POLD1, the catalytic subunit of DNA polymerase delta (Pol δ), is a multi-domain protein (125 kDa) critical for DNA synthesis and proofreading (7). It comprises three key domains: the N-terminal exonuclease domain, the central polymerase domain, and the C-terminal domain with a zinc finger (CysA), a [4Fe-4S] cluster (CysB), and a non-canonical Proliferating Cell Nuclear Antigen (PCNA)-interacting (PIP) box (2). Each domain contributes uniquely to replication fidelity and repair, with distinct structural and functional properties ( Figure 1 ).

The expression and activity of POLD1 are governed by a complex interplay of transcriptional, post-transcriptional, and post-translational mechanisms, which collectively shape its paradoxical roles in maintaining genomic stability and driving oncogenic processes. Transcriptionally, E2F1 and Sp1/Sp3 activate POLD1 during S phase via GC-rich promoter elements, while genotoxic stress triggers p53/p21-mediated repression through Sp1/DNMT1 downregulation and DREAM complex disruption (17–19). CTCF maintains promoter accessibility, linking age-related POLD1 decline to senescence (20).

POLD1 is highly expressed in various cancers, including bladder, breast, endometrial, colorectal, lung, and hepatocellular carcinoma, as evidenced by both clinical studies and TCGA database analyses (16, 21–25) However, its expression patterns yield paradoxical clinical outcomes depending on tumor context. In hepatocellular carcinoma (HCC), TP53 mutations drive POLD1 overexpression, which correlates with advanced tumor stage, vascular invasion, and poor prognosis (22). This aligns with its role in promoting replication under oncogenic stress, where high POLD1 levels exacerbate genomic instability in rapidly proliferating tumors. Strikingly, in clear cell renal cell carcinoma (ccRCC), elevated POLD1 expression paradoxically associates with favorable survival outcomes, potentially reflecting its function in maintaining replication fidelity in slower-growing malignancies with lower baseline replication stress (26). These divergent phenotypes underscore the dual nature of POLD1 in cancer biology: while its overexpression may drive replication-associated mutagenesis in aggressive tumors, it can paradoxically act as a stabilizing force in malignancies where replication demands are less acute.

The spatial distribution of POLD1 mutations across different functional domains determines their distinct pathological consequences through domain-specific mechanisms that affect genomic stability and cellular homeostasis. POLD1 maintained the genomic fidelity through its 3’–5’ exonuclease proofreading activity, making domain-specific mutations particularly consequential for cellular function.

The exonuclease domain mutations (e.g., p.L474P, p.L472P, P286R, S459F) represent the most clinically significant alterations, disrupting proofreading function and leading to catastrophic accumulation of somatic mutations that often exceed 100 mutations per megabase (mut/Mb), a hallmark of ultra-hypermutation (16, 27–31). This genomic instability paradoxically transforms tumors into immunogenic hotspots, creating a unique interplay between mutagenesis and immune recognition. Structural studies reveal that exonuclease domain mutations such as P286R and S459F distort the DNA-binding groove, destabilizing primer-template alignment and exacerbating replication errors (32).

In contrast, polymerase-domain variants (e.g., p.L606M) operate through distinct mechanisms, hijacking replication stress signaling pathways rather than directly compromising proofreading fidelity, potentially favoring tumorigenesis in glioma and polyposis-prone tissues (33). A third category, germline truncating mutations (p.S605del) that spare the exonuclease domain, escape canonical tumorigenesis but provoke MDPL syndrome (mandibular hypoplasia, progeroid lipodystrophy) via impaired PCNA interaction and systemic replication catastrophe (8). POLD1 exonuclease domain mutations induce distinct mutational signatures dominated by single-nucleotide variants (SNVs), particularly TCT>TAT (COSMIC signature SBS10a) and TCG>TTG (SBS10b) transitions, differing from frameshift-driven patterns typical of MSI (34, 35). These mutations are often clonal and early events, preceding other driver mutations, which may explain the widespread genomic heterogeneity observed in these tumors (36).

