DNA-dependent Protein Kinase Inhibitor Research Articles

Radiation and chemo-sensitizing effects of DNA-PK inhibitors are proportional in tumors and normal tissues.

Inhibitors of 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 - 15Gy irradiation ± 3 - 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 5Gy 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 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. Conclusions: DNA-PK inhibitor-mediated sensitization to radiation and DNA-damaging chemotherapy is not 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.

Peposertib, a DNA-PK Inhibitor, Enhances the Anti-Tumor Efficacy of Topoisomerase II Inhibitors in Triple-Negative Breast Cancer Models.

Triple-negative breast cancer (TNBC) remains the most lethal subtype of breast cancer, characterized by poor response rates to current chemotherapies and a lack of additional effective treatment options. While approximately 30% of patients respond well to anthracycline- and taxane-based standard-of-care chemotherapy regimens, the majority of patients experience limited improvements in clinical outcomes, highlighting the critical need for strategies to enhance the effectiveness of anthracycline/taxane-based chemotherapy in TNBC. In this study, we report on the potential of a DNA-PK inhibitor, peposertib, to improve the effectiveness of topoisomerase II (TOPO II) inhibitors, particularly anthracyclines, in TNBC. Our in vitro studies demonstrate the synergistic antiproliferative activity of peposertib in combination with doxorubicin, epirubicin and etoposide in multiple TNBC cell lines. Downstream analysis revealed the induction of ATM-dependent compensatory signaling and p53 pathway activation under combination treatment. These in vitro findings were substantiated by pronounced anti-tumor effects observed in mice bearing subcutaneously implanted tumors. We established a well-tolerated preclinical treatment regimen combining peposertib with pegylated liposomal doxorubicin (PLD) and demonstrated strong anti-tumor efficacy in cell-line-derived and patient-derived TNBC xenograft models in vivo. Taken together, our findings provide evidence that co-treatment with peposertib has the potential to enhance the efficacy of anthracycline/TOPO II-based chemotherapies, and it provides a promising strategy to improve treatment outcomes for TNBC patients.

DNA-PK inhibitor AZD7648 is a more portent radiosensitizer than PARP inhibitor Olaparib in BRCA1/2 deficient tumors

The effectiveness of radiotherapy depends on the sensitivities of ‘normal’ and cancer cells to the administered radiation dose. Increasing the radiosensitivity of cancers by inhibiting DNA damage repair is a goal of much current research, however success depends on avoiding concomitant sensitization of normal tissues inevitably irradiated during therapy. In this study we investigated the mechanisms of radiosensitization for DNA-PK and PARP inhibitors by examining the impacts on proliferating vs quiescent cell populations. Experiments were performed in BRCA1/2null and wild-type parental cancer models in vitro and in vivo. Overall AZD7648 has greater radiosensitizing activity relative to Olaparib, with BRCA2-deficient models showing the greatest sensitivity. However, DNA-PK inhibitor AZD7648 also produced greater toxicity in all irradiated mice. While both DNA-PK and PARP inhibition sensitizes wild type tumor cells to radiation, in BRCA1/2 deficient cells PARP inhibition by Olaparib had limited radiosensitization capacity. Quiescent cells are more radioresistant than proliferating cells, and these were also effectively sensitized by AZD7648 while Olaparib was unable to increase radiation-induced cell kill, even in BRCA1/2null cells. These findings underscore the distinct mechanisms of radiosensitization for DNA-PK and PARP inhibitors. While DNA-PK inhibitors are able to target both proliferating and non-proliferating tumor cells for greater overall anti-cancer benefit, their application is limited by exacerbation of normal tissue toxicities. Conversely, PARP inhibitors exhibit selective activity for proliferating cells, providing a mechanism for targeting activity to cancers, but due to poor activity in non-proliferating cells they have an overall reduced impact on tumor growth control. This study highlights the importance of creating a therapeutic ratio with DNA damage repair inhibition radiation sensitizing strategies.

DNA-PK participates in pre-rRNA biogenesis independent of DNA double-strand break repair.

