Expression of PD-L1, PD-L2, PD-1 and CTLA4 in myelodysplastic syndromes is enhanced by treatment with hypomethylating agents

Abstract

Blockade of immune checkpoints is emerging as a new form of anticancer therapy. We studied the expression of programmed death ligand 1 (PD-L1), PD-L2, programmed death 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA4) mRNA in CD34+ cells from myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML) and acute myeloid leukemia (AML) patients (N=124). Aberrant upregulation (⩾2-fold) was observed in 34, 14, 15 and 8% of the patients. Increased expression of these four genes was also observed in peripheral blood mononuclear cells (PBMNCs) (N=61). The relative expression of PD-L1 from PBMNC was significantly higher in MDS (P=0.018) and CMML (P=0.0128) compared with AML. By immunohistochemical analysis, PD-L1 protein expression was observed in MDS CD34+ cells, whereas stroma/non-blast cellular compartment was positive for PD-1. In a cohort of patients treated with epigenetic therapy, PD-L1, PD-L2, PD-1 and CTLA4 expression was upregulated. Patients resistant to therapy had relative higher increments in gene expression compared with patients who achieved response. Treatment of leukemia cells with decitabine resulted in a dose-dependent upregulation of above genes. Exposure to decitabine resulted in partial demethylation of PD-1 in leukemia cell lines and human samples. This study suggests that PD-1 signaling may be involved in MDS pathogenesis and resistance mechanisms to hypomethylating agents. Blockade of this pathway can be a potential therapy in MDS and AML.

Figure 1. Increased mRNA expression of PD-L1, PD-L2, PD-1 and CTLA4 in MDS, CMML and AML patients

(A) Increased mRNA expression of PD-L1, PD-L2, PD-1 and CTLA4 genes in CD34+ cells from MDS, CMML and AML patients by real-time PCR analysis, normal CD34+ cells were used as normal control. N = number of patients. MDS, myelodysplastic syndromes; CMML, chronic myelomonocytic leukemia; AML, acute myeloid leukemia. (B) Increased mRNA expression of PD-L1, PD-L2, PD-1 and CTLA4 in PBMNCs from MDS, CMML and AML patients, normal PBMNCs were used as normal controls. PBMNC, peripheral blood mononuclear cells. (C) Comparisons of mRNA expression levels of PD-L1, PD-L2, PD-1 and CTLA4 between CD34+ cells and PBMNC from MDS, CMML and AML patients.

Figure 2. Immunohistochemical analysis of PD-L1 and PD-1 membranous expression in CD34+ cell cytospin and bone marrow biopsy samples from MDS, CMML and AML patients

(A) Illustrations of PD-L1 membranous expression in bone marrow biopsy samples from MDS, CMML and AML patients (X1000): a, Representative case show strong PD-L1 membranous expression in blasts; b, Representative case show negative PD-L1 membranous expression in blasts. (B) Illustrations of PD-1 membranous expression in bone marrow biopsy samples from MDS, CMML and AML patients (X1000): a, Representative case show PD-1 membranous expression in blasts; b, Representative case show negative PD-1 membranous expression in blasts, but positive PD-1 expression in stroma/non-blast compartment. (C) Illustrations of PD-L1 membranous expression in CD34+ cell cytospin from MDS, CMML and AML patients (X1000): a, Representative case show strong PD-L1 membranous expression; b, Representative case show negative PD-L1 membranous expression. (D) Illustrations of PD-1 membranous expression in CD34+ cell cytospin from MDS, CMML and AML patients (X1000): a, Representative case show partial PD-1 membranous expression; b, Representative case show negative PD-1 membranous expression.

Figure 3. Induction of PD-L1, PD-L2, PD-1 and CTLA4 expression in patients treated with epigenetic therapy

(A) Summary of dynamics of PD-L1, PD-L2, PD-1 and CTLA4 mRNA expression in 61 patients treated with different forms of epigenetic therapy, expression on day 0 was baseline expression before treatment, most patients received 5 days of hypomethylating agent treatment. (B) Induction of PD-L1, PD-L2, PD-1 and CTLA4 expression in patients treated in a phase 2 trial of vorinostat in combination with azacitidine. C=course, D=days on therapy. Resistance: patients had no response to treatment; Responing: patients acquired complete remission after treatment. (C) Overall survival by comparison of PD-L2 upregulation after treatment in group of patients from a phase 2 trial of vorinostat in combination with azacitidine. Group 0: group of patients without PD-L2 expression induction (≥2 fold) after theatment; Group 1: group of patients acquired PD-L2 expression induction (≥2 fold) after theatment.

Figure 4. Induction of PD-L1, PD-L2, PD-1 and CTLA4 expression in leukemia cell lines KG-1 and THP1 treated with hypomethylating agent

(A) Induction of PD-L1, PD-1 and CTLA4 mRNA expression in KG-1 treated with different concentrations of decitabine and cytarabine. (B) Induction of PD-L1 and PD-L2 mRNA expression in THP1 treated with different concentrations of decitabine and cytarabine. (C) Flow cytometry analysis of PD-1 and PD-L1 protein expression levels in KG-1 treated with different concentrations of decitabine.

Figure 5. PD-1 methylation in leukemia cell lines, MDS and AML patients with and without treatment of hypomethylating agents

(A) PD-1 methylation in leukemia cell lines, AML patients and normal controls. (B) Dynamics of PD-1 methylation in KG-1 leukemia cell line treated with decitabine by pyrosequencing analysis, N=Number. (C) Bisulfite sequencing analysis of dynamics of PD-1 methylation with decitabine treatment of KG-1. (D) Dynamics of PD-1 methylation in AML and MDS patients from group of patients treated with vorinostat in combination with azacitidine, C=Course; D=days on therapy.