The Mechanism Of HO1 Overexpression Induces Immune Evasion Of Acute Myeloid Leukemia Against NK Cells By Inhibiting CD48

Background:Acute myeloid leukemia(AML)is a malignant hematopoietic stem/progenitor cell disease with abnormal differentiation of myeloid primordial immature cells,in which the treatment of relapsed/refractory AML is still a challenge.Our group previously demonstrated that heme oxygenase 1(HO1)is overexpressed in AML,while the functional roles of HO1 remain unclear.Objective:The purpose of this study was to explore the relationships between HO1 expression and the AML immune microenvironment,and to elucidate the detailed molecular mechanism of HO1 overexpression induces immune evasion of AML against NK cells by inhibiting CD48.Methods:1.Clinical sample collection.According to the “Chinese guidelines for the diagnosis and treatment of adult acute myeloid leukemia(not APL)(2021)” and “Chinese guidelines for the diagnosis and treatment of relapsed/refractory acute myelogenous leukemia(2021)”,newly diagnosed and relapsed AML patients were included as the subject of research.Fresh bone marrow blood of patients was collected prior to treatment.Mononuclear cells were purified with Ficoll-Hypaque density centrifugation and then cultured using the Stem Span Leukemic Cell Culture Kit.2.The correlation between HO1 expression and the level of NK cells in the AML immune microenvironment was analyzed.The Tumor Immune Estimate Resource(TIMER)database was used to evaluate the connection between HO1 expression and the AML immune microenvironment;The HO1 expression difference between normal tissues and AML was analyzed via The Cancer Genome Atlas(TCGA)and GenotypeTissue Expression(GTEx)databases;Next,flow cytometry was conducted to assess the association between HO1 levels with four main immune cells(CD4+ T,B,CD8+T,and NK cells)in AML patients to validate the results from bioinformatics databases above.Patients were assigned to two groups based on HO1 expression levels based on flow cytometry results;To explain the underlying mechanism,the associations between HO1 expression and immune checkpoint/ligand genes in AML was analyzed by TCGA database;Then flow cytometry was performed to confirm the findings from TCGA analysis.3.Further analysis in cell lines was conducted to explore how the HO1 in AML cells affects NK cells.HO1 expression in leukemia cell lines was determined by real-time PCR and western blot;Then transduction of cells with HO1 overexpression lentivirus(LV-HO1)upregulated HO1 expression in THP-1,whereas transduction of MV4-11 with HO1 silence lentivirus LV-HO1-RNAi(Si-HO1)downregulated HO1 expression.The transduction ratio was verified by real-time PCR and western blot;The survival of transduced AML cells under the cytotoxic effects of NK cells was detected by the bioluminescent imaging system;The cytotoxic ability of NK cells against the transduced AML cells was detected by LDH release assay;The degranulation(CD107a expression)and activation(CD69 expression)of NK cells against the transduced AML cells was detected by flow cytometry.4.Male 6-8-week-old NOD/SCID IL‐2Rγ null(NPG)mice were selected as the subject of research;By subcutaneous injection of THP-1 cells stably transduced with HO1 lentivirus into the back of mice,the mice were divided into four groups: EV1 group,LV-HO1 group,EV1+NK cells group,and LV-HO1+NK cells group;Mice were treated with NK cells once the tumors were visible or palpable,through injections via the tail vein every three days for a total of 9 treatments;Mice were placed on the InVivo Imaging System platform for observation;Tumors diameter and weights were determined every three days;The survival time of mice was recorded.5.The mechanisms by which HO1 downregulates CD48 in AML cells were analyzed.Western blot was used to determine the acetylation levels after overexpressing HO1 in AML cells;HDAC4/HDAC8/Sirt1 expression levels in HO1 high/low AML specimens as detected by real-time PCR and western blot;Flow cytometry analysis of CD48 expression in THP-1 cells after treated with SRT1720;Flow cytometry analysis of CD48 expression in MV4-11 cells after treated with selisistat;Sirt1 and CD48 expression profiles in THP-1 and MV4-11 transduced cells were detected by real-time PCR,western blot and flow cytometry;Co-immunoprecipitation evaluation of interactions