Mechanism, cellular functions and cancer roles of polymerase-theta-mediated DNA end joining.

Cellular pathways that repair chromosomal double-strand breaks (DSBs) have pivotal roles in cell growth, development and cancer. These DSB repair pathways have been the target of intensive investigation, but one pathway - alternative end joining (a-EJ) - has long resisted elucidation. In this Review, we highlight recent progress in our understanding of a-EJ, especially the assignment of DNA polymerase theta (Polθ) as the predominant mediator of a-EJ in most eukaryotes, and discuss a potential molecular mechanism by which Polθ-mediated end joining (TMEJ) occurs. We address possible cellular functions of TMEJ in resolving DSBs that are refractory to repair by non-homologous end joining (NHEJ), DSBs generated following replication fork collapse and DSBs present owing to stalling of repair by homologous recombination. We also discuss how these context-dependent cellular roles explain how TMEJ can both protect against and cause genome instability, and the emerging potential of Polθ as a therapeutic target in cancer.

MRE11 liberates cGAS from nucleosome sequestration during tumorigenesis.

Oncogene-induced replication stress generates endogenous DNA damage that activates cGAS-STING-mediated signalling and tumour suppression1-3. However, the precise mechanism of cGAS activation by endogenous DNA damage remains enigmatic, particularly given that high-affinity histone acidic patch (AP) binding constitutively inhibits cGAS by sterically hindering its activation by double-stranded DNA (dsDNA)4-10. Here we report that the DNA double-strand break sensor MRE11 suppresses mammary tumorigenesis through a pivotal role in regulating cGAS activation. We demonstrate that binding of the MRE11-RAD50-NBN complex to nucleosome fragments is necessary to displace cGAS from acidic-patch-mediated sequestration, which enables its mobilization and activation by dsDNA. MRE11 is therefore essential for cGAS activation in response to oncogenic stress, cytosolic dsDNA and ionizing radiation. Furthermore, MRE11-dependent cGAS activation promotes ZBP1-RIPK3-MLKL-mediated necroptosis, which is essential to suppress oncogenic proliferation and breast tumorigenesis. Notably, downregulation of ZBP1 in human triple-negative breast cancer is associated with increased genome instability, immune suppression and poor patient prognosis. These findings establish MRE11 as a crucial mediator that links DNA damage and cGAS activation, resulting in tumour suppression through ZBP1-dependent necroptosis.

Stepwise requirements for polymerases δ and θ in theta-mediated end joining.

Timely repair of chromosomal double-strand breaks is required for genome integrity and cellular viability. The polymerase theta-mediated end joining pathway has an important role in resolving these breaks and is essential in cancers defective in other DNA repair pathways, thus making it an emerging therapeutic target1. It requires annealing of 2-6 nucleotides of complementary sequence, microhomologies, that are adjacent to the broken ends, followed by initiation of end-bridging DNA synthesis by polymerase θ. However, the other pathway steps remain inadequately defined, and the enzymes required for them are unknown. Here we demonstrate requirements for exonucleolytic digestion of unpaired 3' tails before polymerase θ can initiate synthesis, then a switch to a more accurate, processive and strand-displacing polymerase to complete repair. We show the replicative polymerase, polymerase δ, is required for both steps; its 3' to 5' exonuclease activity for flap trimming, then its polymerase activity for extension and completion of repair. The enzymatic steps that are essential and specific to this pathway are mediated by two separate, sequential engagements of the two polymerases. The requisite coupling of these steps together is likely to be facilitated by physical association of the two polymerases. This pairing of polymerase δ with a polymerase capable of end-bridging synthesis, polymerase θ, may help to explain why the normally high-fidelity polymerase δ participates in genome destabilizing processes such as mitotic DNA synthesis2 and microhomology-mediated break-induced replication3.

BRCA1 Mutational Complementation Induces Synthetic Viability.

