Graphical abstract

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Highlights

     * •
       ALDOA overexpression in various retinoblastoma cell types and
       tissues
     * •
       ALDOA knockdown reduces retinoblastoma cell viability and alters
       metabolism
     * •
       Itaconate inhibits retinoblastoma proliferation in both in vitro
       and in vivo models
     * •
       Targeting ALDOA holds potential for advancing retinoblastoma
       therapies
     __________________________________________________________________

   Molecular biology; Cancer; Transcriptomics

Introduction

   Retinoblastoma (RB) is the most prevalent ocular malignancy in infants
   and young children. It constitutes 2–4% of all pediatric tumors,
   wielding a profound impact on both the visual acuity and life
   expectancy of affected individuals.[56]^1 The incidence of RB typically
   ranges from 1 in 20,000 to 1 in 15,000, yielding approximately
   8,000–9,000 new cases among children annually.[57]^2 Among the RB
   patients who experience metastasis, an alarming 80% eventually face a
   fatal outcome, with the invasive extraocular extension emerging as
   pivotal contributors to the metastatic process.[58]^3 Recent years have
   witnessed notable progress in the diagnosis and management of RB,
   leading to substantial improvements in both survival and rates of eye
   preservation. However, it is crucial to note that the advanced RB
   remain refractory to current therapeutic modalities, resulting in a
   less favorable prognosis.

   The phenomenon, known as aerobic glycolysis or the Warburg effect, has
   evolved beyond a mere adaptation to the environment, now recognized as
   an integral component of malignant phenotypes and tumor metabolic
   reprogramming.[59]^4 Enhanced glycolysis in tumors not only fuels their
   proliferation but also provides intermediates for synthesizing critical
   biomolecules like nucleotides, lipids, and proteins.[60]^5
   Consequently, targeting tumor glycolysis has arisen as a promising
   strategy in cancer therapy, as tumor cells adjust their energy
   metabolism to meet the demands of growth, biosynthesis, and redox
   reactions. Aldolase is a pivotal enzyme within the glycolysis pathway,
   facilitating the breakdown of 1,6-diphosphate-D-fructose into
   3-phosphate-D-glyceraldehyde, and subsequently interacting with
   α-dihydroxyacetone phosphate.[61]^6 The aldolase family comprises three
   members: ALDOA, ALDOB, and ALDOC. Notably, ALDOA has been observed to
   be overexpressed in a variety of malignant tumors, including gastric
   cancer, hepatocellular carcinoma, colorectal cancer, and cervical
   cancer.[62]^7^,[63]^8^,[64]^9^,[65]^10

   Our recent research into the metabolic microenvironment of RB has
   revealed an upregulation of the glycolytic pathway.[66]^11 In this
   study, we aim to investigate the expression of key glycolytic enzymes
   in RB at the single-cell level. By integrating single-cell RNA
   sequencing (scRNA-seq) datasets from RB and normal retinal tissues, we
   compared gene expression profiles and confirmed the upregulation of
   ALDOA in RB. Subsequent in vitro and in vivo experiments demonstrated
   the effectiveness of targeted ALDOA inhibition in suppressing RB
   growth.

