Mechanism Of RRM1 On Gemcitabine Resistance In NK/T-cell Lymphoma

Background and objective:NK / T-cell lymphoma(NKTL)occurs worldwide and has distinct geographic features,mainly in Asia and Central and South America,accounting for 5%-15% of lymphomas in these countries.It has ever been reported in Europe and North America.NKTL is a highly invasive subtype of non-Hodgkin's lymphoma(NHL),with a rapid progression,short survival and poor prognosis.About two-thirds of NKTL cases are confined to stage I and stage II,mainly in the upper respiratory digestive tract(UADT).Previous NKTL treatment regimens usually used simultaneous or sequential chemotherapy combined with local radiotherapy.The overall response rate was80-90%,and the five-year progression-free survival(PFS)and overall survival(OS)were 60-85%,and 64 to 89%,respectively.However,in the past 20 years,due to the high level of P-glycoprotein expression on NKTL,the doxorubicin and cyclophosphamide used in previous chemotherapy regimens have been intrinsically resistant,so the treatment of NKTL has changed greatly in recent years.At the same time,studies have shown that NKTL cells are sensitive to the chemotherapy regimen containing alpha-asparaginase due to the lack of expression of asparagine synthetase.Even so,the patient's complete response(CR)rate to L-asparaginase remains only50-60%,five-year OS is about 50%.In addition,NKTL patients are susceptible torecurrence after receiving a chemotherapy regimen containing L-asparaginase,and the OS after relapse is only a few months,showing the chemotherapy regimen based L-asparagine enzyme is still not optimistic for NKTL patients.Clinically used SMILE(steroids,methotrexate,ifosfamide,L-asparaginase and etoposide)and MPVIC-P(methotrexate,peplomycin,etoposide,ifosfamide with carboplatin)chemotherapy regimen has a good clinical response to patients with NKTL.However,these therapy including chemotherapy and radiotherapy often produce serious adverse reactions,such as death from severe infections and brain damage caused by radiation therapy.Therefore,the emergence of new chemotherapy regimens for NKTL patients is necessary.Due to the lack of international consensus on standard chemotherapy for NKTL,our center has developed a new chemotherapy regimen of DDGP(cisplatin,dexamethasone,gemcitabine,and pembellase),which has gained consensus in the domestic industry.In the latest clinical trial statistics(NCT01501149),80 patients with NKTL who received chemotherapy with DDGP had longer PFS,OS,and better tolerance than those newly diagnosed patients with SMILE regimen.Therefore,this chemotherapy regimen has become the first-line chemotherapy for the treatment of NKTL in our center.Nucleotide reductase(RNR)is a rate-limiting enzyme in the synthesis of DNA,specifically catalyzing the ribonucleotides into 2-deoxyribonucleotides necessary for DNA synthesis and repair.RNR consists of homodimers,each consisting of two subunits: a large regulatory subunit RRM1 and a small catalytic subunit RRM2.Gemcitabine binds irreversibly to ribonucleotide reductase and inactivates its large subunit(RRM1),so RRM1 is a key molecule in determining the efficacy of gemcitabine.Today,several studies have shown the expression of ribonucleic acid reductase large subunit 1(RRM1)in solid tumors.In patients with pancreatic cancer and lung cancer treated with gemcitabine,patients with low levels of tumor RRM1 expression have higher survival rates;gemcitabine has developed resistance in solid tumors including pancreatic cancer and lung cancer.Studies in multiple solid tumors have shown that high expression of RRM1 is associated with gemcitabine resistance.However,the study on RRM1 and gemcitabine in NK/T-cell lymphoma is lessreported,and its role and mechanism have not been found.The relationship between RRM1 and gemcitabine sensitivity in NKTL and their mechanism needs to be proved through experiments.In the early stage,we found that RRM1 is highly expressed in NKTL gemcitabine-resistant cell lines in the proteomic analysis.The purpose of this study was to investigate the relationship between RRM1 and gemcitabine sensitivity in NKTL and its mechanism.We first detected the expression of RRM1 in NK/T cell lymphoma tumor tissues and cell lines by immunohistochemistry,real-time PCR and Western blot,and analyzed the correlation between RRM1 expression and clinical factors and prognosis of NKTL.The RRM1 high expression cell line(YT-RRM1)and the RRM1 low expression cell line(YT-R-shRRM1)were constructed by lentiviral transfection.CCK-8 and flow cytometry were used to detect the sensitivity and apoptosis of gemcitabine-treated cells.Transcriptome sequencing and Western blot were used to