DNA-dependent Protein Kinase Inhibitor Research Articles

Increasing the Level of Knock-in of a Construct Encoding the HIV-1 Fusion Inhibitor, MT-C34 Peptide, into the <i>CXCR4</i> Locus in the CEM/R5 T Cell Line

The low efficiency of knock-in, especially in primary human cells, limits the use of genome editing technology for therapeutic purposes, which makes it important to develop approaches for increasing knock-in levels. In this work, using a knock-in model of the peptide fusion inhibitor of HIV MT-C34 into the human CXCR4 locus in the CEM/R5 T cell line, we analyzed the effectiveness of several approaches to increasing knock-in levels. First, donor DNA modification aimed at improving the efficiency of plasmid transport into the nucleus was evaluated, namely the introduction into the donor plasmid of the SV40 DNA transport sequence (DTS) or the binding sites for the transcription factor NF-κB, whose effects on knock-in levels have not been described. In the MT-C34 knock-in model into the CXCR4 locus, this modification was ineffective. The second approach, modifying the Cas9 nuclease by introducing two additional nuclear localization signals (NLS), increased the knock-in level by 30%. Finally, blocking DNA repair via the nonhomologous end joining pathway using DNA-dependent protein kinase inhibitors caused a 1.8-fold increase in knock-in. The combination of the last two approaches caused an additive effect. Thus, increasing the number of NLSs in the Cas9 protein and inhibiting DNA repair via the nonhomologous end joining pathway significantly increased the level of knock-in of the HIV-1 peptide fusion inhibitor into the clinically relevant locus CXCR4, which can be used to develop effective gene therapy approaches for the treatment of HIV infection.

An organotypic model for investigating drug-radiation responses in the lung

Background: Established in vivo radiobiological models are commonly used to assess anti-tumor effects and normal tissue toxicity. However, these models have notable limitations, and additional models are necessary to gain a deeper insights into drug-radiation interactions. Objective: This study aimed to develop an organotypic ex vivo model by using precision-cut lung slices (PCLSs) to evaluate radiation-induced residual deoxyribonucleic acid (DNA) damage, both alone and in combination with a pharmacological inhibitor of DNA double-strand break (DSB) repair. Methods: Left lungs from female C57BL/6 mice were dissected, perfused with 4% low-gelling-temperature agarose, and sliced into 250 μm sections. Lung slices were then incubated ex vivo for up to 7 days. The slices were irradiated using 137Cs, either with or without a DNA-dependent protein kinase (DNA-PK) inhibitor (NU7441). Tissue sections were subsequently fixed and stained for γH2AX and 53BP1, which serve as histological markers of DNA DSBs. Results: The established conditions preserved tissue viability for up to 7 days and maintained structural integrity for 2 days. DNA damage, detected through γH2AX and 53BP1 staining, was consistent between lungs irradiated ex vivo and their counterparts irradiated in vivo. In the organotypic model, radiation alone in DNA-PK-deficient SCID mice and radiation combined with DNA-PK inhibition in C57BL/6 mice led to increased residual γH2AX and 53BP1 staining. Conclusion: This study demonstrates that residual DNA damage levels following ionizing radiation in lung tissue are comparable between in vivo and ex vivo tissue slices, suggesting that PCLSs serve as a valuable organotypic model for investigating the effects of drug-radiation combinations.

DNAR-10. PHASE I CLINICAL TRIAL OF PEPOSERTIB PLUS RADIOTHERAPY IN ADULTS WITH NEWLY-DIAGNOSED MGMT-UNMETHYLATED GLIOBLASTOMA

