Decitabine Enhances The Sensitivity Of Leukemia Stem Cells To NK Cell-mediated Killing And The Underlying Mechanisms

BackgroundAcute myeloid leukemia (AML) derived from hematopoietic stem cells, is a serious and often lethal disease with a high relapse rate. The incidence of acute myeloid leukemia in China is 4.17/100,1000 and poses a severe threat to human health. The mortality rate ranks seventh among all the malignant tumors and lies in the top in children as well as adults under the age of 35. Currently, the principal treatment of acute myeloid leukemia consists of chemotherapy and allogeneic hematopoietic stem cell transplantation (Allo-SCT). Allo-SCT is the main way to cure AML. The majority of patients can be completely cured by the method, but there are still about 23% of patients with relapse or death eventually. In addition, suitable HLA-identical donor, high medical expenses, graft-versus host disease (GVHD) and other issues all hamper patients to select the Allo-SCT, most patients have no choice but to receive chemotherapy. Although the effect of chemotherapy regimens has greatly improved and the complete remission rate of the patients can reach to 50%～ 75%, there are still 15%～25% of the patients failed to achieve complete remission (CR) because of drug resistance, and more than 60% die of disease relapse in the end. Bonnet et al.found that the root of recurrence and refractoriness of AML was the leukemia stem cells (LSCs). They first isolated the two kinds of cell subsets of CD34+CD38- and CD34+CD38+ phenotype from the bone marrow of the patients with AML and then separately transplanted those cells to non-obese diabetic/severe combined immunodeficient(SCID) mice through the caudal vein. Finally, they discovered that only the mice being transplanted CD34+CD38- cells were capable of cloning the model of leukemia and the morphology and function of leukemic cells was consistent with patients, which further confirmed the existence of LSCs.LSCs derived from normal hematopoietic stem cells (HSCs), share the common characteristics of unlimited self-renewal of HSCs. Research suggested that 95% of LSCs were in GO stage and the conventional chemotherapeutic regimens could only kill the leukemia cells in the proliferation period rather than the resting LSCs, thereby leading to relapse of leukemia eventually. The existence of LSCs is the root of leukemia relapse. Our earlier studies had found that transplantation of KG1a cells highly expressing CD34+CD38- antigen into NOD/SCID mice could successfully duplicate and establish the animal models of leukemia. Therefore, how to completely kill LSCs is the key factor to cure the AML.Nature Killer cells (NK cells), the crucial components of the innate immune system, are characterized with the broad-spectrum anti-tumor, anti-infection, and immune-regulation action. They are the first line of denfense of the immune system (Do not require prior sensitization and is not restricted by the target cell’s expression of major histocompatibility complex (MHC) molecules) and can spontaneously kill the tumor cells that MHC-1 molecules defect or low expression. In addition, NK cells are also the first implanted lymphocytes after bone marrow transplantation (in the initial three months after bone marrow transplantation, it accounts for more than 70% of human peripheral blood lymphocytes), playing a vital role in killing LSCs. However, several studies had discovered that NKG2D ligands like the human class I-like molecules MICA and MICB, ULBP1, ULBP2 and ULBP3 expressed very low in tumor cells and sometimes can not be detected at all. Enhanced expression of NKG2D ligands can strengthen the cytotoxicity of NK cells. Our previous research also indicated that AML patients had a lower expression of NKG2D ligands (including MICA/B, ULBP1, ULBP2 and ULBP3) on leukemia cells. Therefore, how to enhance the expression of ligands on leukemia cells indicates a new direction for the treatment of AML.Decitabine, also known as 5-aza-2’-deoxycytidine acid, is a natural deoxycytidylate adenosine analogues. It can replace the cytosine in tumour cell and covalently bound to DNA methyltransferase enzyme, inactivate the DNA methyltransferase