analyse changes in downstream proteins and signaling pathways caused by up-regulation or down-regulation of RRM1 to reveal the biological role and mechanism of RRM1-mediated gemcitabine resistance in NK/T cell lymphoma.Finally,by our unique construction method the NK/T mouse model was established.The sensitivity of the RRM1 high expression cell model to gemcitabine after gemcitabine treatment was observed by small animal bioluminescence imaging.The changes of RRM1 downstream protein and signal pathway were identified by immunohistochemistry.The related mechasim is further explored and verified.The results of this study will provide important evidence for determining the relationship between RRM1 and gemcitabine-resistant NK/T cell lymphoma.In addition,the study has verified RASAL3 protein or ERK signaling pathway may be as a target for reversing gemcitabine-resistant NK/T cell lymphoma.Moreover,it offers an additional possibility and choice to t gemcitabine-resistant NKTL patients.Part? Expression and clinical significance of RRM1 in NK/T cell lymphoma Methods:(1)Tumor tissue samples from 62 patients with NK/T cell lymphoma treated with gemcitabine were collected and the expression of RRM1 in NK/T cell lymphoma was detected by immunohistochemistry.(2)The clinical data of 62 patients were collected.The Pearson ?2 test was used to detect the correlation between RRM1 and clinical features,biochemical indicators and staging.(3)The Kaplan–Meier survival curve was used to analyze the correlation between the expression of RRM1 and OS and PFS in patients with NK/T cell lymphoma.Cox proportional hazards regression models were used to determine prognostic factors for OS and PFS of NKTL patients.(4)Real time RCR and Western blot were used to detect RRM1 expression in mRNA and protein level in B cell lymphoma cell lines(LY8,Raji)T cell lymphoma cell lines(Hut78,Karpas,Jurkat),NKTL cell lines(YT,NKYS).Results:(1)In NKTL tumor tissues,RRM1 staining was limited in cytoplasm.62(100%)patients had the RRM1 expression,of which 29(46.8%)patients were highly expressed,and 33(53.2%)patients were under-represented.There were no significant differences in gender,age,Ki67,LDH,EBV-DNA copy number and staging between patients with high RRM1 expression and low RRM1 expression.Of the 33 patients with low RRM1 expression,23(71.9%)had complete or partial remission,and of the29 patients with high RRM1 expression,9(31.0%)had complete or partial remission.The difference in efficiency was statistically significant(P = 0.002).(2)Compared with patients with low RRM1 expression group,PFS and OS were significantly shorter in patients with high RRM1 expression,and the difference was statistically significant(P=0.002).(3)Compared with normal NK cells,B cell lymphoma cell lines(LY8,Raji)and T cell lymphoma cell lines(Hut78,Karpas,Jurkat),RRM1 in NKTL cell lines YT(P=0.0003)and NKYS(P= The RRM1 in 0.0005)is lower in mRNA and protein expression levels.Summary(1)The expression of RRM1 was associated with the efficacy of gemcitabine chemotherapy in patients with NKTL,but not with gender,age,Ki67,LDH,EBV-DNA copy number,IPI score and stage.(2)RRM1 has high expression and low expression in NK/T cell lymphoma tumor tissues,and patients with low expression have better effects in gemcitabine-based chemotherapy.(3)RRM1 is low expressed in NKTL cells(YT and NKYS).Part? Mechanism of RRM1 on gemcitabine resistance in NK/T cell lymphoma Methods:(1)Western blot was used to detect the expression of RRM1 protein in NKTL cell line YT,and gemcitabine-resistant cell line YT-R.(2)YT-RRM1 cells were constructed by over-expressing RRM1 in YT cells using a RRM1 high expression plasmid;YT-R-shRRM1 cells were constructed by silencing RRM1 in YT-R cells using shRNA.All the process was done by lentivirus transfection.(3)CCK8 detects and compares the changes the IC50 value of gemcitabine in YT,YT-RRM1,YT-R and YT-R-shRRM1 cells.(4)Flow cytometry was used to detect and compare the effects of gemcitabine on the apoptosis rate of YT,YT-RRM1,YT-R and YT-R-shRRM1 cells..(5)Transcriptomics sequencing was used to compare the gene expression differences in YT and YT-RRM1.(6)Western blot analysis was performed to compare the changes of RRM1 downstream proteins in YT,YT-RRM1,YT-R and YT-R-shRRM1 cells.Results:(1)Compared with YT cells,the expression of RRM1 was significantly increased in YT-R cells(P<0.001).(2)The IC50 value of gemcitabine-treated YT cells was 0.009 ?g/ml,while the IC50 value of gemcitabine-treated YT-RRM1 cells was 43 ?g/ml;after applying0.009?g/ml gemcitabine to YT and YT-RRM1 cells for 48 h,the ratio of apoptosic cells was 49.2% and 7.37%,respectively(P<0.01).