Results

Elevated ALDOA expression in retinoblastoma

   GSEA of DEGs between retina and RB revealed significant enrichment in
   gene sets related to amide biosynthesis, glycolysis, catalytic
   activity/metabolism, and ATP synthesis ([67]Figure 1A). This suggests a
   significant alteration in energy metabolism in RB, highlighting the
   potential crucial role of the glycolytic pathway in RB. Expression
   patterns of key genes within the glycolytic pathway was showed in
   heatmap ([68]Figure 1B). Through dimensionality reduction and
   subsequent clustering analysis utilizing Uniform Manifold Approximation
   and Projection (UMAP), we visualized the expression profiles of
   Aldolase A (ALDOA) in RB and normal retina ([69]Figure 2A). The
   analysis unveiled a high expression level of ALDOA in RB
   ([70]Figure 2B), particularly elevated in extraocular RB compared to
   intraocular cases ([71]Figure 2C). Branched expression analysis
   modeling unveiled a clear upregulation of ALDOA expression in
   retinoma-like cells as they progressed through pseudotime. However,
   this unique expression profile was not evident in other cell types
   ([72]Figure S1). To validate ALDOA expression in RB, we examined its
   levels in human RB tissue samples and RB cell lines (WERI-RB1, Y79).
   The results demonstrated upregulated ALDOA mRNA and protein levels in
   RB cells compared to ARPE-19 ([73]Figures 3A and 3B). Additionally, we
   analyzed ALDOA mRNA expression using the [74]GSE125903 dataset[75]^12
   from the Gene Expression Omnibus (GEO) database, distinguishing between
   invasive and non-invasive RB cases. The analysis revealed higher ALDOA
   expression in invasive RB cases compared to non-invasive ones
   ([76]Figure 3C). Proteins extracted from tissues of 5 intraocular and 5
   extraocular RB patients revealed higher ALDOA protein expression in
   extraocular RB, as detected by western blotting ([77]Figure 3D). IHC
   results further confirmed significantly higher ALDOA expression in
   extraocular RB compared to intraocular RB and normal retina
   ([78]Figure 3E). Clinicopathological analysis revealed a trend toward
   higher ALDOA expression in cases with high-risk features like scleral
   and optic nerve invasion. Notably, ALDOA expression was significantly
   lower in younger patients (<2 years old) ([79]Figure S2).

Figure 1.

   [80]Figure 1
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   Enrichment analysis and glycolytic pathway expression in retinoblastoma

   (A) Gene set enrichment analysis (GSEA) of differentially expressed
   genes (DEGs) between normal retina and retinoblastoma (RB) samples.

   (B) Heatmap representation of the expression patterns of key genes
   within the glycolytic pathway, highlighting their differential
   expression in RB compared to normal retina.

Figure 2.

   [82]Figure 2
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   Visualization of ALDOA expression profiles in retinoblastoma and normal
   retina

   (A) Visualization of the expression profiles of Aldolase A (ALDOA) in
   retinoblastoma (RB) and normal retina using uniform manifold
   approximation and projection (UMAP).

   (B) Comparison of ALDOA expression between RB and normal retina.

   (C) Comparison of ALDOA expression between extraocular RB and
   intraocular case.

   (D) ALDOA expression across various cell types.

Figure 3.

   [84]Figure 3
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   ALDOA overexpression in various retinoblastoma cell types and tissues

   (A and B) Quantitative PCR (A) and western blot assays (B) revealed
   significantly higher messenger RNA (mRNA) and protein levels of ALDOA
   in retinoblastoma (RB) cells compared to normal cells.

   (C) ALDOA mRNA expression levels in non-invasive and invasive
   retinoblastoma cases from the [86]GSE125903 dataset.

   (D) Western blot analysis of ALDOA protein expression in tissues
   obtained from 5 intraocular and 5 extraocular RB patients,
   demonstrating elevated ALDOA protein levels in extraocular RB.

   (E) Immunohistochemistry (IHC) staining and a statistical analysis of
   ALDOA expression in normal retina, intraocular RB tissue, and
   extraocular RB tissue. R, retina; RB, retinoblastoma; S, sclera; VC,
   vitreous cavity; ON, optic nerve. Data are presented as means ±
   standard deviations. Statistical significance was determined using
   Student’s t test, ∗p < 0.05, ∗∗p < 0.001, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Disruption of retinoblastoma cell growth and energy metabolism by ALDOA
downregulation

