Alternative splicing modulation mediated by G-quadruplex structures in MALAT1 lncRNA.

MALAT1, an abundant lncRNA specifically localized to nuclear speckles, regulates alternative-splicing (AS). The molecular basis of its role in AS remains poorly understood. Here, we report three conserved, thermodynamically stable, parallel RNA-G-quadruplexes (rG4s) present in the 3' region of MALAT1 which regulates this function. Using rG4 domain-specific RNA-pull-down followed by mass-spectrometry, RNA-immuno-precipitation, and imaging, we demonstrate the rG4 dependent localization of Nucleolin (NCL) and Nucleophosmin (NPM) to nuclear speckles. Specific G-to-A mutations that abolish rG4 structures, result in the localization loss of both the proteins from speckles. Functionally, disruption of rG4 in MALAT1 phenocopies NCL knockdown resulting in altered pre-mRNA splicing of endogenous genes. These results reveal a central role of rG4s within the 3' region of MALAT1 orchestrating AS.

Identification and targeting of G-quadruplex structures in MALAT1 long non-coding RNA.

RNA G-quadruplexes (rG4s) have functional roles in many cellular processes in diverse organisms. While a number of rG4 examples have been reported in coding messenger RNAs (mRNA), so far only limited works have studied rG4s in non-coding RNAs (ncRNAs), especially in long non-coding RNAs (lncRNAs) that are of emerging interest and significance in biology. Herein, we report that MALAT1 lncRNA contains conserved rG4 motifs, forming thermostable rG4 structures with parallel topology. We also show that rG4s in MALAT1 lncRNA can interact with NONO protein with high specificity and affinity in vitro and in nuclear cell lysate, and we provide cellular data to support that NONO protein recognizes MALAT1 lncRNA via rG4 motifs. Notably, we demonstrate that rG4s in MALAT1 lncRNA can be targeted by the rG4-specific small molecule, peptide, and L-aptamer, leading to the dissociation of MALAT1 rG4-NONO protein interaction. Altogether, this study uncovers new and important rG4s in MALAT1 lncRNAs, reveals their specific interactions with NONO protein, offers multiple strategies for targeting MALAT1 and its RNA-protein complex via its rG4 structure and illustrates the prevalence and significance of rG4s in ncRNAs.

Immortalization-upregulated protein promotes pancreatic cancer progression by regulating NPM1/FHL1-mediated cell-cycle-checkpoint protein activity.

Immortalization-upregulated protein (IMUP) plays a vital role in cell proliferation and tumor progression. However, its role in pancreatic ductal adenocarcinoma (PDAC) remains unclear. Here, we select IMUP as an alternative gene based on GeneChip analysis of clinical PDAC tissues and transcriptome data from The Cancer Genome Atlas. IMUP expression is upregulated in PDAC tumor tissues. Moreover, high IMUP expression correlates with poor prognosis, while IMUP depletion inhibits PDAC cell proliferation and colony formation capacity in vitro, and decreases xenograft tumor growth in vivo. IMUP downregulation leads to cell-cycle arrest in the S phase. IMUP knockdown increases the expression of four-and-a-half LIM domain protein 1 (FHL1), which regulates the phosphorylation of cell division cycle 25A (CDC25A) by cycle checkpoint kinase 1 (CHK1) and promotes cytoplasmic distribution of CDC25A by interaction with 14-3-3ξ. Furthermore, FHL1 knockdown restores the effects induced by IMUP depletion. Liquid chromatography tandem mass spectrometry and immunoprecipitation analysis further show that IMUP interacts directly with nucleophosmin (NPM1) and enhances its stability. DNA methylation sequencing shows that FHL1 promoter methylation decreases when IMUP is downregulated. Overexpression of NPM1 can increase the methylation level of FHL1, thereby decreasing its expression. Our study provides a novel perspective on IMUP/NPM1/FHL1-mediated cell-cycle arrest by regulating CDC25A phosphorylation in PDAC. These findings may provide a new therapeutic target for PDAC.

The binding between NPM and H2B proteins signals for the diabetes-associated centrosome amplification.

