MOESM1 of HIF-1Îą inhibitor echinomycin reduces acute graft-versus-host disease and preserves graft-versus-leukemia effect

Additional file 1. Additional figures

Increased PRAME-Specific CTL Killing of Acute Myeloid Leukemia Cells by Either a Novel Histone Deacetylase Inhibitor Chidamide Alone or Combined Treatment with Decitabine

<div><p>As one of the best known cancer testis antigens, PRAME is overexpressed exclusively in germ line tissues such as the testis as well as in a variety of solid and hematological malignant cells including acute myeloid leukemia. Therefore, PRAME has been recognized as a promising target for both active and adoptive anti-leukemia immunotherapy. However, in most patients with PRAME-expressing acute myeloid leukemia, PRAME antigen-specific CD8⁺ CTL response are either undetectable or too weak to exert immune surveillance presumably due to the inadequate PRAME antigen expression and PRAME-specific antigen presentation by leukemia cells. In this study, we observed remarkably increased PRAME mRNA expression in human acute myeloid leukemia cell lines and primary acute myeloid leukemia cells after treatment with a novel subtype-selective histone deacetylase inhibitor chidamide <i>in vitro</i>. PRAME expression was further enhanced in acute myeloid leukemia cell lines after combined treatment with chidamide and DNA demethylating agent decitabine. Pre-treatment of an HLA-A0201⁺ acute myeloid leukemia cell line THP-1 with chidamide and/or decitabine increased sensitivity to purified CTLs that recognize PRAME^100–108 or PRAME^300–309 peptide presented by HLA-A0201. Chidamide-induced epigenetic upregulation of CD86 also contributed to increased cytotoxicity of PRAME antigen-specific CTLs. Our data thus provide a new line of evidence that epigenetic upregulation of cancer testis antigens by a subtype-selective HDAC inhibitor or in combination with hypomethylating agent increases CTL cytotoxicity and may represent a new opportunity in future design of treatment strategy targeting specifically PRAME-expressing acute myeloid leukemia.</p></div

Decitabine further enhances chidamide-induced PRAME upregulation and PRAME antigen-specific CTL cytotoxicity of THP-1 cells.

<p>A. AML cell lines were treated with chidamde (1 µM), decitabine (Dac, 0.25 mM), or chidamide/decitabine. PRAME mRNA expression was analyzed by RT-qPCR. Y axis shows folds of PRAME mRNA expression relative to that of non-treated cells. B. THP-1 cells were treated with chidamde, decitabine, or chidamide/decitabine, followed by cytotoxicity analysis with PRAME specific CTLs. *<i>P</i> <0.05, **<i>P</i><0.01.</p

Increased PRAME antigen-specific CTL killing of AML cells after treatment with chidamide.

<p>A. AML cell lines were analyzed for HLA-A2 expression by FACS. In the tested AML cell lines, only THP-1 cells were HLA-A2⁺. We confirmed that THP-1 cells were positive for HLA-A0201 allele by DNA sequencing (data not shown). B. Expansion of HLA-A0201-PRA^100–108 specific CTLs. Number shows the percentage of CD8⁺HLA-A0201-PRA^100–108 positive cells in CD8⁺ T cells. C. Killing of T2 cells pulsed with PRA^100–108 at titration concentrations by PRA^100–108 specific CTLs. D. THP-1 cells treated with chidamide or VPA were analyzed for their sensitivity to CTLs specific for PRA^100–108 or PRA^300–309 at various E/T ratios. T2 cells that were not pulsed with peptide were used as negative control targets. E. Blockade of HLA-A2 abrogated cytotoxicity of THP-1 cells by PRAME-specific CTLs. F. Blockade of CD86 significantly reduced cytotoxicity of THP-1 cells by PRAME-specific CTLs. *<i>P</i><0.05, **<i>P</i><0.01.</p

Information of AML patients.

<p>Information of AML patients.</p

HDAC inhibition induces cell cycle arrest and reduces colony forming ability in AML cells.

