In Vitro And In Vivo Study Of B-AP15 Overcome Imatinib Resistance In Chronic Myeloid Leukemia

Backgrand Chronic granulocytic leukemia(CML),referred to as CML,is related to chromosomal abnormalities in hematopoietic stem cells.The most typical is the translocation of chromosome 9 and chromosome 22 on the long arm of the Philadelphia chromosome.This translocation leads to the production of BCR-ABL fusion gene and produces a large number of BCR-ABL fusion proteins,while tyrosine kinase is constitutively activated.BCR-ABL can alter bone marrow progenitor cells and drive the development of 95% chronic myeloid leukemia(CML)cases.BCR-ABL promotes the development of chronic myelocytic leukemia by activating the downstream pathways that can increase cell proliferation and survival.These pathways include RAF/MEK/ERK,PI3K/AKT,and JAK/STAT.The clinical stages of CML are chronic phase,accelerated phase,and acute phase.Imatinib,a first-line clinical drug for CML and other BCR-ABL-expressing leukemia,can bind to the ABL kinase site and inhibit its substrate phosphorylation.Most patients receive a complete cytogenetic response in the chronic phase of the disease after imatinib therapy.However,molecular studies of imatinib-treated patients in remission showed that high expression of BCR-ABL could still be detected;and if imatinib was discontinued,the disease was easy to relapse.There are often many adverse reactions after imatinib treatment,in which the common side effects are intestinal reactions(nausea,vomiting,diarrhea),edema,muscle spasms,and rash.Myelosupression is more common in patients with CML especially those receiving higher doses.Limited duration of imatinib response is also common in advanced CML patients,and imatinib resistance can occur at any stage of the disease.Imatinib resistance can lead to relapseand deterioration.There are many mechanisms for imatinib resistance,but the most common mechanism is due to the mutation of the ABL gene locus(the most important of which is the T315 I mutation),which eventually makes imatinib unsuccessfully combined with the ABL kinase site and eventually leads to abnormal activation of the downstream pathway.In order to overcome this resistance,second generations of tyrosine kinase inhibitors were subsequently studied,most notably niloticinib and dasatinib.Compared with imatinib,nillotinib is more effective in inhibiting the proliferation of wild-type BCR-ABL cell lines and BCR-ABL mutant cell lines.At the same time,it can reduce the burden of leukaemia(the effective dosage of the two should be smaller than imatinib),prolonging the patient's survival time,but it is ineffective for the T315 I point mutation.But like imatinib,niloticinib can cause side effects: bone marrow suppression at levels 3 to 4,and increase bilirubin and lipase.Dasatinib can be combined with serine / threonine kinase(such as TCL family kinase,mitogen activated protein kinase and receptor tyrosine kinase).Dasatinib is effective in combating imatinib resistant conformational problems(because it has better affinity with tyrosine family kinases).Like nilotinib,dasatinib could inhibit the value added and kinase activity of most of the wild and BCR-ABL mutant cell lines,but the T315 I mutant was still ineffective.At the same time,it also has side effects,especially in the acute phase of disease,which is likely to cause grade 3-4 myelosuppression.Recently approved palatinib has proven to be effective for patients with refractory chronic myelocytic leukemia.Palatinib is the only tyrosine kinase inhibitor that can treat patients with T315 I mutations.But its side effects are also obvious,which is associated with increased risk of arterial hypertension(sometimes severe)and severe arterial occlusion and venous thromboembolism events.The continuous activation of tyrosine leads to several abnormal changes in chronic myelocytic leukemia: changes in cell adhesion,inhibition of cell apoptosis,inhibition of proteasome degradation,and abnormal activation of the mitogen downstream pathway.Therefore,if we start from the downstream channel of BCR-ABL,it may be a good treatment.Ubiquitin proteasome system(UPS)regulates the degradation of proteins in cells.It mainly includes signal transduction,gene expression,cell cycle,replication,differentiation,immune response,cell stress response and so on.The main feature of cancer cells is the loss of cell cycle regulation.The cancer cells lose the DNA repair function and the highly mutated genes.Finally,a large number of misfolded proteins are produced,all of which are cytotoxic substances.To make up for this serious problem,cancer cells enhance the high expression of the proteasome system and eliminate these proteins(that is,the proteasome system of the cancer cells is more active than the UPS of normal cells).Contrary to the non specific lysosome degradation,the proteasome has a high selectivity