   Given the observed ALDOA overexpression in RB, we investigated the
   functional consequences of ALDOA downregulation in RB cell lines. The
   qRT-PCR and western blotting results showed a decrease in ALDOA mRNA
   and protein expression levels after transfection ([87]Figures 4A and
   4B). The CCK-8 assay indicated that upon ALDOA knockdown in Y79 and
   WERI-RB1 cells, noticeable alterations in cell viability were observed
   from the second day, with more pronounced differences becoming evident
   by the third day when compared to the si-NC groups ([88]Figure 4C).
   Additionally, the EdU assay results showed a decrease in the
   proliferation ability of Y79 and WERI-RB1 cells following ALDOA
   knockdown ([89]Figure S3). The colony formation assays revealed a
   significant decrease in RB cells colony formation abilities following
   the knockdown of ALDOA ([90]Figure 4D). Lactate assay results
   demonstrated a marked reduction in extracellular lactate level in RB
   cells following ALDOA knockdown ([91]Figure 4E), accompanied by
   elevated levels of reactive oxygen species and decreased ATP levels
   ([92]Figures 4F and 4G).

Figure 4.

   [93]Figure 4
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   Functional implications of ALDOA downregulation in retinoblastoma cell
   lines

   (A and B) Quantitative reverse transcription-polymerase chain reaction
   (qRT-PCR) (A) and western blotting analysis (B) revealed a substantial
   reduction in ALDOA mRNA and protein expression levels following
   transfection of RB cell lines.

   (C) The CCK-8 assay demonstrated inhibited cell proliferation in
   response to ALDOA downregulation.

   (D) Colony formation experiments indicated a reduced tumorigenic
   potential of RB cell lines after ALDOA knockdown.

   (E) Extracellular lactate analysis exhibited a notable decrease in
   lactate levels in RB cells treated with siALDOA for 48 h compared to
   the negative control.

   (F) Levels of reactive oxygen species (ROS) in RB cells treated with
   siALDOA for 48 h were increased compared to the negative control.

   (G) Intracellular ATP levels in RB cells treated with siALDOA for 48 h
   were reduced in comparison to the negative control. Data are presented
   as means ± standard deviations. Statistical significance was determined
   using Student’s t test, with ∗p < 0.05, ∗∗p < 0.001, and ∗∗∗p < 0.001
   denoting significance.

Signaling alteration in response to ALDOA downregulation in retinoblastoma

   Next, we performed transcriptome sequencing on both the si-ALDOA and
   si-NC WERI-RB1 to explore the downstream alterations resulting from
   ALDOA downregulation. Our transcriptome analysis revealed significant
   changes in gene expression patterns, highlighting 162 upregulated genes
   including PHRF1, SUSD2, IGFN1, UNC79, and 136 downregulated genes
   including PTP4A3, ARHGAP27, MYT1, CLK2 ([95]Figures 5A and 5B). Further
   pathway enrichment analysis of these differentially expressed genes
   (DEGs) indicated potential shifts in biological functions in RB
   following ALDOA downregulation. Significantly, the upregulated DEGs
   were notably enriched in pathways related to “Organic acid transport”,
   “Amino acid transport”, and “Carboxylic acid transport”
   ([96]Figure 5C). Conversely, the downregulated DEGs were predominantly
   associated with pathways involving “DNA-binding transcription activator
   activity” and “histone methyltransferase activity” ([97]Figure 5D).
   Gene set enrichment analysis (GSEA) further revealed that after ALDOA
   knockdown, upregulated DEGs were enriched in pathways such as
   glycosphingolipid biosynthesis, while downregulated DEGs were enriched
   in metabolic pathways like oxidative phosphorylation, glutathione
   metabolism, and fatty acid metabolism ([98]Figure S4). To validate the
   expression levels of the identified DEGs, we conducted qRT-PCR on
   selected genes displaying significant expression differences. The
   results demonstrated a substantial upregulation of SUSD2, ARHGAP27, and
   CLK2 mRNA levels in RB cell lines compared to the ARPE-19
   ([99]Figure 5E). Furthermore, in RB cell lines where ALDOA was knocked
   down, these genes exhibited altered mRNA expression patterns, with
   increased expression of SUSD2 and decreased expression of ARHGAP27 and
   CLK2 ([100]Figure 5F). Western blot results showed that after ALDOA
   knockdown, the protein level of SUSD2 increased while the levels of
   CLK2 and ARHGAP27 decreased in WERI-RB1 cells. However, in Y79 cell
   lines, an increase in SUSD2 protein level was not detected after ALDOA
   knockdown, and the trends for the other two proteins were also
   decreasing ([101]Figure 5G). Furthermore, to individually knock down
   SUSD2, ARHGAP27, and CLK2, three pairs of siRNAs were designed for each
   gene ([102]Figure 5H). CCK8 experiments revealed that knocking down
   ARHGAP27 led to a decrease in the proliferation capacity of RB cells
   ([103]Figure 5I).