(3)Transcriptomic sequencing showed that compared with YT cells,YT-RRM1 had 42 genes up-regulated and 154 genes down-regulated,and the key gene RASAL3 playing a role in the ERK signaling pathway related to drug resistance was down-regulated.Compared with YT cells,YT-RRM1 cells down-regulated RASAL3 protein expression and up-regulated p-ERK protein expression.(4)The IC50 of gemcitabine on YT-R cells was 36 ?g/ml,and the IC50 of gemcitabine on YT-R-shRRM1 cells was 0.003 ?g/ml;after applying 0.003 ?g/ml of gemcitabine,the apoptotic ratio of YT-R and YT-R-shRRM1 cells was 2.63% and46.0%,respectively(p < 0.001).(5)Compared with YT-R,RASAL3 expression was up-regulated and p-ERK expression was down-regulated in YT-R-shRRM1 cells.Summary:(1)Compared with normal NKTL cell lines,the expression of RRM1 was significantly increased in gemcitabine-resistant cells.(2)The sensitivity and apoptosis ratio of normal NKTL cells transfected with RRM1 to gemcitabine were not affected;the sensitivity and apoptosis ratio of gemcitabine-resistant cells to gemcitabine were increased after RRM1 interference.(3)RRM1 may increase the activation of ERK pathway of NKTL by down-regulating the expression of RASAL3,leading to NKTL's resistance to gemcitabine.Part? In vivo validation of the mechanism of RRM1 on gemcitabine resistance in NK/T cell lymphoma Methods:(1)A subcutaneous xenograft mouse model of NKTL was constructed usingNOD/SCID mice and YT,YT-RRM1 cells.When the average diameter of the tumor reached about 0.5 cm,the tumor tissues of three mice were randomly selected from each group for HE staining and immunohistochemical indexes(CD56,Proferin,EBER,RRM1,RASAL3,pERK).The scoring method was used to compare the expression of CD56,Proferin,EBER,RRM1,RASAL3,and p-ERK in the tumor tissues of the YT and YT-RRM1 groups.(2)The remaining mice were divided into four groups,namely YT blank control group and YT gemcitabine group,YT-RRM1 blank control group and YT-RRM1 gemcitabine group.Ensure 6 mice per group.Gemcitabine was administered intraperitoneally twice a week.The growth of tumor-bearing mice in each group was observed daily.The length and diameter of the tumor were measured every 3 days before and during the administration,and small animal imaging was performed to record.The bioluminescence signal of the tumors during the administration of each group of mice was compared,and the changes of tumor volume before and after treatment with gemcitabine in YT and YT-RRM1 mice were compared.Results:(1)The tumor formation rate of NKTL animal model was 100% using YT and YT-RRM1 cells.Immunohistochemistry indicated that NK/T cell marker CD56,cytotoxic particle-related labeling Proferin and EBER were present in each group of tumor tissues.The expression of each index in each group of tumor tissue did not differ significantly,which proved that the models were successfully prepared.(2)Immunohistochemistry results showed that compared with the YT mouse model,the expression of RRM1 and p-ERK in the YT-RRM1 mouse model tissues increased,and the expression of RASAL3 decreased.(3)YT control group mice gradually died from the 24 th day after the intervention.YT-RRM1 control group mice gradually died from the 21 st day after the intervention,YT-RRM1 gemcitabine group mice gradually died from the 33 rd day after the intervention,and there was no death in the YT gemcitabine group.(4)In the YT mouse model,compared with the blank control group,the tumor volume of the gemcitabine treatment group gradually decreased until disappeared,and the tumor bioluminescence signal gradually weakened until disappeared(P<0.01);In the YT-RRM1 mouse model,there was no significant difference in tumor volume and tumor bioluminescence signal change between the gemcitabine-treated group and the blank control group.Summary(1)In vivo experiments showed that gemcitabine can inhibit the tumor growth of normal NKTL,but has no effect in NKTL with high expression of RRM1.(2)Immunohistochemistry results of tumor tissues suggest that RRM1 can increase the activation of ERK pathway by down-regulating the expression of RASAL3,which leads to the resistance of NKTL to gemcitabine.Conclusion1.RRM1 is highly expressed in NK/T cell lymphoma tumor tissues and cell lines.Patients with low RRM1 expression have a better effect on chemotherapy regimens based on gemcitabine.2.The expression of RRM1 is inversely related to the sensitivity of gemcitabine NK/T lymphoma.3.RRM1 increases the activation of the ERK pathway by down-regulating the expression of RASAL3,resulting in the resistance of NKTL to gemcitabine.4.Gemcitabine inhibits tumor growth in the normal NKTL mouse model and does not inhibit tumor growth in the NKTL mouse model with high RRM1 expression...