The ATR-CHK1 pathway plays a fundamental role in the DNA damage response and is therefore an attractive target in cancer therapy. The antitumorous effect of ATR inhibitors is at least partly caused by synthetic lethality between ATR and various DNA repair genes. In previous studies, we have identified members of the B-family DNA polymerases as potential lethal partner for ATR, i.e. POLD1 and PRIM1. In this study, we validated and characterized the synthetic lethality between ATR and POLA1. First, we applied a model of ATR-deficient DLD-1 human colorectal cancer cells to confirm synthetic lethality by using chemical POLA1 inhibition. Analyzing cell cycle and apoptotic markers via FACS and Western blotting, we were able to show that apoptosis and S phase arrest contributed to the increased sensitivity of ATR-deficient cancer cells towards POLA1 inhibitors. Importantly, siRNA-mediated POLA1 depletion in ATR-deficient cells caused similar effects in regard to impaired cell viability and cumulation of apoptotic markers, thus excluding toxic effects of chemical POLA1 inhibition. Conversely, we demonstrated that siRNA-mediated POLA1 depletion sensitized several cancer cell lines towards chemical inhibition of ATR and its main effector kinase CHK1. In conclusion, the synthetic lethality between ATR/CHK1 and POLA1 might represent a novel and promising approach for individualized cancer therapy: First, alterations of POLA1 could serve as a screening parameter for increased sensitivity towards ATR and CHK1 inhibitors. Second, alterations in the ATR-CHK1 pathway might predict in increased sensitivity towards POLA1 inhibitors.

X-linked reticular pigmentary disorder (XLPDR) is a rare condition characterized by skin hyperpigmentation, ectodermal features, multiorgan inflammation, and recurrent infections. All probands identified to date share the same intronic hemizygous POLA1 hypomorphic variant (NM_001330360.2(POLA1):c.1393-354A > G) on the X chromosome. Previous studies have supported excessive type 1 interferon (IFN) inflammation and natural killer (NK) cell dysfunction in disease pathogenesis. Common null polymorphisms in filaggrin (FLG) gene underlie ichthyosis vulgaris and atopic predisposition.
A 9-year-old boy born to non-consanguineous parents developed eczema with reticular skin hyperpigmentation in early infancy. He suffered recurrent chest infections with chronic cough, clubbing, and asthma, moderate allergic rhinoconjunctivitis with keratitis, multiple food allergies, and vomiting with growth failure. Imaging demonstrated bronchiectasis, while gastroscopy identified chronic eosinophilic gastroduodenitis. Interestingly, growth failure and bronchiectasis improved over time without specific treatment.
Whole-genome sequencing (WGS) using Illumina short-read sequencing was followed by both manual and orthogonal automated bioinformatic analyses for single-nucleotide variants, small insertions/deletions (indels), and larger copy number variations. NK cell cytotoxic function was assessed using 51Cr release and degranulation assays. The presence of an interferon signature was investigated using a panel of six interferon-stimulated genes (ISGs) by QPCR.
WGS identified a de novo hemizygous intronic variant in POLA1 (NM_001330360.2(POLA1):c.1393-354A > G) giving a diagnosis of XLPDR, as well as a heterozygous nonsense FLG variant (NM_002016.2(FLG):c.441del, NP_0020.1:p.(Arg151Glyfs*43)). Compared to healthy controls, the IFN signature was elevated although the degree moderated over time with the improvement in his chest disease. NK cell functional studies showed normal cytotoxicity and degranulation.
This patient had multiple atopic manifestations affecting eye, skin, chest, and gut, complicating the presentation of XLPDR. This highlights that common FLG polymorphisms should always be considered when assessing genotype-phenotype correlations of other genetic variation in patients with atopic symptoms. Additionally, while the patient exhibited an enhanced IFN signature, he does not have an NK cell defect, suggesting this may not be a constant feature of XLPDR.