Figure 5.

   [104]Figure 5
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   Effects of ALDOA downregulation on WERI-RB1 cell line

   (A) RNA-seq analysis was employed to examine alterations in the mRNA
   expression profile of WERI-RB1 cells following ALDOA downregulation.

   (B) Heatmap analysis illustrating differentially expressed genes (DEGs)
   in WERI-RB1 cells subjected to ALDOA knockdown.

   (C and D) Pathway enrichment analysis focused on the upregulated DEGs
   (C) and downregulated DEGs (D). Upregulated DEGs were significantly
   associated with pathways related to “Organic acid transport”, “Amino
   acid transport”, and “Carboxylic acid transport”, while downregulated
   DEGs were primarily linked to “DNA-binding transcription activator
   activity” and “Histone methyltransferase activity”.

   (E) Quantitative PCR analysis of selected DEGs (SUSD2, ARHGAP27, and
   CLK2).

   (F) Quantitative PCR analysis of SUSD2, ARHGAP27, and CLK2 in RB cell
   lines with ALDOA knockdown.

   (G) Western blot analysis showing changes in SUSD2, ARHGAP27, and CLK2
   protein levels following ALDOA knockdown.

   (H) Western blot analysis of SUSD2, ARHGAP27, and CLK2 protein
   knockdown.

   (I) Proliferation changes in WERI-RB1 cells following knockdown of
   SUSD2, ARHGAP27, and CLK2. Statistical significance was assessed using
   Student’s t test, with ∗p < 0.05 and ∗∗p < 0.001 indicating
   significance.

Itaconate suppresses retinoblastoma cell proliferation and inhibits WERI-RB1
xenograft growth

   Itaconate is an enzymatic inhibitor of ALDOA, which suppresses the
   catalytic activity of ALDOA in the glycolytic pathway through covalent
   modification of Cys73 and Cys339, without altering the protein
   expression level of ALDOA.[106]^13 Hence, we utilized itaconate as a
   means to inhibit the enzymatic activity of ALDOA, while keeping its
   protein expression level unaltered ([107]Figure 6A). Results from the
   CCK8 assay demonstrate a reduction in RB cell viability upon the
   addition of itaconate ([108]Figure 6B). Moreover, the introduction of
   itaconate led to alterations in energy metabolism within RB cells. Mass
   spectrometry analysis revealed alterations in cellular metabolite
   levels upon treatment with itaconate in RB cell lines. Specifically,
   there was a downward trend in the levels of metabolites involved in
   glycolysis pathways such as 3-Phospho-D-glycerate, Beta-D-Fructose 6
   phosphate, and D-Glucose-6-phosphate, although not statistically
   significant ([109]Figure 6F). However, metabolites associated with
   oxidative phosphorylation pathways including L-Malate acid, Fumarate,
   and Oxaloacetate exhibited a significant decrease ([110]Figure 6G). To
   further investigate, an RB murine model was established by injecting
   WERI-RB1 cells into the eyes of nude mice. 14 days post-grafting, the
   animals were randomized into groups and received intraocular
   administrations of either PBS, itaconate, or melphalan, with
   enucleation performed on day 40 ([111]Figures 7A and 7B). Analysis of
   eye weight revealed that both the itaconate and melphalan treatment
   groups had lower eye weights compared to the control group, although
   these differences were not statistically significant ([112]Figure 7C).
   External examination of the eyes and H&E staining showed significant
   corneal neovascularization and extraocular invasion in the PBS group,
   with extensive tumor cell infiltration in the vitreous body and
   anterior chamber. In contrast, the itaconate treatment group exhibited
   tumor cells primarily located anterior to the retina. The
   melphalan-treated mice had fewer intraocular tumor cells but showed
   signs of ophthalmatrophia and retinal structural abnormalities
   ([113]Figure 7D). Histological analysis revealed ophthalmatrophia in
   40% of the melphalan-treated eyes and 20% of the itaconate-treated
   eyes, while extraocular invasion was observed in 30% of the PBS group
   and 10% of the itaconate group ([114]Figure 7E). TUNEL staining showed
   a higher incidence of TUNEL-positive cells in the tumors of the
   itaconate group compared to PBS controls ([115]Figure 7F).
   Additionally, the itaconate group had a lower proportion of
   TUNEL-positive cells in the retina compared to the melphalan-treated
   group ([116]Figure 7G). These observations indicate that itaconate is
   effective in targeting intraocular RB cells similarly to melphalan,
   while maintaining a more favorable safety profile for retinal tissue.

