Study On The Role Of Differentiation Abnormalities Of Bone Marrow Adipocytes In The Relapse Of Acute Myeloid Leukemia And Its Formation Mechanism

Objective and Significance:We retrospectively study the bone morrow biopsies of AML patients during complete remission(AML-CR)and normal controls by analyzing the morphological characteristics of marrow adipocytes,and explore the correlation of the prognosis of patients with AML-CR,and explore its formation mechanism,which may provide a novel theory basis for the early intervention of leukemia relapse.At the same time,we analyse the expression of WNT11 in the bone marrow biopsies of acute myeloid leukemia patients(AML)and controls,and clarify the regulatory mechanism of bone marrow adipocytes abnormal differentiation,which provides a new therapeutic strategy for prevention the relapse of AML patients.Methods:(1)The IHC staining of fatty-acid binding protein 4(FABP4)was used as a defined marker of adipocytes in the bone marrow biopsies of AML-CR patients and controls,then the grid point method was used to quantitative analyse the adipocyte content in BM sections from 80 AML patients in remission and 71 control groups.Meanwhile,ROC curve and Kaplan-Meier survival analysis were used to further to investigate the relationship between the adipocyte content and the prognosis of AML-CR patients.(2)bone marrow mesenchymal stem cells(MSCs)from AML-CR patients and controls were isolated and induced into adipocytes in vitro,Colony forming experiment(CFU-F)was used to compare MSCs proliferation ability between the two groups.Meanwhile,toxicity assay was used to determine whether chemotherapy drugs have direct toxic effects on MSCs,mature fat cells and adipogenesis differentiation process,by oil red O staining,q RT-PCR and flow analysis(3)The mononuclear cells(MNCs)were isolated from bone marrow of control group,and CD45+ hematopoietic cells(HCs)were sorted by FACS,and then cells were treated with cytarabine(Ara-C),the supernatant of cells was collected and added during the differentiation process of MSCs to investigate their effect on Adipogenic differentiation;(4)Subsequently,gene array was used to selected the key factors,which may regulate the marrow adipocyte volume after chemotherapy,and corresponding intervention measures were adopt to studies the possible regulate mechanism of the decreased adipocyte volume after chemotherapy in vitro.(5)The expression of objective cytokine in the bone marrow hematopoietic cells from AML-CR patients and control group was detected by q PCR and Western Blot.Meanwhile,immunofluorescence assay was used to detect objective cytokine expression in the bone marrow biopsy;Furthermore,the correlation analysis between the expression level of GDF15 in bone marrow aspirates and adipocyte content was analyzed.(6)Immunohistochemical staining was used to detect the expression of WNT11 in the bone marrow biopsies of AML patients and controls.The expression level of WNT11 in leukemia cell lines,bone marrow mesenchymal stem cells(MSCs)and bone marrow mononuclear cells(MNCs)was assayed by PCR.Meanwhile,gene silencing and overexpressing were adopted to validate the regulation function of WNT11 on abnormal differentiation of MSCs in AML patients.Then,Western Blot was used to detect the change of corresponding protein and explicit the molecular mechanisms of WNT11 on the regulation of abnormal differentiation of MSCs in AML patients.Results:(1)Compared with controls,marrow adipocyte volume was significantly lower in the BM section from the AML-CR patients,the average adipocyte content were 21.10% and 27.43%,respectively(p <0.01),Moreover,the marrow adipocyte volume remained significantly lower in the continuous remission patients than in the recurrence group.No significant differences were found between the two groups in the basic characteristics of patients in age,sex or FAB subtype(p<0.05).(2)The correlation analysis between adipocyte content and relapse-free survival(RFS)in AML-CR patients revealed that the area under the curve(AUC)was 0.6927(p<0.0034),and longer RFS and overall survival(OS)in AML patients during CR with marrow adipocyte volume less than AML-CR 21.5%,the reduced adipocyte content was closely related to RFS and OS.(3)The toxicity tests showed that the reduced adipocyte volume was not directly derived from mesenchymal stem cells(MSCs),mature adipocytes or the lipid differentiation process.Instead,the reduced volume was due to GDF15,which was secreted by haematopoietic cells in the BM after chemotherapy and inhibited adipogenesis.(4)Gene array results showed that bone marrow hematopoietic cells treated with Ara-C high expression of GDF15,which inhibit adipogenic differentiation process.When rh GDF15 was added into the culture medium in vitro,the lipid droplets of adipocytes significantly decreased,and the expression of adipogenic genes significantly reduced.In contrast,the human neutralizing anti-GDF15 antibody partially attenuated the inhibitory effect.(5)GDF15 was highly expressed in CD68+ cells from BM sections,which were regarded as macrophage.Furthermore,the regression analysis indicated that the level of extracellular GDF15 in BM aspirates from AML patients during CR was associated with the adipocyte volume.(6)Compared with controls,the expression of WNT11 was higher in the MSCs of AML patients,and result in the abnormal differentiation of BM adipocytes.Moreover,WNT11 regulate the Ca2+ mediated the classic WNT pathways protein PKC,which promote the adipogenic differentiation of MSCs.Conclusion:(1)Compared with controls,marrow adipocyte volume was significantly reduced in the BM section from the AML-CR patients,and the reduced marrow adipocyte volume during CR predicted the good prognosis of AML patients.(2)The experimental results showed that chemotherapy was not directly affect adipogenic differentiation process.Instead,it was due to GDF15,which was secreted by haematopoietic cells in the BM after chemotherapy,which inhibited adipogenesis.And GDF15 expression level is closely related to the content of bone marrow fat cells in patients with AML-CR.(3)WNT11 regulates the non-classic Wnt pathways,and activates the PKC signaling pathway by the second messenger Ca2+,which regulate the abnormal adipogenic differentiation of MSCs in AML patients.