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All‐trans retinoic acid induces leukemia resistance to NK cell cytotoxicity by down‐regulating B7‐H6 expression via c‐Myc signaling

Abstract Background The interaction between activating receptor NKp30 and its major tumor ligand B7‐H6 is important for NK cell‐mediated tumor rejection. However, the regulation of B7‐H6 by tumor therapeutics remains largely unknown. In this study, we investigated the regulation of B7‐H6 by all‐trans retinoic acid (atRA), a terminal differentiation inducer of tumor cells that is extensively used for clinical leukemia therapy. Methods We investigated the role of NKp30:B7‐H6 axis in NK cell‐mediated tumor lysis against leukemia cells and the influence of atRA treatment on the cytotoxicity of NK cells using NK cell lines (NK92 and NKG) and leukemia cell lines (U‐937 and THP‐1). We evaluated the effect of atRA treatment on the expression of B7‐H6 using real‐time PCR, flow cytometry and western blotting. We used CRISPR/Cas9 to knockdown B7‐H6 expression and siRNA to knockdown c‐Myc in U‐937 cells to evaluate the role of B7‐H6 and c‐Myc in atRA‐induced tumor resistance against NK cells. Results NK cell‐mediated U‐937 cell lysis was mainly dependent on NKp30/B7‐H6 interaction. Blockade of B7‐H6 by monoclonal antibody significantly impaired NK cytotoxicity. atRA treatment induced U‐937 resistance to NK cell cytotoxicity by reducing B7‐H6 expression, and showed no effect on NK cytotoxicity against B7‐H6 knockdown U‐937 cells. Epigenetic modifications, such as DNA methylation and histone deacetylase (HDAC), were not responsible for atRA‐mediated B7‐H6 down‐regulation as inhibitors of these pathways could not restore B7‐H6 mRNA expression. On the other hand, atRA treatment reduced c‐Myc expression, which in turn inhibited the transcription of B7‐H6 on leukemia cells. Conclusion atRA treatment promotes tumor cell resistance against NK cell‐mediated lysis by down‐regulating B7‐H6 expression via the c‐Myc signaling pathway, suggesting that more attention needs to be paid to the immunological adverse effects in the clinical use of atRA treatment.

All-trans retinoic acid (atRA)-mediated differentiation therapy is the front-line therapy for acute promyelocytic leukemia (APL). It leads to complete remission rates >90% and long-term remission rates above 80% in combination with chemotherapy [25][26][27][28]. Due to numerous achievements in APL treatment, atRA is tested in non-APL acute myeloid leukemia (AML) and many solid tumors. However, clinical trials using atRA alone or in combination with chemotherapy have somehow shown disappointing results [28].
In this study, we investigated the regulation of B7-H6 expression by atRA treatment in leukemia cells and its influence on tumor resistence to NK cell lysis. We found that NKp30:B7-H6 axis plays an important role in NK cellmediated U-937 cell lysis, and atRA treatment impairs the cytotoxicity of NK cells against U-937 cells by reducing B7-H6 expression through the regulation of c-Myc. Our study revealed potential immunological adverse effects in the clinical use of atRA treatment.

Cell lines
NK cell line NKG was established and maintained in our labratory as previously described [29] and cultured in α-MEM (Thermo Fisher Scientific

Antibody generation and labeling
Mouse anti-human B7-H6 antibody (anti-B7-H6#1, anti-B7-H6#2 and anti-B7-H6#3) were generated and verified in our laboratory. Briefly, BALB/c mice were immunized with B7-H6-hIgG1-Fc fusion protein and spleen cells were fused with Sp/20 cells by Absea-antibody (Beijing, China). Hybridoma culture supernants were selected for the binding with HL60-B7-H6 cells. Antibodies derived from hybridoma cells were tested for their ability to detect B7-H6 as well as to inhibit NK cell-mediated cytotoxicity. Anti-B7-H6#1 and anti-B7-H6#2 were able to inhibit NK cell-mediated tumor lysis in 4 h cytotoxicity assay, and anti-B7-H6#3 was able to detect B7-H6 in western blotting assay. Anti-B7-H6#1 antibody was labeled with Andy Fluor 647 NHS Ester (ABP Biosciences, Rockville, MD, USA) according to Protein Labeling Protocol. 100 μg antibody in 90 μL PBS was added to 1 vial of Andy Fluor 647 NHS Ester and incubated for 30 min at room temperature in a dark space. 10 μL Quench buffer was added to stop labeling and residue flour was removed by centrifuge at 10,000 g, 4°C, using an ultra-filtration vial. The labeled antibody was stored at 1 mg/mL in PBS containing 0.5 mg/mL BSA and 0.02% NaN 3 (Sigma Aldrich, St. Louis, MO, USA) at 4°C.

