Inhibition of cell‐mediated immunity by the histone deacetylase inhibitor vorinostat: Implications for therapy of cutaneous T‐cell lymphoma
Authors: Sasha Stephen, Kelly A. Morrissey, Bernice M. Benoit, Ellen J. Kim, Carmela C. Vittorio, Sunita D. Nasta, Louise C. Showe, Maria Wysocka, Alain H. Rook
Journal: American Journal of Hematology (2011)

Several histone deacetylase inhibitors (HDACi), including vorinostat, have been approved for the therapy of cutaneous T-cell lymphoma (CTCL). Emerging data suggest that HDACi may exert immune suppressive effects which would be disadvantageous for therapy of CTCL. We describe a patient with Sezary syndrome who was monitored for drug-induced immunosuppression while undergoing treatment with vorinostat. Analysis of the patient's natural killer cell function before and after initiation of treatment confirmed inhibition of this important cell-mediated immune function. In addition, the in vitro effects of vorinostat on the immunity of healthy volunteers confirmed that this class of drug can profoundly suppress multiple arms of the cellular immune response. These findings raise concerns of increased susceptibility to infection in this high-risk population. Histone deacetylase inhibitors (HDACi) represent a novel class of anticancer drugs, with two members of the group, vorinostat and romidepsin, approved for the therapy of cutaneous T-cell lymphoma (CTCL). HDACi modulate chromatin structure, and have been shown to promote growth arrest, differentiation, and apoptosis of tumor cells [1]. Specifically, vorinostat has been shown to cause selective apoptosis of malignant T cells by increasing the expression of pro-apoptotic factors p21WAF1, bax, and caspase-3, and decreasing the expression of anti-apoptotic factors such as Stat-6 in CTCL cell lines and patients' peripheral blood mononuclear cells (PBMC) [2]. There is emerging evidence for the potent immunosuppressive properties of HDACi. Studies have shown that HDACi can have therapeutic benefit in autoimmune disease models [3-5] perhaps owing to the enhancement of regulatory T-cell functions [6]. Moreover, we have reported a patient with CTCL with refractory bullous pemphigoid, who experienced rapid resolution of this autoimmune disorder following initiation of therapy with vorinostat [7]. Furthermore, in vitro studies have shown anti-inflammatory properties of HDACi via suppression of cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β [8]. As attempts at preservation of cellular immunity are critical in the management of CTCL, we examined the effects of vorinostat on cellular immunity in a patient undergoing therapy with this agent and in healthy volunteers. A 65-year old female with a past history of erythroderma, lymphadenopathy, and circulating atypical cells was diagnosed with Sezary syndrome in 2001. She experienced a complete clinical response following three years of therapy with photopheresis, interferon gamma, bexarotene, and psoralen and ultraviolet A radiation (PUVA). She was in remission from 2004 until 2007 when she relapsed with recurrent skin lesion and blood involvement. Her disease progressed despite multimodality therapy. In February 2011, she began vorinostat 400 mg/day with marked improvement in skin erythema within one month. Blood was obtained from the patient for study 30 days following initiation of vorinostat. As interaction between natural killer (NK) cells and antigen presenting cells (APCs) is critical in the maintenance of antitumor immunity [9], we examined the effects of vorinostat on these cellular functions. NK cell cytotoxicity from our patient's blood specimen obtained 4 months prior to the initiation of vorinostat therapy was compared to a specimen obtained 30 days after initiation of vorinostat therapy. After culturing cells for 48 hr, NK cytolytic activity was assessed as reported [10]. To assess NK cell responsiveness, the patient's cells were stimulated with 10 μg/mL of a known APC activator Toll-like receptor (TLR) 7/8 agonist, known to indirectly activate NK cells. While stimulation with TLR 7/8 agonist resulted in a significant increase in NK activity from 5.4% to 17.6% K562 lysis in the baseline sample, responsiveness upon stimulation was impaired in the sample obtained during vorinostat therapy, increasing only from 3.2% to 6.1% K562 lysis (Fig. 1). These results indicate that compared to baseline, vorinostat treatment resulted in significant blunting of functional NK activity. Suppression of NK cell cytotoxicity by vorinostat in vivo. PBMC from the patient presented in the case report were collected prior to initiation of systemic CTCL therapy (baseline) and while undergoing treatment with vorinostat (vorinostat) were cultured in medium alone or treated with 10 μg/mL of a synthetic TLR 7/8 agonist 3M-007 for 48 hr, followed by a 4 hr Cr51 release assay using K562 cells as targets. Data represent mean of triplicate of percentage of specific lysis per single NK cell at E:T ratio 50:1. Percentage NK cells/total PBMC defined by flow cytometric analysis. Subsequent in vitro studies of vorinostat on the immunity of healthy volunteers