BRCA1 promotes the DNA end resection and RAD51 loading steps of homologous recombination (HR). Whether these functions can be uncoupled, and whether mutant proteins retaining partial activity can complement one another, is unclear and could affect the severity of BRCA1-associated Fanconi anemia (FA). Here we generated a Brca1CC mouse with a coiled-coil (CC) domain deletion. Brca1CC/CC mice are born at low frequencies, and post-natal mice have FA-like abnormalities, including bone marrow failure. Intercrossing with Brca1Δ11, which is homozygous lethal, generated Brca1CC/Δ11 mice at Mendelian frequencies that were indistinguishable from Brca1+/+ mice. Brca1CC and Brca1Δ11 proteins were individually responsible for counteracting 53BP1-RIF1-Shieldin activity and promoting RAD51 loading, respectively. Thus, Brca1CC and Brca1Δ11 alleles represent separation-of-function mutations that combine to provide a level of HR sufficient for normal development and hematopoiesis. Because BRCA1 activities can be genetically separated, compound heterozygosity for functional complementary mutations may protect individuals from FA.

DNA polymerase θ protects leukemia cells from metabolically induced DNA damage.

Leukemia cells accumulate DNA damage, but altered DNA repair mechanisms protect them from apoptosis. We showed here that formaldehyde generated by serine/1-carbon cycle metabolism contributed to the accumulation of toxic DNA-protein crosslinks (DPCs) in leukemia cells, especially in driver clones harboring oncogenic tyrosine kinases (OTKs: FLT3(internal tandem duplication [ITD]), JAK2(V617F), BCR-ABL1). To counteract this effect, OTKs enhanced the expression of DNA polymerase theta (POLθ) via ERK1/2 serine/threonine kinase-dependent inhibition of c-CBL E3 ligase-mediated ubiquitination of POLθ and its proteasomal degradation. Overexpression of POLθ in OTK-positive cells resulted in the efficient repair of DPC-containing DNA double-strand breaks by POLθ-mediated end-joining. The transforming activities of OTKs and other leukemia-inducing oncogenes, especially of those causing the inhibition of BRCA1/2-mediated homologous recombination with and without concomitant inhibition of DNA-PK-dependent nonhomologous end-joining, was abrogated in Polq-/- murine bone marrow cells. Genetic and pharmacological targeting of POLθ polymerase and helicase activities revealed that both activities are promising targets in leukemia cells. Moreover, OTK inhibitors or DPC-inducing drug etoposide enhanced the antileukemia effect of POLθ inhibitor in vitro and in vivo. In conclusion, we demonstrated that POLθ plays an essential role in protecting leukemia cells from metabolically induced toxic DNA lesions triggered by formaldehyde, and it can be targeted to achieve a therapeutic effect.

TBCRC 039: a phase II study of preoperative ruxolitinib with or without paclitaxel for triple-negative inflammatory breast cancer.

BACKGROUND: Patients with inflammatory breast cancer (IBC) have overall poor clinical outcomes, with triple-negative IBC (TN-IBC) being associated with the worst survival, warranting the investigation of novel therapies. Preclinical studies implied that ruxolitinib (RUX), a JAK1/2 inhibitor, may be an effective therapy for TN-IBC. METHODS: We conducted a randomized phase II study with nested window-of-opportunity in TN-IBC. Treatment-naïve patients received a 7-day run-in of RUX alone or RUX plus paclitaxel (PAC). After the run-in, those who received RUX alone proceeded to neoadjuvant therapy with either RUX + PAC or PAC alone for 12 weeks; those who had received RUX + PAC continued treatment for 12 weeks. All patients subsequently received 4 cycles of doxorubicin plus cyclophosphamide prior to surgery. Research tumor biopsies were performed at baseline (pre-run-in) and after run-in therapy. Tumors were evaluated for phosphorylated STAT3 (pSTAT3) by immunostaining, and a subset was also analyzed by RNA-seq. The primary endpoint was the percent of pSTAT3-positive pre-run-in tumors that became pSTAT3-negative. Secondary endpoints included pathologic complete response (pCR). RESULTS: Overall, 23 patients were enrolled, of whom 21 completed preoperative therapy. Two patients achieved pCR (8.7%). pSTAT3 and IL-6/JAK/STAT3 signaling decreased in post-run-in biopsies of RUX-treated samples, while sustained treatment with RUX + PAC upregulated IL-6/JAK/STAT3 signaling compared to RUX alone. Both treatments decreased GZMB+ T cells implying immune suppression. RUX alone effectively inhibited JAK/STAT3 signaling but its combination with PAC led to incomplete inhibition. The immune suppressive effects of RUX alone and in combination may negate its growth inhibitory effects on cancer cells. CONCLUSION: In summary, the use of RUX in TN-IBC was associated with a decrease in pSTAT3 levels despite lack of clinical benefit. Cancer cell-specific-targeting of JAK2/STAT3 or combinations with immunotherapy may be required for further evaluation of JAK2/STAT3 signaling as a cancer therapeutic target. TRIAL REGISTRATION: www. CLINICALTRIALS: gov , NCT02876302. Registered 23 August 2016.

