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// Aroa Baragano Raneros 1 , Alfredo Minguela Puras 2 , Ramon M. Rodriguez 1 , Enrique Colado 3 , Teresa Bernal 3 , Eduardo Anguita 4 , Adela Vasco Mogorron 2 , Alberto Chaparro Gil 4 , Jose Ramon Vidal-Castineira 1 , Leonardo Marquez-Kisinousky 1 , Paula Diaz Bulnes 1 , Amelia Martinez Marin 5 , Maria Carmen Garcia Garay 6 , Beatriz Suarez-Alvarez 1, * , Carlos Lopez-Larrea 1, * 1 Department of Immunology, Hospital Universitario Central de Asturias, Oviedo, Spain 2 Immunology Service, Instituto Murciano de Investigacion Biosanitaria (IMIB), Hospital Clinico Universitario Virgen de la Arrixaca, Murcia, Spain 3 Department of Hematology, Hospital Universitario Central de Asturias, Oviedo, Spain 4 Hematology Department, Hospital Clinico San Carlos, Instituto de Investigacion Sanitaria San Carlos (IdISSC), Department of Medicine, Universidad Complutense de Madrid (UCM), Madrid, Spain 5 Hematology Service, Hospital Clinico Universitario Virgen de la Arrixaca, Murcia, Spain 6 Hematology Service, Hospital General Universitario Santa Lucia, Cartagena, Murcia, Spain * These authors have contributed equally to this work Correspondence to: Carlos Lopez-Larrea, email: inmuno@hca.es Beatriz Suarez-Alvarez, email: bsuarez@hca.es Keywords: acute myeloid leukemia (AML), DNA methylation, NKG2DL, NKG2D, TIMP3 Received: September 16, 2016 Accepted: March 16, 2017 Published: March 29, 2017 ABSTRACT Acute myeloid leukemia (AML) is a disease with great morphological and genetic heterogeneity, which complicates its prognosis and treatment. The hypomethylating agents azacitidine (Vidaza ® , AZA) and decitabine (Dacogen ® , DAC) have been approved for the treatment of AML patients, but their mechanisms of action are poorly understood. Natural killer (NK) cells play an important role in the recognition of AML blasts through the interaction of the activating NKG2D receptor with its ligands (NKG2DL: MICA/B and ULBPs1-3). However, soluble NKG2DL (sNKG2DL) can be released from the cell surface, impairing immune recognition. Here, we examined whether hypomethylating agents modulate the release of sNKG2DL from AML cells. Results demonstrated that AZA- and DAC-treated AML cells reduce the release of sNKG2DL, preventing downregulation of NKG2D receptor on the cell surface and promoting immune recognition mediated by NKG2D-NKG2DL engagement. We show that the shedding of MICA, MICB and ULBP2 is inhibited by the increased expression of TIMP3, an ADAM17 inhibitor, after DAC treatment. The TIMP3 gene is highly methylated in AML cells lines and in AML patients (25.5%), in which it is significantly associated with an adverse cytogenetic prognosis of the disease. Overall, TIMP3 could be a target of the demethylating treatments in AML patients, leading to a decrease in MICA, MICB and ULBP2 shedding and the enhancement of the lytic activity of NK cells through the immune recognition mediated by the NKG2D receptor.

Acute myeloid leukemia (AML) is a disease with great morphological and genetic heterogeneity, which complicates its prognosis and treatment. The hypomethylating agents azacitidine (Vidaza®, AZA) and decitabine (Dacogen®, DAC) have been approved for the treatment of AML patients, but their mechanisms of action are poorly understood. Natural killer (NK) cells play an important role in the recognition of AML blasts through the interaction of the activating NKG2D receptor with its ligands (NKG2DL: MICA/B and ULBPs1-3). However, soluble NKG2DL (sNKG2DL) can be released from the cell surface, impairing immune recognition. Here, we examined whether hypomethylating agents modulate the release of sNKG2DL from AML cells. Results demonstrated that AZA- and DAC-treated AML cells reduce the release of sNKG2DL, preventing downregulation of NKG2D receptor on the cell surface and promoting immune recognition mediated by NKG2D-NKG2DL engagement. We show that the shedding of MICA, MICB and ULBP2 is inhibited by the increased expression of TIMP3, an ADAM17 inhibitor, after DAC treatment. The TIMP3 gene is highly methylated in AML cells lines and in AML patients (25.5%), in which it is significantly associated with an adverse cytogenetic prognosis of the disease. Overall, TIMP3 could be a target of the demethylating treatments in AML patients, leading to a decrease in MICA, MICB and ULBP2 shedding and the enhancement of the lytic activity of NK cells through the immune recognition mediated by the NKG2D receptor.