Although DNA-PK inhibitors (DNA-PK-i) have been applied in clinical trials for cancer treatment, the biomarkers and mechanism of action of DNA-PK-i in tumor cell suppression remain unclear. Here, we observed that a low dose of DNA-PK-i and PARP inhibitor (PARP-i) synthetically suppresses BRCA-deficient tumor cells without inducing DNA double-strand breaks (DSBs). Instead, we found that a fraction of DNA-PK localized inside of nucleoli, where we did not observe obvious DSBs. Moreover, the Ku proteins recognize pre-rRNA that facilitates DNA-PKcs autophosphorylation independent of DNA damage. Ribosomal proteins are also phosphorylated by DNA-PK, which regulates pre-rRNA biogenesis. In addition, DNA-PK-i acts together with PARP-i to suppress pre-rRNA biogenesis and tumor cell growth. Collectively, our studies reveal a DNA damage repair-independent role of DNA-PK-i in tumor suppression.

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 non-homologous end joining (NHEJ) to repair DNA damage. In this study, we investigated if pharmacological inhibition of the enzyme responsible for NHEJ, 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 AZD-7648 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.

Cell Context is the third axis of synergy for the combination of ATR inhibition and cisplatin in Ewing sarcoma.

The importance of cellular context to the synergy of DNA Damage Response (DDR) targeted agents is important for tumors with mutations in DDR pathways, but less well-established for tumors driven by oncogenic transcription factors. In this study, we exploit the widespread transcriptional dysregulation of the EWS-FLI1 transcription factor to identify an effective DDR targeted combination therapy for Ewing Sarcoma (ES). We used matrix drug screening to evaluate synergy between a DNA-PK inhibitor (M9831) or an ATR inhibitor (berzosertib) and chemotherapy. The combination of berzosertib and cisplatin was selected for broad synergy, mechanistically evaluated for ES selectivity, and optimized for in vivo schedule. Berzosertib combined with cisplatin demonstrates profound synergy in multiple ES cell lines at clinically achievable concentrations. The synergy is due to loss of expression of the ATR downstream target CHEK1, loss of cell cycle checkpoints, and mitotic catastrophe. Consistent with the goals of the project, EWS-FLI1 drives the expression of CHEK1 and five other ATR pathway members. The loss of CHEK1 expression is not due to transcriptional repression and instead caused by degradation coupled with suppression of protein translation. The profound synergy is realized in vivo with a novel optimized schedule of this combination in subsets of ES models leading to durable complete responses in 50% of animals bearing two different ES xenografts. These data exploit EWS-FLI1 driven alterations in cell context to broaden the therapeutic window of berzosertib and cisplatin to establish a promising combination therapy and a novel in vivo schedule.

Discovery of Novel Heterotricyclic Compounds as DNA-Dependent Protein Kinase (DNA-PK) Inhibitors with Enhanced Chemosensitivity, Oral Bioavailability, and the Ability to Potentiate Cancer Immunotherapy.

In this work, a novel series of heterotricyclic DNA-PK inhibitors were rationally designed, synthesized, and assessed for their biological activity. In the DNA-PK biochemical assay, most compounds displayed potent enzymatic activity, with IC50 values between 0.11 and 71.5 nM. Among them, SK10 exhibited the most potent DNA-PK-inhibitory activity (IC50 = 0.11 nM). Studies of the mechanism of action indicated that SK10 could lower γH2A.X expression levels and demonstrate optimal synergistic antiproliferative activity against Jurkat cells (IC50 = 25 nM) when combined with doxorubicin. Importantly, in CT26 and B16-F10 tumor-bearing mouse models, the combination therapies of SK10 with chemotherapeutic drug doxorubicin, a PD-L1 antibody, and SWS1 (a potent PD-L1 small-molecule inhibitor) demonstrated superior synergistic anticancer and potential immunomodulatory effects. Furthermore, SK10 possessed favorable in vivo pharmacokinetic properties [e.g., oral bioavailability (F) = 31.8%]. Taken together, SK10 represents a novel heterotricyclic DNA-PK inhibitor with antitumor immune effects and favorable pharmacokinetics.

Abstract LB222: Elucidating the cellular responses and mechanism of action of 177Lu-based radioligand therapy

