Towards a ‘hot’ tumor phenotype: DKN-01 sensitizes the tumor micro-environment via pro-immune cell cytokine release in vitro and ex vivo

Abstract

<h3>Objectives:</h3> The Wnt/-catenin pathway has been associated with a ‘cold' tumor microenvironment (TME) in ovarian cancer that allows for unregulated tumor progression. We characterized the impact of inhibiting DKK1 - a Wnt pathway target gene - on immune-related molecular responses in ovarian cancer. <h3>Methods:</h3> Data was collected from human cell lines, ascites collected from patients with HGSOC, and tissue from patient tumor samples treated with a DKK-1 inhibitor (DKN-01.) Ascites from 10 high grade serous ovarian cancer (HGSOC) patients were isolated, cultured <i>in vitro,</i> and treated with control or 0.5uL/mL DKN-01 for 48 hours. Cell lysates were collected for multiplex cytokine/chemokine array. Human ES2 epithelial ovarian cancer (EOC) cells were cultured <i>in vitro</i> and treated with control or DKN-01; RNA-sequencing was performed to characterize differential gene expression changes in response to treatment. We also performed H&E staining of human tumor tissue from six patients with recurrent gynecologic malignancies (EEC, MMMT, EOC), pre- and post-DKN-01 monotherapy. <h3>Results:</h3> DKK1 blockade via DKN-01 was associated with an enhanced pro-inflammatory response as exhibited by enhanced inflammatory cytokine/chemokine gene expression and secretion; these findings were associated with improved local immune cell presence. In human ascites, there was a >20% increase in IL-8, CCL5 and CCL4 cytokines in response to DKN-01; these cytokines are associated with enhanced leukocyte, T cell, NK cell, and monocyte recruitment. Additionally, there was a >20% increase in CXCL9 and EGF cytokines (both associated with T cell activation), and GM-CSF (associated with pro-inflammatory/anti-tumor M1 macrophage polarization) in response to DKN-01. RNA-seq analysis of human ovarian tissue from patients with DKN-01 treatment showed a statistically significant increases in gene expression of pro-inflammatory chemokines CXCL8 (p<0.001) and IL-18BP (p<0.05) compared to matched controls. <h3>Conclusions:</h3> DKK1 blockade facilitates a pro-inflammatory TME via increased local immunostimulatory cytokine production and release, providing a mechanism of action for improved clinical outcomes for gynecologic cancer patients by creating a ‘hot' TME. Based on these findings, further studies are needed to investigate how DKK-1 inhibition could prime the TME to sensitize ovarian cancer to immune checkpoint blockade.