HK3 Human

What is HK3 and what is its primary function in human cells?

HK3 (Hexokinase 3) is a member of the hexokinase family that catalyzes the first step of glycolysis - the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. While HK1 and HK2 are ubiquitously expressed, HK3 shows tissue-specific expression, primarily in hematopoietic cells and tissues . Interestingly, recent research indicates that HK3 has functions beyond glycolysis, particularly in cell survival mechanisms and immune regulation .

Methodology: To study HK3's primary function, researchers should compare glycolytic activity in control versus HK3-knockout cells using techniques like extracellular acidification rate (ECAR) measurements with a Seahorse analyzer, while also investigating non-glycolytic functions through protein-protein interaction studies and survival assays.

How does HK3 differ from other hexokinase family members?

Unlike HK1 and HK2, HK3 exhibits highly tissue-specific expression patterns, predominantly in cells of myeloid origin . Functionally, while HK1 and HK2 are critical for glycolytic activity, HK3 appears dispensable for glycolysis in certain cell types like AML cells . CRISPR/Cas9-mediated gene disruption studies have shown that knocking out HK2 significantly reduces basal glycolysis and glycolytic capacity, whereas HK3 knockout does not affect these parameters . Instead, HK3 seems to have evolved non-glycolytic functions, particularly in promoting cell survival, possibly through interactions with pro-apoptotic proteins like BIM .

Where is HK3 predominantly expressed in human tissues?

HK3 expression is predominantly observed in hematopoietic cells and tissues . Within the hematopoietic system, it shows highest expression in cells of myeloid origin, particularly in basophils, neutrophils, and monocytes/macrophages as demonstrated by the Monaco and Schmiedel datasets in the Human Protein Atlas . Single-cell RNA sequencing analysis using the TISCH database confirms that HK3 is predominantly expressed in macrophages in multiple cancer types, including glioblastoma multiforme (GBM) . This cell type-specific expression pattern suggests specialized functions in myeloid cell biology.

What is the prognostic significance of HK3 expression across different cancer types?

• Glioblastoma multiforme (GBM)

• Kidney renal clear cell carcinoma (KIRC)

• Brain lower grade glioma (LGG)

• Thymoma (THYM)

• Uveal melanoma (UVM)

• Kidney chromophobe (KICH)

• Acute myeloid leukemia (LAML)

• Testicular germ cell tumors (TGCT)

• Liver hepatocellular carcinoma (LIHC)

Interestingly, in skin cutaneous melanoma (SKCM), high HK3 expression is associated with favorable OS, highlighting the context-dependent roles of HK3 . These findings suggest that HK3's prognostic significance must be evaluated in a tumor-specific manner.

How is HK3 expression dysregulated in cancer compared to normal tissues?

Analysis of HK3 expression at the pan-cancer level using TIMER2.0 has revealed dysregulation of HK3 expression in 14 types of human cancers . In GBM specifically, HK3 mRNA expression is significantly higher compared to normal brain tissue samples, as demonstrated in both TCGA-GBM and GTEx datasets . At the protein level, immunohistochemistry from the Human Protein Atlas confirms upregulation of HK3 protein in GBM compared to normal tissue . Cell line studies have shown that HK3 mRNA expression is significantly higher in certain GBM cell lines (A172 and U373) compared to normal human astrocytes (NHA), though not all GBM cell lines show this upregulation (U87) .

What biological pathways are enriched in high HK3-expressing tumors?

Gene ontology (GO) and pathway enrichment analyses reveal distinct biological signatures in tumors with high versus low HK3 expression:

In high HK3-expressing GBM tumors, the top enriched GO terms include:

• Activation of immune response

• Acute inflammatory response

• Adaptive immune response

• Adaptive immune response based on somatic recombination of immune receptors

• Alpha-beta T cell activation

The top KEGG pathways enriched in high HK3 expression include:

• Chemokine signaling pathway

• Cytokine-cytokine receptor interaction

• Hematopoietic cell lineage

• Lysosome

• NOD-like receptor signaling pathway

In contrast, low HK3-expressing tumors show enrichment in cell cycle, chromosome segregation, and ribosomal functions . These patterns suggest HK3's involvement in immune-related processes within the tumor microenvironment.

How does HK3 relate to tumor-associated macrophages (TAMs) in the cancer microenvironment?

HK3 shows a remarkably strong correlation with macrophage infiltration in tumors. In GBM, among all genes, HK3 exhibits the strongest correlation with macrophages (p < .001, R = .81) . Further analysis reveals that HK3 expression is specifically associated with M2-like (tumor-promoting) macrophages rather than M1 (anti-tumor) macrophages .

Methodologically, researchers can investigate this relationship using single-cell RNA sequencing, flow cytometry for macrophage markers, and co-culture experiments between HK3-manipulated macrophages and tumor cells.

What is the relationship between HK3 and immune checkpoint molecules?