Abstract BACKGROUND DNA-dependent protein kinase (DNA-PK) is an attractive target in GBM because it promotes unwanted DNA repair and renders cancer cells less vulnerable to DNA damaging agents, such as radiotherapy (RT). Peposertib, a small molecule inhibitor of DNA-PK, has demonstrated pre-clinical efficacy in sensitizing GBM to RT, resulting in tumor regression. METHODS 21 patients with newly-diagnosed MGMT-unmethylated GBM received peposertib plus 6-weeks of RT to determine the maximum-tolerated dose (MTD) based on the Bayesian Optimal Interval design. The dose-limiting toxicity (DLT) period was 10-weeks. Patients received at least 6-cycles of adjuvant temozolomide (NCT04555577). Single-nuclei RNA-sequencing (snRNA-Seq) and spatial transcriptomics (Visium, 10x Genomics) were performed on matched pair primary-recurrent tumors and controls, incorporating tumor cell state and cell type definitions by automated cluster classification and differential expression of marker genes. RESULTS The MTD of peposertib in combination with RT was 300mg per RT fraction day. One DLT (G3 radiation necrosis @300mg) was observed. After a median follow-up of 15.4 months (interquartile-range 8.9-18.5), mPFS was 10.8 months (95%CI 8.33-NR) and mOS was 22.9 months (95%CI 16-NR). 6/21 (29%) patients have undergone re-resection to date. snRNA-Seq on 102,709 cells from 12 primary and 3 recurrent tumors revealed that pre-treatment tumor cell states were progenitor-like, while treatment with peposertib plus RT was associated with differentiated and inflammatory malignant cell states, along with macrophage infiltration. Spatial transcriptomics of 8 matched primary-recurrent tumors (20,793 transcriptomes) confirmed malignant cell differentiation and enrichment of pro-inflammatory macrophages after inhibition of DNA-PK. 5 patients will be enrolled into a window-of-opportunity expansion cohort to evaluate intratumoral drug concentration. CONCLUSION The combination of peposertib and intracranial RT was safe. Survival data for this MGMT-unmethylated GBM population was promising. Single nuclei and spatial transcriptomic analyses revealed enrichment of pro-inflammatory macrophages in the tumor microenvironmentafter DNA-PK inhibition. The study was supported by EMD-Serono (CrossRef Funder ID: 10.13039/100004755).

Disruption of RNA Splicing Increases Vulnerability of Cells to DNA-PK Inhibitors.

DNA-dependent protein kinase (DNA-PK) is a key effector of non-homologous end joining (NHEJ)-mediated double-strand break (DSB) repair. Since its identification, a substantial body of evidence has demonstrated that DNA-PK is frequently overexpressed in cancer, plays a critical role in tumor development and progression, and is associated with poor prognosis in cancer patients. Recent studies have also uncovered novel functions of DNA-PK, shifting the paradigm of the role of DNA-PK in oncogenesis and renewing interest in targeting DNA-PK for cancer therapy. To gain genetic insight into the cellular pathways requiring DNA-PK activity, we used a CRISPR/Cas9 screen to identify genes in which defects cause hypersensitivity to DNA-PK inhibitors. We identified over one hundred genes involved in DNA replication, cell cycle regulation, and RNA processing that promoted cell survival when DNA-PK kinase activity was suppressed. This gene set will be useful for characterizing novel biological processes that require DNA-PK activity and identifying predictive biomarkers of response to DNA-PK inhibition in the clinic. We also validated several genes from this set and reported previously undescribed genes that modulate the response to DNA-PK inhibitors. In particular, we found that compromising the mRNA splicing pathway led to marked hypersensitivity to DNA-PK inhibition, providing a possible rationale for the combined use of splicing inhibitors and DNA-PK inhibitors for cancer therapy.

DNA-PK inhibition shows differential radiosensitization in orthotopic GBM PDX models based on DDR pathway deficits.