These mutations result in TMB levels surpassing those of mismatch repair-deficient (dMMR)/MSI-high (MSI-H) cancers, with median TMB values exceeding 150 mut/Mb in colorectal and endometrial cancers (31, 32). This hypermutated phenotype predominantly affects colorectal and endometrial malignancies, generating SBS10d mutational signatures arising from somatic LOH (loss of heterozygosity, loss of the wild-type allele) (16, 27, 28). Although POLD1 mutations are rare in TCGA(~1–2%) (34), they present a unique paradox: they drive genomic instability while paradoxically enhancing immunogenicity, particularly in MSS tumors traditionally resistant to ICIs. Notably, POLD1-mutant tumors are predominantly MSS across most cancer types, yet exhibit significant tissue-specific variation. For instance, in stomach adenocarcinoma (STAD), >90% show co-occurring MSI-H (see Section 5), while in colorectal and endometrial cancers, >98% maintain MSS status despite ultra-hypermutation (29, 37).

Despite MSS status, POLD1-mutant tumors display robust immune infiltration, including elevated CD8+ T cells and PD-L1 expression (35). The sheer volume of clonal neoantigens generated by ultra-hypermutation likely drives this immunogenicity, bypassing the reliance on MSI-induced frameshift neoantigens (28, 38). For example, POLD1-mutant MSS colorectal cancers exhibit higher tumor-infiltrating lymphocyte density and cytotoxic T-cell markers compared to POLD1 wild-type MSS tumors (16, 34). This functional dichotomy underscores domain-selective vulnerabilities within POLD1: exonuclease perturbations ignite immunogenic mutagenesis through hypermutation, whereas polymerase and C-terminal domain defects disrupt replication homeostasis in developmentally sensitive lineages. The genomic instability paradoxically transforms tumors into immunogenic hotspots, creating a unique interplay between mutagenesis and immune recognition that has significant therapeutic implications.

Germline mutations in POLD1 are present from embryogenesis, leading to systemic effects across multiple tissues throughout development. Variants like p.Ser478Asn and p.Leu474Pro impair Pol δ’s proofreading capacity, increasing base substitution and insertion-deletion mutation rates (39, 40). This continuous accumulation, detectable in normal crypts, endometrial glands, and even sperm, reflects a systemic replication defect originating in the embryo. Variants like S478N, inherited dominantly, elevate mutation rates across all tissues lifelong—152 SBS per year versus 49 in healthy controls—contrasting with the sporadic, tumor-specific onset of somatic mutations (41). This results in an ultramutated phenotype characterized by COSMIC Signature 10 variants, consistent with somatic POLD1-mutant profiles (42).

Molecularly, the apparent contradiction between functional studies suggesting near-haplosufficiency and the dominant inheritance pattern of POLD1-related cancer predisposition (like PPAP) warrants clarification. Functional data indicate that heterozygous exonuclease domain mutations alone cause only a subtle increase in mutation rate (<15%) in somatic cells, implying near-haplosufficiency under normal conditions, potentially due to extrinsic proofreading by wild-type Pol δ complexes (27, 43). However, the dominant cancer risk associated with germline heterozygous mutations strongly suggests that a single mutant allele is sufficient to significantly increase cancer susceptibility. This paradox is resolved by the frequent occurrence of somatic LOH at the POLD1 locus in tumor tissue. LOH eliminates the wild-type allele, creating a functionally homozygous or hemizygous state for the mutant allele, which catastrophically compromises proofreading. This two-hit mechanism (germline mutation + somatic LOH) explains both the dominant inheritance pattern and the tissue-specific vulnerability observed in PPAP, where rapid cell turnover increases the likelihood of acquiring the somatic LOH event (27, 29, 37, 40).

