Design And Optimization Of CRM1-inhibitors And Their Mechanisms Of Eliminating Lymphoma Cancer Cells

Lymphoma is a group of blood cancers that develop from lymphocytes(a type of white blood cell),signs and symptoms may include enlarged lymph nodes,fever,drenching sweats,unintended weight loss,itching and constantly feeling tired.As it is present in the bloodstream,it can spread,or metastasize,to different parts of the body.The two main types of lymphoma are Hodgkin lymphoma and non-Hodgkin lymphoma.Non-Hodgkin lymphoma is the most common type.It’s reported that the happening of lymphoma is often related with the immune system.However,the exactly underlying mechanism is not understood.CRM1 is an important nuclear exportin protein,whose cargo proteins are about 285 and many of them are tumor suppressors,including survivin、p53、p27KIP1、APC.Recently,CRM1 was found upregulated in many different cancers,especially in lymphoma.CRM 1-upregulation was considered as a bad prognosis factor for lymphoma patient.Thus,to discover and design CRM1 inhibitors is a novel strategy for lymphoma therapy.Natural products have many advantages to be a drug screening database,such as its structural and chemical diversity and its biocompatibility.Therefore,to discover CMR1 inhibitors from natural products is feasible,and to design and develop new small molecules from natural product is promising.In this paper,we mainly focus on the following aspects.Previously,our lab found that LFS-01 may be a CRM1 inhibitor.Through cell viability assay,we identified that LFS-01 can selectively eradicate lymphoma cells while sparing normal lymphocytes.MALDI-TOF-MS result showed that LFS-01 can covalently bind to the Cys528 in NES pocket of CRM1.Nuclear-transporting-functional-assay-results showed that RanBP1 and p53,two cargo proteins of CMR1,were trapped in the nucleus by LFS-01.In addition,CRM1 protein levels were down-regulated by LFS-01.Through target mutation analysis,we ensured that CRM1 was a key protein for LFS-01.Furthermore,LFS-01 can induce lymphoma cell cycle arrest at G2-M phase and induce cell apoptosis by activating the intrinsic caspase pathway.Secondly,by employing different experimental methods including the LDH assay,transmission electron microscope,immunofluorescence and Western blot analysis,we found that LFS-01 can trigger autophagy in lymphoma cells.Through co-localization analysis,we further revealed that LFS-01 could induce mitophagy.What’s more,LFS-01 can retain Nrf2 in the nucleus by covalently modulating CRM1 and consequently upregulate p62/SQSTM1,an essential structural component of the autophagosomes during mitophagic process.LFS-01 treatment also stimulated AMPK and thereby inhibited the mTOR pathway by up-regulating AMPK phosphorylation and down-regulating m-TOR phosphorylation.Transcriptomic studies confirmed that 15 autophagy-associated genes such as p62/SQSTM1,VCP and BCL2 were differentially expressed after LFS-01 treatment.Most importantly,LFS-01 exhibited strong efficacy in xenograft mouse model yet with little toxicity to animals.Finally,computational based drug design was conducted using Discovery Studio and a series of CRM 1 inhibitors were obtained.Through virtual screening and molecular dynamic analysis,we discovered a new compound named LFS-25 which has increased cellular viability and is about 50 times stronger than LFS-01.The main reason for its higher activity may stem from its increased interaction with the residues in the NES pocket of CRM1.Interestingly,we found that the ABC-DLBCL cell line U2932 was much more sensitive to LFS-25 than other cell lines.To delineate the underlying mechanism,we conducted a series of experiments including Western blot,Co-IP,nuclear transporting functional assay and flow cytometry.The results showed that the nuclear-export of IκBα was inhibited,the interaction between IκBα and NF-κB was enhanced,and the transcriptional activity of NF-κB was decreased after LFS-25 treatment.What’s more,the caspase substrates were activated,cell cycle was arrested and apoptosis was induced by LFS-25.Collectively,we found that LFS-01 can target CRM1 and suppress the proliferation of lymphoma.A new CMR1 inhibitor LFS-25 was developed based on the structure of LFS-01.And we explained the mechanisms of the growth-inhibitory effect of LFS-25 on ABC-DLBCL cells.Our data provides new insights into developing CRM1 inhibitors for treating human lymphoma diseases.