(2014) 33, 2700–2708 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc

ORIGINAL ARTICLE Coordinated regulation of the subunits by PML/RARa and PU.1 in acute promyelocytic leukemia

X-W Yang1,2, P Wang1,2, J-Q Liu1,2, H Zhang1, W-D Xi1, X-H Jia1 and K-K Wang1

Recognition and elimination of malignant cells by cytotoxic T lymphocytes depends on antigenic peptides generated by . It has been established that impairment of the immunoproteasome subunits, that is, PSMB8, PSMB9 and PSMB10 (PSMBs), is critical for malignant cells to escape immune recognition. We report here the regulatory mechanism of the repression of PU.1-dependent activation of PSMBs by PML/RARa in the pathogenesis of acute promyelocytic leukemia (APL) and the unidentified function of all-trans retinoic acid (ATRA) as an immunomodulator in the treatment of APL. Chromatin immunoprecipitation and luciferase reporter assays showed that PU.1 directly bound to and coordinately transactivated the promoters of PSMBs, indicating that PSMBs were transcriptional targets of PU.1 and PU.1 regulated their basal expression. Analysis of expression profiling data from a large population of acute myeloid leukemia (AML) patients revealed that the expression levels of PSMBs were significantly lower in APL patients than in non-APL AML patients. Further evidence demonstrated that the decrease in their expression was achieved through PML/RARa-mediated repression of both PU.1-dependent transactivation and PU.1 expression. Moreover, ATRA but not arsenic trioxide induced the expression of PSMBs in APL cells, indicating that ATRA treatment might activate the - processing/presentation machinery. Finally, the above observations were confirmed in primary APL samples. Collectively, our data demonstrate that PML/RARa suppresses PU.1-dependent activation of the immunosubunits, which may facilitate the escape of APL cells from immune surveillance in leukemia development, and ATRA treatment is able to reactivate their expression, which would promote more efficient T-cell-mediated recognition in the treatment.

Oncogene (2014) 33, 2700–2708; doi:10.1038/onc.2013.224; published online 17 June 2013 Keywords: immunoproteasome subunits; PML/RARa; PU.1; APL; ATRA

INTRODUCTION genomic binding sites in comparison to wild-type RARa. The Acute promyelocytic leukemia (APL), a subtype of acute myeloid expanded genomic landscape of PML/RARa impacts on the leukemia (AML), is characterized by a typical t(15;17)(q22;q21) regulation of involved in a variety of cellular functions, translocation occurring in almost all of the patients. The including differentiation, proliferation, , translocation results in a rearrangement between the promyelo- progression and – .12 One of the cytic leukemia (PML) and the retinoic acid receptor a (RARa) functions potentially affected, based on the exploration of verified gene, generating the PML/RARa fusion capable of blocking PML/RARa binding sites, is that of the immunoproteasome. the differentiation at the promyelocytic stage.1,2 PML/RARa harbors The presentation of antigenic peptides to cytotoxic T lymphocytes the DNA-binding domain of RARa and recruits corepressor (CTLs) is a crucial prerequisite for successful immune recognition molecules with a high affinity,3 thus acting as a strong and elimination of transformed cells.13 Generally, -g transcriptional repressor for its target genes.4 Clinically, APL has induces the expression of the unique catalytic immunosubunits, progressed from a highly fatal disease to a highly curable one with including PSMB8, PSMB9 and PSMB10.14 The replacement of the the introduction of all-trans retinoic acid (ATRA) and arsenic constitutive subunits by these inducible immunosubunits leads to trioxide (As2O3) in its treatment. As both drugs act by promoting the formation of the immunoproteasome, which is responsible for the degradation of PML/RARa,5,6 the combination of ATRA plus promoting the generation of antigenic peptides and enhancing the 15 As2O3 leads to durable remission of APL. Interestingly, ATRA exerts recognition of target cells by CTLs. However, malignant cells are its effects on APL cells mainly at the transcriptome level, whereas predominantly characterized by the abnormal expression of 7 16 As2O3 exercises its impact mainly at the proteome level. Moreover, immunosubunits, which may contribute to the failure of PML/RARa can interact with other factors such as AP- the immune system to recognize these cells. In APL, several lines 1,8 PU.19 and GATA-2,10 and target genes that are primarily of evidence have demonstrated that human leukocyte regulated by these transcription factors, hence further expanding (HLAs) appear undetectable on the surface of APL cells,17,18 the number of PML/RARa target genes. In addition, PML/RARa suggesting a potential mechanism for these cells to escape exists in multiple complexes, including homodimers, oligomers immune detection. Furthermore, another possible way in which and heterodimers,11 which have been shown to cause a more APL cells avoid the immune surveillance is probably associated with flexible DNA-binding specificity and create numerous additional the disruption of normal PML function. As wild-type PML is