HK3 shows significant positive correlations with multiple immune checkpoint molecules and immunoregulatory genes. Analysis of TCGA and CGGA cohorts reveals positive correlations between HK3 and:

• PD-1 (p < .001, R = .41)

• PD-L1 (p < .001, R = .27)

• CTLA-4 (p < .001, R = .29)

HK3 also positively correlates with most major histocompatibility complex (MHC), immunosuppressive, immune activation, chemokine, and chemokine receptor genes . These correlations suggest potential functional relationships between HK3 and immune regulatory pathways that influence T cell activation and function within the tumor microenvironment.

Tumor Immune Dysfunction and Exclusion (TIDE) analysis reveals that tumors with low HK3 expression have lower TIDE scores, suggesting they might benefit more from immunotherapy compared to tumors with high HK3 expression .

How does HK3 expression change during myeloid cell differentiation?

HK3 expression is significantly upregulated during terminal differentiation of myeloid cells. This upregulation is not restricted to leukemic cell differentiation but also occurs during ex vivo myeloid differentiation of healthy CD34+ hematopoietic stem and progenitor cells . This pattern suggests that HK3 may play important roles in mature myeloid cell function.

In AML cell line models, HK3 is highly upregulated during terminal differentiation . Loss of HK3 through CRISPR/Cas9-mediated gene disruption leads to increased sensitivity to ATRA-induced cell death, suggesting that HK3 promotes cell survival during differentiation .

Methodologically, researchers can track HK3 expression during differentiation using qRT-PCR, western blotting, and single-cell RNA sequencing approaches combined with differentiation assays using agents like ATRA.

How does HK3 promote cell survival independent of its glycolytic function?

While the hexokinase family is known for catalyzing the first step of glycolysis, HK3 appears to promote cell survival through non-glycolytic mechanisms:

• Interaction with pro-apoptotic proteins: HK3 directly interacts with the pro-apoptotic BCL-2 family member BIM, which has previously been shown to shorten myeloid lifespan . This interaction may inhibit BIM's pro-apoptotic functions.

• Protection against oxidative stress: Loss of HK3 leads to accumulation of reactive oxygen species (ROS) and DNA damage during ATRA-induced differentiation of AML cells .

• Regulation of apoptotic pathways: RNA sequencing analysis confirms pathway enrichment for programmed cell death, oxidative stress, and DNA damage response in HK3-null AML cells .

• Chromatin configuration changes: ATAC sequencing shows that loss of HK3 leads to changes in chromatin accessibility, increasing the accessibility of genes involved in apoptosis and stress response .

These findings provide evidence that HK3 promotes cell survival independently of its role in glucose metabolism, potentially making it a unique therapeutic target.

What experimental approaches can distinguish between HK3's glycolytic and non-glycolytic functions?

To accurately differentiate between HK3's glycolytic and non-glycolytic functions, researchers should employ a multi-faceted approach:

• Glycolytic function assessment:

  • Measure glucose uptake using radiolabeled glucose or fluorescent glucose analogs

  • Quantify glycolytic intermediates using mass spectrometry

  • Assess glycolytic flux using extracellular acidification rate (ECAR) measurements

  • Determine hexokinase activity using biochemical assays

• Non-glycolytic function assessment:

  • Evaluate cell survival and apoptosis using flow cytometry (Annexin V/PI staining)

  • Assess ROS levels using fluorescent probes

  • Analyze DNA damage through γH2AX staining or comet assays

  • Investigate protein-protein interactions using co-immunoprecipitation or proximity ligation assays

• Comparative analysis:

  • Compare effects of HK3 manipulation with manipulation of other hexokinases (HK1, HK2)

  • Use metabolic inhibitors to distinguish between glycolytic and non-glycolytic effects

  • Perform rescue experiments with wild-type and mutant HK3 constructs

Current evidence from CRISPR/Cas9-mediated gene disruption studies demonstrates that loss of HK3 has no effect on glycolytic activity in AML cell lines, while knocking out HK2 significantly reduces basal glycolysis and glycolytic capacity .

What is known about HK3's interactions with BIM and implications for apoptosis regulation?

HK3 directly interacts with the pro-apoptotic BCL-2 family member BIM, as demonstrated through isoform-specific pulldown experiments . BIM is a critical mediator of programmed cell death and has been shown to shorten myeloid cell lifespan.

The functional consequences of this interaction appear significant:

• HK3 knockout AML cells show increased sensitivity to ATRA-induced cell death

• Loss of HK3 leads to accumulation of ROS and DNA damage during differentiation

• RNA sequencing analysis confirms enrichment of programmed cell death pathways in HK3-null cells

These findings suggest that HK3 may inhibit BIM's pro-apoptotic functions, thereby promoting cell survival. This represents a novel, non-glycolytic function of HK3 that could be therapeutically targeted.

Methodologically, researchers can further investigate this interaction through detailed mapping of interaction domains, analysis of how this interaction affects downstream apoptotic signaling, and exploration of whether this interaction is regulated by cellular stress or differentiation signals.