Glioblastoma (GBM) remains one of the most therapy-resistant malignancies with frequent local failures despite aggressive surgery, chemotherapy, and ionizing radiation (IR). Small molecule inhibitors of DNA-dependent protein kinase (DNA-PKi's) are potent radiosensitizers currently in clinical trials. Determining which patients may benefit from radiosensitization with DNA-PKi's is critical to avoid unnecessary increased risk of normal tissue toxicity. In this study we used GBM patient derived xenografts (PDXs) in orthotopic murine models to study the relationship between molecular features, pharmacokinetics, and the radiosensitizing potential of the DNA-PKi peposertib. We show that peposertib radiosensitizes established and PDX GBM lines in vitro at 300nM and above, with significant increase in radiosensitization by maintaining post-IR exposure for >12 hours. Radiosensitization by peposertib is mediated by catalytic inhibition of DNA-PK, and knock-down of DNA-PK by short hairpin RNA (shRNA) largely abolished the radiosensitizing effect. Peposertib decreased auto-phosphorylation of DNA-PKcs after IR in a dose-dependent manner with delay in resolution of γH2AX foci at 24 hours. The addition of peposertib to IR significantly increased survival in GBM120 orthotopic xenografts, but not in GBM10. There was no difference in plasma or average tumor concentrations of peposertib in the two cohorts. While the mechanism underpinning this discordant effect in vitro vs. in vivo is not clear, there was an association for greater sensitization in TP53 mutant lines. Transfection of a dominant-negative TP53 mutant in baseline TP53 wildtype GBM lines significantly delayed growth and decreased NHEJ efficiency (but not Homologous Recombination), after peposertib exposure.

DNA-PK inhibition enhances neoantigen diversity and increases T cell responses to immunoresistant tumors.

Effective antitumor T cell activity relies on the expression and MHC presentation of tumor neoantigens. Tumor cells can evade T cell detection by silencing the transcription of antigens or by altering MHC machinery resulting in inadequate neoantigen-specific T cell activation. We identified DNA-PK inhibitor (DNA-PKi) NU7441 as a promising immunomodulator that reduced immunosuppressive proteins while increasing MHC-I expression in a panel of human melanoma cell lines. In tumor-bearing mice, combination therapy using NU7441 and immune adjuvants STING ligand and CD40 agonist (NU-SL40) substantially increased and diversified the neoantigen landscape, antigen presenting machinery, and consequently substantially increased both the number and repertoire of neoantigen-reactive tumor infiltrating lymphocytes (TILs). DNA-PK-inhibition or knockout promoted transcription and protein expression of various neoantigens in human and mouse melanomas and induced sensitivity to ICB in resistant tumors. In patients, PRKDC levels inversely correlated with MHC I expression and CD8 TILs but positively correlated with increased neoantigen loads and improved responses to ICB. These studies suggest that inhibiting DNA-PK activity can restore tumor immunogenicity by increasing neoantigen expression and presentation and broadening the neoantigen-reactive T cell population.

Quantitation of the DNA-dependent protein kinase inhibitor peposertib (M3814) and metabolite in human plasma by LC-MS/MS.

The DNA-dependent protein kinase (DNA-PK) is an abundant nuclear protein that mediates DNA double-strand break repair by nonhomologous end joining (NHEJ). As such, DNA-PK is critical for V(D)J recombination in lymphocytes and for survival in cells exposed to ionizing radiation and clastogens. Peposertib (M3814) is a small molecule DNA-PK inhibitor currently in preclinical and clinical development for cancer treatment. We have developed a high-performance liquid chromatography-mass spectrometry method for quantitating peposertib and its metabolite in 0.1 mL human plasma. After MTBE liquid-liquid extraction, chromatographic separation was achieved with a Phenomenex Synergi polar reverse phase (4μm, 2× 50 mm) column and a gradient of 0.1% formic acid in acetonitrile and water over an 8min run time. Mass spectrometric detection was performed on an ABI SCIEX 4000 with electrospray, positive-mode ionization. The assay was linear from 10 to 3000 ng/mL for peposertib and 1-300 ng/mL for the metabolite and proved to be both accurate (97.3%-103.7%) and precise (<8.9%CV) fulfilling criteria from the Food and Drug Administration (FDA) guidance on bioanalytical method validation. This liquid chromatography-tandem mass spectroscopy (LC-MS/MS) assay will support several ongoing clinical studies by defining peposertib pharmacokinetics.

