DKK3 Gene Linked to Pancreatic Adenocarcinoma Proliferation

Rosa Hwang, MD

An investigational antibody directed against Dickkopf-3 (DKK3) may be the key in suppressing tumor growth and extending survival in patients with pancreatic adenocarcinoma, according to a recent preclinical study published in Science Translational Medicine.^1,2

DKK3 expression was determined to promote growth of pancreatic ductal adenocarcinoma, metastasis, and resistance to systemic therapies. By targeting the gene in an autochthonous model, researchers were able to stop tumor growth, induce CD3⁺ and CD8⁺ T cells, and double survival rates.

“DKK3 stimulates cancer growth, metastasis, and resistance to chemotherapy and immunotherapy,” said Rosa Hwang, MD, lead author and professor at The University of Texas MD Anderson Cancer Center. “Targeting DKK3 in a pancreatic cancer mouse model boosted immune cell infiltration and more than doubled survival.”

Previous efforts focused on depleting the fibrotic stroma of pancreatic ductal adenocarcinoma, but this research suggests that specific inhibition of the DKK3 gene within the pancreatic stellate cells (PSCs) of the fibrotic stroma may be a more effective approach in treating the tumor. Moreover, the researchers identified that a majority of human pancreatic ductal adenocarcinomas carry a high expression of DKK3.

“Pancreatic cancer has a dismal prognosis, and it is unclear if its stromal infiltrate contributes to its aggressiveness,” added Hwang. “We demonstrated that DKK3 is produced by PSCs and is present in the majority of human pancreatic cancer.”

In the study, DKK3 expression was analyzed in patients’ PSCs (HPSCs) and 20 PDAC cell lines through reverse transcription polymerase chain reaction (RT-PCR). RT-PCR revealed a high presence in patients’ PSCs and an absence in the majority of cancer cell lines.

Once DKK3 was localized to patients’ PSCs, researchers compared the gene’s expression in patients with pancreatic ductal adenocarcinoma (n = 10) and without (n = 10). Affymetrix gene expression profiling revealed a higher concentration of DKK3 expression in pancreatic ductal adenocarcinoma than in a normal pancreas. Of those confirmed with DKK3 (118), 58% demonstrated moderate to high expression.

A genetically engineered mouse model was then used to examine DKK3 expression in early-stage KRAS-mutant pancreatic ductal adenocarcinoma. The mouse models developed chronic pancreatitis (CP) and early pancreatic intraepithelial neoplasia (PanIN) lesions within 2 months, CP and late PanIN lesions within 4 months, and invasive pancreatic ductal adenocarcinoma with metastases within 6 months. DKK3 expression was 18 times as high at 2 months, 21 times as high at 4 months, and 20 times as high at 6 months in the tumors of the genetically modified mice as in those of the controlled mice. Similarly to the HPSCs, DKK3 expression was localized to invasive pancreatic tumors as opposed to tangential cancer cell lines.

In the autochthonous model of pancreatic ductal adenocarcinoma, depletion of DKK3 prolonged survival. DKK3-deficient (KPC/DKK3−/−), DKK3-heterozygous (KPC/DKK3+/−), and DKK3 wild-type (KPC/DKK3+/+) mice were examined. Complete (KPC/DKK3−/−) or partial DKK3 depletion (KPC/DKK3+/−) led to a median overall survival of 68 days and 63 days, respectively versus 47 days for mice models with DKK3 wild-type tumors (P = .0002). Moreover, having achieved at least a partial depletion led to an 80% reduction in the risk of death (HR, 0.21; 95% CI, 0.09-0.47) compared with mice models who had DKK3 wild-type tumors (HR, 0.19; 95% CI; 0.08-0.46; P = .0002).

DKK3 blockade was then evaluated with the use of JM6-6-1 and JM8-12-1 clones against human DKK3. HPSC treatment with either clone led to a 70- (P <.01) to 80-fold (P <.001) improvement in cell death and suppressed progression by 5- to 11-fold (P < .0001) compared with the control.

A syngeneic model using luciferase-labeled KPC cells were implanted in DKK3−/− or controlled mice. Immunohistochemistry indicated a 2.4-fold increase in CD3+ T cells in DKK3−/− mice models compared with controls. Mice in both groups were then given isotype control immunoglobulin G (IgG), DKK3 mAb JM6-6-1, anti-cytotoxic T-lymphocyte—associated protein 4 (alpha-CTLA4), or the combination of JM6-6-1 and alpha-CTLA4.

Alpha-CTLA4 and DKK3 mAb JM6-6-1 showed no difference in its inability to stop tumor growth. JM6-6-1 alone showed a suppression of tumor growth at day 18 compared with control IgG or alpha-CTLA4. The combination of JM6-6-1 and alpha-CTLA4 showed the first sign of tumor suppression after 8 days, becoming significant after day 18 (P < .0001). The combination demonstrated the slowest rate of tumor progression at a follow-up of 190 days. The median survival had not yet been reached, though there was a significant improvement in survival (P = .004). By day 726, 80% of the mice treated with the combination were still alive.

Alpha-CTLA4 was then tested in the KPC/DKK3-/- PDAC model for signals of activity. Median survival prior to receiving alpha-CTLA4 indicated a median survival of 57 days versus 43 days (P = .004) in KPC/DKK3−/− and KPC/DKK3+/+ mice, respectively. When KPC/ DKK3−/− mice were treated with alpha-CTLA4, there was an 11-day improvement in survival.

“Previous efforts to target pancreatic cancer stroma were directed at broadly eliminating stromal elements,” concluded Hwang. “Our study shows that a more effective strategy may be to inhibit specific tumor-promoting mechanisms attributed to PSCs, such as DKK3.”

References

1. The University of Texas MD Anderson Cancer Center. Suppression of DKK3 protein thwarts pancreatic tumor progression and prolongs survival. MD Anderson News Release. Published October 24, 2018. Accessed October 29, 2018. https://bit.ly/2OqtPqz.
2. Zhou L, Husted H, Moore T, et al. Suppression of stromal-derived Dickkopf-3 (DKK3) inhibits tumor progression and prolongs survival in pancreatic ductal adenocarcinoma [published online October 24, 2018]. Sci Transl Med. doi: 10.1126/scitranslmed.aat3487.