In contrast, somatic POLD1 mutations, mostly in the exonuclease domain, emerge in adulthood, driving tumorigenesis via proofreading deficits. Studies identify variants like p.S478N in MSS colorectal tumors, recapitulate the ultra-hypermutation and SBS signatures observed in germline contexts, but differ from the insertion-deletion patterns of Lynch syndrome (44, 45). Functional assays in yeast, using the Pol3-C462N strain (corresponding to human p.S478N), reveal a 12-fold increase in mutation rate, confirming that proofreading loss amplifies somatic SBS accumulation (44). This localized effect contrasts sharply with the systemic impact of germline mutations.

The molecular impact of these mutations is highly context-dependent, often synergizing with mismatch repair (MMR) deficiency to produce complex mutational profiles. For instance, in MSS tumors, somatic POLD1 exonuclease domain mutations alone generate hypermutation signatures like SBS10, characterized by C:G→A:T and C:G→T:A transitions (44). However, when combined with MMR defects, they yield distinct signatures such as SBS20, reflecting a compounded effect on genomic stability. A notable example involves a serrated polyposis syndrome (SPS) patient harboring a germline POLD1 frameshift (p.Lys648fs*46) outside the exonuclease domain, where somatic MMR loss led to ultra-hypermutation (TMB > 117 mut/Mb). Despite loss of heterozygosity of the wild-type allele in the tumor, suggesting a haploinsufficient state, this case lacked POLD1-specific signatures (e.g., SBS10c/d), indicating MMR dominance over POLD1’s contribution (40). This demonstrates the hierarchical nature of DNA repair pathway interactions and their relative contributions to tumor mutagenesis.

The functional compartmentalization of POLD1 creates distinct pathogenic mechanisms, where mutations in different domains produce entirely different clinical syndromes, providing compelling evidence for domain-specific functional requirements.

POLD1 mutations drive genomic instability through distinct molecular mechanisms that extend beyond simple proofreading defects. Recent studies have revealed that POLD1 deficiency disrupts the spatial-temporal coordination of replication factories, leading to aberrant origin firing and replication timing (17). Tumini et al. demonstrated that POLD1 depletion significantly reduces the density of active replication origins, forcing cells to rely on fewer origins that must travel longer distances, thereby increasing the probability of replication fork collapse (53). This origin paucity creates regions of under-replicated DNA that persist into mitosis, forming ultrafine anaphase bridges and promoting chromosomal rearrangements. Importantly, POLD1 mutations exhibit allele-specific effects on genomic stability; while exonuclease domain mutations drive point mutation accumulation via misincorporation, polymerase domain variants induce replication fork stalling and double-strand breaks through defective Okazaki fragment maturation (8).

POLD1-driven genomic instability does not operate in isolation but rather interacts synergistically with other DNA repair mechanisms, particularly the MMR pathway. While POLD1 mutations and MMR deficiency can independently induce genomic instability, their co-occurrence creates a synergistic effect that dramatically accelerates mutagenesis (29). This interaction follows a specific evolutionary trajectory: initial POLD1 proofreading deficiency generates a moderate increase in mutation rate, which subsequently leads to the acquisition of secondary mutations in MMR genes, further amplifying genomic instability in a feed-forward loop (35). Specific POLD1 mutations, such as somatic p.S478N and germline p.L474P, impair Pol δ proofreading, leading to ultramutated tumors with tumor mutation frequent loads exceeding 100 variants/Mb and characteristic SBS10d signatures (54, 55). Notably, these mutations frequently co-occur with secondary somatic inactivation of MMR genes, as observed in ultramutated Lynch Syndrome patients, amplifying mutation loads through a synergistic feed-forward mechanism (54). This interaction induces MSI signatures, despite the predominantly MSS phenotype of POLD1-mutated tumors, highlighting a unique evolutionary trajectory where Pol δ infidelity triggers secondary MMR defects (56).

Intriguingly, despite this interaction, most POLD1-mutated tumors maintain MSS, distinguishing them from classical MMR-deficient cancers characterized by microsatellite instability (MSI) (6). This paradoxical observation suggests that POLD1-driven genomic instability follows a distinct evolutionary path, preferentially accumulating single-nucleotide variants rather than the insertion-deletion mutations typical of MMR deficiency. The maintenance of microsatellite stability despite high mutational burden represents a unique feature of POLD1-mutated cancers and has significant implications for their biological behavior and therapeutic responsiveness.

