Non-homologous end joining shapes the genomic rearrangement landscape of chromothripsis from mitotic errors.

Mitotic errors generate micronuclei entrapping mis-segregated chromosomes, which are susceptible to catastrophic fragmentation through chromothripsis. The reassembly of fragmented chromosomes by error-prone DNA double-strand break (DSB) repair generates diverse genomic rearrangements associated with human diseases. How specific repair pathways recognize and process these lesions remains poorly understood. Here we use CRISPR/Cas9 to systematically inactivate distinct DSB repair pathways and interrogate the rearrangement landscape of fragmented chromosomes. Deletion of canonical non-homologous end joining (NHEJ) components substantially reduces complex rearrangements and shifts the rearrangement landscape toward simple alterations without the characteristic patterns of chromothripsis. Following reincorporation into the nucleus, fragmented chromosomes localize within sub-nuclear micronuclei bodies (MN bodies) and undergo ligation by NHEJ within a single cell cycle. In the absence of NHEJ, chromosome fragments are rarely engaged by alternative end-joining or recombination-based mechanisms, resulting in delayed repair kinetics, persistent 53BP1-labeled MN bodies, and cell cycle arrest. Thus, we provide evidence supporting NHEJ as the exclusive DSB repair pathway generating complex rearrangements from mitotic errors.

Analytical propagation model for underwater free-space optical communication through realistic levels of oceanic absorption and scattering.

Optical communications (OC) through water bodies is an attractive technology for a variety of applications. Thanks to current single-photon detection capabilities, OC receiver systems can reliably decode very weak transmitted signals. This is the regime where pulse position modulation is an ideal scheme. However, there has to be at least one photon that goes through the pupil of the fore optics and lands in the assigned time bin. We estimate the detectable photon budget as a function of range for propagation through ocean water, both open and coastal. We make realistic assumptions about the water's inherent optical properties, specifically, absorption and scattering coefficients, as well as the strong directionality of the scattering phase function for typical hydrosol populations. We adopt an analytical (hence very fast) path-integral small-angle solution of the radiative transfer equation for multiple forward-peaked scattering across intermediate to large optical distances. Integrals are performed both along the directly transmitted beam (whether or not it is still populated) and radially away from it. We use this modeling framework to estimate transmission of a 1 J pulse of 532 nm light through open ocean and coastal waters. Thresholds for single-photon detection per time bin are a few km and a few 100 m. These are indicative estimates that will be reduced in practice due to sensor noise, background light, turbulence, bubbles, and so on, to be included in future work.

Preliminary report of a thoracic duct-to-pulmonary vein lymphovenous anastomosis in swine: A novel technique and potential treatment for lymphatic failure.

OBJECTIVE: The thoracic duct is the largest lymphatic vessel in the body, and carries fluid and nutrients absorbed in abdominal organs to the central venous circulation. Thoracic duct obstruction can cause significant failure of the lymphatic circulation (i.e., protein-losing enteropathy, plastic bronchitis, etc.). Surgical anastomosis between the thoracic duct and central venous circulation has been used to treat thoracic duct obstruction but cannot provide lymphatic decompression in patients with superior vena cava obstruction or chronically elevated central venous pressures (e.g., right heart failure, single ventricle physiology, etc.). Therefore, this preclinical feasibility study sought to develop a novel and optimal surgical technique for creating a thoracic duct-to-pulmonary vein lymphovenous anastomosis (LVA) in swine that could remain patent and preserve unidirectional lymphatic fluid flow into the systemic venous circulation to provide therapeutic decompression of the lymphatic circulation even at high central venous pressures. METHODS: A thoracic duct-to-pulmonary vein LVA was attempted in 10 piglets (median age 80 [IQR 80-83] days; weight 22.5 [IQR 21.4-26.8] kg). After a right thoracotomy, the thoracic duct was mobilized, transected, and anastomosed to the right inferior pulmonary vein. Animals were systemically anticoagulated on post-operative day 1. Lymphangiography was used to evaluate LVA patency up to post-operative day 7. RESULTS: A thoracic duct-to-pulmonary vein LVA was successfully completed in 8/10 (80.0%) piglets, of which 6/8 (75.0%) survived to the intended study endpoint without any complication (median 6 [IQR 4-7] days). Initially, 2/10 (20.0%) LVAs were aborted intraoperatively, and 2/10 (20.0%) animals were euthanized early due to post-operative complications. However, using an optimized surgical technique, the success rate for creating a thoracic duct-to-pulmonary vein LVA in six animals was 100%, all of which survived to their intended study endpoint without any complications (median 6 [IQR 4-7] days). LVAs remained patent for up to seven days. CONCLUSION: A thoracic duct-to-pulmonary vein LVA can be completed safely and remain patent for at least one week with systemic anticoagulation, which provides an important proof-of-concept that this novel intervention could effectively offload the lymphatic circulation in patients with lymphatic failure and elevated central venous pressures.

