High microsatellite instability (microsatellite instability-high, MSI-H) is a genomic phenotype resulting from defective DNA mismatch repair. The continuous accumulation of insertion–deletion mutations generates frameshift neoantigens that increase tumor immune visibility while also producing abnormal DNA structures that create a specific dependency on the WRN helicase.

Keywords: MSI-H; mismatch repair; dMMR; frameshift mutation; tumor immunity; WRN; synthetic lethality

1 Molecular Basis of MSI-H

1.1 Microsatellites and Replication Slippage

Microsatellites are short tandem repeat sequences composed of 1–6 nucleotides and are widely distributed throughout coding and noncoding regions. When DNA polymerase passes through these regions, the nascent or template strand can transiently dissociate and mispair, forming insertion–deletion loops; normal cells correct these replication errors through the mismatch repair system, whereas loss of repair function allows gains or losses of repeat units to become fixed, and persistent changes in the lengths of multiple microsatellite loci ultimately produce the MSI-H phenotype.

1.2 Causes of MSI-H

(1) Defective Mismatch Repair

The primary cause of MSI-H is deficient DNA mismatch repair (dMMR). MLH1, MSH2, MSH6, and PMS2 are major components of this system, and inactivation of these key proteins impairs replication-error recognition, removal of the erroneous strand, or DNA resynthesis, allowing microsatellite replication errors to accumulate continuously.

(2) Genetic and Epigenetic Abnormalities

MMR deficiency may result from pathogenic germline variants, biallelic somatic variants, gene deletions, or promoter methylation. MLH1 promoter hypermethylation can suppress MLH1 transcription and destabilize its binding partner PMS2, resulting in the concurrent loss of MLH1 and PMS2.

1.3 Differences Among MSI-H, dMMR, and TMB-H

2 How the Mismatch Repair System Maintains Genomic Stability

2.1 MutS Complexes Recognize Replication Errors

(1) MutSα Complex

MSH2 and MSH6 form the MutSα complex, which primarily recognizes base–base mismatches and small insertion–deletion loops. After binding abnormal DNA, MutSα undergoes an ATP-dependent conformational change and forms a signaling platform capable of moving along DNA.

(2) MutSβ Complex

MSH2 and MSH3 form the MutSβ complex, which preferentially recognizes larger insertion–deletion structures. MutSα and MutSβ have partially complementary functions, but MSH6 loss still markedly impairs the repair of single-base mismatches and errors in short repeat sequences.

2.2 MutLα Mediates Removal of the Error-Containing Strand

MLH1 and PMS2 form the MutLα complex. After being recruited by a MutS complex, MutLα identifies the newly synthesized strand with the assistance of PCNA, RFC, and discontinuities in the replication strand and generates an incision through the endonuclease activity of PMS2; EXO1 then removes the DNA segment containing the mismatch, DNA polymerase δ resynthesizes the missing sequence, and DNA ligase I seals the remaining nick.

2.3 Pairing-Dependent Stability of MMR Proteins

MLH1–PMS2 and MSH2–MSH6 both function as heterodimers. MLH1 and MSH2 are relatively stable core proteins, and their loss frequently causes simultaneous degradation of their binding partners; when PMS2 or MSH6 is lost, MLH1 or MSH2 may remain detectable.

3 How MSI-H Reshapes the Tumor Genome

3.1 Accumulation of Insertion–Deletion Mutations

Following mismatch repair deficiency, replication-slippage products within microsatellites cannot be removed promptly, and mononucleotide and dinucleotide repeats are particularly susceptible to gains or losses of repeat units. When these changes occur in coding regions, they may disrupt proteins involved in growth suppression, apoptosis, DNA repair, and immune regulation, giving MSI-H tumors a mutational spectrum characterized by insertion–deletion mutations.

3.2 Frameshift Mutations and Novel Protein Sequences

Coding-region insertions or deletions that are not multiples of three alter the codon reading frame downstream of the mutation site, replacing the normal amino acid sequence with a new abnormal sequence. Some frameshift transcripts can escape nonsense-mediated mRNA decay and undergo translation, and proteasomal processing of the resulting abnormal proteins can generate frameshift peptides not present in normal tissues.