1State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai, China. Correspondence: Professor K-K Wang, State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine (SJTU-SM), 197 Ruijin Road II, Shanghai 200025, China. E-mail: [email protected] 2These authors contributed equally to this work. Received 23 October 2012; revised 21 March 2013; accepted 8 April 2013; published online 17 June 2013 Regulatory mechanism of the immunosubunits in APL X-W Yang et al 2701 involved in the expression of that has important roles in PML/RARa targets. To verify this finding, we performed chromatin antigen presentation,17 cells with disrupted PML function would be immunoprecipitation quantitative real-time PCR (ChIP-qPCR) assays predicted to be defective in antigen processing and presentation. in APL-derived NB4 cells using primers encompassing the peak Moreover, studies have reported that ATRA can function as an regions of the three genes and antibodies against PML and RARa.As immune modulator via triggering the expression of genes expected, an enrichment of PML/RARa binding was observed at all associated with antigen processing and presentation.19 More promoter regions of PSMBs (Figure 1b), consistent with the results importantly, ATRA treatment can also reactivate the obtained from the ChIP-sequencing analysis. The degree of immunogenicity of APL cells;17,20,21 this is particularly so during enrichment differed among these promoters: ChIP enrichment was ATRA maintenance therapy,21 implicating that restoration of antigen strongest for PSMB8, followed by PSMB10, whereas moderate presentation by ATRA may be essential for effective immune enrichment was observed for PSMB9, indicating that the binding responses in APL treatment. The above observations indicate that affinity might vary among these genes. As PML/RARa selectively the loss of expression of genes involved in antigen presentation targets PU.1-regulated genes,9 we performed ChIP-qPCR assays in contributes to the immune escape of APL cells and effective NB4 cells to determine whether PU.1 binds to the promoters of treatment strategies may enhance APL-specific immune responses. PSMBs in vivo. The chromatin DNA immunoprecipitated by anti-PU.1 While reduced expression of HLAs and impaired PML function antibody was amplified with the same primer sets used in the PML explain, in part, the immune escape of APL cells, there are other and RARa immunoprecipitates. As illustrated in Figure 1c, PU.1 pathways that may also be critically affected. binding was enriched at the same regions bound by PML/RARa.We In this study, we report that PU.1 directly regulates the basal also observed that the enrichment of PU.1 binding was in the order expression of the immunoproteasome subunits PSMBs and that PSMB84PSMB104PSMB9.Takentogether,thedatasuggestthat PML/RARa represses the expression of these subunits in APL. The both PML/RARa andPU.1bindtothepromotersofthe repression is through interfering with PU.1-dependent transacti- immunosubunit genes in APL cells. vation and reducing the PU.1 expression in APL. Furthermore, treatment with ATRA restores the expression of PSMBs and induces the activity of the immunoproteasome. This study PU.1 regulates the basal expression of PSMBs in hematopoietic describes a critical role of PU.1 in the expression of the cells immunoproteasome subunits and suggests that the downregula- Next, we analyzed the nucleotide sequences around the promoter tion of these subunits by PML/RARa is an additional mechanism regions of PSMBs using the TRANSFAC software (Biobase, Braunsch- by which APL cells escape immune surveillance. weig, Germany). As shown in Figure 2a, each of the PSMB promoters harbored at least one putative PU.1 binding site and multiple retinoic acid response element (RARE) half-sites adjacent to the peak center positions in the PML/RARa- and PU.1-enriched ChIP regions. RESULTS The data support the above results that both PU.1 and PML/RARa Both PML/RARa and PU.1 bind to the promoter regions of PSMBs bind to the promoters of PSMBs (Figures 1b and c) and also raise the in APL cells possibility that PU.1 and PML/RARa can coregulate the expression of The development of usually requires that malignant cells PSMBs. To determine whether the promoters of the immunosubunit cannot be effectively recognized by the immune system.16 To genes are primarily targeted by PU.1 or, alternatively, PU.1 is identify genes potentially participating in the immune escape of recruited to these promoters by PML/RARa,weperformedtwo APL cells, we screened the PML/RARa targets that we previously panels of ChIP-qPCR assays in PR9, a PML/RARa-inducible cell line.2 identified from genome-wide studies.9 Interestingly, we observed Chromatin DNA was immunoprecipitated with specific antibodies that PML/RARa binding was enriched in the promoter regions of against PU.1, PML and RARa before and after PML/RARa all three genes encoding the immunosubunits, that is, PSMB8, induction. As shown in Figure 2b, PU.1 binding was enriched at PSMB9 and PSMB10 (Figure 1a), indicating that they were putative the promoters of PSMBs, regardless of the presence or absence

