Spatial and single-cell analyses uncover links between ALKBH1 and tumor-associated macrophages in gastric cancer

STAD predominantly affects populations in Asian and South American countries [1], and is characterized by high levels of intra- and inter-tumor heterogeneity leading to poor overall survival rates worldwide. The disease is often diagnosed at an advanced stage, resulting in a dismal prognosis due to factors such as metastasis, high intra-tumor heterogeneity, and resistance to chemotherapy [2, 3]. Although the classification and terminology of gastric cancer subtypes can vary across different regions, it is widely acknowledged as a fatal disease. Improved diagnosis and precision medicine strategies are urgently needed to address the current challenges and enhance the prognosis for patients [4]. Epigenetic mechanisms involve hundreds of proteins that tightly coordinate to maintain the normal structure of the epigenome. Somatic mutations or alterations in the levels of epigenome regulatory factors play a crucial role in the formation of cancer epigenomes [5].

The AlkB gene found in E. coli codes for a DNA repair enzyme which plays a crucial role in the reversal of DNA lesions, specifically 1-methyladenine (1 mA) and 3-methylcytosine (3mC). This DNA repair enzyme is a member of the AlkB family, which is classified as an Fe (II)- and α-ketoglutarate (αKG)-dependent dioxygenase [6‐8]. However, research over the years has shown that AlkB homologs are widely distributed in eukaryotic cells and function as demethylases on various substrates, including DNA, RNA, and histones [9, 10]. The mammalian AlkB family consists of nine homologs, including ALKBH1-8 and FTO, all of which possess the dioxygenase functional domain [11, 12]. Although they share similar structural features, they are found in different cellular compartments and catalyze different substrates, leading to distinct biological functions [13‐16].

ALKBH1 is a member of the human AlkB family, with a protein structure consisting of 389 amino acids and a molecular structure containing a highly conserved double-stranded β-helix (DSBH) fold, which is a characteristic feature of the ⍺KG-dependent dioxygenase superfamily and a central catalytic core [16, 17]. The primary demethylating enzymatic activity of ALKBH1 is on nucleic acids such as DNA, mRNA, and tRNA [18‐20]. As a DNA demethylase, ALKBH1 primarily targets N6-methyladenine (N6mA) in DNA. The N6mA motif is closely associated with the common heterochromatin marker H3K9me3, leading to alterations in chromatin accessibility, as evidenced by CHIP-seq studies [21, 22].

ALKBH1's interaction with diverse substrates highlighted the significance of genomic DNA's N6mA modification, especially in cancer. In pancreatic ductal adenocarcinoma (PDAC), the downregulation of ALKBH1 disrupted mitochondrial DNA-encoding gene transcription, triggering mitochondrial impairment. Sirtuin4, on the other hand, modulated ALKBH1 stability by deacetylating the HRD1-SEL1L complex, thereby maintaining mitochondrial equilibrium in PDAC cells [23]. Conversely, ALKBH1 was markedly upregulated in colorectal and breast cancers, correlating with metastasis and an unfavorable prognosis [24]. Intriguingly, breast cancer investigations revealed a 7% genetic alteration involving ALKBH1 [25]. In lung cancer, ALKBH1's elevated expression was linked to enhanced invasion and migration of cancer cells in vitro, while its silencing significantly curtailed these abilities. This phenomenon was substantially potentiated by ALKBH1 overexpression [25]. Similarly, a study on tongue squamous cell carcinoma (TSCC) unveiled heightened genomic N6mA levels in TSCC tissues and cultured cells. Suppression of ALKBH1 led to heightened N6mA levels in genomic DNA, promoting tumor colony formation and cell migration [26]. In ovarian cancer, the genetic alteration rate of ALKBH1 stood at 16%. In addition to lowered tumor expression levels, an overall reduction in methylation levels was also observed [27]. ALKBH1's demethylation of 6 mA inhibits NRF1-driven transcription, impacting genes in the AMPK signaling pathway. This inhibition shifts metabolism toward the Warburg effect, promoting STAD tumorigenesis [5].

This study aimed to investigate the theragnostic value of ALKBH1 in STAD using integrated analysis. We conducted differential gene expression, protein correlation, pathway, and prognostic analyses to examine the clinical significance of ALKBH1 in different tumor types and stages. Our results confirmed the clinicopathological significance of ALKBH1 in STAD patients and identified it as a potential prognostic biomarker. We also determined the association between ALKBH1 expression and TIME at the single-cell and whole-tissue levels, and identified a possible mechanism accounting for its tumor-promoting role. Additionally, our study compared ALKBH1 expression with immune-infiltrating cells and correlation of immunomodulatory factors, suggesting its potential as an immunotherapeutic target. Finally, we accessed the Cancer Drug Sensitivity Genomics (GDSC) and Cancer Cell Lineage Encyclopedia (CCLE) cell repositories, and identified ALKBH1 as a validated drug candidate in the context of targeting.