BUB1 regulates non-homologous end joining pathway to mediate radioresistance in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer subtype often treated with radiotherapy (RT). Due to its intrinsic heterogeneity and lack of effective targets, it is crucial to identify novel molecular targets that would increase RT efficacy. Here we demonstrate the role of BUB1 (cell cycle Ser/Thr kinase) in TNBC radioresistance and offer a novel strategy to improve TNBC treatment. METHODS: Gene expression analysis was performed to look at genes upregulated in TNBC patient samples compared to other subtypes. Cell proliferation and clonogenic survivals assays determined the IC50 of BUB1 inhibitor (BAY1816032) and radiation enhancement ratio (rER) with pharmacologic and genomic BUB1 inhibition. Mammary fat pad xenografts experiments were performed in CB17/SCID. The mechanism through which BUB1 inhibitor sensitizes TNBC cells to radiotherapy was delineated by γ-H2AX foci assays, BLRR, Immunoblotting, qPCR, CHX chase, and cell fractionation assays. RESULTS: BUB1 is overexpressed in BC and its expression is considerably elevated in TNBC with poor survival outcomes. Pharmacological or genomic ablation of BUB1 sensitized multiple TNBC cell lines to cell killing by radiation, although breast epithelial cells showed no radiosensitization with BUB1 inhibition. Kinase function of BUB1 is mainly accountable for this radiosensitization phenotype. BUB1 ablation also led to radiosensitization in TNBC tumor xenografts with significantly increased tumor growth delay and overall survival. Mechanistically, BUB1 ablation inhibited the repair of radiation-induced DNA double strand breaks (DSBs). BUB1 ablation stabilized phospho-DNAPKcs (S2056) following RT such that half-lives could not be estimated. In contrast, RT alone caused BUB1 stabilization, but pre-treatment with BUB1 inhibitor prevented stabilization (t1/2, ~8 h). Nuclear and chromatin-enriched fractionations illustrated an increase in recruitment of phospho- and total-DNAPK, and KAP1 to chromatin indicating that BUB1 is indispensable in the activation and recruitment of non-homologous end joining (NHEJ) proteins to DSBs. Additionally, BUB1 staining of TNBC tissue microarrays demonstrated significant correlation of BUB1 protein expression with tumor grade. CONCLUSIONS: BUB1 ablation sensitizes TNBC cell lines and xenografts to RT and BUB1 mediated radiosensitization may occur through NHEJ. Together, these results highlight BUB1 as a novel molecular target for radiosensitization in women with TNBC.

BUB1 regulates non-homologous end joining pathway to mediate radioresistance in triple-negative breast cancer.

