NAP1L5 in acute myeloid leukemia: a prognostic biomarker and potential therapeutic target

Abstract

Background: Nucleosome assembly protein 1-like 5 (NAP1L5), a critical regulator of gene transcription and nucleosome assembly, has been implicated in the progression and poor prognosis of various cancers. However, its specific role and molecular mechanisms in acute myeloid leukemia (AML) remain largely unexplored.

Methods: To identify key genes associated with AML, we analyzed gene expression profiles from AML patients and healthy controls using microarray datasets obtained from the GEO database. Differential expression analysis was performed to identify differentially expressed genes (DEGs), among which NAP1L5 emerged as a critical candidate based on its expression patterns and prognostic relevance, and we validated NAP1L5 expression in clinical AML samples. To elucidate the functional role of NAP1L5, we conducted Gene Set Enrichment Analysis (GSEA) and Gene Ontology (GO) analysis, which revealed its involvement in specific signaling pathways and biological processes. Furthermore, we constructed an interaction network and predictive model for NAP1L5, complemented by an assessment of its role in immune infiltration and drug sensitivity. Finally, we conducted in vitro experiments to explore its biological functions and underlying molecular mechanisms.

Results: In AML, elevated expression of NAP1L5 was significantly associated with reduced overall survival, underscoring its prognostic relevance. GSEA revealed that NAP1L5 was prominently enriched in pathways related to apoptosis and DNA replication. GO analysis further indicated that its co-expressed genes were closely linked to autophagy and stress response mechanisms. Interaction network analysis revealed that NAP1L5 engages in complex regulatory interactions with multiple genes, miRNAs, transcription factors (TFs), and RNA-binding proteins (RBPs). Notably, high NAP1L5 expression correlated with increased infiltration of resting CD4+ memory T cells, implicating its potential influence on the tumor immune microenvironment. A predictive model integrating NAP1L5 expression and clinical AML features exhibited robust prognostic utility. Drug sensitivity analysis identified NAP1L5 overexpression as a marker of resistance to Zibotentan, along with associations with 49 additional therapeutic agents. In vitro functional assays demonstrated that NAP1L5 overexpression promoted cellular proliferation, migration, and colony formation while concurrently inhibiting apoptosis, highlighting its oncogenic potential in AML pathogenesis.

Conclusions: NAP1L5 emerges as a promising prognostic biomarker and therapeutic target in AML, offering potential for improved patient outcomes and precision treatment strategies.

Keywords: NAP1L5; acute myeloid leukemia; biomarker; immune infiltration; prognosis.

Figure 1

This study’s research flow chart.

Figure 2

Identification of differentially expressed genes and association of NAP1L5 with AML. (A) Volcano plots showing DEGs from the GSE24395 and GSE114868 datasets. (B) Venn diagram showing the CDEGs from the GSE24395 and GSE114868 datasets. (C) Differential expression analysis of NAP1L5 in AML patients and healthy controls from the TCGA_GTEx-LAML, GSE24395, and GSE114868 datasets. (D, E) Expression analysis of NAP1L5 mRNA and protein in AML patients using qRT-PCR and Western blot, respectively. (F) K-M curves for OS of AML patients with different NAP1L5 expression levels in the TCGA-LAML database. ***, P < 0.001; ****, P < 0.0001.

Figure 3

Differential expression gene analysis and GSEA of NAP1L5 in TCGA. (A) Analysis of single-gene differences between groups in the TCGA-LAML dataset with high and low NAP1L5 expression. (B) Heatmap of 20 genes co-expressed with NAP1L5. (C) GSEA enrichment analysis reveals four key biological functions primarily involved in NAP1L5 and its enrichment in olfactory transduction, nervous system, apoptosis, and DNA replication pathways. (FDR, false discovery rate; NES, normalized enrichment score; Significant enrichment screening criteria were set at P < 0.05 and FDR P < 0.05).

Figure 4

GO enrichment and interaction network analysis of NAP1L5. (A) GO analysis of NAP1L5 and 20 related genes. (B) NAP1L5’s Protein-Protein Interaction (PPI) network. (C) NAP1L5’s mRNA-miRNA network. (D) NAP1L5’s mRNA-TF network. (E) NAP1L5’s mRNA-RBP network.

Figure 5

Immune infiltration analysis of NAP1L5. (A) Correlation between NAP1L5 expression levels and immune infiltration (CIBERSORT algorithm). (B) Correlation analysis of immune cell infiltration abundance. (C) Lollipop plot of immune cell correlations with NAP1L5 in TCGA-LAML dataset.

Figure 6

Nomogram construction and validation. (A) Forest plot of univariable Cox regression analysis integrating NAP1L5 expression levels and associated clinical factors. (B) Nomograph prediction model based on NAP1L5 expression levels and associated clinical features. (C) Calibration curves with one, two, and three years for the Cox regression prediction model. (D) DCA plots for the Cox regression prognostic model at one, two, and three years. (E) Clinical prognostic model risk score plot. (F) Prognostic KM survival curves stratified by median Risk Score. (G) Time-dependent ROC analysis evaluating the diagnostic performance of Risk Score stratification for AML patient survival. (All based on the TCGA-LAML database).

Figure 7

Immunological evaluation and drug sensitivity analysis of NAP1L5. (A, B) Relationship between NAP1L5 expression levels and TMB. (C, D) Relationship between NAP1L5 expression levels and TIDE score. (E) Relationship between NAP1L5 expression levels and drug sensitivity.

Figure 8

Functional validation of NAP1L5 in AML through in vitro experiments. (A) Western blot analysis of NAP1L5 protein expression in HL60, U937, Kasumi-6, and KG-1 cell lines. (B) Validation of NAP1L5 knockdown efficiency in HL60 cells using lentiviral vectors expressing shRNA targeting NAP1L5 (sh-NAP1L5-1, sh-NAP1L5-2, sh-NAP1L5-3). (C, D) Confirmation of NAP1L5 mRNA and protein expression levels in HL60 cells following knockdown or overexpression. (E) CCK8 assay assessing the impact of NAP1L5 overexpression or silencing on HL60 cell proliferation. (F) Flow cytometry analysis evaluating the effect of NAP1L5 on HL60 cell apoptosis. (G) Transwell assay examining the influence of NAP1L5 on HL60 cell migration. (H) Soft agar colony formation assay assessing the effect of NAP1L5 on HL60 cell colony formation. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.