Defining the role of Tip60 in the DNA damage response of glioma cell lines

Purpose Glioblastomas are resistant to conventional therapies, including radiotherapy. Our previous study proved that epigenetic regulation influences the radiation response of glioma cells. This study evaluated the role of the acetyltransferase Tip60 on the radiation response. Material and Methods Tip60 expression was down-regulated by transfecting specific siRNA’s in A7 and MO59K cells with high and low expression of Tip60, respectively, and its effect on survival was assessed. DNA repair was analyzed by foci scoring (γH2AX, Rad51, 53BP1, pATM). The interaction of Tip60 with ATM and DNA-PK was investigated using the specific inhibitors KU55933 and NU7441, respectively. Results Knockdown of Tip60 significantly (p < .001) reduced survival in both cell lines, but the effect was more pronounced in A7 cells. ATMi and DNA-PKi significantly reduced the surviving fraction following irradiation. However, no further effect of siTip60 on the radiosensitivity of ATMi treated A7 cells was observed. In contrast, DNA-PKi effectively enhanced the sensitizing effect of siTip60. Mechanistically, siTip60 reduced the number of initial Rad51 and ATM foci formation after irradiation and prevented their dissolution at 24 h. siTip60 had no impact on the formation of 53BP1 and γH2AX foci and did not further affect these end-points if combined with ATMi or DNA-PKi. Conclusions Downregulation of Tip60 enhances the radiation sensitivity of both glioma cells and markedly elevates the radiation sensitivity when combined with DNA-PKi. Therefore, treatment with DNA-PK inhibitors represents a promising approach to augment the radiation sensitivity of glioma cell lines with deficient Tip60 activity in a synergistic manner.

154 (PB142): The discovery of imidazo[4,5-c]pyridine-2-ones as selective inhibitors of DNA-dependent protein kinase and effective radiosensitisers

154 (PB142): The discovery of imidazo[4,5-c]pyridine-2-ones as selective inhibitors of DNA-dependent protein kinase and effective radiosensitisers

EP.03F.04 Treatment of Non-Small Cell Lung Carcinoma (NSCLC) Cells with Tumor Treating Fields (TTFields) And DNA-Dependent Protein Kinase (PK) Inhibitors

EP.03F.04 Treatment of Non-Small Cell Lung Carcinoma (NSCLC) Cells with Tumor Treating Fields (TTFields) And DNA-Dependent Protein Kinase (PK) Inhibitors

513LBA (PB-513) LBA Posters: BCCA7693 - A novel, orally bioavailable DNA-PK inhibitor demonstrates efficacy with targeted MAPK and PARP therapeutics by slowing the development of acquired resistance

513LBA (PB-513) LBA Posters: BCCA7693 - A novel, orally bioavailable DNA-PK inhibitor demonstrates efficacy with targeted MAPK and PARP therapeutics by slowing the development of acquired resistance

Impact of Optimized Ku-DNA Binding Inhibitors on the Cellular and In Vivo DNA Damage Response.

Background: DNA-dependent protein kinase (DNA-PK) is a validated cancer therapeutic target involved in DNA damage response (DDR) and non-homologous end-joining (NHEJ) repair of DNA double-strand breaks (DSBs). Ku serves as a sensor of DSBs by binding to DNA ends and activating DNA-PK. Inhibition of DNA-PK is a common strategy to block DSB repair and improve efficacy of ionizing radiation (IR) therapy and radiomimetic drug therapies. We have previously developed Ku-DNA binding inhibitors (Ku-DBis) that block in vitro and cellular NHEJ activity, abrogate DNA-PK autophosphorylation, and potentiate cellular sensitivity to IR. Results and Conclusions: Here we report the discovery of oxindole Ku-DBis with improved cellular uptake and retained potent Ku-inhibitory activity. Variable monotherapy activity was observed in a panel of non-small cell lung cancer (NSCLC) cell lines, with ATM-null cells being the most sensitive and showing synergy with IR. BRCA1-deficient cells were resistant to single-agent treatment and antagonistic when combined with DSB-generating therapies. In vivo studies in an NSCLC xenograft model demonstrated that the Ku-DBi treatment blocked IR-dependent DNA-PKcs autophosphorylation, modulated DDR, and reduced tumor cell proliferation. This represents the first in vivo demonstration of a Ku-targeted DNA-binding inhibitor impacting IR response and highlights the potential therapeutic utility of Ku-DBis for cancer treatment.

