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The Narrow-Spectrum HDAC Inhibitor Entinostat Enhances NKG2D Expression Without NK Cell Toxicity, Leading to Enhanced Recognition of Cancer Cells

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ABSTRACT

Purpose

Natural killer (NK) cell cytotoxicity correlates with the ligation of activating receptors (e.g., NKG2D) by their ligands (e.g., MHC class I–related chains [MIC] A and B) on target cells. Histone deacetylase inhibitors (HDACi) at high concentrations inhibit tumor growth and can increase NKG2D ligand expression on tumor targets, but are widely regarded as toxic to NK cells.

Methods

We investigated the mechanism of entinostat, a benzamide-derivative narrow-spectrum HDACi, in augmenting the cytotoxicity of NK cells against human colon carcinoma and sarcoma by assessing gene and protein expression, histone acetylation, and cytotoxicity in in vitro and murine models.

Results

We observed that entinostat dose- and time-dependent increase in MIC expression in tumor targets and NKG2D in primary human NK cells, both correlating with increased acetylated histone 3 (AcH3) binding to associated promoters. Entinostat pretreatment of colon carcinoma and sarcoma cells, NK cells, or both led to enhanced overall cytotoxicity in vitro, which was reversed by NKG2D blockade, and inhibited growth of tumor xenografts. Lastly, we showed decreased expression of MICA and ULBP2 transcription in primary human osteosarcoma.

Conclusions

Entinostat enhances NK cell killing of cancer cells through upregulation of both NKG2D and its ligands, suggesting an attractive approach for augmenting NK cell immunotherapy of solid tumors such as colon carcinoma and sarcomas.

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Abbreviations

7-AAD:

7-Amino-actinomycin D

AcH3:

acetylated histone 3

AcH4:

acetyl-histone 4

APC:

allophycocyanin

ChIP:

chromatin immunoprecipitation

FFLuc:

firefly luciferase

HDAC:

Histone deacetylase

HDACi:

Histone deacetylase inhibitors

MIC:

MHC class I–related chains

SAHA:

suberoylanilide hydroxamic acid

ULBP:

UL16-binding proteins

VPA:

valproic acid

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ACKNOWLEDGMENTS AND DISCLOSURES

The authors acknowledge Laurence J. N. Cooper for initiating the discussions that led to this work, Joya Chandra for sharing her expertise in epigenetics and critiquing the manuscript, and Shana Palla for guiding our statistical analysis. Financial support for this research was provided to D.A.L. and D.P.M.H. by the Brenda and Howard Johnson Fund, the Physician Scientist Program of MD Anderson Cancer Center, and the Sunbeam Foundation. Financial support to S.Z. was provided by the National Natural Science Foundation of China (81071858; 81273216) and Innovation Program of Shanghai Municipal Education Commission (11ZZ105). STR DNA fingerprinting was done by the Characterized Cell Line Core at MD Anderson Cancer Center, funded by NCI # CA16672. The MSC employed in this work were provided by the Tulane Center for Gene Therapy through a grant from NCRR of the NIH, Grant # P40RR017447.

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Authors and Affiliations

  1. Shanghai University of Traditional Chinese Medicine School of Basic Medical Sciences, Shanghai, China

    Shiguo Zhu

  2. Division of Pediatrics, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA

    Cecele J. Denman, Zehra S. Cobanoglu, Simin Kiany, Dennis P. M. Hughes, Eugenie S. Kleinerman & Dean A. Lee

  3. Hematology/Oncology Section, Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA

    Ching C. Lau

  4. Baylor College of Medicine, Department of Pediatrics Center for Cell and Gene Therapy, Texas Children’s Cancer Center, Houston, Texas, USA

    Stephen M. Gottschalk

  5. Pediatrics Unit #853, University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas, 77030-4009, USA

    Dean A. Lee

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  1. Shiguo Zhu
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Supplemental Fig. 1

Map indicating the sequence and location of human MICA, MICB and DAP10 promoters, including ATG translation start sites and the promoter-specific forward and reverse PCR primers, relative to the transcription initiation sites (TIS). (JPEG 167 kb)

Supplemental Fig. 2

Enhanced expression of GFP-Luc transgene in HT1080-GFP-Luc cells treated 24 h with 0.1 – 1 μM entinostat. (JPEG 41 kb)

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Zhu, S., Denman, C.J., Cobanoglu, Z.S. et al. The Narrow-Spectrum HDAC Inhibitor Entinostat Enhances NKG2D Expression Without NK Cell Toxicity, Leading to Enhanced Recognition of Cancer Cells. Pharm Res 32, 779–792 (2015). https://doi.org/10.1007/s11095-013-1231-0

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