Figure 6.

   [117]Figure 6
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   Itaconate-mediated inhibition of ALDOA and its impact on retinoblastoma
   in vitro and in vivo

   (A) Western blotting analysis Itaconate serves as an enzymatic
   inhibitor of ALDOA while preserving ALDOA’s protein expression level.

   (B) CCK8 assay reveal a reduction in RB cell viability following the
   addition of itaconate.

   (C, D, and E) Introduction of itaconate induces alterations in energy
   metabolism within RB cells, leading to reduced lactate production (C),
   increased generation of reactive oxygen species (ROS) (D), and
   decreased ATP generation (E).

   (F) Metabolite changes in the glycolytic pathway in RB cells treated
   with itaconate.

   (G) Metabolite changes in the oxidative phosphorylation pathway in RB
   cells treated with itaconate. Statistical significance was assessed
   using Student’s t test, with ∗p < 0.05 and ∗∗p < 0.001 indicating
   significance.

Figure 7.

   [119]Figure 7
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   Itaconate treatment in an RB murine model

   (A) Study schematic illustrating the experimental design.

   (B) Gross appearance of eyeballs 40 days post-xenograft.

   (C) Eye weight analysis (n = 10).

   (D) Representative views of enucleated eyes and H&E staining from each
   treatment group.

   (E) Proportion of eyes with ophthalmatrophia and extraocular RB in the
   different treatment groups.

   (F) Representative TUNEL images and quantitative analysis of
   intratumoral cells comparing the itaconate and melphalan groups.

   (G) Representative TUNEL images and quantitative analysis of retinal
   cells comparing the itaconate and PBS groups. Data are presented as
   means ± SD. Statistical significance was determined by unpaired,
   two-tailed Student’s t test, with ∗p < 0.05 and ∗∗p < 0.01 indicating
   significance.

Discussion

   In this study, we integrated the scRNA-seq datasets of normal retinal
   tissue and RB tissue, uncovering a significant upregulation of the
   glycolytic enzyme ALDOA in tumor tissue. Additionally, GSEA identified
   enrichment of gene sets containing ALDOA related to RB, particularly
   those associated with ATP generation, protein synthesis, and
   glycolysis. Next, we confirmed the elevated expression of ALDOA in both
   cell lines and patient tissues, and noted a significant increase in
   extraocular RB. Through the application of siRNA interference and the
   ALDOA inhibitor itaconate, we demonstrated that ALDOA inhibition alter
   tumor energy and metabolism and efficiently diminished the viability of
   RB cells and the tumor progression in the RB mouse model. Furthermore,
   by using transcriptome sequencing, we uncovered possible downstream
   targets under the control of ALDOA, such as SUSD2, ARHGAP27, and CLK2.

