ID2 Human

What is the molecular mechanism of ID2 function in human immune cells?

ID2 functions as a transcriptional repressor that controls the amplitude and temporal dynamics of TCF1 to program natural killer (NK) cell maturation. As a helix-loop-helix protein lacking a DNA-binding domain, ID2 binds to and inhibits basic helix-loop-helix transcription factors, preventing their dimerization and subsequent DNA binding. In NK cells, ID2 sets a threshold for TCF1 expression, thereby controlling the balance between immature and terminally differentiated cells that support future NK cell responses . This mechanism is essential for establishing proper NK cell effector functions, including cytokine-induced IFN-γ production and the ability to clear metastatic melanoma.

How does ID2 expression differ across human cell lineages?

ID2 expression exhibits significant variation across human cell types, with constitutive expression observed in NK cells but differential patterns in other immune and non-immune cells. In neuroblastoma, a striking 20-fold overexpression of ID2 has been documented in anchorage-dependent (AD) cells compared to anchorage-independent (AI) cells . In CD8+ T cells, ID2 shows dynamic expression patterns that correlate with exhaustion states, with different levels observed in progenitor exhausted (Tex prog) versus terminally exhausted (Tex term) subpopulations . The cell-specific expression patterns reflect ID2's context-dependent roles in various developmental and pathological processes.

What signaling pathways regulate ID2 expression in human cells?

Multiple signaling cascades converge to regulate ID2 expression:

ID2 functions partially through the TGF-β pathway, with co-immunoprecipitation studies demonstrating direct binding between ID2 and both TGF-β and Smad2/3 . This interaction represents a crucial regulatory mechanism, as inhibition of TGF-β signaling in ID2-suppressed cells leads to increased apoptosis, indicating that ID2 normally sequesters these factors to modulate their activity.

What is known about the genomic organization of the human ID2 gene?

The human ID2 gene is relatively compact, containing coding regions that specify the protein's functional domains, particularly the conserved helix-loop-helix (HLH) domain that mediates protein-protein interactions. Regulatory elements in the ID2 promoter respond to diverse transcription factors, allowing for context-specific expression patterns. Understanding the genomic architecture of ID2 provides insight into its regulation and the basis for designing targeted genetic manipulations for research purposes.

How does ID2 epigenetically control CD8+ T-cell exhaustion states?

ID2 orchestrates CD8+ T-cell exhaustion through a complex mechanism of transcriptional and epigenetic regulation. Through its HLH domain, ID2 binds and disrupts the assembly of the Tcf3-Tal1 transcriptional regulatory complex, thereby modulating chromatin accessibility at key loci such as the Slamf6 promoter . This mechanism prevents the interaction of transcription factors with regulatory elements, altering the epigenetic landscape. Genetic deletion of ID2 dampens CD8+ T-cell-mediated immune responses and impairs the maintenance of stem-like CD8+ T-cell subpopulations, ultimately suppressing PD-1 blockade efficacy and increasing tumor susceptibility . These findings demonstrate that ID2-mediated transcriptional and epigenetic modification drives hierarchical CD8+ T-cell exhaustion and impacts anti-tumor immunity.

What role does ID2 play in the phenotypic transition of cancer cells?

ID2 functions as a critical regulator of cancer cell phenotypic plasticity, particularly in neuroblastoma. High ID2 expression in anchorage-dependent (AD) neuroblastoma cells promotes proliferation and prevents phenotypic transition. When ID2 is downregulated in these cells, they undergo significant behavioral changes, including:

• Reduced proliferation (demonstrated by decreased BrdU incorporation)

• Increased apoptosis (confirmed by AnnexinV staining)

• Altered cell cycle progression (fewer cells entering S-phase)

• Activation of anoikis resistance pathways (overactivation of Akt, Raf, Erk, and Smad signaling)

Furthermore, stable knockdown of ID2 accelerates the transition from AD to AI phenotype, with ID2-knockdown cells forming dense large spheres much faster than control cells . This mechanism operates partially through the TGF-β pathway, as ID2 normally binds both TGF-β and Smad2/3, preventing activation of anoikis resistance pathways.

How does temporal expression of ID2 affect natural killer cell development from stem cells?

Temporal expression of ID2 dramatically enhances NK cell generation from human pluripotent stem cells (hPSCs). Using CRISPR/Cas9-mediated gene knock-in to create inducible ID2 expression systems, researchers have demonstrated that ID2 overexpression significantly promotes NK cell generation compared with other transcription factors such as NFIL3 and SPI1 . This enhancement occurs under chemically defined, feeder-free culture conditions. The resulting ID2 hPSC-derived NK cells display various mature NK-specific markers and effective tumor-killing activities comparable to NK cells derived from wildtype hPSCs . This finding provides a foundation for developing efficient NK cell production methods for cancer immunotherapy applications.

What is the mechanism behind ID2 antagonism in cancer treatment?