Beyond MMR, POLD1 mutations also impact other DNA repair pathways, including base excision repair and double-strand break repair, where POLD1 normally plays crucial roles in gap-filling and strand displacement synthesis (32). The compromised function of these repair pathways further exacerbates genomic instability, creating a state of “repair crisis” that fuels ongoing mutagenesis and chromosomal aberrations.

POLD1 mutations fundamentally alter replication fork dynamics, inducing chronic replication stress that fuels genomic instability. Under normal conditions, POLD1 coordinates with POLD3 to maintain replication fork stability and processivity (53). However, when POLD1 function is compromised, replication forks exhibit increased stalling, reversed fork structures, and ssDNA accumulation (57). POLD1 transitions between different functional states—from the complete Pol δ4 complex (POLD1, POLD2, POLD3, and POLD4) to the Pol δ3 form (lacking POLD4)—in response to replication challenges ( Figure 2 ) (58, 59). This transition, mediated by the ubiquitin-proteasome pathway and regulated by the ATR-CHK1 signaling axis, represents an adaptive mechanism to navigate replication obstacles (12).

However, POLD1 mutations disrupt this delicate balance, leading to inappropriate activation of error-prone repair mechanisms such as break-induced replication (BIR) (12). BIR, while rescuing stalled replication forks, introduces extensive genomic alterations, including long-tract gene conversions and loss of heterozygosity (LOH). Indeed, LOH at the POLD1 locus is frequently observed in tumors with heterozygous POLD1 mutations, resulting in the complete loss of proofreading capacity and accelerated genomic instability (37).

The replication stress induced by POLD1 mutations also manifests as increased double-strand breaks (DSBs), particularly at common fragile sites and regions with complex secondary structures (36, 60). These DSBs, when repaired through error-prone mechanisms like alternative end-joining, generate chromosomal rearrangements that further destabilize the genome. The accumulation of such structural variations represents a distinct dimension of POLD1-driven genomic instability, complementing the hypermutation phenotype and contributing to the overall genomic instability characteristic of POLD1-mutated cancers.

ICI efficacy typically correlates with TMB and MMR deficiency/MSI-H status, yet POLD1 mutations enable MSS tumors to respond by inducing a hypermutated phenotype ( Figure 3 ) (28, 38). Pan-cancer analyses reveal that patients with POLD1-mutated tumors achieve superior outcomes with ICIs, including prolonged overall survival (34 months for POLD1-mutated vs. 18 months for wild-type MSS tumors) and response rates > 80% in some studies (31, 63). For instance, in colorectal cancer, POLD1 exonuclease domain mutations (e.g., p.S478N, p.L474P) correlate with elevated TMB and robust CD8+ T-cell infiltration, driving durable responses to anti-PD-1 therapy despite MSS status (64). Similarly, in endometrial cancer, the p.D316H variant in the DEDD motif is associated with a T-cell-inflamed gene expression profile, predicting pembrolizumab sensitivity with a complete response in a documented case (11). Remarkably, polymerase domain mutations (e.g., p.L606M) also correlate with ICI efficacy, suggesting that POLD1’s influence transcends proofreading defects (33). The Acsé Nivolumab trial reinforces this, showing mutations in DNA-binding or catalytic regions yield responses rivaling those in traditionally responsive tumors (58.7% vs. 23.8% in wild-type) (65). These findings underscore POLD1’s potential as an independent biomarker for ICI response, broadening the therapeutic horizon beyond MSI-H paradigms.

