Ataxia-Telangiectasia Mutated Loss-of-Function Displays Variant and Tissue-Specific Differences across Tumor Types.

PURPOSE: Mutations in the ATM gene are common in multiple cancers, but clinical studies of therapies targeting ATM-aberrant cancers have yielded mixed results. Refinement of ATM loss of function (LOF) as a predictive biomarker of response is urgently needed. EXPERIMENTAL DESIGN: We present the first disclosure and preclinical development of a novel, selective ATR inhibitor, ART0380, and test its antitumor activity in multiple preclinical cancer models. To refine ATM LOF as a predictive biomarker, we performed a comprehensive pan-cancer analysis of ATM variants in patient tumors and then assessed the ATM variant-to-protein relationship. Finally, we assessed a novel ATM LOF biomarker approach in retrospective clinical data sets of patients treated with platinum-based chemotherapy or ATR inhibition. RESULTS: ART0380 had potent, selective antitumor activity in a range of preclinical cancer models with differing degrees of ATM LOF. Pan-cancer analysis identified 10,609 ATM variants in 8,587 patient tumors. Cancer lineage-specific differences were seen in the prevalence of deleterious (Tier 1) versus unknown/benign (Tier 2) variants, selective pressure for loss of heterozygosity, and concordance between a deleterious variant and ATM loss of protein (LOP). A novel ATM LOF biomarker approach that accounts for variant classification, relationship to ATM LOP, and tissue-specific penetrance significantly enriched for patients who benefited from platinum-based chemotherapy or ATR inhibition. CONCLUSIONS: These data help to better define ATM LOF across tumor types in order to optimize patient selection and improve molecularly targeted therapeutic approaches for patients with ATM LOF cancers.

Quantitative, titratable and high-throughput reporter assays to measure DNA double strand break repair activity in cells.

Repair of DNA damage is essential for the maintenance of genome stability and cell viability. DNA double strand breaks (DSBs) constitute a toxic class of DNA lesion and multiple cellular pathways exist to mediate their repair. Robust and titratable assays of cellular DSB repair (DSBR) are important to functionally interrogate the integrity and efficiency of these mechanisms in disease models as well as in response to genetic or pharmacological perturbations. Several variants of DSBR reporters are available, however these are often limited by throughput or restricted to specific cellular models. Here, we describe the generation and validation of a suite of extrachromosomal reporter assays that can efficiently measure the major DSBR pathways of homologous recombination (HR), classical nonhomologous end joining (cNHEJ), microhomology-mediated end joining (MMEJ) and single strand annealing (SSA). We demonstrate that these assays can be adapted to a high-throughput screening format and that they are sensitive to pharmacological modulation, thus providing mechanistic and quantitative insights into compound potency, selectivity, and on-target specificity. We propose that these reporter assays can serve as tools to dissect the interplay of DSBR pathway networks in cells and will have broad implications for studies of DSBR mechanisms in basic research and drug discovery.

Targeting ATR in Cancer Medicine.

As a key component of the DNA Damage Response, the Ataxia telangiectasia and Rad3-related (ATR) protein is a promising druggable target that is currently widely evaluated in phase I-II-III clinical trials as monotherapy and in combinations with other rational antitumor agents, including immunotherapy, DNA repair inhibitors, chemo- and radiotherapy. Ongoing clinical studies for this drug class must address the optimization of the therapeutic window to limit overlapping toxicities and refine the target population that will most likely benefit from ATR inhibition. With advances in the development of personalized treatment strategies for patients with advanced solid tumors, many ongoing ATR inhibitor trials have been recruiting patients based on their germline and somatic molecular alterations, rather than relying solely on specific tumor subtypes. Although a spectrum of molecular alterations have already been identified as potential predictive biomarkers of response that may sensitize to ATR inhibition, these biomarkers must be analytically validated and feasible to measure robustly to allow for successful integration into the clinic. While several ATR inhibitors in development are poised to address a clinically unmet need, no ATR inhibitor has yet received FDA-approval. This chapter details the underlying rationale for targeting ATR and summarizes the current preclinical and clinical landscape of ATR inhibitors currently in evaluation, as their regulatory approval potentially lies close in sight.

Small-Molecule Polθ Inhibitors Provide Safe and Effective Tumor Radiosensitization in Preclinical Models.