Background: Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer subtype often treated with radiotherapy (RT). Due to its intrinsic heterogeneity and lack of effective targets, it is crucial to identify novel molecular targets that would increase RT efficacy. Here we demonstrate the role of BUB1 (cell cycle Ser/Thr kinase) in TNBC radioresistance and offer a novel strategy to improve TNBC treatment. Methods: Gene expression analysis was performed to look at genes upregulated in TNBC patient samples compared to other subtypes. Cell proliferation and clonogenic survivals assays determined the IC 50 of BUB1 inhibitor (BAY1816032) and radiation enhancement ratio (rER) with pharmacologic and genomic BUB1 inhibition. Mammary fat pad xenografts experiments were performed in CB17/SCID. The mechanism through which BUB1 inhibitor sensitizes TNBC cells to radiotherapy was delineated by γ-H2AX foci assays, BLRR, Immunoblotting, qPCR, CHX chase, and cell fractionation assays. Results: BUB1 is overexpressed in BC and its expression is considerably elevated in TNBC with poor survival outcomes. Pharmacological or genomic ablation of BUB1 sensitized multiple TNBC cell lines to cell killing by radiation, although breast epithelial cells showed no radiosensitization with BUB1 inhibition. Kinase function of BUB1 is mainly accountable for this radiosensitization phenotype. BUB1 ablation also led to radiosensitization in TNBC tumor xenografts with significantly increased tumor growth delay and overall survival. Mechanistically, BUB1 ablation inhibited the repair of radiation-induced DNA double strand breaks (DSBs). BUB1 ablation stabilized phospho-DNAPKcs (S2056) following RT such that half-lives could not be estimated. In contrast, RT alone caused BUB1 stabilization, but pre-treatment with BUB1 inhibitor prevented stabilization (t 1/2 , ∼8 h). Nuclear and chromatin-enriched fractionations illustrated an increase in recruitment of phospho- and total-DNAPK, and KAP1 to chromatin indicating that BUB1 is indispensable in the activation and recruitment of non-homologous end joining (NHEJ) proteins to DSBs. Additionally, BUB1 staining of TNBC tissue microarrays demonstrated significant correlation of BUB1 protein expression with tumor grade. Conclusions: BUB1 ablation sensitizes TNBC cell lines and xenografts to RT and BUB1 mediated radiosensitization may occur through NHEJ. Together, these results highlight BUB1 as a novel molecular target for radiosensitization in women with TNBC.

AXL/WRNIP1 Mediates Replication Stress Response and Promotes Therapy Resistance and Metachronous Metastasis in HER2+ Breast Cancer.

Therapy resistance and metastatic progression are primary causes of cancer-related mortality. Disseminated tumor cells possess adaptive traits that enable them to reprogram their metabolism, maintain stemness, and resist cell death, facilitating their persistence to drive recurrence. The survival of disseminated tumor cells also depends on their ability to modulate replication stress in response to therapy while colonizing inhospitable microenvironments. In this study, we discovered that the nuclear translocation of AXL, a TAM receptor tyrosine kinase, and its interaction with WRNIP1, a DNA replication stress response factor, promotes the survival of HER2+ breast cancer cells that are resistant to HER2-targeted therapy and metastasize to the brain. In preclinical models, knocking down or pharmacologically inhibiting AXL or WRNIP1 attenuated protection of stalled replication forks. Furthermore, deficiency or inhibition of AXL and WRNIP1 also prolonged metastatic latency and delayed relapse. Together, these findings suggest that targeting the replication stress response, which is a shared adaptive mechanism in therapy-resistant and metastasis-initiating cells, could reduce metachronous metastasis and enhance the response to standard-of-care therapies. SIGNIFICANCE: Nuclear AXL and WRNIP1 interact and mediate replication stress response, promote therapy resistance, and support metastatic progression, indicating that targeting the AXL/WRNIP1 axis is a potentially viable therapeutic strategy for breast cancer.

A High-Affinity "Synthavidin" Receptor for Squaraine Dyes.