   The mutation or loss of RB1 is a fundamental etiological factor in the
   development of RB. RB1 encodes the RB protein, which directs cellular
   metabolism reprogramming through E2F-induced transcriptional
   mechanisms, involving processes such as glucose oxidation
   phosphorylation, fatty acid oxidation, and amino acid
   synthesis.[121]^14 Nonetheless, the exact role of metabolic
   reprogramming in tumor progression and the specific metabolic enzymes
   implicated in the development of RB remain incompletely understood.
   ALDOA, a member of the aldolase enzyme family, has been the subject of
   extensive research, shedding light on its intricate interactions with
   hypoxia-inducible factor-1 (HIF-1) and its crucial role in modulating
   the glycolytic pathway and the process of epithelial-mesenchymal
   transition (EMT).[122]^15 It is worth noting that ALDOA expression
   levels have been thoroughly examined across various malignancies,
   revealing a direct link between ALDOA expression and the aggressiveness
   and invasiveness of tumors. Furthermore, comprehensive survival
   analyses consistently emphasize a strong positive correlation between
   increased ALDOA expression and an adverse prognosis.[123]^16 In line
   with previous findings, our research results validate a noticeable
   upregulation of ALDOA in RB tissue. Importantly, in cases of
   extraocular RB, ALDOA demonstrates higher expression levels compared to
   intraocular cases, highlighting the pivotal role of ALDOA in promoting
   the initiation and progression of RB. Recent research has increasingly
   focused on RB invasiveness, identifying targets such as UBE2C, SOX4,
   and the MCM family.[124]^17^,[125]^18^,[126]^19 However, additional
   investigation is needed to clarify the specific role of ALDOA in the
   extraocular invasion of RB. Liquid biopsies, especially aqueous humor
   biopsies, show promise for predicting RB prognosis and assessing
   chemotherapy efficacy.[127]^20^,[128]^21^,[129]^22^,[130]^23
   Investigations utilizing aqueous humor biopsy have shown potential in
   aiding RB prognosis and assessing chemotherapy
   efficacy.[131]^24^,[132]^25 Thus, further exploration of tumor-related
   glycolytic enzymes in aqueous humor or blood samples may provide
   additional support for the diagnostic significance of ALDOA in
   extraocular RB.

   In the course of RB development, distinct cell types play varying
   roles. Our analysis of scRNA-seq dataset reveals a pronounced
   upregulation of ALDOA in specific cell types, particularly cone cell
   precursors, RB-like cells, and retinoma like cells. Cone precursor like
   cells represent an early-stage cell population with the inherent
   potential for differentiation into cones or malignant cells.[133]^26
   RB-like cells embody a highly proliferative malignant cell population
   characterized by a reduction in photoreceptor characteristics.[134]^27
   Retinoma like cells serve as transitional cell stages between
   premalignant cone precursors and fully developed tumor cells.[135]^28
   The specific high expression of ALDOA within these three cell types
   suggests its potential oncogenic role in RB. Apart from tumor cells,
   the tumor microenvironment comprises a complex network of cells,
   stroma, blood vessels, and immune cells surrounding the
   tumor.[136]^29^,[137]^30 Within the tumor microenvironment, the
   expression levels of ALDOA may be regulated, and ALDOA can influence
   the metabolism and survival status of tumor cells. Specifically, ALDOA
   may influence the function and status of other cells within the tumor
   microenvironment by affecting the energy metabolism and signaling
   pathways of tumor cells.[138]^31^,[139]^32 However, in this study, the
   impact of ALDOA on other cells within the RB microenvironment, such as
   immune cells, stromal cells, and endothelial cells, has not yet been
   investigated. Additionally, ALDOA may modulate factors such as oxygen
   concentration, pH balance, and nutrient supply within the tumor
   microenvironment, affecting the growth and chemoresistance of tumor
   cells.[140]^33^,[141]^34^,[142]^35 Moreover, previous studies have
   implicated the POU2F1-ALDOA axis in promoting tumor cell proliferation
   and chemoresistance.[143]^36 In this study, RNA sequencing revealed a
   significant alteration in POU2F1 expression following ALDOA knockdown,
   suggesting that the POU2F1-ALDOA axis may play a critical regulatory
   role in RB.