revealed that this drug can profoundly suppress multiple arms of the cellular immune response. Purified CD56+ NK cells isolated from blood from three healthy donors were cultured in medium with 1 μM or 0.4 μM vorinostat, doses yielding serum concentrations in the range of those achieved with a standard vorinostat regimen of 400 mg daily. Assessment of NK cytotoxicity after 48 hr incubation with vorinostat, showed that vorinostat profoundly suppressed NK activity in a dose-dependent manner (Fig. 2A). Thus, functional NK activity of healthy volunteers was virtually eliminated following a short exposure to therapeutic levels of vorinostat. Vorinostat inhibits NK cell cytotoxicity, APC activation, and IFN-α production in vitro. A: Highly purified NK cells from healthy volunteers (n = 3) were cultured in medium alone or treated with 0.4 μM and 1 μM vorinostat for 48 hr, followed by a 4 hr Cr51 release assay using K562 cells as targets. Data represent mean ± SD of tested individuals and are presented as a percentage of specific lysis at E:T ratio 2:1. *P < 0.05 when compared to medium. B: PBMC from healthy volunteers were cultured in 24-well plates at a density of 2 × 106/mL/well in media alone, with 1 μM vorinostat, or 10 μg/mL synthetic TLR 7/8 agonist 3M-007 for 48 hr, or primed with 1 μM vorinostat for 4 hr followed by activation with TLR 7/8 agonist for further 48 hr. Cells were then harvested, and analyzed for the expression of CD80. Monocytes were identified by anti-CD64 and anti-CD14 staining. Plasmacytoid dendritic cells (pDCs) were defined as lineage-negative cells (Lin 1-FITC cocktail) that co-express HLADR and CD123. Myeloid dendritic cells (mDCs) were defined as lineage-negative cells co-expressing HLADR and CD11c. The figure illustrated one of three representative experiments. C: PBMC from healthy volunteers were cultured in 24-well plates at a density of 2 × 106/mL/well in media alone, with 1 μM vorinostat, or 10 μg/mL synthetic TLR 7/8 agonist 3M-007 for 48 hr, or primed with 1 μM vorinostat for 4 hr followed by activation with TLR 7/8 agonist for further 48 hr. Cell free supernatants were then collected, and IFN-α levels were measured in cell-free supernatants by ELISA. Data represent mean ± SD of tested individuals. *P < 0.05 compared to the TLR 7/8 agonist sample. In addition to depressed NK cell function, other important cellular immune functions appear to be inhibited by vorinostat. Activation of antigen presenting cells measured by the expression of the inducible co-stimulatory molecule CD80 [11] on monocytes, plasmacytoid, and myeloid dendritic cells (DC)s collected from healthy volunteers was markedly inhibited by vorinostat. While stimulation with TLR 7/8 agonist (10 μg/mL for 48 hr) led to significant upregulation of CD80, and, thus activation, across all three cell lineages, this activation was suppressed by vorinostat (Fig. 2B). Furthermore, we observed that interferon-α (IFN-α) production following stimulation of healthy volunteer APCs with a TLR 7/8 agonist was greatly inhibited by 1 μM vorinostat (Fig. 2C). TLR 7/8 stimulation led to significant (766.6 pg/mL) IFN-α production by pDCs, which was markedly inhibited (68.4 pg/mL) by vorinostat. As NK cells require interaction with dendritic cells and monocytes for optimal activation, impairment of APC activation in turn diminishes NK functionality. In summary, while HDACi vorinostat has been shown to have a significant response rate and a high rate of pruritus relief in heavily pretreated, refractory CTCL patients, our findings suggest that vorinostat also potently suppresses multiple arms of the immune system which may contribute to disease progression and lead to greater susceptibility of these patients to opportunistic infections [12]. Our findings highlight the complexity of the effects of vorinostat and the need to balance its anti-tumor effects and immunosuppressive capabilities. Further studies are necessary to determine whether the present findings of immune suppression are representative of the HDACi class as a whole, or solely of vorinostat. Dr. Stephen and Dr. Rook had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Stephen, Showe, Wysocka, Rook. Acquisition of Data: Stephen, Morrissey, Benoit, Nasta. Analysis and Interpretation: Stephen, Morrissey, Kim, Vittorio, Wysocka, Rook. Drafting of the Manuscript: Stephen, Morrissey, Showe, Wysocka, Rook. Critical revision of the manuscript for important intellectual content: Stephen, Morrissey, Benoit, Kim, Vittorio, Nasta. Statistical Analysis: Stephen, Wysocka. Obtained funding: Rook. Administrative, technical, or material support: Benoit, Showe, Wysocka, Rook. Study supervision: Rook Sasha Stephen*, Kelly A. Morrissey*, Bernice M. Benoit*, Ellen J. Kim*, Carmela C. Vittorio*, Sunita D. Nasta , Louise C. Showe , Maria Wysocka*, Alain H. Rook*, * Department of Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania;, Division of Hematology/Oncology, University of Pennsylvania, Philadelphia, Pennsylvania;, Department of Molecular and Cellular Oncogenesis, University of Pennsylvania and the Wistar Institute, Philadelphia, Pennsylvania.