What is the potential of HK3 as a dual-functional biomarker in cancer diagnosis and prognosis?

HK3 shows significant potential as a dual-functional biomarker in cancer:

These multi-faceted associations position HK3 as a promising biomarker not only for diagnosis and prognosis but also for guiding therapeutic decisions.

How might targeting HK3 affect cancer cell survival and immune responses?

Targeting HK3 could affect cancer progression through multiple mechanisms:

• Direct effects on cancer cell survival: In cells where HK3 is expressed, its inhibition could lead to increased sensitivity to apoptosis, potentially by releasing BIM from inhibition . RNA sequencing analysis confirms that HK3-null cells show enrichment in programmed cell death pathways .

• Effects on the tumor microenvironment: Given HK3's predominant expression in macrophages and strong correlation with M2-like TAMs, targeting HK3 might modify macrophage polarization or function within the tumor microenvironment . This could potentially shift the balance from protumoral M2-like macrophages toward antitumoral M1-like phenotypes.

• Immunomodulatory effects: HK3 expression correlates with immune checkpoint molecules like PD-1, PD-L1, and CTLA-4 . Inhibiting HK3 might therefore influence immune checkpoint pathways and potentially enhance anti-tumor immune responses.

• Overcoming drug resistance: Drug sensitivity analysis shows that high HK3 expression correlates with drug resistance . Targeting HK3 might therefore sensitize resistant tumors to conventional therapies.

These multi-faceted effects make HK3 an intriguing therapeutic target, particularly in cancers where its expression is associated with poor outcomes.

What methodological approaches are optimal for evaluating HK3 expression in patient samples?

For comprehensive evaluation of HK3 expression in patient samples, researchers should consider a multi-modal approach:

• Transcriptomic analysis:

  • Quantitative RT-PCR for targeted HK3 mRNA measurement

  • RNA sequencing for genome-wide expression analysis

  • Single-cell RNA sequencing to identify cell type-specific expression patterns

• Protein-level analysis:

  • Immunohistochemistry to assess protein expression and localization in tissue sections

  • Western blotting for semi-quantitative protein level assessment

  • Flow cytometry for cell type-specific protein expression in dissociated samples

• Bioinformatic approaches:

  • Utilization of public databases such as TCGA, CGGA, TISCH, and HPA for comparative analysis

  • Integration of multiple datasets for robust validation

  • Correlation with clinical parameters and outcomes

• Functional assessment:

  • Ex vivo drug sensitivity testing

  • Immune cell profiling in the tumor microenvironment

  • Assessment of glycolytic and non-glycolytic parameters

This comprehensive approach allows for robust evaluation of HK3 expression patterns and their clinical significance, providing valuable insights for personalized medicine approaches.

What are the key unresolved questions regarding HK3's role in human diseases?

Several critical questions about HK3 remain unanswered:

• Structural basis of function: How does the structure of HK3 differ from other hexokinases, and how do these differences contribute to its unique functions?

• Regulatory mechanisms: What factors regulate HK3 expression in different cell types, and how is its expression dysregulated in disease states?

• Interaction network: Beyond BIM, what other proteins interact with HK3, and how do these interactions contribute to its non-glycolytic functions?

• Role in normal physiology: What is the physiological role of HK3 in normal myeloid cell function and homeostasis?

• Cancer-specific functions: How does HK3 contribute to cancer progression in different tumor types, and why does its prognostic significance vary across cancers?

• Therapeutic targeting: Can HK3 be specifically targeted without affecting other hexokinases, and what would be the therapeutic window for such interventions?

• Biomarker validation: How can HK3 expression be effectively integrated into clinical decision-making as a diagnostic, prognostic, or predictive biomarker?

Addressing these questions will require integrated approaches combining structural biology, molecular and cellular biology, immunology, and clinical studies.

How might single-cell technologies advance our understanding of HK3 biology?

Single-cell technologies offer powerful approaches to elucidate HK3 biology:

• Cell type-specific expression patterns: Single-cell RNA sequencing can provide high-resolution maps of HK3 expression across different cell types in normal and diseased tissues. Current evidence already indicates predominant expression in myeloid cells, particularly macrophages .

• Heterogeneity within macrophage populations: Given HK3's strong association with macrophages, single-cell approaches can reveal heterogeneity in HK3 expression among macrophage subpopulations and correlate this with functional states.

• Trajectory analysis: Single-cell trajectory analysis can track how HK3 expression changes during cellular differentiation or activation processes, providing insights into its regulation.

• Spatial context: Spatial transcriptomics and proteomics can reveal how HK3-expressing cells are distributed within tissues and how they interact with neighboring cells.

• Functional genomics at single-cell resolution: Combining CRISPR screens with single-cell readouts can identify genes that regulate HK3 expression or that are regulated by HK3.

These approaches will provide unprecedented resolution of HK3's roles in normal physiology and disease, potentially revealing new therapeutic opportunities and biomarker applications.