   Itaconate has the capability to inhibit the catalytic activity of ALDOA
   in the glycolytic pathway without altering the expression level of the
   ALDOA protein. Initially discovered as a metabolite activated in
   macrophages, itaconate plays a critical role in the negative regulation
   of inflammatory responses by suppressing the production of inflammatory
   factors associated with macrophage activation.[144]^37 This regulatory
   process primarily functions through the alkylation of KEAP1, leading to
   the activation of NRF2.[145]^38 In the research of tumor, there has
   been a hypothesis that itaconate might indirectly influence tumor
   progression by its impact on macrophages.[146]^39 However, it remains
   uncertain whether itaconate directly regulates tumor progression. In
   this study, we found that although the intravitreal injection of
   itaconate did not result in a reduction in the rate of intraocular
   tumorigenesis, there was a notable decrease in the size of intraocular
   tumors, as well as a reduction in structural damage to the eyeball
   following the treatment. These observations suggest the potential of
   itaconate as an adjunctive therapy for RB. One of the primary
   challenges in translating preclinical findings into clinical
   applications is navigating the complex regulatory landscape. For
   itaconate to be considered for clinical use in treating RB or other
   tumors, detailed pharmacokinetic studies must be conducted.
   Additionally, thorough investigation of potential side effects is
   necessary to ensure patient safety.

   Genes such as PHRF1, SUSD2, IGFN1, and UNC79 were identified as the top
   upregulated genes in ALDOA knockdown samples. SUSD2, situated on
   chromosome 22, encodes a transmembrane protein that plays a crucial
   role in cell-cell interactions and cell-matrix adhesion. Studies have
   indicated that SUSD2 acts as a tumor suppressor in RB by regulating the
   occurrence, development, and angiogenesis of RB, making it a potential
   target for RB treatment.[147]^40 Conversely, genes including PTP4A3,
   ARHGAP27, MYT1, and CLK2 were identified as the top downregulated genes
   in ALDOA knockdown samples. ARHGAP27 functions as a Rho
   GTPase-activating protein and is associated with pediatric chronic
   myeloid leukemia as well as ovarian cancer.[148]^41^,[149]^42 CLK2, a
   phosphorylation-involved splicing protein kinase, exerts a promotional
   role in various cancers.[150]^43^,[151]^44 These differentially
   expressed genes might play important roles in RB progression, part of
   which were validated in this study, while the intrinsic interaction
   network in RB still requires further exploration.

   In conclusion, our study illuminates the important role of ALDOA in
   driving tumor growth in RB. These findings not only offer insights into
   the metabolic dynamics of RB but also present potential target
   candidates for the diagnosis and therapy of RB patients.

Limitations of the study

   Our study has certain limitations that warrant discussion. First, the
   relatively small number of clinical samples used may constrain the
   analysis, potentially limiting the generalizability of our findings.
   Expanding the sample size and diversifying patient demographics would
   strengthen the robustness of our conclusions. Second, incorporating
   primary cells into future investigations could better capture the
   heterogeneity of RB, thus enhancing the validity of our results.
   Additionally, further exploration into patient outcomes and correlation
   with relevant clinical parameters would provide deeper insights into
   the clinical significance of our findings. Lastly, while our study
   successfully identifies downstream targets, a more comprehensive
   investigation is needed to elucidate the mechanistic intricacies of how
   ALDOA influences these targets and the broader signaling pathways
   involved.

Resource availability

Lead contact

   Further information and requests for resources should be directed to
   and will be fulfilled by the lead contact, Rong Lu (lurong@gzzoc.com).

Materials availability

   This study did not generate new unique reagents.

Data and code availability

     * •
       The raw sequence data reported in this paper have been deposited in
       the Genome Sequence Archive in National Genomics Data Center, China
       National Center for Bioinformation/Beijing Institute of Genomics,
       Chinese Academy of Sciences (GSA-Human: HRA008175) that are
       publicly accessible at [152]https://ngdc.cncb.ac.cn/gsa-human.
       Original western blot images have been provided in the
       supplementary material.
     * •
       This paper does not report original code.
     * •
       Any additional information required to reanalyze the data reported
       in this paper is available from the [153]lead contact upon request.

Acknowledgments