A comprehensive comparison on cell-type composition inference for spatial transcriptomics data.

Spatial transcriptomics (ST) technologies allow researchers to examine transcriptional profiles along with maintained positional information. Such spatially resolved transcriptional characterization of intact tissue samples provides an integrated view of gene expression in its natural spatial and functional context. However, high-throughput sequencing-based ST technologies cannot yet reach single cell resolution. Thus, similar to bulk RNA-seq data, gene expression data at ST spot-level reflect transcriptional profiles of multiple cells and entail the inference of cell-type composition within each ST spot for valid and powerful subsequent analyses. Realizing the critical importance of cell-type decomposition, multiple groups have developed ST deconvolution methods. The aim of this work is to review state-of-the-art methods for ST deconvolution, comparing their strengths and weaknesses. In particular, we construct ST spots from single-cell level ST data to assess the performance of 10 methods, with either ideal reference or non-ideal reference. Furthermore, we examine the performance of these methods on spot- and bead-level ST data by comparing estimated cell-type proportions to carefully matched single-cell ST data. In comparing the performance on various tissues and technological platforms, we concluded that RCTD and stereoscope achieve more robust and accurate inferences.

Essential Roles for Polymerase θ-Mediated End Joining in the Repair of Chromosome Breaks.

DNA polymerase theta (Pol θ)-mediated end joining (TMEJ) has been implicated in the repair of chromosome breaks, but its cellular mechanism and role relative to canonical repair pathways are poorly understood. We show that it accounts for most repairs associated with microhomologies and is made efficient by coupling a microhomology search to removal of non-homologous tails and microhomology-primed synthesis across broken ends. In contrast to non-homologous end joining (NHEJ), TMEJ efficiently repairs end structures expected after aborted homology-directed repair (5' to 3' resected ends) or replication fork collapse. It typically does not compete with canonical repair pathways but, in NHEJ-deficient cells, is engaged more frequently and protects against translocation. Cell viability is also severely impaired upon combined deficiency in Pol θ and a factor that antagonizes end resection (Ku or 53BP1). TMEJ thus helps to sustain cell viability and genome stability by rescuing chromosome break repair when resection is misregulated or NHEJ is compromised.

Marker-free quantification of repair pathway utilization at Cas9-induced double-strand breaks.

Genome integrity and genome engineering require efficient repair of DNA double-strand breaks (DSBs) by non-homologous end joining (NHEJ), homologous recombination (HR), or alternative end-joining pathways. Here we describe two complementary methods for marker-free quantification of DSB repair pathway utilization at Cas9-targeted chromosomal DSBs in mammalian cells. The first assay features the analysis of amplicon next-generation sequencing data using ScarMapper, an iterative break-associated alignment algorithm to classify individual repair products based on deletion size, microhomology usage, and insertions. The second assay uses repair pathway-specific droplet digital PCR assays ('PathSig-dPCR') for absolute quantification of signature DSB repair outcomes. We show that ScarMapper and PathSig-dPCR enable comprehensive assessment of repair pathway utilization in different cell models, after a variety of experimental perturbations. We use these assays to measure the differential impact of DNA end resection on NHEJ, HR and polymerase theta-mediated end joining (TMEJ) repair. These approaches are adaptable to any cellular model system and genomic locus where Cas9-mediated targeting is feasible. Thus, ScarMapper and PathSig-dPCR allow for systematic fate mapping of a targeted DSB with facile and accurate quantification of DSB repair pathway choice at endogenous chromosomal loci.

