Role Of Folate Receptor 3 In Discontinuation Of Chronic Myeloid Leukemia

Part?Role of folate receptor 3 in discontinuation of chronic myeloid leukemia and its effect on leukemia stem cellObjective:The development and application of tyrosine kinase inhibitor?TKI?has greatly improved the prognosis of patients with chronic myeloid leukemia?CML?.However,long-term medication is accompanied by heavy economic burden and drug side effects,some patients who have achieved long-term deep molecular response?DMR?are eager to stop TKI treatment.Unfortunately,the discontinuation results from the major research centers consistently indicated that only 50%of the patients reached treatment-free remission?TFR?after discontinuation,and no biomarkers or characteristics could accurately predict and prevent relapse.The current view on relapse after discontinuation is that TKI cannot eliminate quiescent leukemia stem cells?LSC?.Based on this,our study started from the transcriptome sequencing on bone marrow of patients who relapsed and non-relapsed to looking for the differentially expressed genes and exploring their effects on CML-LSC and K562 leukemia cells.Methods:We selected bone marrow of 14 CML patients?matched 7 relapsed and 7non-relapsed patients?who discontinued TKIs therapy in our center for transcriptome analysis to discover the differences between relapsers and non-relapsers.Further,we applied colony-forming experiments,LC-MS,subcutaneous tumor models and small animal PET imaging to detect the cell differentiation and tumor metabolism.We also analyzed the cell cycle,apoptosis and DNA synthesis by PI staining,Annexin V/PI staining and EdU probe on flow cytometry,respectively.We measured the cell proliferation by MTT assay and detectd on multifunctional enzyme marking instrument.qRT-PCR was used to determined the expression of genes in cells with different FOLR3 SNP expression.We explored how the differential gene affected the biological function of CML CD34+/K562cells and participated in disease relapse through above methods.Results:We analyzed the differentially expressed genes between relapsers and non-relapsers.Among them,FOLR3 was the most significant one,and FOLR1 and FOLR2did not express in any of our samples.We also searched expression profiles in normal tissues and found that FOLR3 was highly expressed in both bone marrow and blood.In CML datasets,FOLR3 was significantly highly expressed in remission CML samples.Based on TCGA data,patients of other tumors with high level of FOLR3 had poor overall survival.After SNP calling,we identified a rs139130389 SNP in the third exon of the FOLR3 in non-relapsed CML samples.FOLR3 SNP+encodes functional proteins with a complete folate receptor domain,while only a partial folate receptor domain was encoded by wild type FOLR3.The expression level of FOLR3 was positively correlated with the number of mapping reads of rs139130389.Based on evolutionary analysis,we found that almost all non-primate species have the TA insertion,while it was deleted in most primates.The average FOLR3 SNP+genotype proportion in different human populations was11.08%,while in our CML samples,the proportion of FOLR3 SNP+genotype is 8.93%.On the other hand,we found that immune cell populations were not differentially distributed in relapsed and non-relapsed CML samples.So our further studies mainly focused on LSCs.The FOLR3 SNP showed no impact on DNA synthesis and apoptosis.The proliferation and cell cycle activities of FOLR3 SNP+LSC/K562 cells were higher than the control cells.Consistently,the colony-forming capacity of FOLR3 SNP+LSC/K562 cells was significantly higher than that of the other cells.There was no significant difference in the number of cell colony-forming units among the folate-free groups with different FOLR3 SNP expression.The differential metabolites of FOLR3SNP+LSC were enriched in fatty acid metabolism,fatty acid biosynthesis and fatty acid elongation pathways.A comparison between FOLR3 SNP+and FOLR3 SNP-K562 cells revealed that the discriminating factors were glycerophospholipid metabolism,glycerolipid metabolism and fatty acid biosynthesis.An increase in the maximal standard uptake value?SUVmax?of ¹⁸F-FDG was noted in the FOLR3 SNP+group,with a 1.33-fold increase compared with that in control,a 1.65-fold increase for siFOLR3?P=0.0663?and a1.82-fold increase for FOLR3 SNP-?P=0.0579?.Conclusion:FOLR3 rs139130389 SNP is only detected in the TFR group in CML patients who discontinued TKIs treatment.FOLR3 was significantly highly expressed in remission CML samples that treated with TKI for 12 months.While,patients of other tumors with high level of FOLR3 had poor overall survival.These data indicate that FOLR3 plays a specific role in CML disease and is a