Abstract Radioligand therapy (RLT) is emerging as a safe and effective targeted approach for treating several types of cancers. Lutathera® (177Lu-Dotatate) and Pluvicto® (177Lu-PSMA-617) are two examples of FDA-approved 177Lu-based RLT drugs for the treatment of somatostatin receptor-positive gastroenteropancreatic neuroendocrine tumors (GEP-NETs) and of PSMA-positive metastatic castration-resistant prostate cancer (mCRPC), respectively. Despite the clinical success of 177Lu-RLT, there are still patients lacking complete, durable responses. Arguably, elucidating the cellular responses to 177Lu-RLT and its mechanism of action could reveal potential opportunities for the improvement of therapy and outcome for patients. We have investigated the phenotypes and responses of multiple 177Lu- RLT-treated cell lines and observed that 177Lu-RLT induces a plethora of DNA Damage Response (DDR) markers, especially those indicative of double-strand break (DSB) repair by either non-homologous end-joining (NHEJ) or homologous recombination (HR). Furthermore, 177Lu-RLT leads to cell-cycle alterations, accumulation of micronuclei and cell death. In agreement with published reports, we have observed that either the knock-out or inhibition of NHEJ core factors such as the catalytic subunit of DNA-PK (PRKDC) renders cells sensitive to 177Lu-RLT and the combination of both the 177Lu-RLT and DNA-PK inhibitor provides a beneficial tumor growth inhibition in vivo. To further unravel the mechanism of action of 177Lu-RLT, we have conducted ‘DDR-ome’ targeted genetic screens, which also suggest that DSBs are the most cytotoxic form of 177Lu-RLT-induced DNA damage. In addition to DNA-PK, we have identified novel DDR targets that could sensitize to 177Lu-RLT. We are currently testing whether inhibiting these targets in combination with certain 177Lu-RLT could, in addition to potentially achieving greater antitumor response, allow for lower dosing to reduce radiation exposure. Taken together, our work provides better understanding of the cellular responses to 177Lu-based RLT and pinpoints NHEJ as a critical pathway promoting survival to this treatment, which could set the basis for novel combination therapies. Citation Format: Marco Ranzani, Sravanth Hindupur, Alessandro Cicconi, Guillermo Sastre-Moreno, Alexander Kristian, Barbara Schacher Engstler, Benika Pinch, Charlene Hartnagel, Delphine Gorses, Elise Simon, Emeline Mandon, Emilien Schramm, Fanny Schaeffer, Josefine Reber, Louise Barys, Luisa Deberle, Milene Walter, Ralph Bessey, Riccardo Destefani, Xavier Miot, Elias Elinati, Nan Shao, Marie Boursier, Joseph Barlow, Asmita Thapa, Alessandro Galbiati, Diego Grande, Eeson Rajendra, Niall Martin, Graeme Smith, Tobias Schmelzle, Markus Reschke, Helen Robinson, Marta Cortes-Cros. Elucidating the cellular responses and mechanism of action of 177Lu-based radioligand therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB222.

DNA-DEPENDENT PROTEIN KINASE INHIBITOR INDUCES APOPTOSIS IN COLON CANCER CELLS

Objectives: Among all types of DNA damage, DNA double-strand breaks (DSBs) are considered the most deleterious form induced by either endogenous factors (oxi-dative damages, mismatches, altered chromatin structures, and missing, or modified nucleotides) or exogenous factors, i.e., ultraviolet (UV) radiation, ionizing radiation (IR), and chemicals or drugs. DNA-dependent protein kinase (DNA-PK) plays a crucial role in repairing DSBs through non-homologous end joining (NHEJ). Cells lacking DNA-PK exhibit heightened sensitivity to IR and various DNA-damaging agents. The inhibition of DNA-PK further intensifies cellular susceptibility to IR and DNA-damaging agents. Several small molecules that inhibit DNA-PK have been developed. This study aimed to evaluate the effect of DNA-PK inhibitor (DNA-PKi) NU7441 on the HCT116 cell line. Methods: DNA-PKi NU7441 was used to assess the effect on anti-proliferation and induction of apoptosis on the HCT116 colorectal cancer cell line. Cells were cultured under standard conditions; crystal violet and apoptosis assay were applied to evaluate cell proliferation and apoptosis. Data were analysed using GraphPad Prism 8.4. Results: DNA-PKi effectively inhibited HCT116 colon cancer cell growth via crystal violet assay (p < 0.01). In addition, DNA-PKi also induced programmed cell death in the HCT116 cell line (p < 0.05). Conclusion: DNA-PKi NU7441 suppressed cell proliferation and induced apoptosis in the HCT116 colon cancer cell line.