Figure 1. Both PML/RARa and PU.1 bind to the promoters of PSMB8, PSMB9 and PSMB10 in APL cells. (a) Schematic diagram showing the binding of PML/RARa to the promoter regions of PSMBs. ChIP assays were performed in the PML/RARa-inducible PR9 cells using anti-RARa and anti-PML antibodies. The peaks represent the PML/RARa-enriched ChIP regions. PSMB9 is located on the Watson ( þ ) strand of 6, and PSMB8 and PSMB10 are located on the Crick ( À ) strand of 6 and 16, respectively. (b) and (c) PML/RARa (b) and PU.1 (c) bind to the promoters of PSMBs in NB4 cells. ChIP was performed in NB4 cells using anti-RARa, anti-PML, anti-PU.1 or nonspecific (normal immunoglobulin G (IgG)) antibodies. ChIP-qPCR was performed with primers specific for the promoter regions of PSMBs or with negative primers specific for a non-relevant region (NC). Total DNA or chromatin DNA immunoprecipitated with different antibodies was used for PCR amplification. The results are presented as fold enrichment of chromatin normalized to nonspecific IgG. Data represent the mean of three replicates±s.d.

& 2014 Macmillan Publishers Limited Oncogene (2014) 2700 – 2708 Regulatory mechanism of the immunosubunits in APL X-W Yang et al 2702

Figure 2. PU.1 regulates the basal expression of PSMB8, PSMB9 and PSMB10 by targeting the promoter regions of these genes. (a) Schematic representation of the promoter regions of PSMBs. PU.1 binding sites (oval) and RARE half-sites (rectangle) are defined using TRANSFAC with the core and matrix similarity. (b) PML/RARa binds to the promoter regions of PSMBs preoccupied by PU.1. ChIP-qPCR was performed in PR9 cells with or without ZnSO4 treatment using anti-PU.1, anti-RARa and anti-PML antibodies. (c) The promoters of PSMBs are transactivated by PU.1 in a dose-dependent manner. The promoters of PSMBs were individually transfected into 293T cells along with increasing amounts of the PU.1 expression construct. (d) The luciferase activities of the PU.1 site mutant constructs decrease compared with the corresponding wild-type (WT) constructs in U937 cells. Data represent the mean of three replicates±s.d. *Po0.05; **Po0.01, compared to the value of the PSMBs(WT) constructs.