Diabetes not only increases the risk for cancer but also promotes cancer metastasis. Centrosome amplification (CA) is sufficient to initiate tumorigenesis and can enhance the invasion potential of cancer cells. We have reported that diabetes can induce CA, with diabetic pathophysiological factors as the triggers, which involves the signaling of nucleophosmin (NPM). Thus, CA can serve as a candidate biological link between diabetes and cancer. In the present study, we attempted to identify the NPM binding partners and investigated whether the binding between NPM and its partner mediated the CA. We confirmed that high glucose, insulin, and palmitic acid cancer could elicit CA in the HCT16 colon cancer cells and found that the experimental treatment increased the binding between NPM and H2B, but not between p-NPM and H2B. The molecular docking analysis supported the fact that NPM and H2B could bind to each other through various amino acid residues. The treatment also increased the colocalization of NPM and H2B in the cytosol. Importantly, disruption of the NPM1-H2B complex by individual knockdown of the protein level of NPM or H2B led to the inhibition of the treatment-evoked CA. In conclusion, our results suggest that the binding between NPM and H2B proteins signals for the CA by high glucose, insulin, and palmitic acid.

High expression of NPM1 via the Wnt/ß-catenin signalling pathway might predict poor prognosis for patients with prostate adenocarcinoma.

Prostate adenocarcinoma (PRAD) occurs only in males and has a higher incidence rate than other cancers. NPM1 is a nucleocytoplasmic shuttling protein that participates in the development of multiple tumours. The aim of this research was to explore the effect of the upregulation or downregulation of the NPM1 protein on the malignancy of prostate cancer and its possible signalling pathway. Prostate adenocarcinoma cell lines were used in this study, including RWPE-1, PC3, LNCap, and 22RV1 cells. Our research revealed that NPM1 was widely expressed in the PRAD cell lines, as determined by western blotting, and that the levels of NPM1 protein were positively correlated with the degree of malignancy of the PRAD cell lines. Through interference and overexpression experiments, we found that PC3 cell growth was inhibited after NPM1 knockdown and that this inhibition was partly reversed by CTNNB1 overexpression; in contrast, PC3 cells growth was promoted after NPM1 overexpression, and this promotion was partly reversed by CTNNB1 knockdown, suggesting that NPM1 and CTNNB1 play important roles in the progression of prostate cancer cells via the Wnt/ß-catenin signalling pathway. NPM1 may serve as an important biomarker and candidate therapeutic for patients with prostate cancer.

Proteomic Investigation of the Role of Nucleostemin in Nucleophosmin-Mutated OCI-AML 3 Cell Line.

Nucleostemin (NS; a product of the GNL3 gene) is a nucleolar-nucleoplasm shuttling GTPase whose levels are high in stem cells and rapidly decrease upon differentiation. NS levels are also high in several solid and hematological neoplasms, including acute myeloid leukaemia (AML). While a role in telomere maintenance, response to stress stimuli and favoring DNA repair has been proposed in solid cancers, little or no information is available as to the role of nucleostemin in AML. Here, we investigate this issue via a proteomics approach. We use as a model system the OCI-AML 3 cell line harboring a heterozygous mutation at the NPM1 gene, which is the most frequent driver mutation in AML (approximately 30% of total AML cases). We show that NS is highly expressed in this cell line, and, contrary to what has previously been shown in other cancers, that its presence is dispensable for cell growth and viability. However, proteomics analysis of the OCI-AML 3 cell line before and after nucleostemin (NS) silencing showed several effects on different biological functions, as highlighted by ingenuity pathway analysis (IPA). In particular, we report an effect of down-regulating DNA repair through homologous recombination, and we confirmed a higher DNA damage rate in OCI-AML 3 cells when NS is depleted, which considerably increases upon stress induced by the topoisomerase II inhibitor etoposide. The data used are available via ProteomeXchange with the identifier PXD034012.

KPNA2 promotes renal cell carcinoma proliferation and metastasis via NPM.

Karyopherin α2 (KPNA2), involved in nucleocytoplasmic transport, has been reported to be up-regulated in tumorigenesis. However, comprehensive studies of KPNA2 functions in renal cell carcinoma (RCC) are still lacking. In this study, we aim to investigate the roles of KPNA2 in kidney tumour development. Our results showed that down-regulation of KPNA2 inhibited the proliferation and invasion of kidney tumour cell cells in vitro, while the cell cycle arrest and cellular apoptosis were induced once KPNA2 was silenced. Repression of KPNA2 was proved to be efficient to repress tumorigenesis and development of kidney tumour in in nude mice. Furthermore, one related participator, NPM, was identified based on Co-IP/MS and bioinformatics analyses. The up-regulation of NPM attenuates the efficiency of knockdown KPNA2. These results indicated that KPNA2 may regulate NPM to play a crucial role for kidney tumour development.