Mechanistic basis for microhomology identification and genome scarring by polymerase theta.

DNA polymerase theta mediates an end joining pathway (TMEJ) that repairs chromosome breaks. It requires resection of broken ends to generate long, 3' single-stranded DNA tails, annealing of complementary sequence segments (microhomologies) in these tails, followed by microhomology-primed synthesis sufficient to resolve broken ends. The means by which microhomologies are identified is thus a critical step in this pathway, but is not understood. Here we show microhomologies are identified by a scanning mechanism initiated from the 3' terminus and favoring bidirectional progression into flanking DNA, typically to a maximum of 15 nucleotides into each flank. Polymerase theta is frequently insufficiently processive to complete repair of breaks in microhomology-poor, AT-rich regions. Aborted synthesis leads to one or more additional rounds of microhomology search, annealing, and synthesis; this promotes complete repair in part because earlier rounds of synthesis generate microhomologies de novo that are sufficiently long that synthesis is more processive. Aborted rounds of synthesis are evident in characteristic genomic scars as insertions of 3 to 30 bp of sequence that is identical to flanking DNA ("templated" insertions). Templated insertions are present at higher levels in breast cancer genomes from patients with germline BRCA1/2 mutations, consistent with an addiction to TMEJ in these cancers. Our work thus describes the mechanism for microhomology identification and shows how it both mitigates limitations implicit in the microhomology requirement and generates distinctive genomic scars associated with pathogenic genome instability.

Phase 1 trial of adavosertib (AZD1775) in combination with concurrent radiation and cisplatin for intermediate-risk and high-risk head and neck squamous cell carcinoma.

BACKGROUND: Adavosertib (AZD1775) is an inhibitor of the Wee1 kinase. The authors conducted a phase 1b trial to evaluate the safety of adavosertib in combination with definitive chemoradiotherapy for patients with newly diagnosed, intermediate-risk/high-risk, locally advanced head and neck squamous cell carcinoma (HNSCC). METHODS: Twelve patients with intermediate-risk/high-risk HNSCC were enrolled, including those with p16-negative tumors of the oropharynx, p16-positive tumors of the oropharynx with ≥10 tobacco pack-years, and tumors of the larynx/hypopharynx regardless of p16 status. All patients were treated with an 8-week course of concurrent intensity-modulated radiotherapy at 70 grays (Gy) (2 Gy daily in weeks 1-7), cisplatin 30 mg/m2 weekly (in weeks 1-7), and adavosertib (twice daily on Monday, Tuesday, and Wednesday of weeks 1, 2, 4, 5, 7, and 8). The primary objective was to determine the maximum tolerated dose and the recommended phase 2 dose of adavosertib given concurrently with radiation and cisplatin. Secondary objectives were to determine the 12-week objective response rate and progression-free and overall survival. RESULTS: Three patients (25%) experienced a dose-limiting toxicity, including febrile neutropenia (n = 2) and grade 4 thromboembolism (n = 1). Two dose-limiting toxicities occurred with adavosertib at 150 mg. The median follow-up was 14.7 months. The 12-week posttreatment objective response rate determined by positron emission tomography/computed tomography was 100%. The 1-year progression-free and overall survival rates were both 90%. The maximum tolerated dose of adavosertib was 100 mg. CONCLUSIONS: Adavosertib 100 mg (twice daily on Monday, Tuesday, and Wednesday of weeks 1, 2, 4, 5, 7, and 8), in combination with 70 Gy of intensity-modulated radiotherapy and cisplatin 30 mg/m2 , is the recommended phase 2 dose for patients with HNSCC.

TP53 mutations increase radioresistance in rhabdomyosarcoma and Ewing sarcoma.