of PML/RARa (left panel), whereas PML/RARa binding was only expression was inversely correlated with PML/RARa expression. enriched after PML/RARa induction (middle and right panels), Next, we performed luciferase reporter assays on U937 cells in the indicating that PU.1 primarily bound to these promoters. In addition, presence and absence of PML/RARa and found that the relative we found that the degree of enrichment among the three luciferase activities of PSMBs decreased after co-transfection of promoters was consistent with the data from NB4 cells. PML/RARa into U937 cells (Figure 3b), demonstrating that PML/ The above data suggest that PSMBs may be initially bound and RARa repressed promoter activities of the immunosubunit genes. coordinately regulated by PU.1. Next, to investigate whether PU.1 transactivates the promoters of PSMBs, we conducted promoter– Downregulation of PSMBs in APL is achieved through the reporter assays in 293T cells, a non-hematopoietic cell line that repression of PU.1-dependent transactivation of PSMBs and the does not express endogenous PU.1. After co-transfecting increas- reduced PU.1 expression by PML/RARa ing amounts of the PU.1 expression construct, we found that the As the expression of PSMBs was primarily bound and regulated by three promoters were transactivated by PU.1 in a dose-dependent PU.1 (Figures 2b and c), we hypothesized that the reduced manner (Figure 2c), demonstrating that PU.1 directly regulated the expression of PSMBs in APL cells might be mediated by PU.1. To transcription of PSMBs. To further elucidate the role of the PU.1 test this hypothesis, we conducted luciferase reporter assays on sites in the transcriptional regulation of the three promoters in 293T cells to evaluate promoter activities of PSMBs in the presence hematopoietic cells, we mutated these PU.1 sites one by one and and absence of PML/RARa and PU.1. As shown in Figure 4a, PML/ compared promoter activities of these mutants with their RARa alone caused minor/minimal changes in the luciferase corresponding wild-type promoters in U937, which endogenously activities of these promoters, whereas PU.1 significantly enhanced expresses PU.1. As illustrated in Figure 2d, the mutation at the luciferase activities, revealing a PU.1-dependent transactivation of second PU.1 site ( À 1to þ 7 bp) severely impaired promoter the promoters of PSMBs. More importantly, co-transfection of activity of PSMB8, while the mutation at the first PU.1 site ( À 476 PML/RARa effectively suppressed PU.1-dependent transactivation, to À 468 bp) exhibited only moderate effects on promoter indicating that PML/RARa functioned as an effective repressor activity. When both PU.1 sites were mutated, promoter activity only under the presence of PU.1. In addition, as PU.1 expression was abolished, indicating that both sites in the PSMB8 promoter itself can be reduced by PML/RARa in APL cells,9,12,22 we were required for PU.1-dependent transactivation. Similarly, we speculated that the low PU.1 expression in APL might also also observed that the mutation of the PU.1 site resulted in a contribute to the low expression of PSMBs and ectopic expression significant reduction of the PSMB9 and PSMB10 promoter of PU.1 would be able to restore the expression of PSMBs in APL activities, respectively. These results indicate that PU.1 transacti- cells. As shown in Figure 4b, we observed that the expression of vates the promoters of PSMBs and the PU.1 sites located within PSMBs significantly increased after ectopic expression of PU.1. In their promoters are essential for promoter activity. addition, we performed luciferase reporter assays and found that PU.1 overexpression resulted in an obvious increase in promoter PML/RARa represses the expression of the immunosubunit genes activities of PSMBs (Figure 4c). Collectively, the data indicate that downregulation of PSMBs in APL is achieved through PML/RARa- We are further interested in whether PML/RARa binding to the mediated repression of both PU.1-dependent transactivation and promoters of PSMBs influences their expression in APL cells. We PU.1 expression. thus first compared the expression of PSMBs in PML/RARa-positive and PML/RARa-negative hematopoietic cells. As illustrated in Figure 3a, the expression levels of PSMBs in PML/RARa-positive ATRA but not As2O3 induces the expression of PSMBs in APL cells cells (NB4) were significantly lower than those in PML/RARa- ATRA and As2O3 are two drugs in clinical use for APL. To negative cells (U937, HL-60 and Kasumi-1), indicating that their investigate their effects on the immunosubunits, we measured the

Oncogene (2014) 2700 – 2708 & 2014 Macmillan Publishers Limited Regulatory mechanism of the immunosubunits in APL X-W Yang et al 2703

Figure 3. PML/RARa represses the expression of PSMB8, PSMB9 and PSMB10.(a) Endogenous expression of PSMBs is low in PML/RARa-positive leukemia cell lines. The mRNA expression levels of PSMBs were analyzed by qRT–PCR in four leukemia cell lines, including PML/RARa-positive (NB4) and PML/RARa-negative (U937, HL-60 and Kasumi-1) cells. (b) Promoter activities of PSMBs are repressed by PML/RARa. Luciferase reporter assays were performed in U937 cells. ( À ) absence and ( þ ) presence of the indicated plasmid. Data represent the mean of three replicates±s.d. Statistically significant differences are indicated by *.

