HDAC Inhibition Upregulates PD-1 Ligands in Melanoma and Augments Immunotherapy with PD-1 Blockade

Abstract

Expression of PD-1 ligands by tumors and interaction with PD-1-expressing T cells in the tumor microenvironment can result in tolerance. Therapies targeting this coinhibitory axis have proven clinically successful in the treatment of metastatic melanoma, non-small cell lung cancer, and other malignancies. Therapeutic agents targeting the epigenetic regulatory family of histone deacetylases (HDAC) have shown clinical success in the treatment of some hematologic malignancies. Beyond direct tumor cell cytotoxicity, HDAC inhibitors have also been shown to alter the immunogenicity and enhance antitumor immune responses. Here, we show that class I HDAC inhibitors upregulated the expression of PD-L1 and, to a lesser degree, PD-L2 in melanomas. Evaluation of human and murine cell lines and patient tumors treated with a variety of HDAC inhibitors in vitro displayed upregulation of these ligands. This upregulation was robust and durable, with enhanced expression lasting past 96 hours. These results were validated in vivo in a B16F10 syngeneic murine model. Mechanistically, HDAC inhibitor treatment resulted in rapid upregulation of histone acetylation of the PD-L1 gene leading to enhanced and durable gene expression. The efficacy of combining HDAC inhibition with PD-1 blockade for treatment of melanoma was also explored in a murine B16F10 model. Mice receiving combination therapy had a slower tumor progression and increased survival compared with control and single-agent treatments. These results highlight the ability of epigenetic modifiers to augment immunotherapies, providing a rationale for combining HDAC inhibitors with PD-1 blockade.

Figure 1. HDAC inhibitors upregulate PD-L1 in melanoma

(A) B16F10 melanoma cells were cultured for 2 and 24 hours in the presence of indicated HDAC inhibitors. Cells were washed, lysed and analyzed by immunoblotting for acetylated histone 3, total histone 3, acetylated alpha-tubulin and β-actin. (B) Indicated melanoma cell lines were treated with 500nM MS275 (red), 10nM LBH589 (orange), 500nM MGCD0103 (purple) or DMSO control (black) for 72 hours in vitro and PD-L1 expression was evaluated. (C) Indicated melanoma cell lines were plated and treated with 500nM MS275 (triangles), 10nM LBH589 (squares), or 500nM MGCD0103 (diamonds) at 96, 72, 48, or 24 hours prior to evaluation of PD-L1. Expression of DMSO-treated cells was graphed as zero hour treatment. All values are graphed as mean fluorescence intensity (MFI) with autofluorescence values subtracted. Results shown are representative of 2–3 independent experiments.

Figure 2. Inhibition of class I HDACs increases PD-L1 and PD-L2 expression in patient melanomas in a dose dependent manner

Patient melanomas obtained from biopsies and expanded in culture were plated and treated with indicated HDAC inhibitors and concentrations for 24 hours. Cells were then washed and cultured for a further 48 hours. At 72 hours past initial treatment, melanomas were evaluated for expression of (A) PD-L1 and (B) PD-L2. DMSO controls were run in triplicate. MFI values are graphed with autofluorescence values subtracted.

Figure 3. HDAC inhibitors upregulate PD-L1 and PD-L2 expression in vivo

C57BL/6 mice were inoculated subcutaneously with B16F10 melanoma. When tumors were visible, 10 days post inoculation, mice received treatment with 15mg/kg LBH589 or dextrose control (five mice per group) for three consecutive days. On the third day of treatment, tumors were harvested. (A) PD-L1 and (B) PD-L2 expression were evaluated by flow cytometry. *P<0.05, **P<0.01.

Figure 4. HDAC inhibition increases histone acetylation at the PD-L1 and PD-L2 promoters

Indicated melanoma cell lines were treated in vitro for two hours with 12.5nM LBH589 (squares) or DMSO control (circles). Cells were then fixed and chromatin immunoprecipitated for acetylated histone 3 or IgG control. DNA pull-down was quantified by qRT-PCR. Fold enrichment over corresponding IgG pull-down at the (A) PD-L1 and (B) PD-L2 gene regions for WM983A are graphed. Results shown are representative of two independent experiments. Five other cell lines were assessed once for acetylation at the (C) −455 gene region of PD-L1 and (D) +307 gene region of PD-L2. For all graphs, error bars are representative of technical replicates.

Figure 5. PD-L1 mRNA expression increased following HDAC inhibition, correlating with protein expression and gene acetylation

(A) WM983A cells were treated with DMSO or 12.5nM LBH589 for indicated time points. Cells were assessed by qRT-PCR for PD-L1 expression. (B) Indicated cell lines were treated with DMSO or 12.5nM LBH589 for six hours and subsequently assessed by qRT-PCR for PD-L1 expression. For all graphs, error bars are representative of technical replicates. Correlations of (C) PD-L1 surface expression versus gene acetylation, (D) PD-L1 surface expression versus gene expression and (E) PD-L1 gene acetylation versus gene expression were assessed for various melanoma cell lines at basal state (DMSO control). Acetylated H3 was graphed as fold enrichment over corresponding IgG pull-down at the −455 region of PD-L1 gene. Gene expression was determined by qRT-PCR and calculated as fold units relative to 18S endogenous ribosomal RNA. Flow cytometry analysis of PD-L1 surface expression was indicated as mean fluorescence intensity (MFI).

Figure 6. Combining HDAC inhibition with PD-1 blockade in vivo results in delayed tumor growth and enhanced survival

C57BL/6 mice were inoculated subcutaneously with B16F10 melanoma. Seven days after inoculation mice began treatment with LBH589 (15mg/kg, triangles) (Monday, Wednesday and Friday), PD-1 blocking antibody (3mg/kg, squares) (Tuesday and Thursday), a combination of these agents (diamonds) or dextrose control (circles) for three weeks. (A) Tumor growth was measured and (B) survival monitored. Log rank test of survival curve differences was p<0.05. Ten mice were assessed per group and results shown are representative of two independent experiments. *p<0.05.