Treatment Of Aplastic Anemia With LS001 Through LKB1-MARK2 Pathway

Aplastic anemia?AA?is caused by hematopoietic stem cells?HSCs?and/or hematopoietic microenvironment injury,normal bone marrow replaced by adipose tissue and functional abnormalities.The clinical manifestations of AA are 3-leanage blood cells reduction.The major clinical manifestations are severe anemia,infection and bleeding.Aplastic anemia is a rare blood disease,the incidence rate in China is about 0.765/100,000,lower than the incidence in western countries?about 0.2/100,000?and patients are mostly teenagers.Aplastic anemia especially severe aplastic anemia?SAA?has a high mortality rates,patients with AA are mostly because of exposure to toxic or immune abnormalities.In recent years,incidence of AA was significantly increased.The clinical and pathological manifestations of aplastic anemia is very typical,with detailed laboratory tests in time,it is not difficult to diagnose.Currently the clinical treatment options are include bone marrow stem cell transplantation,immunosuppressive therapy and supportive care treatment.With the progress in recent years,the survival rate of patients with aplastic anemia has been improved,but there are still enormous challenges.These challenges are mainly as follows:?1?Because of aplastic anemia is a rare disease,so it is more difficult to carry out large randomized controlled studies on clinical samples,which limited the progress of treatment to some extent.?2?A considerable number of patients due to HLA matching failure can not be treated with bone marrow stem cell transplantation.?3?Immuno-suppressive drugs are macromolecular drugs,only about 50%of the patients are effective,and the side effects include normal immune function suppressed,risk of tumor,severe liver and kidney toxicity.?4?Stem cell transplantation,immunosuppressive agents,blood transfusion and other treatment are expensive,which is not suitable for China's national conditions.Seeking new economic and safe drugs for the treatment of AA has become imperative.In recent years,with the rapid development of bioinformatics and pharmacogenomics,"big data"analysis has been an effective means of medical research and drug development,and especially for the development of rare diseases and"orphan drug",it has very practical and precious value.Human disease is often not single-gene disease,mostly due to the abnormal genomic changes and drug treatment is also by affecting the whole genome,which is the core philosophy of pharmacogenomics.ConnectivityMap?cMap?as a pharmacogenomics tool was developed by the renowned Broad Institute.By comparison analysis of the drug-related genomes and disease characteristics of the transcriptome,it can filter out small molecule compounds for treatment of diseases.Using online open resources,we retrieved the aplastic anemia-related microarray data,analyzed by cMap,we found ls001 may have therapeutic effects for aplastic anemia.LS001 as a small molecule compound has been clinically used to treat other diseases,which has the advantages of low cost and less side effects.Currently there is no research about LS001 treating aplastic anemia,but the latest research shows Liver Kinase B1?LKB1?plays a key role of HSCs maintainingnormal conditions,and LS001 has a close association with LKB1.This rsearch attempts to confirm LS001 protective effect of aplastic anemia on both the overall level and cell level and elucidate the molecular mechanism.We hypothesized:LS001 treatment for aplastic anemia is through LKB1-MARK2 signaling pathway.Accordingly this hypothesis,we conducted experiments.RESULTSLS001 may has a therapeutic effect for AA:the prediction of cMapWe retrieved AA gene expression microarray data GSE3807 in Gene Expression Omnibus database;Using GeneSpring GX software,we seeked the differentially