The potential role of nucleophosmin (NPM1) in the development of cancer.

Nucleophosmin (NPM1) is a well-known nucleocytoplasmic shuttling protein that performs several cellular functions such as ribosome biogenesis, chromatin remodeling, genomic stability, cell cycle progression, and apoptosis. NPM1 has been identified to be necessary for normal cellular functions, and its altered regulation by overexpression, mutation, translocation, loss of function, or sporadic deletion can lead to cancer and tumorigenesis. In this review, we focus on the gene and protein structure of NPM1 and its physiological roles. Finally, we discuss the association of NPM1 with various types of cancer including solid tumors and leukemia.

The role of tumor necrosis factor receptor superfamily member 4 (TNFRSF4) gene expression in diagnosis and prognosis of acute myeloid leukemia.

OBJECTIVES: Acute myeloid leukemia (AML) is still challenging in predicting the prognosis due to its high heterogeneity. Molecular aberrations and abnormalities play a significant prognostic role in AML patients. Our aim of the study was to investigate the prognostic role of TNFRSF4 gene expression in AML patients and its potential effect on treatment protocols. METHODS: Bone marrow mononuclear cells were analyzed for TNFRSF4 expression by real-time quantitative PCR as well as of FLT3/ITD and NPM1 mutations in 80 newly diagnosed AML patients and 80 control subjects. RESULTS: TNFRSF4 was significantly overexpressed in the AML patients (p < 0.001). TNFRSF4 expression was associated with unfavorable clinical outcomes including treatment response, relapse free survival, and overall survival. On multivariate testing, TNFRSF4 high expression proved to be an independent prognostic marker for clinical remission and relapse free survival but not overall survival. CONCLUSION: TNFRSF4 expression was revealed as an unfavorable prognostic marker and might be a target for immunotherapy in the future.

The acetylation of STAT3 at K685 attenuates NPM-ALK-induced tumorigenesis.

Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), a fusion protein generated by a chromosomal translocation, is a causative gene product of anaplastic large cell lymphoma (ALCL). It induces cell proliferation and tumorigenesis by activating the transcription factor, signal transducer and activator of transcription factor 3 (STAT3). We herein demonstrated that STAT3 underwent acetylation at K685 in a manner that was dependent on the kinase activity of NPM-ALK. To investigate the role of STAT3 acetylation in NPM-ALK-induced oncogenesis, we generated Ba/F3 cells expressing NPM-ALK in which STAT3 was silenced by shRNA, named STAT3-KD cells, and then reconstituted wild-type STAT3 or the STAT3 K685R mutant into these cells. The phosphorylation level of the K685R mutant at Y705 and S727 was significantly higher than that of wild-type STAT3 in STAT3-KD cells. The expression of STAT3 target genes, such as IL-6, Pim1, Pim2, and Socs3, was more strongly induced by the reconstitution of the K685R mutant than wild-type STAT3. In addition, the proliferative ability of STAT3-KD cells reconstituted with the K685R mutant was slightly higher than that of STAT3-KD cells reconstituted with wild-type STAT3. In comparisons with the inoculation of STAT3-KD cells reconstituted with wild-type STAT3, the inoculation of STAT3-KD cells reconstituted with the K685R mutant significantly enhanced tumorigenesis and hepatosplenomegaly in nude mice. Collectively, these results revealed for the first time that the acetylation of STAT3 at K685 attenuated NPM-ALK-induced oncogenesis.

Phosphorylation Code of Human Nucleophosmin Includes Four Cryptic Sites for Hierarchical Binding of 14-3-3 Proteins.

