Preliminary Study On The Mechanism And Clinical Application Of Chidamide In Acute Myeloid Leukemia (AML)

Objective: t(8;21) Acute Myeloid Leukemia (AML) is a heterogeneous hematological malignancy. Although the standard chemotherapy regimen is better, there are still 20～40%patients can not achieve complete remission (CR), 60% of patients eventually relapse [1].The 5 year survival rate of young patients was only 40～45%, while the elderly patients were less than 10%[2],so it is very important to study the targeted therapy of this kind of leukemia. Histone deacetylases inhibitors (HDACi) are a class of small molecules with a wide range of anti-tumo activity, it has been reported that HDACi can inhibit the proli-feration of t (8; 21) AML cells, induce its apoptosis, promote the differentiation and block the cycle[3], and it has entered the stage of clinical trials of leukemia [4]. Chidamide (CS055)is a selective HDACi developed in China. The aim of this study was to investigate the effect of Chidamide on t (8; 21) AML cells and to explore its mechanism.Methodology: In this study, CCK-8 and flow cytometry were used to detect the effect of Chidamide on the proliferation, apoptosis and cell cycle of AML1-ETO positive and negative AML cells. Bone marrow samples of AML patients were collected, and primary AML cells were treated with Chidamide to observe the effect on cell proliferation.Screening the best Chidamide treatment conditions, and to ensure that its concentration accords with human blood concentration, gene expression chip screening, The results of the chip were verified by Western blot assay, and to observe the effect of Chidamide on the expression of AML1/ETO fusion protein and C-KIT protein. AML1/ETO positive AML cells were treated with MEK1/2 specific inhibitor U0126, to observe the cell proliferation and the effect on AML1/ETO fusion protein and C-KIT protein expression. The expression of AML1-ETO and C-KIT mRNA was detected by q-PCR after treatment with AML1-ETO positive AML cells.Resμlts: 1. Chidamide can effectively inhibit the proliferation of AML cells in a dose-and time-dependent manner. The inhibition rate of AML1/ETO positive AML cells was significantly higher than that of AML1/ETO negative AML cells. Chidamide can inhibit the proliferation of primary AML cells, and the inhibition of time and dose-dependent.2. Genes function were screened using gene expression.AML1/ETO-positive Kasumi-1 cells were treated with 0.25μM of Chidamide for 48h, molecular functional clustering analysis showed that mitogen-activated protein kinase (MAPK) signal pathway related genes have undergone important changes One of the important members of the MAPK family is the main molecular function of extracellular signal-regulated kinase(ERK1/2), which regulates cell proliferation and apoptosis.ERK1/2 pathway involved in apoptosis of 16 genes, suggesting that Chidamide induced AML1/ETO positive cell apoptosis of the main molecular pathway may be inhibition of ERK1/2 pathway.3. The results of Western blot showed that the phosphorylation level of ERK1/2 protein was down-regulated in the AML cells, and the effect of Chidamide on AML1/ETO positive AML cells was more pronounced and longer.The expression of AML1/ETO fusion protein and C-KIT protein was down regulated in the AML1/ETO positive cells at the same time of up regulation of acetylated histone H3.4. Compared with the control group, the AML 1-ETO fusion gene and the C-KIT mRNA transcription level of the AML1-ETO positive AML cells were not significantly statistically significant.5. After AML 1/ETO positive AML cells were treated with U0126, the proliferation and the expression level of AML1/ETO and C-KIT protein were down regulated, but the effects of AML1/ETO and C-KIT mRNA were not obvious.Conclusions: Chidamide can inhibit the phosphorylation of ERK 1/2, and then inhibition of t (8;21)AML cell proliferation by inducing apoptosis, blocking cell cycle, and at the same time, reduce the expression of AML1/ETO and C-KIT protein, which provides a new idea and theoretical basis for the clinical targeted therapy for t (8; 21) AML.Backgroud: With