Functional screening in human HSPCs identifies optimized protein-based enhancers of Homology Directed Repair

Homology Directed Repair (HDR) enables precise genome editing, but the implementation of HDR-based therapies is hindered by limited efficiency in comparison to methods that exploit alternative DNA repair routes, such as Non-Homologous End Joining (NHEJ). In this study, we develop a functional, pooled screening platform to identify protein-based reagents that improve HDR in human hematopoietic stem and progenitor cells (HSPCs). We leverage this screening platform to explore sequence diversity at the binding interface of the NHEJ inhibitor i53 and its target, 53BP1, identifying optimized variants that enable new intermolecular bonds and robustly increase HDR. We show that these variants specifically reduce insertion-deletion outcomes without increasing off-target editing, synergize with a DNAPK inhibitor molecule, and can be applied at manufacturing scale to increase the fraction of cells bearing repaired alleles. This screening platform can enable the discovery of future gene editing reagents that improve HDR outcomes.

Abstract 416: Development of substituted oxindole derivatives as a Ku-targeted DNA-PK inhibitors for cancer therapy

Abstract DNA double strand breaks (DSBs) are the most cytotoxic type of DNA damage. DSB repair pathway deficiency, often observed in cancer cells, results in translocations and genetic mutations that contribute to genomic instability. The generation of DNA DSBs is the primary mechanism of numerous chemo- and radio-therapeutic strategies used to treat various cancers. Modulating DSB repair pathways can have a profound impact on the clinical efficacy of DNA damaging therapies. DNA-dependent protein kinase (DNA-PK), which is composed of a DNA-PK catalytic subunit (DNA-PKcs) and Ku80-Ku70 heterodimer, acts as the molecular sensor for DSB and plays a prime role in DSB repair through non-homologous end joining (NHEJ). DNA-PK inhibitors become an attractive therapeutic target for cancer in combination with DSB-inducing radiotherapy and chemotherapy. There are at least 6-7 DNA-PK inhibitors in the preclinical and clinical stages which exhibit potential as treatment for various cancers. However, we have taken a unique and innovative approach to inhibiting DNA-PK via blocking the Ku70/80 heterodimer interaction with DNA, an essential step in DNA-PK activation and phosphorylation activity. Exploiting this unique mechanism of kinase activation, we have expanded our structure-activity relationships of pyrrolone derivatives. Modified synthetic schemes have been developed to allow a series of modifications to the X80 core structure. We have prepared furan, thiophene and pyrrole derivatives that enabled the identification of pharmacophores that impact in vitro potency and cellular off-target toxicity. Towards addressing cellular uptake and bioavailability, we identified substituted oxindole derivatives as potent Ku and DNA-PK inhibitors. These oxindole derivatives exhibited a better cellular uptake while retaining potent Ku inhibitory activity. Our data demonstrate that the cellular effects observed are a function of Ku inhibition and that this novel class of DNA-PK inhibitors can be further developed as anti-cancer therapeutics that can be used as an adjuvant to, or concomitant with radiotherapy and other cancer therapies that induce DNA damage. Citation Format: Dineshsinha Chauhan, Pamela L. Mendoza-Munoz, Narva Deshwar Kushwaha, Hariprasad Aruri, John J. Turchi, Navnath S. Gavande. Development of substituted oxindole derivatives as a Ku-targeted DNA-PK inhibitors for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 416.

Abstract 3585: DNA-PK inhibition to enhance radiation sensitivity in metastatic pancreatic neuroendocrine cancer