BACKGROUND: p53 plays a key role in the DNA repair process and response to ionising radiation. We sought to determine the clinical phenotype of TP53 mutations and p53 pathway alterations in patients with rhabdomyosarcoma (RMS) and Ewing sarcoma (ES) treated with radiation. METHODS: Of patients with available genomic sequencing, we identified 109 patients with RMS and ES treated to a total of 286 radiation sites. We compared irradiated tumour control among tumours with TP53 mutations (n = 40) to those that were TP53 wild-type (n = 246). We additionally compared irradiated tumour control among tumours with any p53 pathway alteration (defined as tumours with TP53 mutations or TP53 wild-type tumours identified to have MDM2/4 amplification and/or CDKN2A/B deletion, n = 78) to those without such alterations (n = 208). RESULTS: The median follow-up was 26 months from radiation. TP53 mutations were associated with worse irradiated tumour control among the entire cohort (hazard ratio, HR = 2.8, P < 0.0001). Tumours with any p53 pathway alteration also had inferior irradiated tumour control (HR = 2.0, P = 0.003). On multivariable analysis, after controlling for tumour histology, intent of radiation, presence of gross disease, and biologically effective dose, TP53 mutations continued to be associated with a radioresistant phenotype (HR = 7.1, P < 0.0001). CONCLUSIONS: Our results show that TP53 mutations are associated with increased radioresistance in RMS and ES. Novel strategies to overcome this radioresistance are important for improved outcomes in p53 disruptive RMS and ES.

The Mre11 complex suppresses oncogene-driven breast tumorigenesis and metastasis.

The DNA damage response (DDR) is activated by oncogenic stress, but the mechanisms by which this occurs, and the particular DDR functions that constitute barriers to tumorigenesis, remain unclear. We established a mouse model of sporadic oncogene-driven breast tumorigenesis in a series of mutant mouse strains with specific DDR deficiencies to reveal a role for the Mre11 complex in the response to oncogene activation. We demonstrate that an Mre11-mediated DDR restrains mammary hyperplasia by effecting an oncogene-induced G2 arrest. Impairment of Mre11 complex functions promotes the progression of mammary hyperplasias into invasive and metastatic breast cancers, which are often associated with secondary inactivation of the Ink4a-Arf (CDKN2a) locus. These findings provide insight into the mechanism of DDR engagement by activated oncogenes and highlight genetic interactions between the DDR and Ink4a-Arf pathways in suppression of oncogene-driven tumorigenesis and metastasis.

Evidence that the human cell cycle is a series of uncoupled, memoryless phases.

The cell cycle is canonically described as a series of four consecutive phases: G1, S, G2, and M. In single cells, the duration of each phase varies, but the quantitative laws that govern phase durations are not well understood. Using time-lapse microscopy, we found that each phase duration follows an Erlang distribution and is statistically independent from other phases. We challenged this observation by perturbing phase durations through oncogene activation, inhibition of DNA synthesis, reduced temperature, and DNA damage. Despite large changes in durations in cell populations, phase durations remained uncoupled in individual cells. These results suggested that the independence of phase durations may arise from a large number of molecular factors that each exerts a minor influence on the rate of cell cycle progression. We tested this model by experimentally forcing phase coupling through inhibition of cyclin-dependent kinase 2 (CDK2) or overexpression of cyclin D. Our work provides an explanation for the historical observation that phase durations are both inherited and independent and suggests how cell cycle progression may be altered in disease states.

Mechanisms of Immune Sensing of DNA Damage.

Genomic stability relies on a multifaceted and evolutionarily conserved DNA damage response (DDR). In multicellular organisms, an integral facet of the DDR involves the activation of the immune system to eliminate cells with persistent DNA damage. Recent research has shed light on a complex array of nucleic acid sensors crucial for innate immune activation in response to oncogenic stress-associated DNA damage, a process vital for suppressing tumor formation. Yet, these immune sensing pathways may also be co-opted to foster tolerance of chromosomal instability, thereby driving cancer progression. This review aims to provide an updated overview of how the innate immune system detects and responds to DNA damage. An improved understanding of the regulatory intricacies governing this immune response may uncover new avenues for cancer prevention and therapeutic intervention.

Mediators of vascular remodelling co-opted for sequential steps in lung metastasis.

