Background: Therapies targeting anti-tumor T-cell responses have proven successful in the treatment of a variety of malignancies. However, as most patients still fail to respond, approaches to augment immunotherapeutic efficacy are needed. Here, we investigated the ability of histone deacetylase 6 (HDAC6)-selective inhibitors to decrease immunosuppression and enhance immune function of melanoma patient T-cells in ex vivo cultures. Methods: T-cells were harvested from peripheral blood or tumor biopsies of metastatic melanoma patients and cultured in the presence of pan-, class-specific or class-selective histone deacetylase (HDAC) inhibitors. Changes in cytokine production were evaluated by Luminex and intracellular flow cytometry staining. Expression of surface markers, transcription factors, protein phosphorylation, and cell viability were assessed by flow cytometry. Changes in chromatin structure were determined by ATAC-seq. Results: T-cell viability was impaired with low doses of pan-HDAC inhibitors but not with specific or selective HDAC inhibitors. The HDAC6-selective inhibitors ACY-1215 (ricolinostat) and ACY-241 (citarinostat) decreased Th2 cytokine production (i.e. IL-4, IL-5, IL-6, IL-10 and IL-13). Expansion of peripheral blood T-cells from melanoma patients in the presence of these inhibitors resulted in downregulation of the Th2 transcription factor GATA3, upregulation of the Th1 transcription factor T-BET, accumulation of central memory phenotype T-cells (CD45RA-CD45RO + CD62L + CCR7+), reduced exhaustion-associated phenotypes (i.e. TIM3 + LAG3 + PD1+ and EOMES+PD1+), and enhanced killing in mixed lymphocyte reactions. The frequency, FOXP3 expression, and suppressive function of T regulatory cells (Tregs) were decreased after exposure to ACY-1215 or ACY-241. Higher frequencies of T-cells expressing CD107a + IFNγ+ and central memory markers were observed in melanoma tumor-infiltrating lymphocytes (TIL), which persisted after drug removal and further expansion. After ACY-1215 treatment, increased chromatin accessibility was observed in regions associated with T-cell effector function and memory phenotypes, while condensed chromatin was found in regions encoding the mTOR downstream molecules AKT, SGK1 and S6K. Decreased phosphorylation of these proteins was observed in ACY-1215 and ACY-241-treated T-cells. AKT- and SGK1-specific inhibition recapitulated the increase in central memory frequency and decrease in IL-4 production, respectively, similar to the observed effects of HDAC6-selective inhibition. Conclusions: HDAC6-selective inhibitors augmented melanoma patient T-cell immune properties, providing a rationale for translational investigation assessing their potential clinical efficacy.
Bibliographical noteFunding Information:
Andressa S. Laino: Received one-year of grant funding from Acetylon Pharmaceuticals, in 2016. Brian C. Betts: The author declares no potential conflicts of interest. Anandharaman Veerapathran: The author declares no potential conflicts of interest. Igor Dolgalev: The author declares no potential conflicts of interest. Amod Sarnaik: Paid consultant for Iovance Biotherapeutics Inc. and B4CC Inc., and has received research funding support from Provectus, Iovance and Genentech. Steven N. Quayle: Former employee and stockholder of Acetylon Pharmaceuticals. Simon S. Jones: Former employee and stockholder of Acetylon Pharmaceuticals. Jeffrey S. Weber: Has stock or other ownership in Altor BioScience, Celldex, Biond and CytomX Therapeutics. Has honoraria in Bristol-Myers Squibb, Merck, Genentech, AbbVie, AstraZeneca, Daiichi Sankyo, GlaxoSmithKline, Eisai, Altor BioScience, Amgen, Roche, Ichor Medical Systems, Celldex, CytomX Therapeutics, Nektar, Novartis, Array, WindMIL, Takeda and Sellas. Has consulting or advisory role in Celldex, Ichor Medical Systems, Pieris Pharmaceuticals, Altor BioScience, Bristol-Myers Squibb, Merck, Genentech, Roche, Amgen, AstraZeneca, GlaxoSmithKline, Daiichi Sankyo, AbbVie, Eisai, CytomX Therapeutics, Nektar, Novartis, Array, WindMIL, Takeda and Sellas. Received research funding (to the Institution) from Bristol-Myers Squibb, Merck, Glax-oSmithKline, Genentech, Astellas Pharma, Incyte, Roche and Novartis. Received one-year of grant funding from Acetylon Pharmaceuticals. Received travel, accommodations, expenses from Bristol-Myers Squibb, GlaxoSmithK-line, Daiichi Sankyo, Roche, Celldex, Amgen, Merck, AstraZeneca, Genentech, Novartis, Incyte, WindMIL and Takeda. David M. Woods: Received research funding from Mirati Therapeutics, in 2016 and 2017. Has stock in Bristol-Myers Squibb, Merck, GlaxoSmithKline, Seattle Genetics, Mirati Therapeutics, Iovance Biotherapeutics, Cue Biopharma, Fate Therapeutics, Atra Biotherapeutics, and Fortress Biotech.
We extend our appreciation to Dr. Anders Berglund at Moffitt for his scientific input. We also extend our appreciation to the Genome Technology Center and Dr. Kasthuri Kannan at NYU Langone Medical Center (New York, NY), and the Flow Cytometry Core Facility and Jodi Kroger at Moffitt for their assistance. 1P50CA168536-01A1 (National Cancer Institute); R01 CA175732-01 (National Cancer Institute).
© 2019 The Author(s).
- Central memory
- HDAC inhibitor
- Regulatory T-cell