PURPOSE: DNA polymerase theta (Polθ, encoded by the POLQ gene) is a DNA repair enzyme critical for microhomology mediated end joining (MMEJ). Polθ has limited expression in normal tissues but is frequently overexpressed in cancer cells and, therefore, represents an ideal target for tumor-specific radiosensitization. In this study we evaluate whether targeting Polθ with novel small-molecule inhibitors is a feasible strategy to improve the efficacy of radiotherapy. EXPERIMENTAL DESIGN: We characterized the response to Polθ inhibition in combination with ionizing radiation in different cancer cell models in vitro and in vivo. RESULTS: Here, we show that ART558 and ART899, two novel and specific allosteric inhibitors of the Polθ DNA polymerase domain, potently radiosensitize tumor cells, particularly when combined with fractionated radiation. Importantly, noncancerous cells were not radiosensitized by Polθ inhibition. Mechanistically, we show that the radiosensitization caused by Polθ inhibition is most effective in replicating cells and is due to impaired DNA damage repair. We also show that radiosensitization is still effective under hypoxia, suggesting that these inhibitors may help overcome hypoxia-induced radioresistance. In addition, we describe for the first time ART899 and characterize it as a potent and specific Polθ inhibitor with improved metabolic stability. In vivo, the combination of Polθ inhibition using ART899 with fractionated radiation is well tolerated and results in a significant reduction in tumor growth compared with radiation alone. CONCLUSIONS: These results pave the way for future clinical trials of Polθ inhibitors in combination with radiotherapy.

Modulating mutational outcomes and improving precise gene editing at CRISPR-Cas9-induced breaks by chemical inhibition of end-joining pathways.

Gene editing through repair of CRISPR-Cas9-induced chromosomal breaks offers a means to correct a wide range of genetic defects. Directing repair to produce desirable outcomes by modulating DNA repair pathways holds considerable promise to increase the efficiency of genome engineering. Here, we show that inhibition of non-homologous end joining (NHEJ) or polymerase theta-mediated end joining (TMEJ) can be exploited to alter the mutational outcomes of CRISPR-Cas9. We show robust inhibition of TMEJ activity at CRISPR-Cas9-induced double-strand breaks (DSBs) using ART558, a potent polymerase theta (PolÏ´) inhibitor. Using targeted sequencing, we show that ART558 suppresses the formation of microhomology-driven deletions in favor of NHEJ-specific outcomes. Conversely, NHEJ deficiency triggers the formation of large kb-sized deletions, which we show are the products of mutagenic TMEJ. Finally, we show that combined chemical inhibition of TMEJ and NHEJ increases the efficiency of homology-driven repair (HDR)-mediated precise gene editing. Our work reports a robust strategy to improve the fidelity and safety of genome engineering.

Addressing the dauntingness of formulation: The value of considering empathy as "grasping".

AIMS AND OBJECTIVES: To explore the value of using Karl Jaspers' lived experience concept of 'grasping' in remediating the reported dauntingness of formulation. CONCLUSIONS: Formulation can be construed as both the process and explication of understanding why a patient is presenting in a particular way. In an automatic process of abduction, 'feeling into' the mind of the other, hypotheses are posted to consciousness with little mental effort as meaningful connections are grasped. Subsequent more deliberative reasoning is synthesised continuously and with surprisingly little mental effort into the best explanation(s). Karl Jaspers' introduction to Psychiatry of the concepts involved, empathy and understanding, and his aim of making their use more scientific established the ongoing, often fierce debate about the ontology of Psychiatry; empirical versus interpretive. Trainees must resolve this for themselves in explicating a formulation, risking exposure of their prejudices. Jaspers' emphasis on the lived experience of empathic understanding that psychiatrists bring to their work found him often using the term 'grasp' rather than 'empathise'. 'Grasping' seems to convey more vividly and meaningfully the role that empathy plays in the initial ascertainment of mood and the subsequent hypothesis discovery, testing and synthesis.

POLQ seals post-replicative ssDNA gaps to maintain genome stability in BRCA-deficient cancer cells.