siRNA and transfection
Small interfering RNA (siRNA) was used for transient knockdown of c-Myc. c-Myc siRNA (sc-29226) and con-trol siRNA (sc-37007) were purchased from the Santa Cruz Biotechnology (Santa Cruz, CA, USA) and used at a final concentration of 100 nmol/L. The cells were transfected using Amaxa R Cell Line Nucleofector R Kit C (VCA-1004, Lonza, Basel, Switzerland) according to the manufacturer's instructions. Transfection efficiency was determined by real-time PCR, flow cytometry and western blot.

Statistical analysis
Statistical analysis and figure creation were performed using GraphPad Prism 6.0 (La Jolla, CA, USA) and SPSS Statistics 21 (IBM, Armonk, NY, USA). Comparisons between two groups were analyzed using the unpaired ttest. Comparisons between three or more groups were analyzed using either the one-way or two-way ANOVA test.
Results are expressed as means ± SEM. Values of P <0.05 were considered statistically significant.

atRA treatment promotes tumor cell resistance against NK cell cytotoxicity
To define the influence of atRA treatment on NK cell cytotoxicity against tumor cells, we treated U-937 and THP-1 cells with 10 μmol/L atRA for 24 h and tested their sensitivity to NK cells in a 4-hour cytotoxicity assay. Lysis of U-937 cells mediated by NKG cells was significantly impaired by atRA treatment at E:T ratios of 8:1 and 16:1 (P <0.001; Figure 2A), while lysis of U-937 cells mediated by NK92 cells was significantly impaired by atRA treatment at all the E:T ratios tested (all P <0.05; Figure 2B). Furthermore, atRA treatment significantly impaired the lysis of THP-1 cells mediated by NK92 cells at E:T ratios of 2:1 and 8:1 (P < 0.05; Supplementary Figure S2). Taking together, these data suggest that atRA treatment promoted tumor resistance against NK cell cytotoxicity.

F I G U R E 1 Blockade of B7-H6 significantly impairs the cytotoxicity of NK cells against U-937 cells. Lysis of U-937 cells by NKG (A) or NK92 cells (B) was tested in a 4-hour cytotoxicity assay. 5
μg/mL mouse anti-human B7-H6 antibodies (anti-B7-H6#1 and anti-B7-H6#2) or control antibodies (mIgG) was added to the culture medium. Data were analyzed for significance by the one-way ANOVA test. Results are representative of at least three independent experiments with a minimum of two technical replicates and expressed as the mean±SEM. E:T, effector/target; ****, P <0.0001.

atRA treatment down-regulates B7-H6 expression in U-937 and THP-1 cells
To explore the influence of atRA treatment on B7-H6 expression, we treated U-937 and THP-1 cells with 10 μmol/L atRA for 24 h respectively, and analyzed B7-H6 cell-surface expression by flow cytometry. atRA treatment significantly down-regulated B7-H6 cell-surface expression in both U-937 and THP-1 cells (P <0.0001; Figure 3A and B). To explore whether atRA treatment downregulates B7-H6 at the transcriptional level, tumor cells were treated with 10 μmol/L atRA and subjected to analysis by real-time PCR. 24 h and 48 h of atRA treatment both resulted in the reduced B7-H6 mRNA level in U-937 cells (P <0.0001; Figure 4A). We further treated U-937 cells with various concentrations (0.039, 0.156, 0.625, 2.5, and 10 μmol/L) of atRA for 24 h and found that atRA downregulated B7-H6 mRNA level in a dose-dependent manner ( Figure 4B). The influence of atRA treatment on THP-1 cells was also investigated, 2.5 or 10 μmol/L atRA treatment significantly reduced B7-H6 mRNA level in THP-1 cells F I G U R E 2 atRA induces U-937 cell resistance against NK cell cytotoxicity. U-937 cells were treated with 10 μmol/L atRA for 24 h or left untreated (Control) and subjected to NKG (A) or NK92 (B)mediated lysis in a 4-hour cytotoxicity assay. Data were analyzed for significance by the unpaired t-test. Results are representative of at least three independent experiments with a minimum of two technical replicates and expressed as the mean±SEM. E:T, effector/target; *, P <0.05; ***, P <0.001; ns, not significant.
( Figure 4C). However, atRA treatment showed no negative effect on B7-H6 mRNA level in human immortalized myelogenous leukemia cell line K562 (Supplementary Figure  3A). Taking together, these data suggest that atRA treatment reduced B7-H6 expression in both U-937 and THP-1 cells.