Cocaine-induced DNA-PK relieves RNAP II pausing by promoting TRIM28 phosphorylation.

Drug abuse continues to pose a significant challenge in HIV control efforts. In our investigation, we discovered that cocaine not only upregulates the expression of DNA-dependent protein kinase (DNA-PK) but also augments DNA-PK activation by enhancing its phosphorylation at S2056. Moreover, DNA-PK phosphorylation triggers the translocation of DNA-PK into the nucleus. The finding that cocaine promotes nuclear translocation of DNA-PK further validates our observation of enhanced DNA-PK recruitment at the HIV long terminal repeat (LTR) following cocaine exposure. By activating and facilitating the nuclear translocation of DNA-PK, cocaine effectively orchestrates multiple stages of HIV transcription, thereby promoting HIV replication. Additionally, our study indicates that cocaine-induced DNA-PK promotes hyper-phosphorylation of RNA polymerase II (RNAP II) carboxyl-terminal domain (CTD) at Ser5 and Ser2 sites, enhancing both initiation and elongation phases, respectively, of HIV transcription. Cocaine's enhancement of transcription initiation and elongation is further supported by its activation of cyclin-dependent kinase 7 (CDK7) and subsequent phosphorylation of CDK9, thereby promoting positive transcriptional elongation factor b (P-TEFb) activity. We demonstrate for the first time that cocaine, through DNA-PK activation, promotes the specific phosphorylation of TRIM28 at Serine 824 (p-TRIM28, S824). This modification converts TRIM28 from a transcriptional inhibitor to a transactivator for HIV transcription. Additionally, we observe that phosphorylation of TRIM28 (p-TRIM28, S824) promotes the transition from the pausing phase to the elongation phase of HIV transcription, thereby facilitating the production of full-length HIV genomic transcripts. This finding corroborates the observed enhanced RNAP II CTD phosphorylation at Ser2, a marker of transcriptional elongation, following cocaine exposure. Accordingly, upon cocaine treatment, we observed elevated recruitment of p-TRIM28-(S824) at the HIV LTR. Overall, our results have unraveled the intricate molecular mechanisms underlying cocaine-induced HIV transcription and gene expression. These findings hold promise for the development of highly targeted therapeutics aimed at mitigating the detrimental effects of cocaine in individuals living with HIV.

Simultaneous inhibition of ATM, ATR, and DNA-PK causes synergistic lethality

Here we report that simultaneous inhibition of the three primary DNA damage recognition PI3 kinase-like kinases (PIKKs) —ATM, ATR, and DNA-PK— induces severe combinatorial synthetic lethality in mammalian cells. Utilizing Chinese hamster cell lines CHO and V79 and their respective PIKK mutants, we evaluated effects of inhibiting these three kinases on cell viability, DNA damage response, and chromosomal integrity. Our results demonstrate that while single or dual kinase inhibition increased cytotoxicity, inhibition of all three PIKKs results in significantly higher synergistic lethality, chromosomal aberrations, and DNA double-strand break (DSB) induction as calculated by their synergy scores. These findings suggest that the overlapping redundancy of ATM, ATR, and DNA-PK functions is critical for cell survival, and their combined inhibition greatly disrupts DNA damage signaling and repair processes, leading to cell death. This study provides insights into the potential of multi-targeted DDR kinase inhibition as an effective anticancer strategy, necessitating further research to elucidate underlying mechanisms and therapeutic applications.