POLQ is a key effector of DSB repair by microhomology-mediated end-joining (MMEJ) and is overexpressed in many cancers. POLQ inhibitors confer synthetic lethality in HR and Shieldin-deficient cancer cells, which has been proposed to reflect a critical dependence on the DSB repair pathway by MMEJ. Whether POLQ also operates independent of MMEJ remains unexplored. Here, we show that POLQ-deficient cells accumulate post-replicative ssDNA gaps upon BRCA1/2 loss or PARP inhibitor treatment. Biochemically, cooperation between POLQ helicase and polymerase activities promotes RPA displacement and ssDNA-gap fill-in, respectively. POLQ is also capable of microhomology-mediated gap skipping (MMGS), which generates deletions during gap repair that resemble the genomic scars prevalent in POLQ overexpressing cancers. Our findings implicate POLQ in mutagenic post-replicative gap sealing, which could drive genome evolution in cancer and whose loss places a critical dependency on HR for gap protection and repair and cellular viability.

Discovery, Characterization, and Structure-Based Optimization of Small-Molecule In Vitro and In Vivo Probes for Human DNA Polymerase Theta.

Human DNA polymerase theta (Polθ), which is essential for microhomology-mediated DNA double strand break repair, has been proposed as an attractive target for the treatment of BRCA deficient and other DNA repair pathway defective cancers. As previously reported, we recently identified the first selective small molecule Polθ in vitro probe, 22 (ART558), which recapitulates the phenotype of Polθ loss, and in vivo probe, 43 (ART812), which is efficacious in a model of PARP inhibitor resistant TNBC in vivo. Here we describe the discovery, biochemical and biophysical characterization of these probes including small molecule ligand co-crystal structures with Polθ. The crystallographic data provides a basis for understanding the unique mechanism of inhibition of these compounds which is dependent on stabilization of a "closed" enzyme conformation. Additionally, the structural biology platform provided a basis for rational optimization based primarily on reduced ligand conformational flexibility.

Why is formulation daunting? A response to Bagster et al.'s 'instructions' for developing skills in formulation.

OBJECTIVE: To explore the theme identified by Bagster et al.1 in their selective psychiatric literature review that formulation can appear daunting. CONCLUSION: Formulation is understandably daunting, even though it occurs in all human encounters. The plural nature of mental symptoms is such that anxiety-provoking intuitive judgement is required at all points in both the process and explication of formulation, a type of instinctive guessing. There are no rules for this, because the laws of vertical integration of systems are not established. Guidelines are more appropriate than 'instructions'. Much of the wider mental health and clinical reasoning literature addresses intuitive judgement, but the current psychiatric literature tends to focus on pattern recognition as a deliberative cognitive act of Type 2 processes. Arguably this reductionism adds to the dauntingness. Anxiety detected about the intuitive judgement involved can be addressed in supervision, taking into account the psychological mindedness of the trainee.

Loss of nuclear DNA ligase III reverts PARP inhibitor resistance in BRCA1/53BP1 double-deficient cells by exposing ssDNA gaps.

Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, preclinical and clinical research with PARPi has revealed multiple resistance mechanisms, highlighting the need for identification of novel functional biomarkers and combination treatment strategies. Functional genetic screens performed in cells and organoids that acquired resistance to PARPi by loss of 53BP1 identified loss of LIG3 as an enhancer of PARPi toxicity in BRCA1-deficient cells. Enhancement of PARPi toxicity by LIG3 depletion is dependent on BRCA1 deficiency but independent of the loss of 53BP1 pathway. Mechanistically, we show that LIG3 loss promotes formation of MRE11-mediated post-replicative ssDNA gaps in BRCA1-deficient and BRCA1/53BP1 double-deficient cells exposed to PARPi, leading to an accumulation of chromosomal abnormalities. LIG3 depletion also enhances efficacy of PARPi against BRCA1-deficient mammary tumors in mice, suggesting LIG3 as a potential therapeutic target.

Development and evaluation of a nanopore 16S rRNA gene sequencing service for same day targeted treatment of bacterial respiratory infection in the intensive care unit.