POLA1 encodes a subunit of the DNA polymerase alpha, a key enzyme for the initiation of DNA synthesis. In males, hemizygous hypomorphic variants in POLA1 have been identified as the cause of X-linked pigmentary reticulate disorder (XLPDR) and a novel X-linked neurodevelopmental disorder termed Van Esch-O\'Driscoll syndrome (VEODS), while female carriers have been reported to be healthy. Nullisomy for POLA1 was speculated to be lethal due to its crucial function, while the effect of loss of one allele in females remained unknown. Here, we report on a three-generation family harboring a deletion of POLA1 in females showing subfertility as the only phenotype. Our findings show that heterozygous deletions or truncating variants in females with skewed X inactivation do not cause VEODS and support the hypothesis of very early embryonic lethality in males with POLA1 nullisomy.

POLA1 encodes the catalytic unit of DNA polymerase α, which together with the Primase complex launches the DNA replication process. While complete deficiency of this essential gene is presumed to be lethal, at least two conditions due to partial POLA1 deficiency have been described. The first genetic syndrome to be mapped to POLA1 was X-linked reticulate pigmentary disorder (XLPDR, MIM #301220), a rare syndrome characterized by skin hyperpigmentation, sterile multiorgan inflammation, recurrent infections, and distinct facial features. XLPDR has been shown to be accompanied by profound activation of type I interferon signaling, but unlike other interferonopathies, it is not associated with autoantibodies or classical autoimmunity. Rather, it is accompanied by marked Natural Killer (NK) cell dysfunction, which may explain the recurrent infections seen in this syndrome. To date, all XLPDR cases are caused by the same recurrent intronic mutation, which results in gene missplicing. Several hypomorphic mutations in POLA1, distinct from the XLPDR intronic mutation, have been recently reported and these mutations associate with a separate condition, van Esch-O\'Driscoll syndrome (VEODS, MIM #301030). This condition results in growth retardation, microcephaly, hypogonadism, and in some cases, overlapping immunological features to those seen in XLPDR. This review summarizes our current understanding of the clinical manifestations of POLA1 gene mutations with an emphasis on its immunological consequences, as well as recent advances in understanding of its pathophysiologic basis and potential therapeutic options.

It has been long assumed that normally leading strand synthesis must proceed coordinated with the lagging strand to prevent strand uncoupling and the pathological accumulation of single-stranded DNA (ssDNA) in the cell, a dogma recently challenged by in vitro studies in prokaryotes. Here, we report that human DNA polymerases can function independently at each strand in vivo and that the resulting strand uncoupling is supported physiologically by a cellular tolerance to ssDNA. Active forks rapidly accumulate ssDNA at the lagging strand when POLA1 is inhibited without triggering a stress response, despite ssDNA formation being considered a hallmark of replication stress. Acute POLA1 inhibition causes a lethal RPA exhaustion, but cells can duplicate their DNA with limited POLA1 activity and exacerbated strand uncoupling as long as RPA molecules suffice to protect the elevated ssDNA. Although robust, this uncoupled mode of DNA replication is also an in-built weakness that can be targeted for cancer treatment.

Replicating the human genome efficiently and accurately is a daunting challenge involving the duplication of upward of three billion base pairs. At the core of the complex machinery that achieves this task are three members of the B family of DNA polymerases: DNA polymerases α, δ, and ε. Collectively these multimeric polymerases ensure DNA replication proceeds at optimal rates approaching 2 × 103 nucleotides/min with an error rate of less than one per million nucleotides polymerized. The majority of DNA replication of undamaged DNA is conducted by DNA polymerases δ and ε. The DNA polymerase α-primase complex performs limited synthesis to initiate the replication process, along with Okazaki-fragment synthesis on the discontinuous lagging strand. An increasing number of human disorders caused by defects in different components of the DNA-replication apparatus have been described to date. These are clinically diverse and involve a wide range of features, including variable combinations of growth delay, immunodeficiency, endocrine insufficiencies, lipodystrophy, and cancer predisposition. Here, by using various complementary approaches, including classical linkage analysis, targeted next-generation sequencing, and whole-exome sequencing, we describe distinct missense and splice-impacting mutations in POLA1 in five unrelated families presenting with an X-linked syndrome involving intellectual disability, proportionate short stature, microcephaly, and hypogonadism. POLA1 encodes the p180 catalytic subunit of DNA polymerase α-primase. A range of replicative impairments could be demonstrated in lymphoblastoid cell lines derived from affected individuals. Our findings describe the presentation of pathogenic mutations in a catalytic component of a B family DNA polymerase member, DNA polymerase α.