ALKBH1 is a multifaceted enzyme that has been implicated in various biological processes, including DNA and RNA demethylation. Among its substrates, N6-methyladenosine (N6mA) on genomic DNA is considered the most critical for cancer effects [41]. This study highlights that Copy Number Variations (CNV) and mutation status associated with the ALKBH1 gene regulate the progression of STAD, concurrently influencing immune cell infiltration in the tumor microenvironment. We will comprehensively explore the roles played by ALKBH1 gene variations in various cancers. In glioblastoma, N6mA levels are significantly elevated and co-localized with heterochromatin histone modifications, mainly H3K9me3. Downregulation of ALKBH1 leads to increased levels of N6mA in genomic DNA, which coordinates with H3K9me3 to reduce chromatin accessibility and silence transcription of some oncogenes [18]. In a patient-derived human glioblastoma model, the deliberate reduction of ALKBH1 expression through targeted knockdown proved to be highly effective. This intervention resulted in a substantial inhibition of tumor cell proliferation, subsequently leading to a significant extension in the survival of mice involved in the study. These findings hold promise for the development of novel therapeutic strategies in the context of glioblastoma treatment [21]. However, the function of ALKBH1 in cancer seems to vary depending on the tissue type. For instance, in squamous cell carcinoma of the tongue (TSCC), both TSCC tissues and cultured cells exhibit heightened levels of genomic N6mA [17]. Surprisingly, silencing ALKBH1 increases N6mA levels in genomic DNA, ultimately amplifying tumor colony formation and cell migration [42]. In contrast, targeted knockouts of N6AMT1 and METTL4, two methyltransferases that typically counteract the effects of ALKBH1, significantly reduced genomic N6mA levels. This reduction markedly inhibited the ability of TSCC cells to form colonies and migrate. These findings underscored the complex regulatory network involving these methyltransferases and suggested their potential as therapeutic targets for TSCC [13].

On the other hand, the knockdown of N6AMT1 and METTL4, which are methyltransferases with opposing functions to ALKBH1, results in a reduction of genomic N6mA levels, leading to the inhibition of TSCC cell colony formation and cell migration [26]. Similar results were observed in the study of HCC, where the overexpression of N6AMT1, a methyltransferase, increased N6mA levels, resulting in enhanced cell viability, reduced apoptosis, and increased cell migration and invasion, whereas the overexpression of ALKBH1 had the opposite effect [43]. Additionally, overexpression of ALKBH1 was found to reverse the inhibitory effect of miRNA-339-5p, an upstream gene of ALKBH1, on the migration and proliferation of STAD [44]. Apart from its role as a genomic DNA N6mA demethylase, ALKBH1 can also bind mRNA and act as a demethylase. In both lung cancer tissues and cultured cells, there was an observed upregulation in the expression of ALKBH1. Silencing ALKBH1 in these lung cancer cells led to a significant inhibition of their in vitro invasion and migration capabilities. Conversely, when ALKBH1 was overexpressed, it notably enhanced these invasive and migratory properties. These findings shed light on the critical role of ALKBH1 in lung cancer progression and its potential as a therapeutic target [45]. Similarly, ALKBH1 overexpression promotes metastasis in CRC through modifying METTL3 mRNA m1A levels, resulting in reduced protein translation, increased m6A demethylation of SMAD7 mRNA, and enhanced tumor migration and invasion. Silencing SMAD7 can significantly reverse cell migration and invasion defects caused by depletion of ALKBH1 and METTLE3 [24].

The present study investigates the potential of ALKBH1 as a diagnostic and prognostic biomarker for immune infiltration in STAD. It is well established that the tumor microenvironment and immune cell infiltration play crucial roles in the host's immune response to cancer cells. In this research, we have showcased the power of single-cell RNA sequencing (RNA-seq) technology, which facilitates the profiling of transcriptomes at the individual cell level. This cutting-edge technology offers unprecedented capabilities to dissect intricate aspects of tumorigenesis and cancer progression. By employing these advanced techniques, we can precisely sequence transcripts, thereby enhancing our comprehension of the diverse cellular composition within the STAD tumor microenvironment. Furthermore, it allows us to unravel the intricate interactions among cells within the complex and heterogeneous landscape of cancer tissues. Our findings suggest that the ALKBH1 cluster network is involved in inflammatory and immune-related pathways and may serve as a new diagnostic window for monitoring the tumor microenvironment. We validated our hypothesis using a multicomponent approach that analyzed data from TCGA, spatial transcription from GEO database, and single-cell sequencing datasets. Our analysis revealed a positive correlation between macrophage infiltration and ALKBH1 expression in the STAD infiltration cohort, suggesting that ALKBH1 may reflect immune status in addition to disease prognosis. The pathway enrichment analysis of the ALKBH1 positive and negative correlation clustering network supports this observation. Thus, these findings suggest that ALKBH1 is involved in the immune infiltration of STAD and may serve as a prognostic biomarker of the immune response to these cancers. Importantly, our study provides clinical implications for the prognostic evaluation and follow-up management of immunotherapy.

This research is not without its limitations. We conducted a drug screening process, explored various cell lines using pharmacogenomics, and identified six potential targets for ALKBH1 inhibition in STAD cells. Furthermore, there is a need to recruit a substantial number of patients with STAD for the examination of ALKBH1 and the analysis of its correlation with tumor progression. While targeting the ALKBH1 signaling axis could potentially yield dual benefits by suppressing genes and enhancing the immunotherapeutic response in STAD, it's important to note that further experimental validation is required to delve into the molecular mechanisms linked to ALKBH1 in STAD. In summary, our study contributes to a deeper comprehension of ALKBH1's role in STAD, particularly from the standpoint of clinical tumor samples, and paves the way for the exploration of novel immunotherapeutic strategies.

In summary, this study unveils crucial insights into the intricate molecular biology of ALKBH1, particularly in the context of STAD. Elevated ALKBH1 expression is associated with unfavorable outcomes in STAD, impacting both the tumor microenvironment and macrophage infiltration. ALKBH1 stands out as a promising candidate for diagnostic, prognostic, and immune-related therapeutic interventions in STAD (Fig. 11). However, further investigations are necessary to validate our findings and unravel the immunomodulatory role and underlying mechanisms of ALKBH1 in STAD. Concurrently, the six drugs identified through pharmacogenomics predictions show potential for inhibiting STAD development. This study underscores the significant correlation between ALKBH1 and macrophages, pointing towards genes that merit deeper exploration for advancing diagnostic precision, treatment strategies, and prognostication for STAD patients.