OBJECTIVES: Assess the feasibility and impact of nanopore-based 16S rRNA gene sequencing (Np16S) service on antibiotic treatment for acute severe pneumonia on the intensive care unit (ICU). METHODS: Speciation and sequencing accuracy of Np16S on isolates with bioinformatics pipeline optimisation, followed by technical evaluation including quality checks and clinical-reporting criteria analysing stored respiratory samples using single-sample flow cells. Pilot service comparing Np16S results with all routine respiratory tests and impact on same-day antimicrobial prescribing. RESULTS: Np16S correctly identified 140/167 (84%) isolates after 1h sequencing and passed quality control criteria including reproducibility and limit-of-detection. Sequencing of 108 stored respiratory samples showed concordance with routine culture in 80.5% of cases and established technical and clinical reporting criteria. A 10-week same-day pilot Np16S service analysed 45 samples from 37 patients with suspected community (n=15) or hospital acquired (n=30) pneumonia. Np16S showed concordance compared with all routine culture or molecular tests for 27 (82%) of 33 positive samples. It identified the causative pathogen in 32/33 (97%) samples and contributed to antimicrobial treatment changes for 30 patients (67%). CONCLUSIONS: This study demonstrates feasibility of providing a routine same-day nanopore sequencing service that makes a significant contribution to early antibiotic prescribing for bacterial pneumonia in the ICU.

Functional and regulatory profiling of energy metabolism in fission yeast.

BACKGROUND: The control of energy metabolism is fundamental for cell growth and function and anomalies in it are implicated in complex diseases and ageing. Metabolism in yeast cells can be manipulated by supplying different carbon sources: yeast grown on glucose rapidly proliferates by fermentation, analogous to tumour cells growing by aerobic glycolysis, whereas on non-fermentable carbon sources metabolism shifts towards respiration. RESULTS: We screened deletion libraries of fission yeast to identify over 200 genes required for respiratory growth. Growth media and auxotrophic mutants strongly influenced respiratory metabolism. Most genes uncovered in the mutant screens have not been implicated in respiration in budding yeast. We applied gene-expression profiling approaches to compare steady-state fermentative and respiratory growth and to analyse the dynamic adaptation to respiratory growth. The transcript levels of most genes functioning in energy metabolism pathways are coherently tuned, reflecting anticipated differences in metabolic flows between fermenting and respiring cells. We show that acetyl-CoA synthase, rather than citrate lyase, is essential for acetyl-CoA synthesis in fission yeast. We also investigated the transcriptional response to mitochondrial damage by genetic or chemical perturbations, defining a retrograde response that involves the concerted regulation of distinct groups of nuclear genes that may avert harm from mitochondrial malfunction. CONCLUSIONS: This study provides a rich framework of the genetic and regulatory basis of energy metabolism in fission yeast and beyond, and it pinpoints weaknesses of commonly used auxotroph mutants for investigating metabolism. As a model for cellular energy regulation, fission yeast provides an attractive and complementary system to budding yeast.

AnGeLi: A Tool for the Analysis of Gene Lists from Fission Yeast.

Genome-wide assays and screens typically result in large lists of genes or proteins. Enrichments of functional or other biological properties within such lists can provide valuable insights and testable hypotheses. To systematically detect these enrichments can be challenging and time-consuming, because relevant data to compare against query gene lists are spread over many different sources. We have developed AnGeLi (Analysis of Gene Lists), an intuitive, integrated web-tool for comprehensive and customized interrogation of gene lists from the fission yeast, Schizosaccharomyces pombe. AnGeLi searches for significant enrichments among multiple qualitative and quantitative information sources, including gene and phenotype ontologies, genetic and protein interactions, numerous features of genes, transcripts, translation, and proteins such as copy numbers, chromosomal positions, genetic diversity, RNA polymerase II and ribosome occupancy, localization, conservation, half-lives, domains, and molecular weight among others, as well as diverse sets of genes that are co-regulated or lead to the same phenotypes when mutated. AnGeLi uses robust statistics which can be tailored to specific needs. It also provides the option to upload user-defined gene sets to compare against the query list. Through an integrated data submission form, AnGeLi encourages the community to contribute additional curated gene lists to further increase the usefulness of this resource and to get the most from the ever increasing large-scale experiments. AnGeLi offers a rigorous yet flexible statistical analysis platform for rich insights into functional enrichments and biological context for query gene lists, thus providing a powerful exploratory tool through which S. pombe researchers can uncover fresh perspectives and unexpected connections from genomic data. AnGeLi is freely available at: www.bahlerlab.info/AnGeLi.

Widespread exon skipping triggers degradation by nuclear RNA surveillance in fission yeast.