expressed genes between control group and AA patients.515 genes significantly differentially expressed,including 202 up-regulated genes and 313 down-regulated genes.Using the Connectivity Map online platform,we predicted LS001 may has therapeutic effects for AA.LS001 to treat AA on overall levelICR mice were irradiated by 7.0 Gy ⁶⁰Co-?ray and administered with a dose of 300mg/kg of LS001.LS001 can significantly prolong the survival time of mice?Log-rank P<0.05?.Compared to the control group of mice,LS001 group platelets count significantly improved?1286±135×10⁹/l vs 1055±143×10⁹/l,P<0.01?,immature reticulocyte ratio significantly increased?10.31±9.83%vs 2.53±3.85%,P<0.01?,the number of nucleated bone marrow cells significantly increased?P<0.01?,bone marrow area recovered significantly?P<0.05?.LS001 to treat AA on cell levelPrimary bone marrow cells were irradiated by 8.0 Gy⁶⁰Co-?ray and treated with a dose of 1 mM LS001.LS001 can reduce the percentage of apoptotic cells significantly,which was detected by flow cytometry.The flow cytometry results were 23.5±3.8%vs39.3±5.6%?P<0.01?.LS001 treatment of AA is dependent on LKB1Lkb1 knockout mice were irradiated by 6.5 Gy ⁶⁰Co-?ray and administered with a dose of 300 mg/kg of LS001.In wild type mice,LS001 can significantly prolong the survival time of mice?Log-rank P<0.01?;in homozygous mice LS001 cannot prolong the survival time?Log-rank P>0.05?.Lkb1 knockout mice were irradiated by 6.5 Gy⁶⁰Co-?ray and administered with a dose of 300 mg/kg of LS001.Blood test shows LS001 failed to improve lkb1?-/-?mice PLT count?986±216×10⁹/l vs 965±205×10⁹/l,P>0.05?;failed to improve lkb1?-/-?RET ratio?0.19±0.08%vs 0.20±0.05%,P>0.05?.Blood test shows LS001 can significantly improve lkb1?+/+?mice platelet count?PLT 1628±97×10⁹/l vs 1395±202×10⁹/l,P<0.05?;significantly improve lkb1?+/+?RET mice ratio?0.45±0.10%vs 0.3±0.06%,P<0.01?.Primary bone marrow cells from normal ICR mice were cultured for 24 h,then treated with LS001 at 30 min,1 h,2 h,3 h,6 h,12 h and 24 h,respectively.WB detected the expression of p-LKB1 and LKB1.Compared with the internal reference GAPDH expression,LKB1 expression did not change significantly at each time point,the expression of p-LKB1 significantly increased at 30 min,and at about 6 h after dosing the expression reached a peak,and then gradually decreased.Protective effects of LKB1 for AA not dependent on AMPKMeg01 cells were cultured to a suitable density.Treatment group were given 0.1 mM and 1 mM of LS001,the control group received an equal volume of PBS,after 24 h,the cells were collected and lysed and immunoprecipitation was processed.The results show:with the increase of concentration of LS001,LKB1-AMPK?2 binding increased significantly,but LKB1-AMPK?1 binding not changed.We use ampk?2 knockout mice to verify whether the AMPK?2 participate the protective effect of AA.Ampk?2 knockout mice were irradiated by 6.5 Gy ⁶⁰Co-?ray and administered with a dose of 300 mg/kg of LS001.Blood test showed LS001 can improve ampk?2?-/-?mice PLT count?1243±106×10⁹/l vs 936±99×10⁹/l,P<0.01?;improve ampk?2?-/-?RET proportion of mice?0.29±0.06%vs 0.21±0.05%,P<0.01?.At the same time LS001 can significantly improve ampk?2?+/+?mice platelet count?PLT1533±85×10⁹/l vs 1310±132×10⁹/l,P<0.01?;significantly improved ampk?2?