Nucleophosmin (NPM1) is the 46th most abundant human protein with many functions whose dysregulation leads to various cancers. Pentameric NPM1 resides in the nucleolus but can also shuttle to the cytosol. NPM1 is regulated by multisite phosphorylation, yet molecular consequences of site-specific NPM1 phosphorylation remain elusive. Here we identify four 14-3-3 protein binding sites in NPM1 concealed within its oligomerization and α-helical C-terminal domains that are found phosphorylated in vivo. By combining mutagenesis, in-cell phosphorylation and PermaPhos technology for site-directed incorporation of a non-hydrolyzable phosphoserine mimic, we show how phosphorylation promotes NPM1 monomerization and partial unfolding, to recruit 14-3-3 dimers with low-micromolar affinity. Using fluorescence anisotropy we quantified pairwise interactions of all seven human 14-3-3 isoforms with four recombinant NPM1 phosphopeptides and assessed their druggability by fusicoccin. This revealed a complex hierarchy of 14-3-3 affinities toward the primary (S48, S293) and secondary (S106, S260) sites, differentially modulated by the small molecule. As three of these 14-3-3 binding phosphosites in NPM1 reside within signal sequences, this work suggests a mechanism of NPM1 regulation by which NPM1 phosphorylation can promote 14-3-3 binding to affect NPM1 shuttling between cell compartments. It also provides further evidence that phosphorylation-induced structural rearrangements of globular proteins serve to expose otherwise cryptic 14-3-3-binding sites that are important for cellular function.

Nucleolar condensates: A cellular machinery necessary for T cell activation.

Naive T cells must shift from a state of quiescence to an active metabolic state. To do this, T cells must ramp up their production of ribosomes. In this issue, Zhou et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202201096) identify DDB1 and Cul4-associated factor 13 (DCAF13) as a T cell activation-induced nucleolar protein that functions to enhance ribosome biosynthesis. DCAF13 binds to nucleophosmin 1 (NPM1) to form a biomolecular condensate that functions, in part, by recruiting the endonuclease UTP23 into the nucleolus.

A clinicopathological experience in acute myeloid leukemia: Effects of clinical data and status of FLT3, CEBPA and NPM1 on prognosis.

Background: The purpose of the study was to analyze the expression of nucleophosmin (NPM1), CCAT/enhancer-binding protein alpha (CEBPA), and FMS-like tyrosine kinase 3 (FLT3) with immunohistochemistry and evaluate the relationship with clinicopathologic data with special emphasis on prognosis in bone marrow biopsy specimens diagnosed with acute myeloid leukemia (AML). Materials and Methods: Bone marrow biopsies of 104 patients who were diagnosed with AML were re-evaluated for diagnosis and subclassification. Immunohistochemically, anti-NPM1, anti-CEBPA, and anti-FLT3 antibodies were applied to slides prepared from formalin-fixed paraffin-embedded tissues. Sixty-three of these patients had their follow-up in our institutional hematology clinic and these patients' clinical, biochemical, and radiological data were obtained and analyzed from patient files. These data were analyzed with survival times statistically. Results: Except for age, no significant effect of clinical data on prognosis was detected. Immunohistochemical results were also statistically compared with clinical data. No correlation was found between overall survival and disease-free survival with the expression of anti-CEBPA or anti-NPM1 antibodies. However, immunohistochemical reactivity for anti-FLT3 antibody was found to be a poor prognostic factor and statistically significant. Also, when the expression of FLT3 was analyzed with that of NPM1 or CEBPA, a correlation (dependent on the expression of FLT3) was found with disease-free survival. Conclusions: FLT3 is an independent prognostic factor for AML. CEBPA and NPM1 should be considered as good prognostic factors only in the absence of FLT3 abnormalities.

Type C mutation of nucleophosmin 1 acute myeloid leukemia: Consequences of intrinsic disorder.

BACKGROUND: Nucleophosmin 1 (NPM1) protein is a multifunctional nucleolar chaperone and its gene is the most frequently mutated in Acute Myeloid Leukemia (AML). AML mutations cause the unfolding of the C-terminal domain (CTD) and the protein delocalizing in the cytosol (NPM1c+). Marked aggregation endowed with an amyloid character was assessed as consequences of mutations. SCOPE: Herein we analyzed the effects of type C mutation on two protein regions: i) a N-terminal extended version of the CTD, named Cterm_mutC and ii) a shorter polypeptide including the sequences of the second and third helices of the CTD, named H2_mutC. MAJOR CONCLUSIONS: Both demonstrated able to self-assembly with different kinetics and conformational intermediates and to provide fibers presenting large flexible regions. GENERAL SIGNIFICANCE: The present study adds a new piece of knowledge to the effects of AML-mutations on structural biology of Nucleophosmin 1, that could be exploited in therapeutic interventions targeting selectively NPMc+.

Non-coding small nucleolar RNA SNORD17 promotes the progression of hepatocellular carcinoma through a positive feedback loop upon p53 inactivation.