Exon skipping is considered a principal mechanism by which eukaryotic cells expand their transcriptome and proteome repertoires, creating different splice variants with distinct cellular functions. Here we analyze RNA-seq data from 116 transcriptomes in fission yeast (Schizosaccharomyces pombe), covering multiple physiological conditions as well as transcriptional and RNA processing mutants. We applied brute-force algorithms to detect all possible exon-skipping events, which were widespread but rare compared to normal splicing events. Exon-skipping events increased in cells deficient for the nuclear exosome or the 5'-3' exonuclease Dhp1, and also at late stages of meiotic differentiation when nuclear-exosome transcripts decreased. The pervasive exon-skipping transcripts were stochastic, did not increase in specific physiological conditions, and were mostly present at less than one copy per cell, even in the absence of nuclear RNA surveillance and during late meiosis. These exon-skipping transcripts are therefore unlikely to be functional and may reflect splicing errors that are actively removed by nuclear RNA surveillance. The average splicing rate by exon skipping was ∼ 0.24% in wild type and ∼ 1.75% in nuclear exonuclease mutants. We also detected approximately 250 circular RNAs derived from single or multiple exons. These circular RNAs were rare and stochastic, although a few became stabilized during quiescence and in splicing mutants. Using an exhaustive search algorithm, we also uncovered thousands of previously unknown splice sites, indicating pervasive splicing; yet most of these splicing variants were cryptic and increased in nuclear degradation mutants. This study highlights widespread but low frequency alternative or aberrant splicing events that are targeted by nuclear RNA surveillance.

LaSSO, a strategy for genome-wide mapping of intronic lariats and branch points using RNA-seq.

Both canonical and alternative splicing of RNAs are governed by intronic sequence elements and produce transient lariat structures fastened by branch points within introns. To map precisely the location of branch points on a genomic scale, we developed LaSSO (Lariat Sequence Site Origin), a data-driven algorithm which utilizes RNA-seq data. Using fission yeast cells lacking the debranching enzyme Dbr1, LaSSO not only accurately identified canonical splicing events, but also pinpointed novel, but rare, exon-skipping events, which may reflect aberrantly spliced transcripts. Compromised intron turnover perturbed gene regulation at multiple levels, including splicing and protein translation. Notably, Dbr1 function was also critical for the expression of mitochondrial genes and for the processing of self-spliced mitochondrial introns. LaSSO showed better sensitivity and accuracy than algorithms used for computational branch-point prediction or for empirical branch-point determination. Even when applied to a human data set acquired in the presence of debranching activity, LaSSO identified both canonical and exon-skipping branch points. LaSSO thus provides an effective approach for defining high-resolution maps of branch-site sequences and intronic elements on a genomic scale. LaSSO should be useful to validate introns and uncover branch-point sequences in any eukaryote, and it could be integrated into RNA-seq pipelines.

Revisiting formulation: part 1. The tasks of formulation: their rationale and philosophic basis.

OBJECTIVE: This paper notes the continuing problems that Royal Australian and New Zealand College of Psychiatry (RANZCP) candidates and other professionals have with the task of formulation. It re-establishes this as a problematic to be understood and reviews its intellectual history, its rationale, the tools of reasoning that it requires and the nature of the challenges that it can pose to individuals. Its premise is that an understanding of the theoretical basis of formulation is required prior to application of pedagogical tools in teaching and remediation. Four tasks of formulation implied in the definition provided in the RANZCP Formulation Guidelines for Candidates are identified, and their rationale and theoretical status reviewed. CONCLUSION: Task 1, classification, must address both diagnoses and problems. Task 2, using theory to infer meaning, ideally requires multiple models and theories in development of a set of hypotheses, using inductive inference (plausibility). Task 3, prioritising hypotheses, requires abduction, defined as inference to the best explanation. Task 4, integrating hypotheses, may aim for reconciling systems (syncretism), but is likely to use the more problematic eclecticism. The task of formulation is thus challenging but well prescribed by philosophers for thousands of years.

Revisiting formulation: part 2. The task of addressing the concept of the unique individual. Remediating problems with formulation.