Strong-binding host-guest pairings in aqueous media have potential as "supramolecular glues" in biomedical techniques, complementing the widely-used (strept)avidin-biotin combination. We have previously found that squaraine dyes are bound very strongly by tetralactam macrocycles possessing anthracenyl units as cavity walls. Here we show that replacing the anthracenes with pentacyclic 5,7,12,14-tetrahydro-5,7,12,14-tetraoxapentacene (TOP) units generates receptors which bind squaraines with increased affinities (around Ka =1010  m-1 ) and improved selectivities. Binding can be followed through changes to squaraine fluorescence and absorbance. The TOP units are easy to prepare and potentially variable, while the TOP-based receptor shows improved photostability, both in itself and in complex with squaraines. The results suggest that this system could prove valuable in the further development of practical "synthavidin" chemistry.

The cancer testes antigen, HORMAD1, limits genomic instability in cancer cells by protecting stalled replication forks.

Tumors anomalously induce the expression of meiotic genes, which are otherwise restricted only to developing gametes. If and how these aberrantly expressed meiotic proteins influence DNA metabolism is not clear, but could have important implications for how tumors acquire and mitigate genomic instability. HORMAD1 is a highly conserved meiotic protein that is frequently expressed in lung adenocarincoma where its expression correlates with reduced patient survival and increased mutation burden. Here, we find that HORMAD1 associates with the replisome and is critical for protecting stalled DNA replication forks. Loss of HORMAD1 leads to nascent DNA strand degradation, an event which is mediated by the MRE11-DNA2-BLM pathway. We find that these phenotypes are due to limited RAD51 loading onto stalled replication forks in the absence of HORMAD1. Ultimately, loss of HORMAD1 leads to increased DNA breaks and chromosomal defects, which is exacerbated dramatically by induction of replication stress. Tumor cells proliferate despite encountering chronic replication stress, placing them on the precipice of catastrophic genomic damage. Our data support the hypothesis that the aberrant expression of HORMAD1 is engaged to attenuate the accumulation of excessive DNA damage due to chronic replication stress, which may otherwise lead to accumulation of toxic levels of genomic instability.

DNA-PK is activated by SIRT2 deacetylation to promote DNA double-strand break repair by non-homologous end joining.

DNA-dependent protein kinase (DNA-PK) plays a critical role in non-homologous end joining (NHEJ), the predominant pathway that repairs DNA double-strand breaks (DSB) in response to ionizing radiation (IR) to govern genome integrity. The interaction of the catalytic subunit of DNA-PK (DNA-PKcs) with the Ku70/Ku80 heterodimer on DSBs leads to DNA-PK activation; however, it is not known if upstream signaling events govern this activation. Here, we reveal a regulatory step governing DNA-PK activation by SIRT2 deacetylation, which facilitates DNA-PKcs localization to DSBs and interaction with Ku, thereby promoting DSB repair by NHEJ. SIRT2 deacetylase activity governs cellular resistance to DSB-inducing agents and promotes NHEJ. SIRT2 furthermore interacts with and deacetylates DNA-PKcs in response to IR. SIRT2 deacetylase activity facilitates DNA-PKcs interaction with Ku and localization to DSBs and promotes DNA-PK activation and phosphorylation of downstream NHEJ substrates. Moreover, targeting SIRT2 with AGK2, a SIRT2-specific inhibitor, augments the efficacy of IR in cancer cells and tumors. Our findings define a regulatory step for DNA-PK activation by SIRT2-mediated deacetylation, elucidating a critical upstream signaling event initiating the repair of DSBs by NHEJ. Furthermore, our data suggest that SIRT2 inhibition may be a promising rationale-driven therapeutic strategy for increasing the effectiveness of radiation therapy.

Non-invasive diffuse optical monitoring of cerebral physiology in an adult swine-model of impact traumatic brain injury.

In this study, we used diffuse optics to address the need for non-invasive, continuous monitoring of cerebral physiology following traumatic brain injury (TBI). We combined frequency-domain and broadband diffuse optical spectroscopy with diffuse correlation spectroscopy to monitor cerebral oxygen metabolism, cerebral blood volume, and cerebral water content in an established adult swine-model of impact TBI. Cerebral physiology was monitored before and after TBI (up to 14 days post injury). Overall, our results suggest that non-invasive optical monitoring can assess cerebral physiologic impairments post-TBI, including an initial reduction in oxygen metabolism, development of cerebral hemorrhage/hematoma, and brain swelling.