3.3 Abnormal Repeat Sequences Increase Replication Stress

(1) Formation of Non-B DNA Structures

Expanded repeat sequences can form hairpins, slipped loops, cruciform structures, or other non-B DNA conformations. These structures impede replication polymerase progression, causing replication-fork stalling and generating extended regions of single-stranded DNA.

(2) ATR–CHK1 Checkpoint Response

After binding exposed single-stranded DNA, RPA recruits ATR and its accessory factors, leading to CHK1 activation, cell-cycle delay, and replication-fork stabilization. If abnormal structures are not resolved promptly, stalled replication forks may undergo reversal, nuclease cleavage, or breakage, resulting in DNA double-strand damage.

4 Immune Vulnerabilities of MSI-H Tumors

4.1 Frameshift Neoantigens Increase Immune Visibility

Proteasomal degradation of frameshift proteins generates short peptides, and those capable of binding HLA molecules and being presented on the cell surface can be recognized by CD8⁺ T cells. Compared with single-amino-acid substitutions, frameshift mutations can produce continuous novel sequences with the potential to form highly immunogenic epitopes, although the generation of an effective neoantigen ultimately depends on transcript stability, protein processing, HLA-binding capacity, and T-cell receptor recognition.

4.2 DNA Damage Activates cGAS–STING Signaling

Replication-fork collapse, chromosome breakage, and micronuclear rupture can expose DNA to the cytoplasm. After recognizing cytoplasmic DNA, cGAS synthesizes 2′,3′-cGAMP and activates STING–TBK1–IRF3 signaling, promoting type I interferon expression, dendritic-cell maturation, and effector T-cell recruitment; this pathway can provide an auxiliary immune-activation mechanism in addition to frameshift neoantigens.

4.3 IFN-γ–JAK–STAT1 Amplifies Immune Responses

Activated T cells and natural killer cells can release IFN-γ. Following receptor binding, IFN-γ activates JAK1, JAK2, and STAT1, increasing antigen processing, MHC expression, and immune effector molecules while also inducing negative-feedback molecules such as PD-L1, causing MSI-H tumors to display both immune activation and adaptive immune suppression.

4.4 PD-1/PD-L1 Restricts T-Cell Effector Function

Persistent antigen stimulation can induce the expression of inhibitory receptors such as PD-1 on tumor-infiltrating T cells. Binding of PD-L1 to PD-1 suppresses downstream T-cell receptor signaling, cytokine release, and cytotoxic activity; immune checkpoint blockade relieves established immune suppression, but its efficacy still depends on effective neoantigen presentation, T-cell infiltration, and recoverable immune effector function.

4.5 Immune Evasion in MSI-H Tumors

(1) Impaired Antigen Presentation

B2M loss, HLA abnormalities, or defects in antigen-processing components can block the presentation of frameshift neoantigens, allowing tumor cells to evade CD8⁺ T-cell recognition. Even when a tumor has a high mutational burden, disruption of the antigen-presentation pathway can prevent effective immune clearance.

(2) Interferon Signaling and Immune Exclusion

Inactivation of JAK1 or JAK2 can impair tumor-cell responses to IFN-γ, while the accumulation of immunosuppressive cells and T-cell exclusion can also restrict immune effects. MSI-H indicates a higher probability of immunogenicity but does not guarantee an immune response.

5 WRN Dependency in MSI-H Tumors

5.1 Molecular Structure and Functions of WRN

WRN is a member of the RecQ family and contains an N-terminal 3′→5′ exonuclease domain, a central ATPase/3′→5′ helicase domain, and DNA-binding domains including RQC and HRDC. WRN participates in replication-fork recovery, homologous recombination, telomere stability, and the processing of complex DNA structures and can resolve hairpins, forked structures, and other abnormal nucleic acid conformations that obstruct DNA replication.

5.2 TA Repeats Form Structural Replication Barriers

Certain long TA dinucleotide repeats in MSI-H cells can undergo persistent expansion. The resulting palindrome-like sequences readily undergo intrastrand base pairing and form cruciform or other non-B DNA structures; WRN resolves these abnormal structures and permits continued replication-fork progression, making it an important survival factor for maintaining replication stability in MSI-H cells.