OBJECTIVE: The purpose of this paper is to review the theoretical basis of addressing the concept of the unique individual, one of the tasks prescribed in the Royal Australian and New Zealand College of Psychiatrists (RANZCP) Formulation guidelines for candidates, and to propose a rational basis for remediation of the problems that many candidates and other professionals have with formulation. CONCLUSION: The difficulty that RANZCP candidates and other mental health professionals have in producing a completely integrated account of an individual is multi-determined, but is partly explainable on theoretical grounds. Understanding why this task (and other tasks of formulation) is problematic requires knowledge of its intellectual history, its rationale, the tools of reasoning that it requires and the nature of the challenges that it can pose to individuals. The paper argues that a rational plan for remediation of a professional's problems with formulation requires first a 'formulation' of those problems, drawing on the theories discussed and acknowledging the uniqueness of that individual. In answering the question, 'Why is this person having difficulty formulating this patient at this time?', one needs to address cultural, social and systemic factors, and psychological factors such as the degree of psychological mindedness of the person seeking remediation. Hypotheses based on the formulation about the professional's difficulty should be developed and tested by interventions.

1-substituted (Dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H-chromen-4-ones endowed with dual DNA-PK/PI3-K inhibitory activity.

Analogues of (dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H-chromen-4-one (NU7441), a potent inhibitor of DNA-dependent protein kinase (DNA-PK; IC50 = 42 ± 2 nM), have been synthesized in which water-solubilizing groups [NHCO(CH2)nNR¹R², where n = 1 or 2 and the moiety R¹R²N was derived from a library of primary and secondary amines, e.g., morpholine] were placed at the 1-position. Several of the newly synthesized compounds exhibited high potency against DNA-PK and potentiated the cytotoxicity of ionizing radiation (IR) in vitro 10-fold or more (e.g., 2-(4-ethylpiperazin-1-yl)-N-(4-(2-morpholino-4-oxo-4H-chromen-8-yl)dibenzo[b,d]thio-phen-1-yl)acetamide, 39; DNA-PK IC50 = 5.0 ± 1 nM, IR dose modification ratio = 13). Furthermore, 39 was shown to potentiate not only IR in vitro but also DNA-inducing cytotoxic anticancer agents, both in vitro and in vivo. Counter-screening against other members of the phosphatidylinositol 3-kinase (PI-3K) related kinase (PIKK) family unexpectedly revealed that some of the compounds were potent mixed DNA-PK and PI-3K inhibitors.

Chemosensitization of cancer cells by KU-0060648, a dual inhibitor of DNA-PK and PI-3K.

DNA double-strand breaks (DSB) are the most cytotoxic lesions induced by topoisomerase II poisons. Nonhomologous end joining (NHEJ) is a major pathway for DSB repair and requires DNA-dependent protein kinase (DNA-PK) activity. DNA-PK catalytic subunit (DNA-PKcs) is structurally similar to PI-3K, which promotes cell survival and proliferation and is upregulated in many cancers. KU-0060648 is a dual inhibitor of DNA-PK and PI-3K in vitro. KU-0060648 was investigated in a panel of human breast and colon cancer cells. The compound inhibited cellular DNA-PK autophosphorylation with IC(50) values of 0.019 µmol/L (MCF7 cells) and 0.17 µmol/L (SW620 cells), and PI-3K-mediated AKT phosphorylation with IC(50) values of 0.039 µmol/L (MCF7 cells) and more than 10 µmol/L (SW620 cells). Five-day exposure to 1 µmol/L KU-0060648 inhibited cell proliferation by more than 95% in MCF7 cells but only by 55% in SW620 cells. In clonogenic survival assays, KU-0060648 increased the cytotoxicity of etoposide and doxorubicin across the panel of DNA-PKcs-proficient cells, but not in DNA-PKcs-deficient cells, thus confirming that enhanced cytotoxicity was due to DNA-PK inhibition. In mice bearing SW620 and MCF7 xenografts, concentrations of KU-0060648 that were sufficient for in vitro growth inhibition and chemosensitization were maintained within the tumor for at least 4 hours at nontoxic doses. KU-0060648 alone delayed the growth of MCF7 xenografts and increased etoposide-induced tumor growth delay in both in SW620 and MCF7 xenografts by up to 4.5-fold, without exacerbating etoposide toxicity to unacceptable levels. The proof-of-principle in vitro and in vivo chemosensitization with KU-0060648 justifies further evaluation of dual DNA-PK and PI-3K inhibitors.