atRA treatment promotes tumor cell resistance to NK cell-mediated cytotoxicity by reducing B7-H6 expression
To explore and verify the underlying mechanism, we generated B7-H6 knockdown U-937 cells (U-937-δB7H6) by transducing U-937 cells with Crispr/Cas9 plasmid expressing B7-H6 sgRNA ( Figure 5A). U-937 and U-937-δB7H6 cells were then treated with 10 μmol/L atRA for 24 h and subjected to NK92 mediated lysis in a 4-hour cytotoxicity assay. The cytotoxicity of NK92 cells against U-937-δB7H6 cells was lower than that against U-937 cells ( Figure 5B and C). Furthermore, the use of atRA treatment impaired NK92 cytotoxicity against U-937 cells, however, it showed no effect on the lysis of U-937-δB7H6 cells ( Figure 5B and C), suggesting that atRA treatment promoted tumor cell resistance to NK cell-mediated cytotoxicity by regulating B7-H6 expression.

atRA-mediated B7-H6 down-regulation does not depend on DNA methylation or HDAC
atRA has been reported to regulate gene expression through epigenetic modifications such as DNA methylation and HDAC [28,[30][31][32]. To investigate whether atRA-mediates B7-H6 down-regulation relies on epigenetic modifications, we treated U-937 cells with DNA methylation inhibitors and HDAC inhibitor, and then analyzed B7-H6 mRNA by real-time PCR. DNA methylation inhibitors, including Azacitidine, Zebularine, and RG-108, inhibit DNA methylation through different mechanisms and therefore were all tested. None of these inhibitors restored B7-H6 mRNA level which was suppressed by atRA treatment (Figure 6A-C), suggesting that down-regulation of B7-H6 mRNA by atRA treatment was not dependent on DNA methylation.
To study whether HDAC is involved, U-937 cells were treated with HDAC inhibitor Romidepsin. Romidepsin alone reduced B7-H6 mRNA level (data not shown), consistent with the report that HDAC inhibitors (suberoylanilide hydroxamic acid, trichostatin A, and valproic acid) down-regulate B7-H6 transcription in HeLa cells [15]. The combination of atRA and Romidepsin further reduced B7-H6 mRNA level compared with atRA treatment alone ( Figure 6D). Together, these data suggest that the downregulation of B7-H6 by atRA treatment was neither dependent on DNA methylation nor HDAC.

atRA inhibits B7-H6 transcription by down-regulating c-Myc
It was recently reported that the oncogenic transcription factor c-Myc drives B7-H6 transcription in tumor cells [24,33]. We further investigated whether the transcriptional factor c-Myc was involved in B7-H6 transcriptional regulation in leukemia cell lines. We transfected U-937 cells with c-Myc siRNA and then analyzed B7-H6 mRNA expression. As shown in Figure 7A and B, c-Myc knockdown significantly reduced B7-H6 mRNA and protein levels (P <0.0001). To study whether atRA down-regulates c-Myc expression, we treated U-937 cells with 10 μmol/L atRA for 24 h and 48 h respectively, and then analyzed c-Myc mRNA level by real-time PCR. Both 24 h and 48 h of atRA treatment significantly reduced c-Myc level in U-937 cells (P <0.0001; Figure 7C). However, atRA treatment showed no effect on the c-Myc mRNA level in K562 cells (Supplementary Figure S3B). Furthermore, atRA treatment significantly reduced c-Myc protein expression in U-937 cells ( Figure 7D). THP-1 cells were treated with either 2.5 μmol/L or 10 μmol/L atRA for 24 h and c-Myc mRNA expression were evaluated by real-time PCR. Both 2.5 μmol/L and 10 μmol/L atRA treatment significantly reduced c-Myc mRNA in THP-1 cells ( Figure 7E). Taking together, these data suggest that atRA treatment downregulated B7-H6 transcription possibly through inhibiting the c-Myc signaling pathway.