Figure 4. Downregulation of PSMBs by PML/RARa requires PU.1. (a) Promoter activities of PSMBs are repressed by PML/RARa. Luciferase reporter assays were performed in 293T cells. ( À ) absence and ( þ ) presence of the indicated plasmid. (b) Overexpression of PU.1 restores the expression of PSMBs in NB4 cells. The mRNA levels of PU.1 and PSMB8, PSMB9 and PSMB10 were detected in NB4 cells 48 h post-transfection with the control plasmid (migR1) or PU.1 expression plasmid (migR1-PU.1) by qRT–PCR. (c) The promoters of PSMB8, PSMB9 and PSMB10 are transactivated by PU.1 in NB4 cells. The promoter constructs were individually co-transfected with migR1 or migR1-PU.1. Data represent the mean of three replicates±s.d. Statistically significant differences are indicated by *. expression of PSMBs following treatment of NB4 cells with ATRA PU.1 expression influenced by the two drugs. We thus further or As2O3. As shown in Figures 5a and b, the expression of PSMBs measured PU.1 expression after ATRA or As2O3 treatment. As was upregulated at both mRNA and protein levels after ATRA shown in Figure 5c, ATRA significantly increased PU.1 expression, treatment and the increase was maintained up to 72 h. In contrast, whereas As2O3 hardly affected its expression. We also performed As2O3 only caused minor/minimal changes in their expression. As luciferase reporter assays in NB4 cells to verify the responsiveness the expression of PSMBs is dependent on PU.1, the difference in of the promoters of PSMBs to ATRA or As2O3. As shown in the effects of ATRA and As2O3 is probably due to the difference in Figure 5d, an obvious increase in promoter activities of PSMBs was

& 2014 Macmillan Publishers Limited Oncogene (2014) 2700 – 2708 Regulatory mechanism of the immunosubunits in APL X-W Yang et al 2704

Figure 5. ATRA but not As2O3 induces the expression of PSMB8, PSMB9 and PSMB10.(a) and (b) The expression of PSMBs following treatment of NB4 cells with ATRA or As2O3 in NB4 cells. The mRNA (a) and protein levels (b) of PSMBs were detected by qRT–PCR and western blot, respectively. The mRNA levels of PSMB8, PSMB9 and PSMB10 were normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (c) The expression of PU.1 following treatment of NB4 cells with ATRA or As2O3 in NB4 cells. (d) Promoter activities of PSMBs following treatment of NB4 cells with ATRA or As2O3. Luciferase reporter assays were performed in NB4 cells. (e) The peptidase activity of the immunosubunits increases in NB4 cells following ATRA treatment. The data in (a), (c) and (d) show the mean±s.d. of three replicates. Statistically significant differences are indicated by *.

observed after ATRA treatment, whereas their promoter activities four fresh human UCB specimens. As shown in Figure 6b, the were less influenced or even inhibited by As2O3 treatment, expression of PSMBs was significantly lower in primary APL consistent with the above observation (Figures 5a and b). samples than in normal hematopoietic cells, including white Induction of the expression of immunosubunits may result in a blood cells, mononuclear cells, granulocytes and immature change in proteasome activity. Therefore, we compared the progenitor cells. The results indicated that the immunosubunits peptidase activity of the immunosubunits in NB4 cells before and were suppressed in primary APL patients, confirming the inverse after ATRA treatment. As illustrated in Figure 5e, a significant correlation between PML/RARa and the immunosubunits. To increase in peptidase activity was detected after 48 h of ATRA further test this concept in a large cohort of samples, we retrieved treatment and maintained throughout the rest of the experi- three data sets of the AML expression profiling,23–25 including 73 mental period. The data indicate that ATRA but not As2O3 has the APL patients and 640 AML patients with other subtypes, and ability to induce the expression of the immunosubunits and compared the expression of PSMBs between PML/RARa-positive relieve the PML/RARa-mediated repression, which might activate and PML/RARa-negative AML patient samples. As shown in the antigen-processing/presentation machinery. Figure 6c, the large-scale analysis clearly revealed that PSMBs were expressed at a lower level in APL patients than in non-APL AML patients. We also detected PU.1 Evidence for the targeting and regulation of PSMBs by PU.1 and expression in this cohort of patient samples. As expected, PU.1 PML/RARa in primary APL cells expression was significantly repressed in APL samples. The data Using a series of cell lines, we determined that PSMBswere are compliant with our findings from the cell line that the primarily bound and regulated by PU.1, and PML/RARa targeted expression of the immunosubunits is downregulated by PML/ and repressed their expression. To further verify these observa- RARa in primary APL patients and the repression is mediated by tions in primary clinical samples, we isolated mononuclear cells PU.1 (Figures 3 and 4). As AML is a group of diseases from two newly diagnosed primary APL patient samples with characterized by the abnormal development of malignant high percentages of blasts (91% and 98%, respectively) and myeloid cells, we are interested in the expression of PSMBs and performed ChIP-qPCR with antibodies against RARa and PU.1. As PU.1 in the other subtypes, including those with normal and shown in Figure 6a, both PML/RARa and PU.1 bound to the abnormal karyotypes. We divided the AML samples into nine promoters of PSMBs, consistent with the data shown in Figures 1b groups according to the French–American–British classification and c. Next, to investigate whether this binding interferes with and karyotype status (that is, normal vs abnormal), and evaluated the expression of the immunosubunits in primary APL patients, the expression of PSMBsandPU.1 in each specific group. we compared the mRNA expression of PSMBs using qRT–PCR in Interestingly, we found that the expression of PSMBs and PU.1 blast cells from four primary APL patient samples with the was lower in M3 (PML/RARa-positive APL) than in other subtypes peripheral blood cells from six healthy volunteers and normal whether the karyotype of the specimen was normal or not hematopoietic stem cell-enriched CD34 þ cells separated from (Supplementary Figure S1), further confirming that the expression

