1 treatment, the ORR was 50% and the disease control rate (DCR) was 80%. Among the 6 GEJ/GC patients who were refractory to PD-1/PD-L1 treatment, 3 patients with high serum DKK1 levels had stable disease, while Three patients with low serum DKK1 levels progressed. These results indicate that DKK1 inhibitory therapy significantly improves the efficacy of patients with high serum DKK1 levels, and conclude that the combination of DKK1 inhibitors and PD1/PDL-1 immune checkpoint therapy is a promising approach in patients with tumors with high DKK1 expression. strategies for better outcomes. There are currently multiple DKK1 inhibitors in the research and development stage, most of which are neutralizing antibodies to DKK1, and there are also a small number of small molecules and recombinant peptides, small nucleic acid drugs, etc.

For this important tumor immunity target, we also promptly developed a cell screening model for screening DKK1 inhibitors, which can be used for functional screening and testing of active DKK1 blocking antibodies to facilitate the development of related drugs.

DKK-1 Effector Reporter Cell RQP74200

Figure 1. Dose Responese of DKK-1 Neutralization Ab to Rescue the Inhibition of Recombinant hWnt-3a Induced Reporter Activity by DKK-1 in DKK-1 Effector Reporter Cells (C11).