between HO1 and Sirt1 in AML cells.Results:1.In the present study,a significant positive correlation was found between HO1 gene levels and: ESTIMATE(r=0.788),immune(r=0.763),as well as stromal(r=0.689)scores in acute myeloid leukemia(LAML).HO1 expression was significantly higher in AML than in normal tissues.Next,flow cytometry was conducted in AML patients to validate the results from the above findings.Our results showed that the number of NK cells in the HO1-high group was significantly lower than that in the HO1-low group.Further studies showed that HO1 expression was significantly correlated with CTLA4,CD48,CD200R1(CD200R),HAVCR2(CD366),PDCD1LG2(CD273),TNFRSF8(CD30),VSIR(VISTA),CD40,CD86 and TNFRSF9(CD137)in AML.Then flow cytometry was performed in AML patients to confirm the findings from the TCGA analysis.Our results indicated that the HO1-high group exhibited suppressed CD48 levels in tumor cells relative to the HO1-low group.2.On the one hand,overexpression of HO1 protected AML cells from the cytotoxic effects of NK cells,while silencing HO1 in AML cells increased the sensitivity of AML cells to the killing effects of NK cells.On the other hand,in the co-culture system,AML cells overexpressing HO1 inhibited the killing activity of NK cells,while silencing HO1 in AML cells increased the killing activity of NK cells.Besides,CD69 and CD107 a expression of NK cells in the LV-HO1 group were markedly reduced relative to NK cells in the EV1 group.The addition of CD48 to cells in the LV-HO1 group partly upregulated NK cell CD69 and CD107 a expression.On the contrary,knockdown of HO1 in MV4-11 cells upregulated CD69 and CD107 a expression of NK cells in the group.Moreover,blocking 2B4 inhibited CD69 and CD107 a expression of NK cells in the Si-HO1 group to some extent.3.In the AML mice model,HO1 overexpression caused an increase in tumor proliferation relative to the EV1 group.Furthermore,HO1 overexpression effectively promoted an increase in tumor volume,tumor weight,and cell proliferation compared with the EV1 group.Notably,treatment with NK cells caused a significant decrease in tumor burden.Analysis of the four groups’ survival data revealed that the “EV1+NK cells” group exhibited longer survival,whereas the other three groups had a similar and shorter survival time.4.A low pan-acetylation level was observed in THP-1 cells overexpressing HO1.Further studies showed that Sirt1 was increased in HO1-high expressing AML patients in protein levels.Next,a series of experiments was conducted in AML cells to validate the results from the above findings.The results indicated that SRT1720 significantly downregulated the expression of CD48 in THP-1,whereas selisistat upregulated CD48 expression in MV4-11.In THP-1 cells,HO1 overexpression significantly increased Sirt1 protein levels and downregulated CD48 m RNA/protein expression.Notably,the addition of selisistat rescued CD48 expression in cells expressing LV-HO1 to some extent by weakening the negative regulation of CD48 by HO1.Sirt1 protein level was downregulated,whereas CD48 m RNA/protein expression was upregulated by the downregulation of HO1 in MV4-11 cells.The addition of SRT1720 decreased CD48 expression in Si-HO1 by restoring the negative regulation of CD48 by HO1.Mechanically,HO1 could form a complex with endogenous Sirt1 in THP-1 and MV4-11 cells.Conclusions:1.HO1 expression is correlated with the immune microenvironment of AML.And targeting HO1 is a potential immunotherapy option for AML.In AML patients,the number of NK cells in the HO1-high group was significantly lower than that in the HO1-low group.Furthermore,the HO1-high group exhibited suppressed CD48 levels in tumor cells relative to the HO1-low group.2.Cell lines in vitro: Overexpression of HO1 in AML cells inhibits NK cell cytotoxicity via targeting the CD48-2B4 axis,thereby inducing the immune evasion of AML cells against NK cells.Animals in vivo: HO1 overexpression promoted AML cell growth and inhibited the cytotoxic activity of NK cells in the AML mice model,thereby inducing the immune evasion of AML cells against NK cells in vivo.3.Mechanically,HO1 directly interacted with Sirt1 and increased its expression in AML cells,enabling histone deacetylation to suppress CD48 transcription and expression,finally inducing the immune evasion of AML cells against NK cells.