+/+?mice RET ratio?0.51±0.10%vs 0.33±0.06%,P<0.01?.LS001 treatment of AA dependent on MARK2,but not MARK3Meg01 cells were first transfected with lv-GFP,lv-MARK2,lv-MARK3over-expression lentivirus,then irradiated by 10.0 Gy ⁶⁰Co-?ray.After 24 h,using Annexin-V/PI staining,cell apoptosis was detected by flow cytometry within 1 h.Compared with the control group lv-GFP,the over-expression of MARK2 can significantly reduce the percentage of apoptosis?21.2±2.3%vs 38.6±7.5%,P<0.01?;over-expression MARK3 cannot reduce the Meg01 apoptosis proportion?37.3±7.3%vs38.6±7.5%,P>0.05?..Meg01 cells transfected with sh-Scramble,sh-MARK2,sh-MARK3 lentivirus,irradiated by 10.0 Gy ⁶⁰Co-?ray,24 h after irradiation underwent Annexin-V/PI staining and flow cytometry to detect apoptotic cells.Compared with the PBS+sh-MARK2 group,LS001+sh-MARK2 not significantly reduced the proportion of Meg01 apoptotic cells?42.9±2.5%vs 40.1±3.4%,P>0.05?.The interaction between LKB1 and MARK2Meg01 cells were cultured to a suitable density,treatment group were given 0.1 mM and 1 mM of LS001,the control group received an equal volume of PBS.Lysed cells were collected 24 h after treatment and immunoprecipitation was processed.The results show:with the increase of concentration of LS001,LKB1-MARK2 binding significantly increased.Bone marrow primary cells from mark2 knockout mice,were transfected with lv-GFP,and lv-LKB1 lentivirus,underwent 10 Gy-irradiation modeling.Compared with wild-type MARK2+lv-GFP group,the wild-type MARK2+lv-GFP group apoptotic cells were significantly reduced?flow cytometry:24.2±2.2%vs 30.1±1.9%,P<0.01?.Compared with homozygous MARK2+lv-GFP group,homozygous MARK2+lv-GFP group apoptosis had no significant changes?flow cytometry:43.5±5.7%vs 42.2±6.3%,P>0.05?.Preliminary studies related to cell apoptosis.5 ICR mice were taken bone marrow cells in primary culture,cells were grown to be an appropriate density and were divided into two groups,control group and radiation group.Exposure dose was 7.0 Gy.After 24 h,total protein was extracted,then the expression of apoptotic signals were measured.After modeling,the mitochondrial pathway signals Bcl2and Bax,and the death receptor pathway signal Cle-caspase8 no significant change;a significant increase was seen in the expression of the endoplasmic reticulum signal Cle-caspase12 and terminal signal Cle-caspase3.MARK2 knockout mice and wild-type homozygotes each four,were taken bone marrow cells in primary culture and cells were grown to be an appropriate density.Then cells were divided into two groups,namely control group and irradiation group,the two groups were divided into 2 groups again before modeling which were given PBS and LS001.Exposure dose 6.5 Gy.After 24 h total protein was extracted,we measured the expression of apoptotic signals.MARK2 wild-type mice after administration with LS001,Cle-caspase12 and Cle-caspase3 were significantly decreased.In homozygous mice after administration with LS001,the expression of Cle-caspase12 and Cle-caspase3 not significant changed.LKB1 knockout mice homozygous for four,were taken bone marrow cells in primary culture,cells were grown to an appropriate density to be later divided into three groups,namely control group,lv-GFP group and lv-MARK2 group,to be stably transfected virus.Exposure dose 6.5 Gy.24 h after irradiation total protein was extracted,we measured the expression of apoptotic signals.LKB1 knockout mice homozygous bone marrow cells transfected with lv-MARK2 virus,the expression of Cle-caspase12 and Cle-caspase3 were significantly decreased.MARK2 knockout mice homozygous for four,were taken bone marrow cells in primary culture,cells were grown to be an appropriate density,divided into three groups,namely control group,lv-GFP group and lv-LKB1 group,to be stably transfected virus.Exposure dose 6.5 Gy.24 h after irradiation total protein was extracted,we measured the expression of apoptotic signals.MARK2 knockout mice homozygous transfected with the lv-LKB1 viruses,the expression of Cle-caspase12 and Cle-caspase3 not significant changed.Conclusions:?A?LS001 has definite protective effect for AA.?B?LS001 protective effect for AA is dependent on LKB1-MARK2 pathway.?C?The protective effect of LS001 on aplastic anemia via endoplasmic reticulum pathway:Cle-caspase 12 and apoptotic terminal signal Cle-caspase 3.