enzyme and reach demethylation, thereby inhibiting the proliferation and promoting the apoptosis of tumour cells. On the other hand, the DNA demethylation also can inhibit the synthesis of killer cell inhibitory receptors (KIR) on tumor cells and correspondingly activate the killer cell activatory receptors (KAR), and ultimately enhance the killing effect of NK cell on tumor cells. In addition, some evidences demonstrated that decitabine had immunomodulatory effects, it could increase the expression of NKG2D ligands on the surface of tumor cells and improve the cytotoxicity of NK cells against tumor cells. Thus, we conjecture that whether decitabine can enhance the killing effect of allo-NK cells to LSCs by increasing the expression of NKG2D ligands on LSCs. Based on our preliminary research, current study centering on the CD34+ CD38-LSCs intends to investigate the immunomodulatory effects of decitabine and provide a theoretical basis and experimental evidence for the treatment of AML.MethodChapter Ⅰ Sorting leukemia stem cells from KG1a cell lineCD34+CD38- cells were separated from KGla cell line by magnetic activated cell sorting method, and flow cytometry was used to detect the purity of CD34+CD38-LSCs.Chapter Ⅱ Decitabine enhanced the cytotoxicity of NK cell lines to leukemia stem cells1) Using lymphocyte separation medium to isolate peripheral blood mononuclear cells (PBMC), and then induced the NK cell formation by human rIL-2 and human rIL-15. Cell Counting Kit-8 (CCK-8) method was used to analyze the viabilities of LSCs after decitabine treatment with different concentration. Cytotoxicity of NK cell lines (NK-92 cell and NK cell) against K562 cells and LSCs before and after decitabine treatment at different effector to target ratios were measured by LDH releasing assay. Flow cytometry was applied to analyze the kill rate of NK cell lines (NK-92 cell and NK cell) against K562 cells and LSCs before and after decitabine treatment at the effector to target ratio of 10:1.2) Using SPSS 20.0 software for data analysis, and the date were shown as mean ± standard deviation (X±s). Cytotoxicity of NK cell lines (NK-92 cell and NK cell) against K562 cells and LSCs was assessed using factorial design analysis of variance. If the variance is homogeneous, the multiple comparisons between groups using LSD method, otherwise using Dunnertt T3 method. P<0.05 is considered statistically significant.Chapter Ⅲ The mechanisms of the cytotoxicity of NK cell lines to leukemia stem cells enhanced by decitabine1) The expression of NKG2D ligands of K562 cells and LSCs before and after decitabine treatment were tested by flow cytometry. Western blotting was used to detect the expression of apoptosis related proteins.2) Using SPSS 20.0 software for data analysis, and the date were shown as mean ± standard deviation (X±s).The expression of NKG2D ligands between K562 cells and LSCs before and after decitabine treatment and the expression of apoptosis related proteins before and after decitabine treatment were using independent sample t test. P<0.05 is considered statistically significant.ResultChapter Ⅰ Sorting leukemia stem cells from KG la cell lineWe had successfully seperated CD34+CD38-LSCs cells from KGla cell line by magnetic activated cell sorting method and the purity of CD34+CD38-LSCs were up to 99.95%.Chapter Ⅱ Decitabine enhanced the cytotoxicity of NK cell lines to leukemia stem cells1) The morphology of NK cellsThe PBMC isolated from peripheral blood of healthy volunteers were small and bright and they began to proliferate on the second day after the stimulation of Human rIL-2 and Human rIL-15. NK cells grew very fast and the microscopic observation of them are small, round and bright, showing a cluster like growth.2) The sensitivity of LSCs to decitabineCCK-8 experiment showed that 24h after the treatment of decitabine at different concentrations (0-60umol/L), the activity of LSCs were still over 60%, indicating that LSCs were not sensitive to decitabine. LSCs presented a resistance to decitabine.3) Cytotoxic activity of NK cell linesLDH releasing assay showed that K562 cells were sensitive to NK cell lines. The Killing rates of NK-92 cells to K562 cells at the effector