Abstract Gastroenteropancreatic (GEP) neuroendocrine tumors (NETs) are neoplasms originating from the gastrointestinal track and pancreas. Unfortunately, most patients at the time of diagnosis have extensive metastatic disease and are not candidates for curative surgical resection of metastatic tumors. In such cases, targeted radiation therapy (RT) can slow the progression of metastatic disease, but eventually tumors develop resistance to radiation therapy. In this study, we examined whether DNA-PK inhibition could sensitize pancreatic NETs to radiation therapy and enhance the radiation therapy effect in a preclinical model of pNET metastasis. Methods. We used 96-well clonogenic assays to evaluate the efficacy of the DNA-PK inhibitor peposertib in combination with RT in vitro in QGP-1 and BON pancreatic NET cell lines. Double-strand break-induced DNA repair was visualized with confocal laser scanning microscopy using γ-H2AX immunofluorescence after peposertib treatment alone or in combination with RT. Western blot analysis was used to confirm inhibition of DNA-PKcs (pDNA-PKcs S2056) combination RT. The efficacy of peposertib in combination with RT was evaluated in QGP-1 and BON human xenograft models in athymic nude mice and a BON preclinical lung metastasis model. Results. Clonogenic assays demonstrated absence of cytotoxicity from peposertib alone at doses below 1000 nM and a strong radiosensitizing effect at 200 nM in both QGP-1 and BON cell lines. Pretreatment with peposertib or treatment within 2 h after RT suppressed colony formation; treatment at later timepoints did not enhance radiotherapy. Immunofluorescence analysis revealed high numbers of γH2AX foci with peposertib therapy alone at 96 h or when given 2 h after RT. Western blot analysis confirmed inhibition of DNA-PK Ser2056 after RT in combination with peposertib. DNA-PK inhibition in combination with RT led to remarkable repression of pNET growth both in subcutaneous xenografts (>90%) and in the preclinical lung metastasis model (>90%). Conclusions. Our results showed several advantages of targeting DNA-PK to enhance radiation sensitivity in high-grade pNETs: (1) DNA-PK inhibition alone disrupts DNA damage response in pNETs as demonstrated by strong H2AX phosphorylation. (2) DNA-PK inhibition is most effective at enhancing radiotherapy either right before or after radiation therapy administration. This demonstrates that extended DNA-PK inhibition is not necessary to achieve marked responses during pNET radiotherapy. (3) DNA-PK inhibition 30 min before radiation therapy is sufficient to greatly reduce pNET tumor growth or metastasis progression in pNET preclinical models. In summary, selective DNA-PK inhibition provides a potent therapeutic strategy for disruption of non-homologous end joining DNA double-strand break repair and may offer a novel therapeutic approach in advanced NET radiotherapy. Citation Format: Piotr G. Rychahou, Aman Chauhan, Zeta Chow, Tadahide Izumi, Quan Chen, Eun Y. Lee, Dana Napier, Lowell Brian Anthony, Michael J. Cavnar, Courtney M. Townsend, B. Mark Evers. DNA-PK inhibition to enhance radiation sensitivity in metastatic pancreatic neuroendocrine cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3585.

Abstract 132: DNA-PK inhibition to enhance etoposide therapy in neuroblastoma

Abstract Introduction. Neuroblastoma is the most common extracranial solid tumor in children and accounts for ~15% of all pediatric cancer deaths. DNA-PK, a key regulator of repair responses to double-strand DNA breakages induced by radiation or induction chemotherapy is used in the treatment of high-risk neuroblastomas. Here, we have demonstrated that high DNA-PK expression is associated with poor overall survival in a patient population, DNA-PK is highly activated in neuroblastoma cell lines, and inhibition of DNA-PK enhances therapeutic effects of DNA damaging therapies. Methods. DNA double-strand break damage was induced with etoposide, a topoisomerase II inhibitor. DNA-PK activity was inhibited with peposertib or DNA-PK siRNA. DNA-PK Ser2056 phosphorylation was analyzed in MYCN amplified (SK-NDZ, BE(2)-C, LAN-1) and MYCN non-amplified (SK-N-AS, SK-N-SH) cell lines. Clonogenic and proliferation assays were used to evaluate the efficacy of peposertib in combination with etoposide in BE-2-C and SK-N-DZ neuroblastoma cell lines. DSB-induced DNA repair was visualized with confocal laser scanning microscopy using γ-H2AX immunofluorescence. Western blot was used to confirm inhibition of DNA-PKcs (pDNA-PKcs S2056) and analyze cleaved PARP expression in combination groups. Results. DNA-PK inhibition resulted in enhancement of etoposide cytotoxic therapy (BE-2-C IC50 ET = 2.77 ± 0.2, ET+Pep 0.76 ± 0.04; SK-N-DZ IC50 ET = 0.55 ± 0.06, IC50 ET+Pep = 0.11 ± 0.02) in both BE-2-C and SK-N-DZ cell lines. Immunofluorescence analysis detected γH2AX foci increase in the etoposide and peposertib combination group. DNA-PK inhibition in combination with etoposide treatment significantly increased PARP cleavage in BE-2-C and SK-N-DZ cell lines. The effect of DNA-PK knockdown on neuroblastoma cell survival was even more pronounced compared to DNA-PK inhibition with peposertib. DNA-PK knockdown by siRNA alone increased PARP cleavage to the levels comparable to 2 µM etoposide treatment. The combination of etoposide and DNA-PK knockdown enhanced PARP cleavage even further in neuroblastoma cells. Conclusions. High DNA repair activity is commonly associated with cancer drug resistance. We have shown that DNA-PK activation is continuously supported in both MYCN amplified and MYCN non-amplified neuroblastoma cell lines. The importance of DNA-PK activity in neuroblastoma cell lines is confirmed with DNA-PK knockdown that leads to apoptosis in MYCN amplifiedneuroblastoma cell lines. In addition, the combination of DNA-PK inhibition or DNA-PK knockdown with DNA-damaging therapies stalled DNA damage response and enhanced neuroblastoma cell apoptosis. These findings demonstrate the significance of high DNA-PK activity in neuroblastoma cells and provide a potent therapeutic strategy to enhance neuroblastoma cell apoptosis with selective DNA-PK inhibition. Citation Format: Mahnaz Norouzi, Beibei Zhu, Tadahide Izumi, Eric J. Rellinger, B. Mark Evers, Piotr G. Rychahou. DNA-PK inhibition to enhance etoposide therapy in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 132.