Oncogene (2014) 2700 – 2708 & 2014 Macmillan Publishers Limited Regulatory mechanism of the immunosubunits in APL X-W Yang et al 2705

Figure 6. Validation in primary cells. (a) The promoters of PSMBs are targeted by PML/RARa and PU.1 in fresh APL samples. ChIP-qPCR was performed on two primary APL samples, using the anti-RARa and anti-PU.1 antibodies, respectively. (b) The expression of PSMBs is downregulated in primary APL patients compared to normal hematopoietic cells. The mRNA expression of PSMBs was analyzed in primary samples, including normal CD34 þ cells (n ¼ 4), white blood cells (n ¼ 6, WBCs), monocytes (n ¼ 5, MNCs), granulocytes (n ¼ 5) and APL patient samples (n ¼ 4, APL#3, APL#4, APL#5, APL#5_relapse). The expression values are the absolute intensities after log-transformation. (c) The expression levels of PSMBs and PU.1 are lower in APL patients compared with non-APL AML patients. A total of 713 AML patient samples including 73 APL patients were analyzed. (d) The expression of PSMBs increases in ATRA-treated primary APL patient samples. qRT–PCR assays were performed to detect the mRNA levels of PSMBs in APL blast cells in the absence or presence of ATRA for 24 and 48 h. Data represent the mean of three replicates±s.d. of the immunosubunits were specifically downregulated with the enhancing the expression of the costimulatory molecules on the expression of PML/RARa and reduced PU.1 expression in APL. In cell surface. PU.1 increases the expression of CD80 and CD86, two addition, we also treated samples from two newly diagnosed APL important cell surface markers on dendritic cells, through directly patients with ATRA and found their expression levels were binding to their promoter regions, facilitating the CTL-mediated upregulated after ATRA treatment (Figure 6d), in accordance with activation.33 In addition to the signals from costimulatory the results observed in NB4 cells. Taken together, the results from molecules, the effective recognition and activation of antigen- primary clinical samples validate the observations obtained with presenting cells also require a stronger major histocompatibility the cell lines. complex (MHC)/antigen activation signal from antigen-presenting cells.34 The level of MHC molecules largely depends on the expression of the functional immunoproteasome. Here, we DISCUSSION demonstrated that PU.1 participated in regulating the expression The impairment of antigen presentation is one of the main of antigen presentation genes. The expression levels of the mechanisms that malignant cells use to escape the CTL-mediated immunosubunits were primarily regulated by PU.1 through direct immune surveillance. The immunosubunits that are composed of binding in hematopoietic cells (Figure 2b). Our results indicate that PSMB8, PSMB9 and PSMB10 function as the proteolytically active the concerted upregulation of the immunosubunits by PU.1 may subunits of the immunoproteasome, and the coordinated enhance the density of the MHC complex on the cell surface, incorporation of the three immunosubunits into the proteasome yielding an elevated efficacy of CTL activation. complex has a pivotal role in antigen generation/processing. In Previous studies have demonstrated that antigen presentation this study, we demonstrated that all three immunosubunits were processes are defective in APL cells. For example, HLAs are hardly primarily bound and regulated by PU.1, and then targeted and detected on the surface of APL cells.17,35,36 Studies on the repressed by the PML/RARa fusion protein in APL. expression of the immunosubunits have shown that the APL cell Our results provide new insights into the regulatory mechanism line NB4 exhibits barely detectable PSMB8 and PSMB9 levels.17 of the basal expression of the immunosubunit genes. It has been PSMB10 expression data were absent from that study because reported that the expression of the immunosubunits can be PSMB10, unlike PSMB8 and PSMB9, is located outside of the MHC upregulated by inducing signals such as interferon-g and tumor cluster,37 and was therefore not identified during the investigation. necrosis factor a,26 yet the key transcription factors that are However, the critical mechanism by which the expression of the essential for their basal expression have not been identified. Here, immunosubunits is inhibited remains unclear. We revealed that we observed that the hematopoietic specific PML/RARa bound the promoter regions of PSMBs (Figures 1b and PU.1 was required for the basal expression of the immunosubunits 2b), resulting in the repression of PU.1-dependent transactivation in hematopoietic cells. PU.1 is believed to contribute predo- of PSMBs. In contrast, PML/RARa alone exerted minor effects on minantly to hematopoietic development. PU.1-deficient mice the repression of these promoters (Figure 4a). Furthermore, exhibit maturation arrest of macrophages and granulocytes.27 through comparing the PML/RARa-positive and PML/RARa-nega- Recently, growing evidence has suggested a role for PU.1 in the tive AML patient samples, we observed the low expression levels generation of innate and adaptive immune cells.28,29 For instance, of PSMBs in PML/RARa-positive APL samples, confirming the PU.1 is capable of functioning as a transcriptional regulator in inverse correlation between PML/RARa and the immunosubunits. determining the fate of immunocytes such as lymphocyte and These results suggest that the inhibition of PSMBs may result in the dendritic cell lineages.30–32 Moreover, PU.1 critically participates in defects of antigen presentation in APL and affect recognition of