to target ratio of 5:1,10:1, 20:1 were 46.00±3.73%,53.58± 3.10%,66.51 ± 1.70% separately, which were much higher than the rates to LSCs (24.13 ± 1.26%,27.88 ± 2.04%,34.92 ± 4.22%) at the corresponding effector to target ratio. The difference of killing rates between two groups at different effector to target ratio cells were statistically significant (F=4.327, P=0.038). The killing rates of NK cells to K562 cells were 44.38± 2.81%, 64.77 ± 3.66%,73.91 ± 3.54%, which were higher than the rates to LSCs (22.08 ± 2.07%,28.99 ± 3.13%,36.44 ± 2.40%), the difference between two groups was also statistically significant at different effector to target ratio (F=11.588, P=0.002). All of the above suggested that K562 cells were sensitive to NK cell lines while LSCs were resistant. The killing rates of NK-92 cells to LSCs after the intervention with decitabine at 10 umol/L were significantly reinforced, they were 40.29 ± 1.72%, 55.47 ± 1.86%,66.91 ± 2.08% at the effector to target ratio of 5:1,10:1,20:1 respectively, both of them were higher than the corresponding effector to target ratio on LSCs, the differences between two groups on different effector to target ratio were statistically significant (F=13.845, P=0.001); The killing rates of NK cells to LSCs were 60.52 ± 3.52%,73.93 ± 2.33%,83.08 ± 1.32%, which were also higher than the non-intervened groups at the identical effector to target ratio, the difference between two groups at different effector to target ratio was statistically significant (F-4.276, P=0.04).Flow cytometry showed that at the effector to target cell ratio of 10:1, the killing rate of NK-92 cells to K562 cells was 7.33 ± 1.08%, which was higher than that of the non-intervened LSCs groups (2.43 ± 0.60%), so did NK cells to K562 cells (the killing rate was 14.65 ± 1.98% vs 3.33 ± 0.64%), which were consistent with the LDH releasing assay. Both the differences between two groups were statistically significant (t= 6.863, P= 0.002; t= 9.446, P= 0.001). After the treatment of LSCs by decitabine at the concentration of 10 umol/L, the killing rate of NK-92 cells to LSCs was 7.85 ± 0.88% while the NK cells was 7.84 ± 0.34%, all of them were higher than that of non-intervened group (2.43 ± 0.60%,3.33 ± 0.64%), the difference between two groups were statistically significant respectively (t=8.864, P=0.001, t=10.821,P=0.000).Chapter Ⅲ The mechanisms of the cytotoxicity of NK cell lines to leukemia stem cells enhanced by decitabine1) The expression of NKG2D ligands of K562 cells and LSCs before and after the intervention of decitabineFlow cytometry showed that the NKG2D ligands of K562 cells sensitive to NK cell lines, like MICA/B, ULBP1, ULBP2, ULBP3, were 2.45 ± 0.92%,16.85 ± 1.78%,1.94 ± 0.90%,4.73 ± 0.58%, they were higher than that of LSCs (0.17 ± 0.07%,0.28 ± 0.10%,1.24 ± 0.12%,0.48 ± 0.08%), the difference between two groups were statistically significant (P<0.05). After the treatment of LSCs by decitabine at the concentration of 10 umol/L, the expression of NKG2D ligands increased to 2.88 ± 0.10%,1.26 ± 0.35%,2.16 ± 0.34%,3.37 ± 0.82%, the difference between two groups were statistically significant (P<0.05), all above showed that decitabine could enhance the expression of NKG2D ligands of LSCs.2) The influence of decitabine on mitochondrial apoptosis pathway-related proteinsThe data of Western-blot test revealed that decitabine could contribute to the activation of caspase-9, caspase-3 and PARP (decrease in pro-caspase-9 and pro-caspase-3, improve in cleave-PARP) and the down-regulation of expression of anti-apoptotic Bcl-2 proteins (Bcl-2 and Bcl-xl), while the pro-apoptotic protein Bax did not change.Conclusion1) KGla cells are rich in CD34+CD38-LSCs and the LSCs with high purity can be acquired for subsequent related research; 2) LSCs presents a resistance to decitabine;3) LSCs were resistant to NK cell lines (NK-92 cell and NK cell) and decitabine could enhance the killing rate of NK cell lines against LSCs;4) Decitabine could enhance the killing rate of NK cell lines against LSCs, possibly by upregulating the expression of NKG2D ligands;5)Decitabine might upregulate the expression of NKG2D ligands through mitochondrial apoptosis pathway.