Abstract LB-268: Entinostat increases susceptibility of osteosarcoma cells to NK cell lysis without suppressing NK cell cytolytic activity
Authors: Kiany Simin , Yu Ling , Guindani Michele , Wu Jimin , Stewart John , Kleinerman Eugenie S 
Published: May 2015

Introduction:

Osteosarcoma (OS) is the most common primary malignant tumor of the bone in pediatric patients. Lungs are the most common site of metastasis. Patients with recurrent and metastatic OS are resistant to chemotherapy and have very poor survival rates. Our goal is to find an alternate more effective therapy for OS lung metastasis. Allogeneic natural killer (NK) cells are emerging as a promising treatment strategy for cancers. Although data from our lab and other labs show that OS cells are susceptible to NK cell cytotoxicity, NK cell therapy by itself may not be effective enough to cure OS since tumor cells can escape immunotherapy. One way to augment cytotoxicity of NK cell is using HDAC inhibitors (HDACi). Several studies have shown that HDACis (including entinostat) sensitize tumor cells for cytotoxic effects of NK cells mostly by inducing expression of ligands for NK cells on tumor cells. In this study we sought to investigate whether entinostat would augment the cytotoxic effects NK cells on OS cells.

Methods:

In our in vitro study we investigated whether entinostat would increase expression of ligands for NK cell receptors on OS cells and as a result would enhance OS cells’ susceptibility to the cytotoxic effects of NK cells. Since the combination of NK cells and entinostat will be used in our in vivo study (a mouse model with OS lung metastasis) we determined the effect of the drug on NK cell viability, receptor expression, and cytotoxicity. NK cells were isolated from healthy donor buffy coats and were expanded ex vivo for 4 weeks in RPMI 1640 media supplemented with artificial antigen-presenting cells (K562) and IL-2.

Results:

We demonstrated that 2μM entinostat (≤ IC50) for 48hr upregulates expression of NK cell ligands (ULBP1, ULBP2/5/6, and MIC A/B) on OS cell lines (LM7, KRIB, CCHOSD, and CCHOSO). Upregulated ligands on OS cells are stable for at least 24hr after drug removal from the cultured media. Moreover, entinostat treatment increased susceptibility of OS cell lines to cytotoxic killing by NK cells. By using blocking antibodies for NK receptors we confirmed that NK cell cytotoxicity toward OS cells is dependent on receptor and ligand interaction. Treatment of NK cells with up to 2μM entinostat did not affect the viability of NK cells. This drug did not affect the expression of NK cell receptors (NKG2D, NKp30, NKp44, NKp46, and DNAM-1) within the first 24hr; however, after 48hr 1μM and 2μM of the drug decreased expression of receptors except DNAM-1. Pre-treatment of NK cells with entinostat did not decrease cytotoxicity of NK cell to OS cell lines.

Conclusion:

Our results demonstrate that entinostat immunosensitizes OS cells to NK cell lysis by inducing upregulation of ligands for NK cells on OS cells. We also show that up to 2μM treatment of entinostat for 24 hr does not have any adverse effect on NK cells. These results tell us the time point at which to administer NK cells and entinostat in combination to mice in our in vivo. These results support our hypothesis, saying that combining entinostat with NK cell therapy would augment the cytotoxicity effect of NK cells on osteosarcoma lung metastasis.

Citation Format: Simin Kiany, Ling Yu, Michele Guindani, Jimin Wu, John Stewart, Eugenie S Kleinerman. Entinostat increases susceptibility of osteosarcoma cells to NK cell lysis without suppressing NK cell cytolytic activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-268. doi:10.1158/1538-7445.AM2014-LB-268