1. Study On The Effect Of 2B4-CD48 Interaction On NK Cell Apoptosis 2. Study On The Mechanism Of CCND2 Mutation Cooperating With AML1-ETO Fusion Gene In Inducing The Occurrence And Development Of Acute Myeloid Leukemi

Background:Decades after the identification of natural killer(NK)cells as potential effector cells against malignant transformed cells,an increasing number of researches suggest that NK cells are prospective lymphocytes for cancer immunotherapy.Previous researches indicated that in the context of hematopoietic stem cell transplantation(HSCT),donorderived alloreactive NK cells can effectively eliminate recipient tumor cells.Recent studies have led to a breakthrough in the use of HSCT in combination with allogeneic NK cells for the treatment of malignant tumors.However,the short lifespan of NK cells in patients is one of the factors affecting their efficacy.Therefore,efficient access to longer survival of NK cells will promote the application of NK cell immunotherapy.As we have known that NK cells utilize "missing-self" mechanism to lyse target cells and exert their functions through a wide array of activating,co-stimulatory and inhibitory receptors.Our previous study has suggested that CD244(2B4),one of the co-stimulatory receptors,could improve the function of chimeric antigen receptor(CAR)NK cells.However,the underlying mechanism of how 2B4 engaged in the function of NK cells needs to be further investigated.In addition,due to the NK cells’ natural antiviral function,their transduction efficiency is poor,and the transduction process influences their activity.So here we establish a feeder cell with the expression of CD48,the ligand of 2B4,to investigate the function of 2B4-CD48 axis in NK cells and,in the meantime,to explore whether a newly generated feeder cell can improve the function of ex vivo expanded NK cells.Methods:Firstly,K562 cells overexpressing 4-1BBL and membrane bound IL21(mbIL21)were constructed(K562-41BBL-mbIL21),and were sorted to generate the single clone.This widely-used feeder cells(K562-41BBL-mbIL21)were named as Basic Feeder hereinafter.Based on the Basic feeder,CD48 was overexpressed and named as CD48 Feeder.Then the genetically modified feeder cells were used to expand primary NK cells and the proliferation,apoptosis,phenotype and cytotoxicity of NK cells were evaluated.Results:Compared with Basic Feeders,CD48 Feeders can promote the proliferation of primary NK cells,whether from peripheral blood or umbilical cord blood,and reduce NK cell apoptosis by activating the p-ERK/BCL2 pathway both in vitro and in vivo without affecting overall phenotype.Furthermore,NK cells expanded via CD48 Feeders have stronger anti-tumor capability and infiltration ability into the tumor microenvironment.Conclusions:In this preclinical study,the engagement of the 2B4-CD48 axis can inhibit the apoptosis of NK cells through the p-ERK/BCL2 signal pathway,leading to an improvement in therapeutic efficiency.Background:The translocation between chromosome 8 and 21 resulting in the formation of AML1-ETO fusion gene is one of the most frequent cytogenetic abnormalities in acute myeloid leukemia(AML).AML1-ETO impairs myeloid differentiation and increases the selfrenewal of hematopoietic stem cells(HSCs).However,AML1-ETO alone is not sufficient to drive leukemia,secondary events are required for leukemogenesis.The spectrum of cooperating mutations varies in different cohorts.The common mutations such as KIT.NRAS,FLT3-ITD had been well studied.However,factors beyond these common mutated genes which cooperate to induce myeloid malignancies remain largely uncharacterized.In some cohorts,additional mutations such as CCND2 have also been reported.CCND2 gene,located on chromosome12,encodes the cyclin D2 cell cycle protein.It is a critical cell cycle regulator as well as a key member of the CyclinD2-CDK4/6 complex.CCND2 can phosphorylate retinoblastoma(RB)family of proteins including RBI.Phosphorylation of RBI allows the transcription factor E2F to dissociate from the RB/E2F complex,which is crucial for controlling GI/S transition in the cell cycle.In hematopoietic malignancies,CCND2 mutations(with a conserved mutated hotpot on Thr280)have been found in AML especially in core binding factor(CBF)leukemias.The mutated CCND2 protein is more stable and is resistant to proteasomal degradation.Furthermore.a previous study also identified that CCND2 is a transcriptional target of AML1-ETO,to be specific,AML1ETO can promote the CCND2 expression.But whether mutated CCND2 can cooperate with AML1-ETO fusion gene to drive leukemia initiation and progression hasn’t been studied.Methods:In our previous study,we established the conditional AML 1-ETO knock-in mouse model with Mx1-cre(hereafter called AML 1/ETO mice),the AML 1-ETO fusion gene was labeled with m-cherry tag.The coding sequences of CCND2wt and CCND2mut(Thr280AIa)were cloned into the retroviral vector MSCV-IRES-GFP respectively.Then the corresponding viruses were generated and used to infect the AML1/ETO mice fetal liver cells which were harvested from the embryos at embryonic day(E=12.5).AML1-ETO and CCND2wt or CCND2mut co-expressing cells were injected into lethally irradiated(9Gy)C57BL/6 mice through tail vein with supportive cells.All the mice were divided into three groups,which was named as Vector(MSCV-IRES-GFP,Vec hereinafter).CCND2wt(MSCV-IRES-CCND2wt-GFP)and CCND2mut(MSCV-IRES-CCND2mut-GFP),respectively.Then the onset of AML in three groups of mice were closely monitored.Results:The mice of CCND2wt and CCND2mut mice were succumbed to leukemia.And compared to Vec group,the mice in CCND2wt and CCND2mut group showed accelerated cell cycle.The mTOR pathway was enriched.Everolimus,the mTOR inhibitor,was able to prolong the survival of mice in some degree both in CCND2wt and CCND2mut groupConclusion:The CCND2 mutation can cooperate with AML1-ETO to drive leukemia progression as a second hit.