Inhibition of DCLK1 with DCLK1-IN-1 Suppresses Renal Cell Carcinoma Invasion and Stemness and Promotes Cytotoxic T-Cell-Mediated Anti-Tumor Immunity.

Ling Ding,Qin Lu,Xiaohui Xu,Xuzheng Chen,Sassan Hafizi,Jian Du,Yuning Yang,Yang Ge,Jiannan Yao,Jian Liu,Zixing Yan,Zhiyun Cao,Nathaniel Weygant

doi:10.3390/cancers13225729

Abstract

Simple SummaryIn this study, we found that the novel small molecule kinase inhibitor DCLK1-IN-1 not only inhibited DCLK1 phosphorylation, stemness, and EMT-related properties of RCC cells but also revealed its potential as an immunotherapy agent and potential combination therapy with anti-PD1 against RCC in immune co-culture experiments.The approval of immune checkpoint inhibitors has expanded treatment options for renal cell carcinoma (RCC), but new therapies that target RCC stemness and promote anti-tumor immunity are needed. Previous findings demonstrate that doublecortin-like kinase 1 (DCLK1) regulates stemness and is associated with RCC disease progression. Herein, we demonstrate that small-molecule kinase inhibitor DCLK1-IN-1 strongly inhibits DCLK1 phosphorylation and downregulates pluripotency factors and cancer stem cell (CSC) or epithelial-mesenchymal transition (EMT)-associated markers including c-MET, c-MYC, and N-Cadherin in RCC cell lines. Functionally, DCLK1-IN-1 treatment resulted in significantly reduced colony formation, migration, and invasion. Additionally, assays using floating or Matrigel spheroid protocols demonstrated potent inhibition of stemness. An analysis of clinical populations showed that DCLK1 predicts RCC survival and that its expression is correlated with reduced CD8+ cytotoxic T-cell infiltration and increases in M2 immunosuppressive macrophage populations. The treatment of RCC cells with DCLK1-IN-1 significantly reduced the expression of immune checkpoint ligand PD-L1, and co-culture assays using peripheral blood monocytes (PBMCs) or T-cell expanded PBMCs demonstrated a significant increase in immune-mediated cytotoxicity alone or in combination with anti-PD1 therapy. Together, these findings demonstrate broad susceptibility to DCLK1 kinase inhibition in RCC using DCLK1-IN-1 and provide the first direct evidence for DCLK1-IN-1 as an immuno-oncology agent.

Highlights

Early stage, unilateral renal cell carcinoma (RCC) is generally curable via radical nephrectomy, it is highly intractable in the advanced stages
To confirm the efficacy of doublecortin-like kinase 1 (DCLK1)-IN-1, we performed Western blotting using a specific antibody to detect the phosphorylation of Serine 337 in the 82 kDa isoform of DCLK1 (Uniprot O15074-2; long-α) in three human RCC cell lines: ACHN, 786-O and CAKI-1
These findings demonstrate that DCLK1-IN-1 has anticlonogenic effects in RCC cell lines at non-toxic doses ranging from 1 to 10 μM

Summary

Introduction

Unilateral renal cell carcinoma (RCC) is generally curable via radical nephrectomy, it is highly intractable in the advanced stages. A variety of treatment options are available including IL-2 cytokine therapy, angiogenesis inhibitors (sunitinib, axitinib, and others), mTOR inhibitors (everolimus and temsirolimus), and immune checkpoint inhibitors (ICIs; ipilimumab and nivolumab). Antiangiogenic or mTOR inhibitors are generally used as first-line chemotherapies and ICIs are provided as a second-line chemotherapy upon resistance or relapse. In 2021, on the basis of improvements in progression-free survival (PFS), the FDA approved two combinations of ICIs and receptor tyrosine kinase inhibitors as first-line therapies (cabozantinib + nivolumab; levatinib + pembrolizumab). Only the PD1-targeted ICI nivolumab has been shown to significantly prolong overall survival (OS) [2].

Methods

Results

Discussion

Conclusion