manifestation of tumor heterogeneity,which is expected to be used as CML withdrawal monitoring and stratified treatment for patients.In vitro and in vivo,we demonstrated that FOLR3 SNP promoted proliferation,differentiation and fatty acid metabolism,activated cell cycle in CML-LSC and K562 cells likely through increasing the uptake of folic acid.Part ? Mechanism of folate receptor 3 affecting the function of CML-LSCObjective: The survival of leukemia stem cells does not depend on the activity of BCR-ABL1 tyrosine kinase and always in quiescence,so it cannot be eliminated by TKI.Minimal residual disease?MRD?,especially LSC in the CML patients are responsible for the post-cessation relapse.CML-LSC can be detected in most patients before drug withdrawal,but there was no statistical difference in the number of CML-LSC between relapsers and non-relapsers.The most likely explanation is that the inherent properties of LSC maybe diverse in different patients.Therefore,the discussion on the mechanism of discontinuation lies in the difference in the function of CML-LSC between relapsers and TFR.So we further explored the mechanism of FOLR3 SNP affecting LSC differentiation,and the outcome of continuously differentiated LSC and the relationship between FOLR3 SNP and TFR after TKI withdrawal on the basis of the preliminary work.Methods: We established different FOLR3 SNP expression subtypes in newly diagnosed CML LSC and K562 cells through lentiviral transfection,and performed m RNA-seq for three groups of LSC and one group of K562 cells with differentially expressed FOLR3 SNP.The results were analyzed to find the differentially expressed genes for pathway enrichment.In vitro experiments,we identified the mitochondrial morphology by transmission electron microscopy;the mitochondrial function was assessed by measuring the changes in maximum oxygen consumption by cell mitochondrial stress test.Mitochondrial content was assessed by staining LSC and K562 cells with Mito Tracker Green.Mitochondrial membrane potential was measured by JC-1 staining.Intracellular ROS were detected by staining cells with DCFH-DA.All samples were read on flow cytometry.Data were analyzed with Flow Jo version 10.K562 cells were treated with drugs at different concentration gradients for 48 h.Intracellular ATP was determined using an ATP assay kit.We also applied clony-forming assay,q RT-PCR to detect the ending of differentiated LSC.Results: We identified 428?220 upregulated and 208 downregulated?differentially expressed genes?DEGs?of FOLR3 SNP+ LSC samples.The GO enrichment analysis of these DEGs indicated that mitochondrion-related processes,such as ATP hydrolysis,ATPase activity and mitochondrial electron transport were significantly enriched.370 DEGs of the FOLR3 SNP+ K562 sample were also enriched in mitochondrion-related processes,such as mitochondrial translation,NAD binding and electron carrier activity.In addition,the expression density analysis of mitochondrion?from Mito Carta?,oxidative phosphorylation?from KEGG?and ATP synthesis?from GO?showed that samples of FOLR3 SNP+ and SNP-had a higher MGE than that of si FOLR3 in CML LSC group.The numbers of mitochondrion in the si FOLR3 and FOLR3 SNP-groups were less than those in the control and FOLR3 SNP+ groups.FOLR3 SNP+ LSC/K562 cells had much higher oxygen consumption rates?OCR?throughout the detection process compared to those of the remaining groups,In terms of individual indicators,except for basal respiration,the maximal respiration and spare respiratory capacity of FOLR3 SNP+ LSC cells were significantly higher than those in other experimental groups.In K562 cells,the FOLR3 SNP significantly increased the maximal respiration and proton leak.Mitochondrial mass and mitochondrial membrane potential of FOLR3 SNP+ cells increased in different degrees.AICAR and DAC could elevate ATP production in K562 cells in a concentration-dependent manner within certain limitations.In the later stage,the colony-forming capacity of FOLR3SNP+ LSC cells was remarkably decreased.In addition,ROS levels and cell cycle arrest in FOLR3 SNP+ LSC cells were substantially higher.In FOLR3 SNP+ LSC,IL-6 and MMP9 were increased;SIRT7 and Cyclin E2 were decreased.Conclusion: We demonstrated that FOLR3 rs139130389 SNP promotes differentiation via increased mitochondrial activity.Continuous differentiation of CML-LSC shows an aging phenotype.AICAR and DAC treatment could elevate mitochondrial activity and potentially assist FOLR3 SNP-cells achieve similar biological characteristics to FOLR3 SNP+ cells.We demonstrated that FOLR3 SNP+ CML patients are the priority selection for TKIs cessation and activating mitochondrial function-induced senescence is an alternative novel approach to ‘eradicate' CML-LSCs.