Abstract 4540: SY-7021, a novel DNA-PK inhibitor, exhibits significant anti-tumor activity in vitro and in vivo

Abstract Cancer therapies such as ionizing radiation or topoisomerase II inhibitors (for example, doxorubicin) induce DNA double-strand breaks (DSBs), which can subsequently be repaired by homologous recombination or non-homologous end joining (NHEJ). DNA-dependent protein kinase (DNA-PK), a nuclear serine/threonine protein kinase complex, is a pivotal component of the NHEJ process that maintain genome integrity. The critical role of DNA-PK in DNA damage response (DDR) and the dysregulated DNA-PK expression in cancer make it an intriguing therapeutic target for cancer, especially when combined with DSBs agents. Here, we identified a highly potent and selective DNA-PK inhibitor, SY-7021, with an IC50 value of 0.242 nM on DNA-PK kinase and high selectivity on ATM and ATR (greater than 400-fold) in biochemical assays. In a reporter assay for NHEJ repair, SY-7021 was able to dose-dependently reduce cellular NHEJ efficiency. Functionally, SY-7021 inhibited cell proliferation in multiple cancer cell lines alone or combined with doxorubicin. Basically, combination of SY-7021 and doxorubicin induced significant G2/M phase arrest and cell apoptosis, as well as enhanced the phosphorylation on Ser139 of γH2AX, Tyrosine 68 of CHK2 and Serine15 of p53 in MDA-MB-468 cells. In a subcutaneous NCI-H1703 xenograft tumor model, SY-7021 administered orally twice daily achieved dose-dependent tumor growth inhibition in vivo, with a tumor growth inhibition (TGI) of 105.6% at 60 mg/kg and no significant weight loss was observed. In addition, SY-7021 demonstrates good PK properties and acceptable safety profiles in in vivo studies. Collectively, SY-7021, a potent and selective DNA-PK inhibitor, shows significant inhibition on tumor growth in in vitro and in vivo studies, providing a rationale treatment for multiple tumors in monotherapy or in combination with other agents. Citation Format: Kai Zhang, Zhihua Liu, Yuhao Gao, Chang Lu, Shikang Cheng, Xijie Liu, Hong Luo, Yinghui Sun. SY-7021, a novel DNA-PK inhibitor, exhibits significant anti-tumor activity in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4540.

Abstract 701: Preclinical data of a novel DNA-PK inhibitor in combination with radiation therapy shows promise in the treatment of established GBM and lung carcinoma cell lines