Recent evidence suggests that small nucleolar RNAs (snoRNAs) are involved in the progression of various cancers, but their precise roles in hepatocellular carcinoma (HCC) remain largely unclear. Here, we report that SNORD17 promotes the progression of HCC through a positive feedback loop with p53. HCC-related microarray datasets from the Gene Expression Omnibus (GEO) database and clinical HCC samples were used to identify clinically relevant snoRNAs in HCC. SNORD17 was found upregulated in HCC tissues compared with normal liver tissues, and the higher expression of SNORD17 predicted poor outcomes in patients with HCC, especially in those with wild-type p53. SNORD17 promoted the growth and tumorigenicity of HCC cells in vitro and in vivo by inhibiting p53-mediated cell cycle arrest and apoptosis. Mechanistically, SNORD17 anchored nucleophosmin 1 (NPM1) and MYB binding protein 1a (MYBBP1A) in the nucleolus by binding them simultaneously. Loss of SNORD17 promoted the translocation of NPM1 and MYBBP1A into the nucleoplasm, leading to NPM1/MDM2-mediated stability and MYBBP1A/p300-mediated activation of p53. Interestingly, p300-mediated acetylation of p53 inhibited SNORD17 expression by binding to the promoter of SNORD17 in turn, forming a positive feedback loop between SNORD17 and p53. Administration of SNORD17 antisense oligonucleotides (ASOs) significantly suppressed the growth of xenograft tumors in mice. In summary, this study suggests that SNORD17 drives cancer progression by constitutively inhibiting p53 signaling in HCC and may represent a potential therapeutic target for HCC.

Conformational Freedom and Topological Confinement of Proteins in Biomolecular Condensates.

The emergence of biomolecular condensation and liquid-liquid phase separation (LLPS) introduces a new layer of complexity into our understanding of cell and molecular biology. Evidence steadily grows indicating that condensates are not only implicated in physiology but also human disease. Macro- and mesoscale characterization of condensates as a whole have been instrumental in understanding their biological functions and dysfunctions. By contrast, the molecular level characterization of condensates and how condensates modify the properties of the molecules that constitute them thus far remain comparably scarce. In this minireview we summarize and discuss the findings of several recent studies that have focused on structure, dynamics, and interactions of proteins undergoing condensation. The mechanistic insights they provide help us identify the relevant properties nature and scientists can leverage to modulate the behavior of condensate systems. We also discuss the unique environment of the droplet surface and speculate on effects of topological constraints and physical exclusion on condensate properties.

Histone Chaperone Nucleophosmin Regulates Transcription of Key Genes Involved in Oral Tumorigenesis.

Nucleophosmin (NPM1) is a multifunctional histone chaperone that can activate acetylation-dependent transcription from chromatin templates in vitro. p300-mediated acetylation of NPM1 has been shown to further enhance its transcription activation potential. Acetylated and total NPM1 pools are increased in oral squamous cell carcinoma. However, the role of NPM1 or its acetylated form (AcNPM1) in transcriptional regulation in cells and oral tumorigenesis is not fully elucidated. Using ChIP-seq analyses, we provide the first genome-wide profile of AcNPM1 and show that AcNPM1 is enriched at transcriptional regulatory elements. AcNPM1 co-occupies marks of active transcription at promoters and DNase I hypersensitive sites at enhancers. In addition, using a high-throughput protein interaction profiling approach, we show that NPM1 interacts with RNA Pol II, general transcription factors, mediator subunits, histone acetyltransferase complexes, and chromatin remodelers. NPM1 histone chaperone activity also contributes to its transcription activation potential. Further, NPM1 depletion leads to decreased AcNPM1 occupancy and reduced expression of genes required for proliferative, migratory and invasive potential of oral cancer cells. NPM1 depletion also abrogates the growth of orthotopic tumors in mice. Collectively, these results establish that AcNPM1 functions as a coactivator during during RNA polymerase II-driven transcription and regulates the expression of genes that promote oral tumorigenesis.

HO-1 nuclear accumulation and interaction with NPM1 protect against stress-induced endothelial senescence independent of its enzymatic activity.