DNA-PK inhibition enhances gene editing efficiency in HSPCs for CRISPR-based treatment of X-linked hyper IgM syndrome

DNA-PK inhibition enhances gene editing efficiency in HSPCs for CRISPR-based treatment of X-linked hyper IgM syndrome

Identification of 6-Anilino Imidazo[4,5-c]pyridin-2-ones as Selective DNA-Dependent Protein Kinase Inhibitors and Their Application as Radiosensitizers.

The dominant role of non-homologous end-joining in the repair of radiation-induced double-strand breaks identifies DNA-dependent protein kinase (DNA-PK) as an excellent target for the development of radiosensitizers. We report the discovery of a new class of imidazo[4,5-c]pyridine-2-one DNA-PK inhibitors. Structure-activity studies culminated in the identification of 78 as a nM DNA-PK inhibitor with excellent selectivity for DNA-PK compared to related phosphoinositide 3-kinase (PI3K) and PI3K-like kinase (PIKK) families and the broader kinome, and displayed DNA-PK-dependent radiosensitization of HAP1 cells. Compound 78 demonstrated robust radiosensitization of a broad range of cancer cells in vitro, displayed high oral bioavailability, and sensitized colorectal carcinoma (HCT116/54C) and head and neck squamous cell carcinoma (UT-SCC-74B) tumor xenografts to radiation. Compound 78 also provided substantial tumor growth inhibition of HCT116/54C tumor xenografts in combination with radiation. Compound 78 represents a new, potent, and selective class of DNA-PK inhibitors with significant potential as radiosensitizers for cancer treatment.

Radiation and Chemo-Sensitizing Effects of DNA-PK Inhibitors Are Proportional in Tumors and Normal Tissues.

Inhibitors of DNA-dependent protein kinase (PRKDC; DNA-PK) sensitize cancers to radiotherapy and DNA-damaging chemotherapies, with candidates in clinical trials. However, the degree to which DNA-PK inhibitors also sensitize normal tissues remains poorly characterized. In this study, we compare tumor growth control and normal tissue sensitization following DNA-PK inhibitors in combination with radiation and etoposide. FaDu tumor xenografts implanted in mice were treated with 10 to 15 Gy irradiation ± 3 to 100 mg/kg AZD7648. A dose-dependent increase in time to tumor volume doubling following AZD7648 was proportional to an increase in toxicity scores of the overlying skin. Similar effects were seen in the intestinal jejunum, tongue, and FaDu tumor xenografts of mice assessed for proliferation rates at 3.5 days after treatment with etoposide or 5 Gy whole body irradiation ± DNA-PK inhibitors AZD7648 or peposertib (M3814). Additional organs were examined for sensitivity to DNA-PK inhibitor activity in ATM-deficient mice, where DNA-PK activity is indicated by surrogate marker γH2AX. Inhibition was observed in the heart, brain, pancreas, thymus, tongue, and salivary glands of ATM-deficient mice treated with the DNA-PK inhibitors relative to radiation alone. Similar reductions are also seen in ATM-deficient FaDu tumor xenografts where both pDNA-PK and γH2AX staining could be performed. DNA-PK inhibitor-mediated sensitization to radiation and DNA-damaging chemotherapy are not only limited to tumor tissues, but also extends to normal tissues sustaining DNA damage. These data are useful for interpretation of the sensitizing effects of DNA damage repair inhibitors, where a therapeutic index showing greater cell-killing effects on cancer cells is crucial for optimal clinical translation.

Stimulating macropinocytosis of peptide-drug conjugates through DNA-dependent protein kinase inhibition for treating KRAS-mutant cancer