& 2014 Macmillan Publishers Limited Oncogene (2014) 2700 – 2708 Regulatory mechanism of the immunosubunits in APL X-W Yang et al 2706

APL cells by CTLs, thereby allowing leukemia cells to escape from (Sigma, St Louis, MO, USA), ATRA (Sigma) and As2O3 (Sigma) were used at the host immune response. final concentrations of 100, 1 and 1 mM, respectively. ATRA and As2O3, as two drugs in clinical use for APL, both show favorable curative effects on the treatment of APL. ATRA plus Plasmid constructions and site-directed mutagenesis conventional chemotherapy is currently the standard treatment 38 The promoter regions of PSMBs were amplified from U937 cells by PCR strategy. As2O3 is also considered as front-line therapy for APL in with the corresponding primers and the resulting PCR fragments were 39 some areas. More importantly, a combination of ATRA plus cloned between the XhoI and HindIII sites of the luciferase reporter vector As2O3 performs even better than treatment with ATRA alone or pGL4-basic (Promega, Madison, WI, USA), generating the plasmids PSMBs 40 As2O3 alone and represents the most successful therapeutic (wild-type). Mutations of the PU.1 binding sites in the three promoters strategy for a hematopoietic malignant disease. Recent studies were made using the QuikChange Site-directed Mutagenesis Kit (Strata- gene, La Jolla, CA, USA) following the manufacturer’s protocol. Detailed have demonstrated that As2O3 may prevent relapse of APL by eradicating leukemia-initiating cells,41 providing evidence that the primer information is available in Supplementary Table S1. combination is better than ATRA treatment alone. Our data likely indicate that ATRA but not As2O3 has the ability to induce the RNA extraction, qRT–PCR, western blot and proteolytic activity expression of the immunosubunits, which may lead to the assays activation of the antigen-processing/presentation machinery and RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) ultimately elicit a host response to kill malignant cells. Thus, from a and cDNA was reverse transcribed using the SuperScript II Reverse clinical point of view, our finding supports the use of combination Transcriptase (Invitrogen) with random hexamer primers according to the therapy, rather than As O alone, as the added potential for manufacturer’s protocol. The qRT–PCR assays were performed using the 2 3 SYBR Green PCR Master Mix (Toyobo, Osaka, Japan) and the ABI 7900 HT immune stimulation is likely to improve overall outcome. Sequence Detection System (Applied Biosystems Inc., Foster City, CA, USA). Numerous studies have demonstrated that ATRA exerts Glyceraldehyde 3-phosphate dehydrogenase was used as an internal pleiotropic effects on a variety of biological processes. Among control. The relative mRNA expression level of the target gene in each them, the roles in differentiation induction, arrest and sample was analyzed according to the comparative cycle time (Ct) apoptosis are well documented.42–45 However, sparse data are method.49 Each experiment was performed in triplicate. Detailed primer available regarding its effects on the immune response. We information is available in Supplementary Table S2. To measure the protein demonstrated here that ATRA was also able to function as an levels of PSMBs, NB4 cell nuclear extracts were prepared and western blotting was performed. The detailed procedure was described pre- immune modulator, which could contribute to the clinical 50 outcome by immune elimination of leukemic cells. This viously. The proteolytic activity of the immunoproteasome was analyzed using the Proteasome-GloTM Cell-Based Assay (Promega) according to the observation is consistent with a previous study on ATRA-treated manufacturer’s instructions. neuroblastoma,19 where ATRA induced the systematic modulation of antigen presentation through upregulating multiple steps in the MHC class I presentation pathway in non-professional antigen- Transient transfection and luciferase assays presenting cells transformed from cancer cells. Moreover, the Suspension cells (U937 and NB4) were electrotransfected using