Heme oxygenase-1 (HO-1) has attracted accumulating attention for its antioxidant enzymatic activity. However, the exact regulatory role of its non-enzymatic activity in the cardiovascular system remains unaddressed. Here, we show that HO-1 was accumulated in the nuclei of stress-induced senescent endothelial cells, and conferred protection against endothelial senescence independent of its enzymatic activity. Overexpression of ΔHO-1, a truncated HO-1 without transmembrane segment (TMS), inhibited H2O2-induced endothelial senescence. Overexpression of ΔHO-1H25A, the catalytically inactive form of ΔHO-1, also exhibited anti-senescent effect. In addition, infection of recombinant adenovirus encoding ΔHO-1 with three nuclear localization sequences (NLS), alleviated endothelial senescence induced by knockdown of endogenous HO-1 by CRISPR/Cas9. Moreover, repression of HO-1 nuclear translocation by silencing of signal peptide peptidase (SPP), which is responsible for enzymatic cleavage of the TMS of HO-1, exacerbated endothelial senescence. Mechanistically, nuclear HO-1 interacted with NPM1 N-terminal portion, prevented NPM1 translocation from nucleolus to nucleoplasm, thus disrupted NPM1/p53/MDM2 interactions and inhibited p53 activation by NPM1, finally resisted endothelial senescence. This study provides a novel understanding of HO-1 as a promising therapeutic strategy for vascular senescence-related cardiovascular diseases.

A Curious Novel Combination of Nucleophosmin (NPM1) Gene Mutations Leading to Aberrant Cytoplasmic Dislocation of NPM1 in Acute Myeloid Leukemia (AML).

Nucleophosmin (NPM1) mutations occurring in acute myeloid leukemia (AML) (about 50 so far identified) cluster almost exclusively in exon 12 and lead to common changes at the NPM1 mutants C-terminus, i.e., loss of tryptophans 288 and 290 (or 290 alone) and creation of a new nuclear export signal (NES), at the bases of exportin-1(XPO1)-mediated aberrant cytoplasmic NPM1. Immunohistochemistry (IHC) detects cytoplasmic NPM1 and is predictive of the molecular alteration. Besides IHC and molecular sequencing, Western blotting (WB) with anti-NPM1 mutant specific antibodies is another approach to identify NPM1-mutated AML. Here, we show that among 382 AML cases with NPM1 exon 12 mutations, one was not recognized by WB, and describe the discovery of a novel combination of two mutations involving exon 12. This appeared as a conventional mutation A with the known TCTG nucleotides insertion/duplication accompanied by a second event (i.e., an 8-nucleotide deletion occurring 15 nucleotides downstream of the TCTG insertion), resulting in a new C-terminal protein sequence. Strikingly, the sequence included a functional NES ensuring cytoplasmic relocation of the new mutant supporting the role of cytoplasmic NPM1 as critical in AML leukemogenesis.

Nucleophosmin/B23 promotes endometrial cancer cell escape from macrophage phagocytosis by increasing CD24 expression.

Despite recent therapeutic breakthroughs, advanced and/or recurrent endometrial cancer still poses a significant health burden globally. While immunotherapy can theoretically lead to durable responses, the benefits to patients remain limited. In an effort to identify novel immunotherapeutic targets, we specifically focused on the potential role of nucleophosmin (NPM, also known as B23) - a nucleolar phosphoprotein involved in tumorigenesis - in cancer immune evasion. Expression profiling with oligonucleotide microarrays was conducted to identify differentially expressed genes in NPM/B23-silenced endometrial cancer cells. CD24 - a heat-stable antigen commonly overexpressed in solid tumors and a target for cancer immunotherapy - was identified as one of the key NPM/B23-regulated molecules. We found that NPM/B23 was capable of inducing CD24 expression, with the Sp1 binding site in the CD24 promoter being essential for NPM/B23-mediated transcriptional activation. Interestingly, NPM/B23 silencing in endometrial cancer cells enhanced phagocytic removal by macrophages through a decreased exposure of CD24 on the cell surface. Conversely, restoration of CD24 expression in NPM/B23-silenced endometrial cancer cells inhibited macrophage-mediated phagocytosis. These results indicate that NPM/B23-driven CD24 overexpression enables endometrial cancer cells to evade from phagocytosis. We further suggest that CD24 may serve as a novel target for endometrial cancer immunotherapy. KEY MESSAGES: NPM/B23 induced CD24 expression in endometrial tumorigenesis. Sp1 binding site in the CD24 promoter is essential for the activation. NPM/B23 silencing enhanced phagocytosis by macrophages through decrease of CD24 on cancer cells. Restoration of CD24 expression in NPM/B23-silenced cancer cells inhibited macrophage-mediated phagocytosis.