DISCUSSION
The cytotoxicity of NK cells depends on the balance between activating and inhibitory signals. The natural cytotoxicity receptors (NCRs) and NKG2D are the major activating receptors involved in tumor cell surveillance F I G U R E 5 atRA shows no effect on the cytotoxicity of NK cells against B7-H6 knockdown U-937 cells. A. B7-H6 was knockdown in U-937 cells (U-937-δB7H6) by transducing the cells with Crispr/Cas9 plasmid expressing B7-H6 sgRNA. B7-H6 cell-surface expression of U-937 and U-937-δB7H6 cells was verified through flow cytometry. B-C. U-937 (B) and U-937-δB7H6 (C) cells were treated with 10 μmol/L atRA for 24 h or left untreated (Control) and subjected to NK92 mediated lysis in a 4-hour cytotoxicity assay. Data were analyzed for significance by the unpaired t test. Results are representative of at least three independent experiments with a minimum of two technical replicates and are expressed as the mean±SEM. **, P <0.01; ***, P <0.001; ns, not significant. [14]. The interaction between NKp30 and tumor cellderived B7-H6 results in the production of cytotoxic mediators, such as perforins, granzymes, TNF-α, and interferon-γ (IFN-γ) [34]. Our study showed that NK cellmediated lysis of tumor cells (U-937 and THP-1) was mainly dependent on NKp30/B7-H6 interaction and atRA treatment induced U-937 and THP-1 resistance against NK cell cytotoxicity by reducing the expression of NKp30 ligand B7-H6. It has been reported that transcriptional factor c-Myc is responsible for driving B7-H6 expression in tumor cells, our study showed that atRA treatment down-regulated B7-H6 transcription by reducing c-Myc expression.
The epigenetic modifications by atRA have been deeply investigated. The receptor of atRA, retinoic acid receptor (RAR), forms a heterodimer with retinoic X receptor (RXR) and binds to the RA response element (RARE) of the promoter region [35]. In the absence of ligand binding, RAR/RXR forms a complex with co-repressors including epigenetic modifiers that mediate DNA methylation or HDACs and suppress gene expression [28,32,31]. However, our data indicate that atRA-mediated B7-H6 downregulation was neither dependent on DNA methylation nor DHAC.
c-Myc reduction is one of the hallmarks of atRA-induced terminal differentiation [36]. However, c-Myc is not F I G U R E 6 atRA-mediated B7-H6 down-regulation is neither dependent on DNA methylation nor HDAC. U-937 cells were treated with 10 μmol/L atRA, 10 μmol/L atRA in combination with various concentrations of Azacitidine (A), Zebularine (B), RG-108 (C), or Romidepsin (D) for 24 h. B7-H6 mRNA expression was analyzed by real-time PCR. U-937 cells treated with 10 μmol/L atRA or left untreated (Control) were used as control. Data were analyzed for significance by the two-way ANOVA test. Results are representative of at least three independent experiments with a minimum of two technical replicates and are expressed as the mean±SEM. ****, P <0.0001; ns, not significant. down-regulated in atRA-resistant tumors upon atRA treatment. It has been reported that atRA alone does not lead to c-Myc reduction in solid tumor cells, such as MCF-7, while the combination of atRA and type I IFN leads to c-Myc down-regulation [33,37]. As c-Myc drives the B7-H6 transcription in tumor cells and c-Myc mRNA level is not affected by atRA treatment in K562 cells, atRA resistance explains the unaffected B7-H6 expression in tumor cell line K562.
In conclusion, we found that lysis of U-937 cells by NK92 or NKG cells was largely dependent on NKp30/B7-H6 interaction, blockade of B7-H6 by mAb significantly impaired NK cytotoxicity. atRA treatment impaired U-937 lysis by NK92 or NKG cells and reduced B7-H6 expression in U-937 and THP-1 cells on both protein and mRNA levels. Furthermore, atRA treatment showed no effect on NK cytotoxicity against B7-H6 knockdown U-937 cells. Epigenetic modifications such as DNA methylation and HDAC were not responsible for atRA-mediated down-regulation of B7-H6 expression, as inhibitors of these pathways could not restore B7-H6 expression. On the other hand, atRA treatment reduced c-Myc expression, the transcriptional factor driving B7-H6 expression in tumor cells, and therefore inhibited the transcription of B7-H6 in leukemia cells. Our study suggests that atRA impaired NK cell-mediated tumor rejection through down-regulating B7-H6 expression via the c-Myc signaling pathway.

D E C L A R AT I O N S E T H I C S A P P R O VA L A N D C O N S E N T T O PA R T I C I PAT E
Not applicable.

C O N S E N T F O R P U B L I C AT I O N
Not applicable.

AVA I L A B I L I T Y O F D ATA A N D M AT E R I A L S
The data that support the findings of this study are available from the corresponding author upon reasonable request.

A C K N O W L E D G M E N T S
Not applicable.

C O M P E T I N G I N T E R E S T S
The authors declare that they have no competing interests.

A U T H O R S ' C O N T R I B U T I O N S
GC and RS designed the study. GC and YC performed the experiments and analyzed the data. GC and HS wrote and revised the manuscript. ZT, HW, RS, and XZ supervised the study. RS and HS critically reviewed the manuscript. All authors read and approved the final manuscript.