the increased expression of MHC class I molecules on the surface of electroporation generator ECM830 system (BTX, Holliston, MA, USA); adherent cells (293T) were transfected using Lipofectamine 2000 (Invitro- target cells subsequently promoted T-cell-mediated recognition gen) according to the manufacturer’s instructions. The detailed procedure and killing. Indeed, using an APL-transplantable mouse model, 51 20 was described previously. Luciferase assays were performed with the Padua et al. have shown that ATRA treatment combined with Dual-Luciferase Reporter Assay (Promega) 24 h after transfection. DNA can activate the T-cell response and increase the Luciferase activities were corrected for transfection efficiency by co- release, whereas the CTL responses cannot be detected in transfecting a plasmid expressing Renilla luciferase. APL mice only with DNA vaccine treatment. In summary, our findings support the fact that the restoration of the ChIP analysis immunosubunits by ATRA is a crucial prerequisite in the effective ChIP was performed according to the Affymetrix protocol as described,52 antigen presentation and leukemia-specific CTL responses. with the following antibodies: anti-RARa (C-20 X; Santa Cruz Biotechnology, Our findings not only reveal a previously unidentified role of Santa Cruz, CA, USA), anti-PML (H238 X; Santa Cruz), anti-PU.1 (T-21 X; Santa ATRA in APL treatment but also suggest a potential clinical use of Cruz) and the rabbit immunoglobulin G (ab46540; Abcam, Cambridge, UK). ATRA in the treatment of non-APL AML. The potential significance qPCR was performed using the SYBR Green PCR Master Mix (Toyobo) and of our study is indeed supported by clinical trials, in which ATRA the ABI 7900 HT Sequence Detection System (Applied Biosystems Inc.). The fold enrichment of chromatin-immunoprecipitated PCR product relative to given after intensive chemotherapy significantly improved the 9 outcome of older patients with non-APL AML.46 Our study may the input PCR product was calculated as described previously. Detailed present an additional layer of mechanisms of ATRA as an immune primer information is available in Supplementary Table S3. modulator in treating non-APL AML, in addition to the reported potential mechanisms such as through repression of the Patient samples and normal hematopoietic specimens antiapoptotic protein BCL247 or survivin.48 Taken together, the This study was approved by the institutional ethics committee of Ruijin immunomodulating property of ATRA may be one of the potential Hospital, Shanghai Jiao Tong University School of Medicine and was mechanisms underlying effective treatment of APL, and the performed in accordance with the Declaration of Helsinki. Informed identification of therapeutic concepts that contribute to the consent was obtained from all patients and healthy donors. APL blasts success of APL therapy will be vital to the further improvement of were obtained at initial diagnosis from patient bone marrow samples. Peripheral blood specimens were obtained from healthy volunteers. the treatment protocols and even to its potential application in Detailed information about APL patients is presented in Supplementary treating other . Table S4.

Gene expression analysis Three transcriptome data sets for AML patients performed by Wouters MATERIALS AND METHODS et al.23 (GSE14468), Tomasson et al.25 (GSE10358) and Ross et al.24 (St Jude) Cell culture and reagents were pooled to compare the expression levels of immunosubunits Kasumi-1, U937, U937-PR9, HL-60 and NB4 cells were cultured in RPMI 1640 between APL and non-APL patient samples. GSE14468 and GSE10358 (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (Gibco). The were obtained from the Gene Expression Omnibus. St Jude was obtained 293T cells were cultured in DMEM (Gibco) containing 10% fetal bovine from the website at http://www.stjuderesearch.org/data/AML1. To perform serum. Cells were grown in an incubator at 37 1C and 5% CO2. ZnSO4 interarray comparisons, the CEL files were analyzed by Affymetrix MAS 5.0

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