for the degradation of the protein in the body,and only the proteins that have been labeled by the small molecules of the small molecules can be degraded.Ubiquitin proteasome degrading protein is a complex process,which involves ubiquitin,3 enzymes and proteasomes.Among them,ubiquitin is a highly conserved protein with 76 lysine residues.Ubiquitin is combined with lysine of substrates to form a hetero peptide bond,that is to form a single ubiquitination substrate.Subsequently,the substrate can be marked by more ubiquitin,forming polyubiquitin chain.Ubiquitin molecules have 7 lysine,but lysine 48 residues are bound to the substrate protein,which ultimately leads to the degradation of target protein.Other residues of ubiquitin are responsible for protein entry,nucleation,nuclear DNA repair,gene expression,signal transduction,and endocytosis.The first step in protein degradation is the formation of ubiquitin and ubiquitin activating enzyme E1 chain,which requires ATP hydrolysis to provide energy.Then the activated ubiquitin is transported to the enzyme E2(ubiquitin binding enzyme),and the complex E2 enzyme activates the ubiquitin and combines with a specific E3(ubiquitin ligase)and transtransport ubiquitin to the corresponding substrate.The last step is the proteasome degradation process.Proteasome is a protein hydrolysate,both in the nucleus and cytoplasm.Proteasome is composed of three parts,the main unit is 20 S in the center of the empty columnar multienzyme nucleus.20 S consists of 4 rings,including 2 identical non catalytic subunit alpha rings,2 catalytic subunits beta rings,together to form an ????structure,and 20 S is responsible for the degradation of the ubstrate.The other 2 regulating units(19S)are the same,which are located at the 2 end of 20 S.19S consists of 2 parts: an outer cover,which identifies ubiquitin ubiquitin substrates and removes ubiquitin.The internal(basal)part of 19 S is responsible for the denaturation of related substrates through the action of 6 ATP subunits.During the final degradation process,the albumin was identified by 19 S and removed by 19 S to the polyubiquitin chain,then transferred to the 20 S hydrolysis center,and the target protein was eventually degraded into a short peptide structure of 10-12 amino acids.Borteal Zomi(PS-341)is the first line proteasome inhibitor approved by FAD.It is a boron-dipeptide structure that can reversibly inhibit the activity of chymotrypsin on 20 S and also slightly inhibit the activity of caspase on 20S(that is,bortezomizomi is a 20 S proteasome inhibitor).From the point of view of mechanics,bortezomib,a acidic substance,covalently formed the nucleophilic hydroxyl side chain of the beta subunit.Bortezomib was initially approved for newly diagnosed multiple myeloma,relapsed / refractory multiple myeloma and mantle cell lymphoma.Bortezomib has a variety of anti-tumor mechanisms,including: inhibiting the NF-k B signaling pathway,inhibiting the anti apoptotic target genes,inhibiting several anti apoptotic proteins(such as Bcl-XL,BCL 2 and STAT-3),downregulating the expression of DNA repair related proteins,inducing endoplasmic reticulum stress(ER)and apoptosis stimulating protein response(UPR).Bortezomib has a good chemotherapy effect and can overcome traditional resistance problems when combined with other drugs.Although bortezomib has achieved good therapeutic effect in the malignant tumor of the blood,but many patients relapse easily after the disease improves.At the same time,there are many cytotoxicity(such as neurotoxicity)after bortezomizomib treatment,and the phenomenon of drug resistance is also present.These factors have prompted the study to search for other proteasome inhibitors that can overcome bortezomib resistance,and have better performance,have smaller cytotoxicity,and also have a wider spectrum of anticancer antistomach.Subsequently,second generation of bortezomib derivatives with better performance: Carfilzomib were developed.In 2012,Carfilzomib was approved by FAD for the treatment for recurrent and refractory multiple myeloma.carfilzomib forms irreversible covalent bonds with he ?5 subunit thus inhibiting the chymotrypsin-like activity of 20 S.In addition to carfilzomib,several other second generation proteasome inhibitors have been developed and are now being actively tested in clinical studies:marizomib.Marizomib is a derivative of natural lactoin.Marizomib is a natural product derivative that resembles lactacystin.In contrast to bortezomib and carfilzomib,marizomib irreversibly binds all three enzymatically active subunits of the proteasome(?1,?2,and ?5),thus providing a durable and strong inhibition of both chymotrypsin-like and trypsin-like activities.It is worth mentioning that Marizomib can overcome the resistance of multiple myeloma and chronic lymphoma to bortezomib.Compared with bortezomib,this clinical feature of Marizomib