ID2 antagonism represents a promising therapeutic approach, particularly for glioma treatment. Through pharmacophore-based virtual screening, researchers have identified novel ID2 antagonists, with compound AK-778-XXMU emerging as a potent inhibitor with therapeutic potential . These antagonists likely function by disrupting the protein-protein interactions mediated by ID2's HLH domain, thereby preventing its inhibitory effects on tumor-suppressive transcription factors. This approach could potentially restore normal differentiation programs in cancer cells or modulate immune cell functions. The development of ID2 antagonists demonstrates the feasibility of targeting transcriptional regulators that were previously considered "undruggable."

What are the optimal techniques for measuring ID2 protein-protein interactions in primary human cells?

For investigating ID2 protein-protein interactions in primary human cells, researchers should employ a multi-faceted approach:

• Co-immunoprecipitation (Co-IP): This technique has successfully demonstrated ID2 binding to both TGF-β and Smad2/3 . For primary cells with limited abundance, micro-scale Co-IP protocols with high-sensitivity detection methods are recommended.

• Proximity ligation assay (PLA): This technique enables visualization of protein interactions in situ with single-molecule sensitivity, which is particularly valuable for rare primary cell populations.

• FRET/BRET analysis: These approaches can detect real-time interactions in living cells, providing insights into the dynamics of ID2 interactions under various stimulation conditions.

• Mass spectrometry-based interactomics: For unbiased identification of the complete ID2 interactome, immunoprecipitation followed by mass spectrometry can reveal novel interaction partners.

• ChIP-reChIP: This method can assess complex formation on chromatin, particularly important for understanding how ID2 disrupts transcriptional complexes such as the Tcf3-Tal1 complex at specific genomic loci .

Each method offers distinct advantages, and combining multiple approaches provides the most comprehensive understanding of ID2's interaction network in different cellular contexts.

How can researchers effectively manipulate ID2 expression in human primary NK cells?

Manipulating ID2 expression in primary NK cells requires specialized approaches due to the challenges of transfecting these cells:

For temporal control of ID2 expression, inducible systems such as tetracycline-responsive promoters provide the most precise regulation. This approach has been effectively employed in stem cell models, where CRISPR/Cas9-mediated gene knock-in generated hPSCs with inducible ID2 expression . For primary NK cells, optimized nucleofection protocols with mRNA or ribonucleoprotein complexes offer the best balance of efficiency and cell viability. Post-manipulation, comprehensive phenotypic and functional assessment should include monitoring of maturation markers, cytotoxicity assays, and cytokine production profiles.

What experimental design is most appropriate for studying ID2's role in epigenetic regulation?

To comprehensively investigate ID2's epigenetic functions, a sequential experimental design is recommended:

• Baseline epigenetic profiling:

  • ATAC-seq to map chromatin accessibility changes

  • ChIP-seq for histone modifications (H3K4me3, H3K27ac, H3K27me3)

  • DNA methylation analysis at regulatory regions

• ID2 manipulation experiments:

  • Controlled ID2 knockdown/overexpression with time-course sampling

  • Conditional systems for temporal precision

  • Rescue experiments with wildtype vs. mutant ID2 (particularly HLH domain mutations)

• Protein-chromatin interaction analysis:

  • ID2 ChIP-seq (or CUT&RUN for higher resolution)

  • ChIP-seq for ID2-interacting partners (e.g., Tcf3, Tal1)

  • Sequential ChIP to identify co-occupancy patterns

• Functional validation:

  • Site-directed epigenetic editing at ID2-regulated loci

  • Reporter assays with wildtype and mutated regulatory elements

  • Single-cell approaches to capture heterogeneity in responses

This experimental framework has successfully revealed how ID2 modulates chromatin accessibility at the Slamf6 promoter by preventing the interaction of the Tcf3-Tal1 transcriptional complex , demonstrating its utility for dissecting epigenetic mechanisms.

What are the methodological considerations for developing ID2-targeting therapeutics?

Development of ID2-targeting therapeutics requires rigorous methodological approaches:

• Target validation phase:

  • Genetic approaches (CRISPR-Cas9, shRNA) to confirm ID2 dependency

  • Domain-specific mutations to identify critical functional regions

  • Patient-derived models to establish clinical relevance

• Screening methodologies:

  • Pharmacophore-based virtual screening (successfully used to identify compound AK-778-XXMU)

  • Fragment-based screening for protein-protein interaction inhibitors

  • Targeted library design based on structural insights

• Lead optimization considerations:

  • Structure-activity relationship studies

  • Cell-type specific activity profiling

  • Assessment of effects on other ID family members

• Preclinical evaluation:

  • Pharmacokinetic/pharmacodynamic modeling

  • Biomarker development for target engagement

  • Combination studies with standard-of-care therapies

Special attention must be paid to the context-dependent roles of ID2 across different cell types, as inhibition beneficial in tumor cells might have detrimental effects on immune cell function. This necessitates careful therapeutic window determination and potentially cell-type selective delivery approaches.