the continuous improvement of combined chemotherapy and hematopoietic stem cell transplantation (HSCT), the effect of AML was improved obviously, but there are still 20～40% of patients can not achieve complete remission (CR),50～70% of patients with CR eventually relapse[1].The treatment of these diseases has become a difficult problem and hot spot in leukemia clinical research.Objective:For the treatment of relapsed/refractory AML, NCCN guidelines recommended clinical trials, "acute myeloid leukemia (relapsed/refractory) Chinese guidelines 2011 edition" recommended FLAG, CAG,HAA and other programs in the clinic to obtain certain curative effect.However, for patients with poor risk stratification and repeated recurrence, more effective treatment strategies should be explored. Based on previous in vitro and in vivo laboratory studies, we combined Chidamide and DCAG(DCCAG) regimen. The aim of this study was to investigate the clinical characteristics of recurrent/refractory AML, and to evaluate the efficacy and safety of the DCCAG regimen in the treatment of relapsed/refractory leukemic patients.Methods: Patients were screened according to WHO diagnosis, clinical study enrollment and exclusion criteria. Patients in the, group were treated with DCCAG regimen for 2 cycles. After first cycles, the curative effect of PR and CR was continued in the second cycle, patients with NR were determined by the investigators whether to enter second cycles of treatment. 4 weeks after the end of treatment to evaluate the efficacy. The efficacy was divided into: CR, partial remission (PR), no remission (NR) and OR (overall response rate). At the same time, adverse reactions of patients were observed, which were classified according to WHO adverse drug reactions.Resμlts: 1. Into the group:Screening out 43 patients, 20 cases relapsed more than 2 times,and 1 case relapsed after HSCT.According to the NCCN risk stratification standard(2016),there were 13 cases (30.2%) with poor-risk, 16 cases (37.2%) with intermediate-risk and 5 cases (11.6%) with favorable-risk.The proportion of gene mutations associated with poor prognosis was high, FLT3-ITD in 11 cases(28.9%), RUNX1 in 7 cases (18.4%),GATA2 in 6 cases (15.8%), TP53 in 2 cases (5.2%), and ASXL in 1 cases (2.6%).28 cases were treated with other relapsed/refractory AML regimens, and 11 cases had received more than 8 cycles of chemotherapy.2. Efficacy evaluation: 4 cases were withdrawn, and 39 cases were evaluated.Short-term efficacy: the overall CR rate was 41.0%, OR was 59.0% (23/39). AML1/ETO positive AML CR rate of 75% (1/4), a good prognosis CR rate of 100% . CR 36.0%and OR 52.0% (13/25) in patients who had previously received treatment with other refractory/relapsed regimens. Of the 5 patients who had previously used the CAG regimen, 4 had CR and 1 had PR.Of the 6 patients who had previously used the DCAG regimen, 2 had CR and 1 had PR.3. Adverse reaction: 60.5% of patients had treatment-related granulocytopenia,granulocyte deficiency (<0.5×109/L) duration of 16.5 (2-31) d. 51.2% of the patients with thrombocytopenia associated with treatment, thrombocytopenia (<20 X 109/L)duration of 17 (2-48) d.A total of 5 patients died, 2 cases died of disease progression, 1 case died of sepsis, 1 patient died of pulmonary infection and respiratory failure, the median OS 45.5 (36 -65) d.Conclusions: In this study,39 patients with relapsed/refractory AML were treated with DCCAG regimen, and more than 60% of the patients were effective.In patients with repeated recurrence, adverse prognostic factors, previously received other highly effective relapse refractory AML regimen treatment, OR 50% or more. Major adverse reactions were myelosuppression and infection.The preliminary results show that DCCAG regimen has a certain effect on the poor prognosis of refractory AML patients,and further treatment opportunities for such patients.The bone marrow function of these patients was poor, immune function was low, prolong the lack of granulocytes, the risk of infection was significantly increased, should pay attention to strengthen the prevention and treatment of infection.