5.3 WRN Dependency Arises Primarily from Its Helicase Function

(1) ATPase/Helicase Activity

Functional complementation studies have shown that WRN mutants lacking ATPase or helicase activity cannot effectively restore MSI-H cell survival, indicating that the core function of WRN is to use ATP hydrolysis to drive the unwinding of abnormal DNA structures rather than merely serving as a scaffold at DNA damage sites.

(2) Exonuclease Activity Is Not the Primary Dependency

Defective exonuclease activity generally has a weaker effect on MSI-H cell survival than defective helicase activity, indicating that synthetic lethality with WRN depends mainly on its ATPase/helicase function. This feature also makes the WRN helicase catalytic region and its conformational regulatory regions important targets for inhibitor development.

5.4 WRN Loss Triggers Replication Catastrophe

(1) Persistent Replication-Fork Stalling

Following WRN inhibition or depletion, abnormal structures formed by TA repeats cannot be resolved promptly, causing replication forks to remain stalled in these regions. Single-stranded DNA exposure and RPA recruitment increase, and the ATR–CHK1 checkpoint becomes persistently activated.

(2) MUS81–EME1 Cleavage of Abnormal Structures

Persistently stalled replication structures can be cleaved by structure-specific endonucleases such as MUS81–EME1, generating extensive DNA double-strand breaks. The resulting damage is further manifested by increased γH2AX, formation of 53BP1 foci, chromosome fragmentation, and mitotic abnormalities, ultimately leading to cell-cycle arrest or death.

5.5 Synthetic Lethality Between WRN and MSI-H

MMR deficiency alone permits tumor-cell survival but continuously increases microsatellite mutations and abnormal DNA structures; when WRN remains functional, cells can still process some of these structural replication barriers. When MMR deficiency and WRN inactivation coexist, abnormal DNA structures cannot be resolved and replication damage exceeds cellular repair capacity, resulting in synthetic lethality.

5.6 Two Therapeutically Actionable Vulnerabilities of MSI-H

(1) Immunological Vulnerability

Frameshift mutations and cytoplasmic DNA signaling increase tumor immune visibility, making some MSI-H tumors more sensitive to immune checkpoint blockade, although this effect depends on intact antigen-presentation and immune effector pathways.

(2) Replication-Structure Vulnerability

Abnormal repeat sequences make MSI-H cells dependent on WRN for replication-fork stability, and WRN inhibition can directly trigger replication catastrophe without requiring T-cell recognition. MSI-H thus creates an immunological vulnerability driven by frameshift neoantigens and a WRN dependency driven by abnormal DNA structures.

6 Products

6.1 Products for MMR and WRN Research

6.2 Products for DNA Replication and Damage-Response Research

6.3 Products for Tumor Immunity and Antigen-Presentation Research

6.4 Products for Cell Proliferation and Death Research

MSI-H creates an immunological vulnerability through frameshift neoantigen formation and a WRN dependency through abnormal repeat sequences. Stratified interventions targeting immune checkpoints and the WRN helicase can exploit the molecular defects of MSI-H at both the tumor-immunity and replication-structure levels.

References

[1] Chen Q, Wen Y. Research Progress on the Synthetic Lethal New Target WRN in Microsatellite Instability Malignant Tumors. Chinese Journal of Modern Applied Pharmacy. 2023;40(18):2600–2607. DOI: 10.13748/j.cnki.issn1007-7693.20231166.

[2] Jiricny J. The multifaceted mismatch-repair system. Nat Rev Mol Cell Biol. 2006;7(5):335–346.

[3] Boland CR, Goel A. Microsatellite instability in colorectal cancer. Gastroenterology. 2010;138(6):2073–2087.e3.

[4] Germano G, et al. Inactivation of DNA repair triggers neoantigen generation and impairs tumour growth. Nature. 2017;552(7683):116–120.

[5] Le DT, et al. Mismatch-repair deficiency predicts response of solid tumors to PD-1 blockade. Science. 2017;357(6349):409–413.

[6] Motwani M, Pesiridis S, Fitzgerald KA. DNA sensing by the cGAS-STING pathway in health and disease. Nat Rev Genet. 2019;20(11):657–674.

For more related articles, please see below:

[1] DNA Damage and Repair

[2] Research on Cell Cycle and DNA Damage Response Mechanisms