may be attributed to its stronger specificity for Caspase-8 induced apoptosis.19 S is used to regulate granules.Its function is to remove ubiquitin from target proteins,and then transport target proteins to 20 S core particles for degradation.If the deubiquitination process of the protein is blocked,then the protein can not be degraded,because the narrow channel on the 20 S core particles does not allow the polyubiquitin protein to enter the catalytic site.Thus,the removal of ubiquitin inhibitors may interfere with proteasome activity.Deubiquitinase plays an important role in many diseases,such as inheritance cancer and neurodegenerative disease,which are involved in the process of DNA loss stress,DNA repair and transduction.De ubiquitination enzymes can be classified into 2 groups according to their structures: metalloproteinases and cysteine proteinases.All metalloproteinases(including POH1)contain the same ubiquitin protease domain JAMM.There are 4 types of cysteine: USP,UCH,OTU and MJD.The classification of these two enzymes reflects their specific affinity for the ubiquitin chain.Among them,the USP family(such as USP14)specifically recognizes the two glycine of the ubiquitin free hydroxyl terminus.Therefore,USP14 can only remove single ubiquitin,but not the whole ubiquitin chain of target protein.On the other hand,UCH37,which is a member of the UCH family,can specifically recognize two ubiquitin ubiquitin on the substrate.POH1 can excise the whole ubiquitination chain that stretches out of the protease substrate.Further studies have shown that knocking out POH1 will interfere with the assembly of nzyme bodies,while UCH37 or USP14 will only affect the degradation of protein substrates.This indicates that in the process of proteasome degrading proteins,POH1 enzymes are most important for deubiquitination,while UCH37 and USP14 play a more specific role.However,after double knockout of UCH37 and USP14,proteasome activity is also inhibited,which makes them candidates for drug candidates.Although no inhibitor of POH1 has been found,but a novel selective inhibitor of UCHL5 and USP14,b-AP15,has been developed recently.b-AP15 was initially found in the screening of small molecules,which can induce apoptosis in lysosomes,and this process does not depend on tumor suppressor inhibitor p53.This compound caused severe inhibition of the DUB activity against Ub-AMC substrate,but it does not affect the activity of 20 S hydrolyzed protein centers.In vivo studies have shown that b-AP15 can affect tumor progression.In addition,b-AP15 can also inhibit the increase in the growth of imatinib resistant multiple myeloma cells.At the same time,it also has a good effect in the treatment of multiple solid tumors.Because this small molecule inhibitor has made such a good effect and its clinical side effects are relatively small,researchers are looking for a deeper exploration to see if it can treat more diseases and,of course,to cure more diseases.To this end,the purpose of our study is to explore the role of b-AP15 in imatinib sensitive and imatinib resistant cells and to provide a theoretical basis for the application of b-AP15 in CML.Rerults 1.b-AP15 decreases viability of Bcr-Abl and Bcr-Abl T315 I cells.2.b-AP15 induces apoptosis in both Bcr-Abl-WT and Bcr-Abl T315 I cells.3.b-AP15 induces apoptosis is associated with caspase activation and decreased expression of anti-apoptotic protein in CML cells.4.b-AP15 suppressed proteasome function acompany with apoptosis in Bcr-Abl-WT and Bcr-Abl T315 I cells.5.b-AP15-induced proteasome inhibition is associated with ER-stress and cell apoptosis in CML cells.6.b-AP15 downregulates Bcr-Abl protein and inhibits its downstream signaling.7.b-AP15 inhibited the level of USP14 protein and reduced the level of beta-catenin,resulting in the decrease of Bcr-Abl and the death of CML cells.8.The effect of b-AP15 on primary monocytes from patients with CML in ex vivo,acompany with apoptosis and decreased viability.9.b-AP15 inhibits the growth of Bcr-Abl-WT and Bcr-Abl T315 I mutant xenografts in nude mice.Conclutions 1.b-AP15 inhibits viability and induces apototic in Bcr-Abl-WT and Bcr-Abl T315 I mutation cells.b-AP15 activate caspase signaling trigger cell death through downregulation of Bcr-Abl transcription and Bcr-Abl protein cleavage mediated by proteasome inhibition-induced caspase activation,meanwhilem,19 S proteasome-associated DUB inhibition plays an important role in the event.We here propose an alternative strategy to overcome IM resistance by enhancing Bcr-Abl downregulation,which should have great clinical significance in IM-resistant cancer therapy.2.b-AP15 inhibits USP14 and reduces the level of beta-catenin in the WNT pathway,resulting in the decrease of Bcr-Abl protein level and finally the cell death.3.b-AP15 suppressed the growth of Bcr-Abl-WT and Bcr-Abl T315 I xenografts in nude mice.