ATM phosphorylates the FATC domain of DNA-PKcs at threonine 4102 to promote non-homologous end joining.

Ataxia-telangiectasia mutated (ATM) drives the DNA damage response via modulation of multiple signal transduction and DNA repair pathways. Previously, ATM activity was implicated in promoting the non-homologous end joining (NHEJ) pathway to repair a subset of DNA double-stranded breaks (DSBs), but how ATM performs this function is still unclear. In this study, we identified that ATM phosphorylates the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a core NHEJ factor, at its extreme C-terminus at threonine 4102 (T4102) in response to DSBs. Ablating phosphorylation at T4102 attenuates DNA-PKcs kinase activity and this destabilizes the interaction between DNA-PKcs and the Ku-DNA complex, resulting in decreased assembly and stabilization of the NHEJ machinery at DSBs. Phosphorylation at T4102 promotes NHEJ, radioresistance, and increases genomic stability following DSB induction. Collectively, these findings establish a key role for ATM in NHEJ-dependent repair of DSBs through positive regulation of DNA-PKcs.

DNA-PKcs and ATM modulate mitochondrial ADP-ATP exchange as an oxidative stress checkpoint mechanism.

DNA-PKcs is a key regulator of DNA double-strand break repair. Apart from its canonical role in the DNA damage response, DNA-PKcs is involved in the cellular response to oxidative stress (OS), but its exact role remains unclear. Here, we report that DNA-PKcs-deficient human cells display depolarized mitochondria membrane potential (MMP) and reoriented metabolism, supporting a role for DNA-PKcs in oxidative phosphorylation (OXPHOS). DNA-PKcs directly interacts with mitochondria proteins ANT2 and VDAC2, and formation of the DNA-PKcs/ANT2/VDAC2 (DAV) complex supports optimal exchange of ADP and ATP across mitochondrial membranes to energize the cell via OXPHOS and to maintain MMP. Moreover, we demonstrate that the DAV complex temporarily dissociates in response to oxidative stress to attenuate ADP-ATP exchange, a rate-limiting step for OXPHOS. Finally, we found that dissociation of the DAV complex is mediated by phosphorylation of DNA-PKcs at its Thr2609 cluster by ATM kinase. Based on these findings, we propose that the coordination between the DAV complex and ATM serves as a novel oxidative stress checkpoint to decrease ROS production from mitochondrial OXPHOS and to hasten cellular recovery from OS.

ATM phosphorylates the FATC domain of DNA-PK cs at threonine 4102 to promote non-homologous end joining.

Ataxia-telangiectasia mutated (ATM) drives the DNA damage response via modulation of multiple signal transduction and DNA repair pathways. Previously, ATM activity was implicated in promoting the non-homologous end joining (NHEJ) pathway to repair a subset of DNA double strand breaks (DSBs), but how ATM performs this function is still unclear. In this study, we identified that ATM phosphorylates the DNA-dependent protein kinase catalytic subunit (DNA-PK cs ), a core NHEJ factor, at its extreme C-terminus at threonine 4102 (T4102) in response to DSBs. Phosphorylation at T4102 stabilizes the interaction between DNA-PK cs and the Ku-DNA complex and promotes assembly and stabilization of the NHEJ machinery at DSBs. Ablating phosphorylation at this site results in decreased NHEJ, radiosensitivity, and increased radiation-induced genomic instability. Collectively, these findings establish a key role for ATM in NHEJ-dependent repair of DSBs through positive regulation of DNA-PK cs .

The Cancer Testes Antigen, HORMAD1, is a Tumor-Specific Replication Fork Protection Factor.