Metastasis entails numerous biological functions that collectively enable cancerous cells from a primary site to disseminate and overtake distant organs. Using genetic and pharmacological approaches, we show that the epidermal growth factor receptor ligand epiregulin, the cyclooxygenase COX2, and the matrix metalloproteinases 1 and 2, when expressed in human breast cancer cells, collectively facilitate the assembly of new tumour blood vessels, the release of tumour cells into the circulation, and the breaching of lung capillaries by circulating tumour cells to seed pulmonary metastasis. These findings reveal how aggressive primary tumorigenic functions can be mechanistically coupled to greater lung metastatic potential, and how such biological activities may be therapeutically targeted with specific drug combinations.

Polymerase θ inhibition steps on the cGAS pedal.

Deficiencies in homologous recombination (HR) repair lead to an accumulation of DNA damage and can predispose individuals to cancer. Polymerase theta (Pol θ, encoded by POLQ) is overexpressed by HR-deficient cancers and promotes cancer cell survival by mediating error-prone double-stranded break (DSB) repair and facilitating resistance against poly-ADP ribose polymerase inhibitor treatment. In this issue of the JCI, Oh, Wang, et al. report on the impact of Pol θ inhibition on activation of antitumor immunity. The authors used pancreatic ductal adenocarcinoma (PDAC) cell and mouse models characterized by HR-associated gene alterations and POLQ overexpression. POLQ knockdown showed synthetic lethality in combination with gene mutations involving DNA repair, including BRCA1, BRCA2, and ATM. Notably, Pol θ deficiency or inhibition suppressed tumor growth, increased the accumulation of unrepaired DNA damage, and enhanced T cell infiltration via the cGAS/STING pathway. These findings suggest a broader scope for Pol θ inhibition in HR-deficient cancers.

Cell composition inference and identification of layer-specific spatial transcriptional profiles with POLARIS.

Spatial transcriptomics (ST) technology, providing spatially resolved transcriptional profiles, facilitates advanced understanding of key biological processes related to health and disease. Sequencing-based ST technologies provide whole-transcriptome profiles but are limited by the non-single cell-level resolution. Lack of knowledge in the number of cells or cell type composition at each spot can lead to invalid downstream analysis, which is a critical issue recognized in ST data analysis. Methods developed, however, tend to underuse histological images, which conceptually provide important and complementary information including anatomical structure and distribution of cells. To fill in the gaps, we present POLARIS, a versatile ST analysis method that can perform cell type deconvolution, identify anatomical or functional layer-wise differentially expressed (LDE) genes, and enable cell composition inference from histology images. Applied to four tissues, POLARIS demonstrates high deconvolution accuracy, accurately predicts cell composition solely from images, and identifies LDE genes that are biologically relevant and meaningful.

Calibration of cell-free DNA measurements by next-generation sequencing.

OBJECTIVES: Accurate monitoring of disease burden depends on accurate disease marker quantification. Although next-generation sequencing (NGS) is a promising technology for noninvasive monitoring, plasma cell-free DNA levels are often reported in misleading units that are confounded by non-disease-related factors. We proposed a novel strategy for calibrating NGS assays using spiked normalizers to improve precision and to promote standardization and harmonization of analyte concentrations. METHODS: In this study, we refined our NGS protocol to calculate absolute analyte concentrations to (1) adjust for assay efficiency, as judged by recovery of spiked synthetic normalizer DNAs, and (2) calibrate NGS values against droplet digital polymerase chain reaction (ddPCR). As a model target, we chose the Epstein-Barr virus (EBV) genome. In patient (n = 12) and mock (n = 12) plasmas, NGS and 2 EBV ddPCR assays were used to report EBV load in copies per mL of plasma. RESULTS: Next-generation sequencing was equally sensitive to ddPCR, with improved linearity when NGS values were normalized for spiked DNA read counts (R2 = 0.95 for normalized vs 0.91 for raw read concentrations). Linearity permitted NGS calibration to each ddPCR assay, achieving equivalent concentrations (copies/mL). CONCLUSIONS: Our novel strategy for calibrating NGS assays suggests potential for a universal reference material to overcome biological and preanalytical variables hindering traditional NGS strategies for quantifying disease burden.