Abstract Radiation therapy (RT) is a cornerstone of oncologic therapy for the majority of solid malignancies, and unrepaired DNA double-strand breaks are responsible for radiation-induced cytotoxicity. RT-induced DNA lesions can be repaired by multiple mechanisms including homologous recombination (HR) and non-homologous end joining (NHEJ), and disruption of either of these pathways can enhance cytotoxicity. A key component of the NHEJ pathway is the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), and loss of kinase activity can significantly increase radiosensitivity. Herein we describe the results of our medicinal chemistry campaign to develop a potent and selective DNA-PKcs inhibitor, WNC0901. WNC0901 inhibits DNA-PKcs kinase activity in a cell free system with an IC50 of 0.071 nM and demonstrated at least 30-fold higher sensitivity than other family members (ATM, ATR, mTOR, PI3K). WNC0901 has limited aldehyde oxidase (AO) liability with a T1/2 >2000 min in liver cytosol and is stable for over 120 minutes in the presence and absence of an AO inhibitor. A preliminary pharmacokinetic analysis in wista han rat (IV=2mg/kg, PO=20mg/kg) demonstrated 116% apparent absolute oral bioavailability, 2.6 hour terminal half-life, 33.0 mL/min/kg clearance, and a 2.6% unbound brain-to-plasma partition coefficient (Kpuu). WNC0901 also had favorable pharmacokinetic properties in beagle dog (IV=2mg/kg, PO=20mg/kg) with moderate clearance (8.5 mL/min/kg), high apparent absolute oral bioavailability (131%) with good exposure (AUC=36092 h*ng/mL), moderate half-life (4.28 h), and low protein binding (74.3% fraction unbound). The volume of distribution was moderate in both species (Vss = 1.32 L/kg in wistar han rat and 1.87 L/kg in beagle dog). In cell culture, WNC0901 inhibited autophosphorylation of DNA-PKcs in HT29 cells irradiated with 10 Gy with an IC50 of 32.7 nM and robustly inhibited autophosphorylation in both U251 glioma and A549 lung cancer cell lines at 300 nM in combination with 5Gy. In a clonogenic assay, 5Gy irradiation (10% survival) combined with 100nM WNC0901 demonstrated modestly enhanced cell killing (1.5% survival), and maximal effects were seen at 300nM (0.04% survival, p<0.01). Similar radiosensitizing effects with 300 nM WNC0901 were seen in the A549 cell line (0.2% survival with combination compared to 19% with 5Gy alone, p<0.01). In summary, WNC0901 inhibits DNA-PK kinase activity and provides potent radiosensitization in a GBM and lung cancer cell line. Future studies will assess the combination of WNC0901 and RT in GBM patient-derived xenograft models in vivo. Citation Format: Ann C. Mladek, Danielle L. Burgenske, William F. Elmquist, Wei Zhong, Jann N. Sarkaria. Preclinical data of a novel DNA-PK inhibitor in combination with radiation therapy shows promise in the treatment of established GBM and lung carcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 701.

Abstract 7128: Novel Ku-DNA binding inhibitors impact on the cellular and in vivo DNA damage response to radio- and radiomimetic-therapy

Abstract DNA-PK, the DNA-dependent protein kinase, is a validated target for cancer therapeutics that plays a central role in the non-homologous end joining (NHEJ) DNA repair pathway and the DNA damage response (DDR). NHEJ is a key mechanism of DNA double-strand breaks (DSB) repair, especially those induced by ionizing radiation (IR). Ku 70/80 heterodimer is required as initiator in this process, acting as an essential DNA-binding component of DNA-PK that senses DNA damage. Blocking the DNA-PK kinase activity in combination with DSB inducing agents has been widely used as a therapeutic strategy to drive clinical efficacy of radiation therapy for cancer treatment. We have reported a unique approach of DNA-PK inhibition by developing Ku-DNA binding inhibitors (Ku-DBi’s), small molecule inhibitors that target the interaction between Ku70/80 and DNA. Ku-DBi’s demonstrated a direct interaction with Ku70/80, a potent inhibition of Ku-DNA binding and DNA-PK catalytic activity, and in vitro and cellular NHEJ inhibitory activity. This results in the potentiation of non-small cell lung cancer (NSCLC) cellular sensitivity to DSB generating therapies, as a function of inhibiting DNA-PKcs autophosphorylation and dysregulation of signaling to the DDR. In this study, we have expanded our structure activity relationship analyses to focus on optimizing selectivity, cellular uptake and Ku inhibitory activity. The new series of Ku-DBi’s include oxindole derivatives of the X80 core structure that achieve these goals. These novel compounds display increased cellular uptake while retaining potent Ku inhibitory activity and enhanced cellular inhibition on DNA-DSB repair. Ku-DBi’s optimization has enabled in vivo analyses of Ku-DBi - ionizing radiation combination therapy in human xenograft models of non-small cell lung cancer. The impact of Ku-DBi treatment on therapeutic efficacy, toxicity, Ku/DNA-PK inhibition, and DDR signaling will be presented. These data represent a significant advance in the development of Ku-DNA binding inhibitors and their therapeutic intervention for cancer treatment. Citation Format: Pamela L. Mendoza-Munoz, Dineshsinha Chauhan, Navnath S. Gavande, Joseph R. Dynlacht, Joy E. Garrett, John J. Turchi. Novel Ku-DNA binding inhibitors impact on the cellular and in vivo DNA damage response to radio- and radiomimetic-therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7128.

ARF4-mediated retrograde trafficking as a driver of chemoresistance in GBM.