Tumors frequently activate the expression of genes that are only otherwise required for meiosis. HORMAD1, which is essential for meiotic recombination in multiple species, is expressed in over 50% of human lung adenocarcinoma cells (LUAD). We previously found that HORMAD1 promotes DNA double strand break (DSB) repair in LUAD. Here, we report that HORMAD1 takes on an additional role in protecting genomic integrity. Specifically, we find HORMAD1 is critical for protecting stalled DNA replication forks in LUAD. Loss of HORMAD1 leads to nascent DNA degradation, an event which is mediated by the MRE11-DNA2-BLM pathway. Moreover, following exogenous induction of DNA replication stress, HORMAD1 deleted cells accumulate single stranded DNA (ssDNA). We find that these phenotypes are the result of a lack of RAD51 and BRCA2 loading onto stalled replication forks. Ultimately, loss of HORMAD1 leads to increased DSBs and chromosomal aberrations in response to replication stress. Collectively, our data support a model where HORMAD1 expression is selected to mitigate DNA replication stress, which would otherwise induce deleterious genomic instability.

Tricyclic octaurea "Temples" for the recognition of polar molecules in water.

Two water-soluble tricyclic "Temple" macrocycles featuring pyrene roof/floor units and bis-urea spacers have been synthesised and studied as receptors for aromatic compounds in aqueous media. The tricycles show good selectivity for methylated purine alkaloids such as caffeine versus unsubstituted heterocycles such as adenine and indole. Binding is signalled by major changes in fluorescence, apparently due to the break-up of intramolecular excimers. The formation of excimers implies cavity collapse in the absence of guests explaining why, unlike an earlier relative, these receptors do not bind carbohydrates. Naphthalenediimides (NDIs) have also been studied as geometrically complementary guests, and indeed bind especially strongly (Ka > 107 M-1); this powerful and selective association suggests potential applications in supramolecular self-assembly.

Photon path distributions in optically thin slabs.

The probability distribution function of photon path length in a scattering medium contains valuable information on that medium. While strongly scattering optically thick media have been extensively studied, in particular, with resort to the diffusion approximation, optically thin media have received much less attention. Here, we derive the probability distribution functions for the lengths of singly- and twice-scattered photon paths in an isotropically scattering slab of optical thickness τ, for both reflected and transmitted photons. We show that, in the case of an optically thin slab, these photons dominate the overall response of the medium. We confirm that the second moment of the distribution deviates from the ballistic limit in the case of collimated illumination. Interestingly, we show that under diffuse illumination, the second moment of the distribution is dominated by unscattered transmitted photons, hence is proportional to lnτ, and independent of the phase function. Higher moments of order n (≥3) scale as Hnτn-2. When only reflected or transmitted photons are considered, the second moment scales as H2τ-1, whatever the illumination and viewing conditions. This provides direct access to τ. These theoretical results are extensively supported by Monte Carlo ray-tracing simulations. Extension to anisotropic scattering using these same simulations shows that the results hold, given a scaling factor for collimated illumination, and without any dependence on the phase function for diffuse illumination. These results overall demonstrate that the optical thickness of an optically thin slab can be estimated from the second moment of the distribution. Along with the fact that under diffuse illumination the geometrical thickness can be derived from the first moment of the distribution, this proves that the extinction coefficient of the medium can be estimated from the combination of both moments. This study thus opens new perspectives for non-invasive characterization of optically thin media either in the laboratory or by remote sensing.

Vancomycin mimicry: towards new supramolecular antibiotics.

Vancomycin is the best-known of the glycopeptide group antibiotics (GPAs), a family of agents which operate by binding the C-terminal deptide D-Ala-D-Ala. This anionic epitope is an interesting target because it plays a central role in bacterial cell wall synthesis, and is not readily modified by evolution. Accordingly, vancomycin has been in use for >60 years but has only provoked limited resistance. Agents which mimic vancomycin but are easier to synthesise and modify could serve as valuable weapons against pathogenic bacteria, broadening the scope of the GPAs and addressing the resistance that does exist. This article gives an overview of vancomycin's structure and action, surveys past work on vancomycin mimicry, and makes the case for renewed effort in the future.