<p>THP-1 cells were treated with chidamide at 1 µM or VPA at 1 mM for 24 h or 48 h. A. Cell cycle of THP-1 cells was determined by FACS analysis based on DNA content. Cell cycle was presented as percentage of cells in G1/S/G2&M phase. B. Western blot analysis on CDK2 and CDK4. C, D. Apoptosis of THP-1 cells after chidamide or VPA treatment. E, F. Colony forming analysis of THP-1 cells. Number of clone (≥50 cells) per well (E) as well as representative photographs in each group (F) were shown. *<i>P</i><0.05, **<i>P</i><0.01.</p

HDAC inhibition upregulates CD86 expression in AML cell lines.

<p>AML cells were treated with chidamide (1 µM) or VPA (1 mM) for 24 h. Cell surface expression of HLA-I, CD80, and CD86 were analyzed by FACS analysis.</p

Chidamide and/or decitabine treatment does not impair CTL cytotoxic functions and chidamide inhibits proliferation of activated T cells.

<p>A. Percentage of IFN-γ producing CD8⁺ T cells activated by PMA/Ionomycin following treatment with chidamide at various concentrations for 24 h (left), decitabine (Dac), or chidamide/decitabine (right). B. PRAME antigen-specific killing of chidamide treated THP-1 cells by chidamide, decitabine or chidamide/decitabine treated CTLs. C. PBMCs from healthy donors were treated with chidamide, decitabine or chidamide/decitabine at the indicated concentrations. Cells were washed in PBS to remove any residual drugs and suspended in fresh medium, followed by activation with anti-CD3 and anti-CD28 mAbs (1 µg/ml each) at the presence of recombinant human IL-2 (50 IU/ml). PBMCs cultured with only IL-2 were used as control. On day7 of culture, cells in each well were stained with anti-CD8 FITC and anti-CD3 PE, suspended in 500 µl PBS and acquired in flow cytometer for 30 seconds. Dead cells were excluded by gating out high side scatter cells, and number of CD4⁺ T cells (CD3⁺CD8⁻) and CD8⁺ T cells (CD3⁺CD8⁺) were divided by that of control CD4⁺ or CD8⁺ T cells to obtain expansion folds. For combined treatment with chidamide and decitabine in A and B, CTLs were treated with chidamide at 1 µM for 24 h, in combination to decitabine (250 nM) supplemented in culture media twice for 48 h at 24 h interval. In B, chidamide treated THP-1 cells (1 µM for 24 h) were used as target cells, and an E/T ratio of 20/1 was used. *<i>P</i><0.05, **<i>P</i><0.01.</p

Upregulation of PRAME expression in human AML cells after HDAC inhibition.

<p><b>A.</b> PRAME mRNA expression in human leukemia cell lines after chidamide of VPA treatment. Human AML cell lines Kasumi, NB4, U937, THP-1, and K562, ALL cell lines Hut78, Molt-4, and Z-138 were treated <i>in vitro</i> with chidamide at 1 µM or VPA at 1 mM for 24 h. PRAME mRNA expression was analyzed by real-time quantitative RT-PCR (SYBRgreen). <b>B.</b> THP-1 cells were treated <i>in vitro</i> with chidamide at various concentrations for 24 h. PRAME mRNA expression was determined by real-time quantitative RT-PCR. <b>C.</b> PRAME mRNA expression of THP-1 cells at various time points after chidamide treatment at 1 µM for 24 h. <b>D.</b> Western blot analysis of PRAME protein expression of THP-1 cells that were not treated or treated <i>in vitro</i> with chidamide at 1 µM or VPA at 1 mM for 48 h. <b>E.</b> H3 histone acetylation in untreated, chidamide treated, or VPA treated AML cell lines THP-1 and U937. <b>F.</b> Bone marrow cells from patients with PRAME-expressing AML were treated <i>ex vivo</i> with chidamide (1 µM for 24 h), followed by real-time quantitative RT-PCR analysis of PRAME mRNA. See Table1 for patient information. In D and E, Contr = Control; Chida = Chidamide. In A, B, C and F, Y axis shows folds of PRAME mRNA expression relative to that of non-treated cells. *<i>P</i><0.05, **<i>P</i><0.01.</p