KRAS-mutant cancers, due to their protein targeting complexity, present significant therapeutic hurdles. The identification of the macropinocytic phenotype in these cancers has emerged as a promising alternative therapeutic target. Our study introduces MPD1, an macropinocytosis-targeting peptide-drug conjugates (PDC), which is developed to treat KRAS mutant cancers. This PDC is specifically designed to trigger a positive feedback loop through its caspase-3 cleavable characteristic. However, we observe that this loop is hindered by DNA-PK mediated DNA damage repair processes in cancer cells. To counter this impediment, we employ AZD7648, a DNA-PK inhibitor. Interestingly, the combined treatment of MPD1 and AZD7648 resulted in a 100% complete response rate in KRAS-mutant xenograft model. We focus on the synergic mechanism of it. We discover that AZD7648 specifically enhances macropinocytosis in KRAS-mutant cancer cells. Further analysis uncovers a significant correlation between the increase in macropinocytosis and PI3K signaling, driven by AMPK pathways. Also, AZD7648 reinforces the positive feedback loop, leading to escalated apoptosis and enhanced payload accumulation within tumors. AZD7648 possesses broad applications in augmenting nano-sized drug delivery and preventing DNA repair resistance. The promising efficacy and evident synergy underscore the potential of combining MPD1 with AZD7648 as a strategy for treating KRAS-mutant cancers.

Up-Regulation of Non-Homologous End-Joining by MUC1.

Ionizing radiation (IR) and chemotherapy with DNA-damaging drugs such as cisplatin are vital cancer treatment options. These treatments induce double-strand breaks (DSBs) as cytotoxic DNA damage; thus, the DSB repair activity in each cancer cell significantly influences the efficacy of the treatments. Pancreatic cancers are known to be resistant to these treatments, and the overexpression of MUC1, a member of the glycoprotein mucins, is associated with IR- and chemo-resistance. Therefore, we investigated the impact of MUC1 on DSB repair. This report examined the effect of the overexpression of MUC1 on homologous recombination (HR) and non-homologous end-joining (NHEJ) using cell-based DSB repair assays. In addition, the therapeutic potential of NHEJ inhibitors including HDAC inhibitors was also studied using pancreatic cancer cell lines. The MUC1-overexpression enhances NHEJ, while partially suppressing HR. Also, MUC1-overexpressed cancer cell lines are preferentially killed by a DNA-PK inhibitor and HDAC1/2 inhibitors. Altogether, MUC1 induces metabolic changes that create an imbalance between NHEJ and HR activities, and this imbalance can be a target for selective killing by HDAC inhibitors. This is a novel mechanism of MUC1-mediated IR-resistance and will form the basis for targeting MUC1-overexpressed pancreatic cancer.

Enhancing Standard of Care Chemotherapy Efficacy Using DNA-Dependent Protein Kinase (DNA-PK) Inhibition in Preclinical Models of Ewing Sarcoma.

Disruption of DNA damage repair via impaired homologous recombination is characteristic of Ewing sarcoma (EWS) cells. We hypothesize that this disruption results in increased reliance on nonhomologous end joining to repair DNA damage. In this study, we investigated if pharmacologic inhibition of the enzyme responsible for nonhomologous end joining, the DNA-PK holoenzyme, alters the response of EWS cells to genotoxic standard of care chemotherapy. We used analyses of cell viability and proliferation to investigate the effects of clinical DNA-PK inhibitors (DNA-PKi) in combination with six therapeutic or experimental agents for EWS. We performed calculations of synergy using the Loewe additivity model. Immunoblotting evaluated treatment effects on DNA-PK, DNA damage, and apoptosis. Flow cytometric analyses evaluated effects on cell cycle and fate. We used orthotopic xenograft models to interrogate tolerability, drug mechanism, and efficacy in vivo. DNA-PKi demonstrated on-target activity, reducing phosphorylated DNA-PK levels in EWS cells. DNA-PKi sensitized EWS cell lines to agents that function as topoisomerase 2 (TOP2) poisons and enhanced the DNA damage induced by TOP2 poisons. Nanomolar concentrations of single-agent TOP2 poisons induced G2M arrest and little apoptotic response while adding DNA-PKi-mediated apoptosis. In vivo, the combination of AZD7648 and etoposide had limited tolerability but resulted in enhanced DNA damage, apoptosis, and EWS tumor shrinkage. The combination of DNA-PKi with standard of care TOP2 poisons in EWS models is synergistic, enhances DNA damage and cell death, and may form the basis of a promising future therapeutic strategy for EWS.