Annotation of the Complete Genome Sequences of Bacteriophages Sara and Birdfeeder.

Sara is a siphovirus with a linear 17,362bp genome containing 25 genes. Birdfeeder is a podovirus with a circularly permuted 53,897bp genome containing 52 genes. Sara and Birdfeeder were isolated from environmental samples in Plattsburgh, NY, USA and Forest Hill, MD, USA, respectively, using Microbacterium foliorum NRRL B-24224.

DNA-PKcs-dependent phosphorylation of RECQL4 promotes NHEJ by stabilizing the NHEJ machinery at DNA double-strand breaks.

Non-homologous end joining (NHEJ) is the major pathway that mediates the repair of DNA double-strand breaks (DSBs) generated by ionizing radiation (IR). Previously, the DNA helicase RECQL4 was implicated in promoting NHEJ, but its role in the pathway remains unresolved. In this study, we report that RECQL4 stabilizes the NHEJ machinery at DSBs to promote repair. Specifically, we find that RECQL4 interacts with the NHEJ core factor DNA-PKcs and the interaction is increased following IR. RECQL4 promotes DNA end bridging mediated by DNA-PKcs and Ku70/80 in vitro and the accumulation/retention of NHEJ factors at DSBs in vivo. Moreover, interaction between DNA-PKcs and the other core NHEJ proteins following IR treatment is attenuated in the absence of RECQL4. These data indicate that RECQL4 promotes the stabilization of the NHEJ factors at DSBs to support formation of the NHEJ long-range synaptic complex. In addition, we observed that the kinase activity of DNA-PKcs is required for accumulation of RECQL4 to DSBs and that DNA-PKcs phosphorylates RECQL4 at six serine/threonine residues. Blocking phosphorylation at these sites reduced the recruitment of RECQL4 to DSBs, attenuated the interaction between RECQL4 and NHEJ factors, destabilized interactions between the NHEJ machinery, and resulted in decreased NHEJ. Collectively, these data illustrate reciprocal regulation between RECQL4 and DNA-PKcs in NHEJ.

Evaluation of the Response of HNSCC Cell Lines to γ-Rays and 12C Ions: Can Radioresistant Tumors Be Identified and Selected for 12C Ion Radiotherapy?

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. Thirty percent of patients will experience locoregional recurrence for which median survival is less than 1 year. Factors contributing to treatment failure include inherent resistance to X-rays and chemotherapy, hypoxia, epithelial to mesenchymal transition, and immune suppression. The unique properties of 12C radiotherapy including enhanced cell killing, a decreased oxygen enhancement ratio, generation of complex DNA damage, and the potential to overcome immune suppression make its application well suited to the treatment of HNSCC. We examined the 12C radioresponse of five HNSCC cell lines, whose surviving fraction at 3.5 Gy ranged from average to resistant when compared with a larger panel of 38 cell lines to determine if 12C irradiation can overcome X-ray radioresistance and to identify biomarkers predictive of 12C radioresponse. Cells were irradiated with 12C using a SOBP with an average LET of 80 keV/µm (CNAO: Pavia, Italy). RBE values varied depending upon endpoint used. A 37 gene signature was able to place cells in their respective radiosensitivity cohort with an accuracy of 86%. Radioresistant cells were characterized by an enrichment of genes associated with radioresistance and survival mechanisms including but not limited to G2/M Checkpoint MTORC1, HIF1α, and PI3K/AKT/MTOR signaling. These data were used in conjunction with an in silico-based modeling approach to evaluate tumor control probability after 12C irradiation that compared clinically used treatment schedules with fixed RBE values vs. the RBEs determined for each cell line. Based on the above analysis, we present the framework of a strategy to utilize biological markers to predict which HNSCC patients would benefit the most from 12C radiotherapy.