Cellular functions hinge on the meticulous orchestration of protein transport, both spatially and temporally. Central to this process is retrograde trafficking, responsible for targeting proteins to the nucleus. Despite its link to many diseases, the implications of retrograde trafficking in glioblastoma (GBM) are still unclear. To identify genetic drivers of TMZ resistance, we conducted comprehensive CRISPR-knockout screening, revealing ADP-ribosylation factor 4 (ARF4), a regulator of retrograde trafficking, as a major contributor. Suppressing ARF4 significantly enhanced TMZ sensitivity in GBM patient-derived xenograft (PDX) models, leading to improved survival rates (p<0.01) in both primary and recurrent lines. We also observed that TMZ exposure stimulates ARF4-mediated retrograde trafficking. Proteomics analysis of GBM cells with varying levels of ARF4 unveiled the influence of this pathway on EGFR signaling, with increased nuclear trafficking of EGFR observed in cells with ARF4 overexpression and TMZ treatment. Additionally, spatially-resolved RNA-sequencing of GBM patient tissues revealed substantial correlations between ARF4 and crucial nuclear EGFR (nEGFR) downstream targets, such as MYC, STAT1, and DNA-PK. Decreased activity of DNA-PK, a DNA repair protein downstream of nEGFR signaling that contributes to TMZ resistance, was observed in cells with suppressed ARF4 levels. Notably, treatment with DNA-PK inhibitor, KU57788, in mice with a recurrent PDX line resulted in prolonged survival (p<0.01), highlighting the promising therapeutic implications of targeting proteins reliant on ARF4-mediated retrograde trafficking. Our findings demonstrate that ARF4-mediated retrograde trafficking contributes to the development of TMZ resistance, cementing this pathway as a viable strategy to overcome chemoresistance in GBM.

204P Maintenance of anti-tumour innate immune response in SCLC with DNA-PK inhibition

204P Maintenance of anti-tumour innate immune response in SCLC with DNA-PK inhibition

An improved synthetic strategy for the multigram-scale synthesis of DNA–PK inhibitor AZD7648

An improved synthetic strategy for the multigram-scale synthesis of DNA–PK inhibitor AZD7648

Different Impacts of DNA-PK and mTOR Kinase Inhibitors in Combination with Ionizing Radiation on HNSCC and Normal Tissue Cells.

Despite substantial advancements in understanding the pathomechanisms of head and neck squamous cell carcinoma (HNSCC), effective therapy remains challenging. The application of kinase inhibitors (KIs) in HNSCC, specifically mTOR and DNA-PK inhibitors, can increase radiosensitivity and therefore presents a promising strategy when used simultaneously with ionizing radiation (IR) in cancer treatment. Our study focused on the selective DNA-PK-inhibitor AZD7648; the selective mTOR-inhibitor Sapanisertib; and CC-115, a dual inhibitor targeting both mTOR and DNA-PK. The impact of these KIs on HNSCC and normal tissue cells was assessed using various analytical methods including cell death studies, cell cycle analysis, real-time microscopy, colony-forming assays and immunohistochemical staining for γH2AX and downstream mTOR protein p-S6. We detected a strong inhibition of IR-induced DNA double-strand break (DSB) repair, particularly in AZD7648-treated HNSCC, whereas normal tissue cells repaired DNA DSB more efficiently. Additionally, AZD7648 + IR treatment showed a synergistic decline in cell proliferation and clonogenicity, along with an elevated G2/M arrest and cell death in the majority of HNSCC cell lines. CC-115 + IR treatment led to an elevation in G2/M arrest, increased cell death, and a synergistic reduction in cell proliferation, though the effect was notably lower compared to the AZD7648 + IR- treated group. Sapanisertib led to a high cellular toxicity in both HNSCC and normal tissue cells, even in non-irradiated cells. Regarding cell proliferation and the induction of apoptosis and necrosis, Sapanisertib + IR was beneficial only in HPV+ HNSCC. Overall, this study highlights the potential of AZD7648 as a radiosensitizing agent in advanced-stage HPV-positive and negative HNSCC, offering a promising therapeutic strategy. However, the dual mTOR/DNA-PK-I CC-115 did not provide a distinct advantage over the use of selective KIs in our investigations, suggesting limited benefits for its application in KI + IR therapy. Notably, the selective mTOR-inhibitor Sapanisertib was only beneficial in HPV+ HNSCC and should not be applied in HPV- cases.