Despite advances in therapeutic strategies, colorectal cancer (CRC) remains the third cause of cancer-related deaths with a relatively low survival rate. Resistance to standard chemotherapy represents a major hurdle in disease management; therefore, developing new therapeutic agents demands a thorough understanding of their mechanisms of action. One of these compounds is ST1926, an adamantyl retinoid that has shown potent antitumor activities in several human cancer models. Here, we show that ST1926 selectively suppressed the proliferation of CRC cells while sparing normal counterparts, and significantly reduced tumor volume in a xenograft cancer mouse model. Next, we investigated the effects of ST1926 in CRC cells and observed early DNA damage, S-phase arrest, dissipation of mitochondrial membrane potential, and apoptosis induction, in a p53 and p21-independent manner. To address the underlying mechanism of resistance to ST1926, we generated ST1926-resistant HCT116 cells and sequenced DNA polymerase α (POLA1), which was reported to be a direct target to the drug\'s parent molecule, CD437. We identified similar mutations in POLA1 that conferred resistance to ST1926 and CD437. These mutations were absent in 5-fluorouracil-resistant HCT116 cells, clearly validating the specificity of these mutations to the lack of DNA damage and acquired resistance to ST1926. ST1926 also inhibited POLA1 activity and reduced its protein expression levels. Further, in silico analysis of normal and malignant tissue expression data demonstrated that POLA1 levels are elevated in CRC cells and tissues compared to normal counterparts as well as to other cancer types. Our findings highlight previously uncharacterized mechanisms of action of ST1926 in CRC and suggest that elevated POLA1 expression is a pertinent molecular feature and an attractive target in CRC.

Transcriptomics is increasingly used to assess biological responses to environmental stimuli and stressors such as aquatic pollutants. However, fundamental studies characterizing individual variability in mRNA levels are lacking, which currently limits the use of transcriptomics in environmental monitoring assessments. To address individual variability in transcript abundance, we performed a meta-analysis on 231 microarrays that were conducted in the fathead minnow (FHM), a widely used toxicological model. The mean variability for gene probes was ranked from most to least variable based upon the coefficient of variation. Transcripts that were the most variable in individual tissues included NADH dehydrogenase flavoprotein 1, GTPase IMAP family member 7-like and v-set domain-containing T-cell activation inhibitor 1-like while genes encoding ribosomal proteins (rpl24 and rpl36), basic transcription factor 3, and nascent polypeptide-associated complex alpha subunit were the least variable in individuals across a range of microarray experiments. Gene networks that showed high variability (based upon the variation in expression of individual members within the network) included cell proliferation, metabolism (steroid, lipids, and glucose), cell adhesion, vascularization, and regeneration while those that showed low variability (more stability) included mRNA and rRNA processing, regulation of translational fidelity, RNA splicing, and ribosome biogenesis. Real-time PCR was conducted on a subset of genes for comparison of variability collected from the microarrays. There was a significant positive relationship between the two methods when measuring individual variability, suggesting that variability detected in microarray data can be used to guide decisions on sample sizes for measuring transcripts in real-time PCR experiments. A power analysis revealed that measuring estrogen receptor ba (esrba) requires fewer biological replicates than that of estrogen receptor bb (esrbb) in the gonad and samples sizes required to detect a 50% change for reproductive-related transcripts is between 12 and 20. Characterizing individual variability at the molecular level will prove necessary as efforts are made toward integrating molecular tools into environmental risk assessments.