How can ID2 expression be leveraged to improve cancer immunotherapy outcomes?

ID2's dual roles in cancer cells and immune cells present unique opportunities for immunotherapy enhancement:

• NK cell manufacturing optimization:

  • Temporal ID2 overexpression significantly improves NK cell generation from human pluripotent stem cells

  • The resulting NK cells exhibit mature markers and effective tumor-killing activities

  • This approach provides a platform for efficient off-the-shelf NK cell production

• T cell exhaustion modulation:

  • ID2 inhibition may prevent terminal exhaustion of tumor-infiltrating lymphocytes

  • Genetic deletion of ID2 dampens CD8+ T-cell responses and suppresses PD-1 blockade efficacy

  • Targeted, temporal ID2 modulation could enhance existing checkpoint inhibitor therapies

• Cancer cell vulnerability targeting:

  • ID2 antagonists (such as AK-778-XXMU) have shown potential against glioma

  • ID2 inhibition could potentiate immune-mediated killing by altering cancer cell phenotypes

Strategic approaches might include sequential or cell-type specific ID2 modulation, where initial targeting in tumor cells is followed by immune cell ID2 optimization to maximize therapeutic benefit while minimizing antagonistic effects.

What biomarkers related to ID2 function could predict response to immunotherapy?

Several ID2-related biomarkers show potential for predicting immunotherapy response:

The ID2-TCF1 axis is particularly promising as it controls the balance of immature and terminally differentiated NK cells . Similarly, in T cells, ID2's role in regulating the progenitor exhausted (Tex prog) to terminally exhausted (Tex term) transition suggests that measuring this axis could predict sustainability of anti-tumor responses and response duration to checkpoint inhibition .

How can knowledge of ID2 biology inform combination therapy approaches?

Understanding ID2 biology reveals several rational combination strategies:

• Sequencing considerations:

  • ID2 antagonism followed by checkpoint inhibition could prime the immune microenvironment

  • TGF-β pathway inhibitors combined with ID2 targeting might provide synergistic effects

  • Cell cycle modulators paired with ID2 inhibition could enhance anti-proliferative effects

• Cell-specific targeting approaches:

  • Tumor-directed ID2 inhibition combined with immune cell ID2 enhancement

  • Stromal-targeted therapies to modify ID2-dependent niche interactions

  • Sequential modulation of ID2 in different cellular compartments

• Temporal considerations:

  • Pulsed ID2 inhibition to prevent compensatory mechanisms

  • Maintenance strategies following initial response to ID2-targeted therapy

  • Adaptive approaches based on monitoring ID2-dependent biomarkers

Research has shown that ID2 functions partially through the TGF-β pathway , suggesting that combined inhibition of both pathways could overcome resistance mechanisms and enhance therapeutic efficacy.

What are the key considerations for designing clinical trials of ID2-targeting agents?

Clinical trial design for ID2-targeting agents requires careful consideration of several factors:

• Patient selection strategies:

  • Molecular profiling to identify ID2-dependent tumors

  • Assessment of immune cell ID2 status and exhaustion markers

  • Stratification based on prior treatment history and response patterns

• Endpoint selection:

  • Primary: Traditional response and survival metrics

  • Secondary: Immune cell phenotyping, TCF1/ID2 ratios, T cell exhaustion markers

  • Exploratory: Spatial immune profiling, clonal dynamics, epigenetic reprogramming

• Monitoring considerations:

  • Serial liquid biopsies for circulating tumor cells and cell-free DNA

  • Immune monitoring panels focused on ID2-regulated cell populations

  • On-treatment biopsies to assess pharmacodynamic effects

• Special design elements:

  • Adaptive designs to adjust dosing based on biomarker changes

  • Basket trial approaches grouping ID2-dependent tumors across histologies

  • Window-of-opportunity studies to assess biological effects

Given ID2's role in both cancer cells and immune populations, special attention must be paid to potential antagonistic effects between anti-tumor activity and immune suppression, necessitating careful dose finding and scheduling optimization.

How might single-cell technologies advance our understanding of ID2 function?

Single-cell technologies offer unprecedented opportunities to dissect ID2's context-dependent roles:

• Single-cell RNA-seq can reveal:

  • Cell-specific ID2 expression patterns across heterogeneous populations

  • Transcriptional consequences of ID2 activity with single-cell resolution

  • Rare cell populations with unique ID2-dependent states

• Single-cell ATAC-seq provides insights into:

  • Cell-specific chromatin accessibility changes mediated by ID2

  • Regulatory element usage across developmental trajectories

  • Correlation between ID2 expression and genome-wide accessibility patterns

• Multi-omics approaches combining:

  • Protein, transcriptome, and epigenome data at single-cell resolution

  • Spatial information to contextualize ID2 function within tissue architecture

  • Lineage tracing to follow ID2-expressing cells through development or disease progression

These technologies would be particularly valuable for understanding how ID2 controls the balance between immature and terminally differentiated NK cells and the heterogeneity in T cell exhaustion states .