The therapeutic implications of POLD1 mutations are amplified by their interplay with co-occurring genetic alterations. Notably, co-mutations with PBRM1, a tumor suppressor, enhance ICI efficacy in POLD1-mutated tumors. In advanced solid tumors, POLD1/PBRM1 co-mutations yielded higher response rates (70% vs. 40% for POLD1 alone), driven by elevated TMB and a T-cell-permissive microenvironment (66). PBRM1 loss sensitizes tumor cells to interferon-γ-mediated killing, complementing the neoantigen-driven immunogenicity of POLD1 mutations (67). This synergy suggests that POLD1/PBRM1 co-mutation could serve as a composite biomarker, identifying a subset of MSS patients likely to benefit from immunotherapy.

POLD1 also frequently co-occurs with oncogenic drivers like APC, BRCA2, and MYC, notably in colorectal and bladder cancers (16, 36). In bladder cancer, POLD1 stabilizes MYC by inhibiting its degradation via FBXW7, promoting proliferation and metastasis (16). While this pro-tumorigenic axis complicates prognosis, it also hints at combinatorial strategies: targeting MYC alongside ICIs could exploit POLD1-driven immunogenicity while curbing tumor growth. These interactions highlight the need for comprehensive genomic profiling to tailor therapies based on POLD1’s mutational context.

Several clinical trials are investigating the therapeutic potential of POLD1 and POLE mutations, particularly their role in predicting ICI response in hypermutated tumors. Table 1 summarizes key ongoing or recently completed trials focused on POLD1/POLE mutations and their impact on immunotherapy outcomes.

Despite its promise, the prognostic and therapeutic significance of POLD1 remains contentious, varying across cancer types. In HCC, POLD1 overexpression, often driven by TP53 mutations, correlates with advanced staging, vascular invasion, and poor survival, suggesting a tumor-promoting role resistant to conventional therapies (22). Similarly, in triple-negative breast cancer and endometrial carcinoma, high POLD1 expression is linked to aggressive phenotypes and reduced survival, potentially reflecting its role in sustaining replication under oncogenic stress (68). Conversely, in ccRCC elevated POLD1 expression paradoxically associates with favorable survival, possibly due to preserved replication fidelity in less aggressive tumors (26). This dichotomy underscores a critical challenge: POLD1’s therapeutic relevance is highly context-dependent, influenced by tumor biology, mutation type, and coexisting alterations.

The predictive value of POLD1 mutations for ICI efficacy also faces scrutiny. While high TMB in POLD1-mutated tumors enhances immunogenicity, the lack of MSI-H signatures in most cases raises questions about the mechanisms driving immune recognition. Some studies argue that clonal neoantigens, rather than frameshift mutations typical of MSI-H, underlie this effect, yet the precise neoantigen profiles remain poorly characterized (69). Moreover, the rarity of POLD1 mutations (1-2% in TCGA cohorts) limits large-scale validation, necessitating prospective trials to confirm its utility as a standalone biomarker.

POLD1’s biology inspires innovative approaches. Its mutational signatures (e.g., SBS10a, SBS10b) suggest neoantigen vaccines targeting specific base transitions, potentially boosting T-cell responses in MSS tumors (34, 35). Second, POLD1’s role in replication stress opens avenues for synthetic lethality. Mutations like p.S605del in the polymerase domain, which impair DNA synthesis and induce double-strand breaks (DSBs), render cells reliant on ATR-CHK1 signaling for fork stabilization (70). Inhibitors of ATR or CHK1, such as berzosertib or prexasertib, could selectively kill POLD1-mutant tumor cells by exacerbating replication collapse, a strategy already showing promise in POLE-mutated cancers (71). Combining these agents with ICIs might further enhance efficacy by increasing tumor cell death and neoantigen release. Finally, targeting POLD1’s regulatory network offers indirect therapeutic leverage. In bladder cancer, POLD1’s stabilization of MYC suggests that MYC inhibitors (e.g., KJ-Pyr-9) could disrupt this axis, while miR-155 agonists might suppress POLD1 expression by downregulating FOXO3a, reducing replication fidelity in tumor cells (16, 72). These approaches, though speculative, illustrate how POLD1’s molecular dependencies could be exploited to design combination therapies.