DNA-dependent Protein Kinase Activity Research Articles

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 B068: Modulation of the DNA damage response by novel Ku-DNA binding inhibitors enhances cellular effects of DNA-double strand break inducing agents

Abstract The DNA-dependent protein kinase (DNA-PK) plays a critical role in the non-homologous end joining (NHEJ) double-strand break (DSB) repair pathway and the DNA damage response (DDR). Consequently, blocking DNA-PK kinase activity is being pursued as a therapeutic strategy for the treatment of cancer in combination with DNA-double strand break inducing agents. We have previously reported the development of Ku-DNA binding inhibitors (Ku-DBi’s) that act via a novel mechanism of action to inhibit DNA-PK catalytic kinase activity by targeting the Ku 70/80 heterodimer interaction with DNA. Ku-DBi’s display nanomolar activity in vitro, possess cellular DNA-PK and NHEJ inhibitory activity, and sensitize non-small cell lung cancer (NSCLC) cells to DSB generating therapies bleomycin, etoposide and ionizing radiation. In this study, we have expanded our structure activity relationship analyses to focus on optimizing cellular uptake and reducing protein binding. We present the synthesis and analysis of a new series of Ku-DBi’s which demonstrated that chemical modifications of the X80 core structure can be implemented to achieve these goals while retaining Ku inhibitory activity. Furthermore, novel relationships with in vitro inhibitory activity were identified as a function of new pharmacophore additions. In addition, these novel compounds display reduced non-specific protein binding, increased cellular uptake, and enhanced cellular inhibition on DNA-double strand break repair. These novel compounds enable cellular studies to be conducted in serum contain media and the pursuit of in vivo analysis and represent a significant advance in the development of Ku-DNA binding inhibitors. Citation Format: Pamela L Mendoza-Munoz, Dineshsinha Chauhan, Navnath S Gavande, John J Turchi. Modulation of the DNA damage response by novel Ku-DNA binding inhibitors enhances cellular effects of DNA-double strand break inducing agents [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B068.

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

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

PTEN potentiation of oncolytic HSV therapy for glioblastoma

PTEN potentiation of oncolytic HSV therapy for glioblastoma

UbcH5c-dependent activation of DNA-dependent protein kinase in response to replication-mediated DNA double-strand breaks

Camptothecin (CPT) exhibits strong cytotoxicity by inducing DNA double-strand breaks (DSBs) through DNA replication. Unlike radiation-induced DSBs, which have two DNA ends, CPT-induced DSBs are considered to have only one DNA end. However, the differences in cellular responses to one-ended and two-ended DSBs are not well understood. Our previous study showed that proteasome inhibitor treatment suppressed CPT-induced activation of DNA-PK, a factor required for non-homologous end-joining in DSB repair, suggesting that the ubiquitin-proteasome pathway is involved in DNA-PK activation in response to one-ended DSBs. To identify the ubiquitination factors required for DNA-PK activation, we screened an siRNA library against E2 ubiquitin-conjugating enzymes and identified UbcH5c. Knockdown of UbcH5c suppressed DNA-PK activation caused by CPT, but not by the radio-mimetic drug neocarzinostatin. UbcH5c-dependent DNA-PK activation occurred independent of DNA end resection. Furthermore, loss of UbcH5c reduced DNA-PK-dependent chromosomal aberrations and suppressed the activation of cell cycle checkpoint in response to CPT. These results suggest that UbcH5c regulates DNA-PK activation in response to one-ended DSBs caused by replication fork collapse. To our knowledge, this is the first report of a DSB repair-related factor that is differentially involved in the response to one- and two-ended DSBs.

Synergistic Targeting of DNA-PK and KIT Signaling Pathways in KIT Mutant Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the most common and aggressive form of acute leukemia, with a 5-year survival rate of just 24%. Over a third of all AML patients harbor activating mutations in kinases, such as the receptor tyrosine kinases FLT3 (receptor-type tyrosine-protein kinase FLT3) and KIT (mast/stem cell growth factor receptor kit). FLT3 and KIT mutations are associated with poor clinical outcomes and lower remission rates in response to standard-of-care chemotherapy. We have recently identified that the core kinase of the non-homologous end joining DNA repair pathway, DNA-PK (DNA-dependent protein kinase), is activated downstream of FLT3; and targeting DNA-PK sensitized FLT3-mutant AML cells to standard-of-care therapies. Herein, we investigated DNA-PK as a possible therapeutic vulnerability in KIT mutant AML, using isogenic FDC-P1 mouse myeloid progenitor cell lines transduced with oncogenic mutant KIT (V560G and D816V) or vector control. Targeted quantitative phosphoproteomic profiling identified phosphorylation of DNA-PK in the T2599/T2605/S2608/S2610 cluster in KIT mutant cells, indicative of DNA-PK activation. Accordingly, proliferation assays revealed that KIT mutant FDC-P1 cells were more sensitive to the DNA-PK inhibitors M3814 or NU7441, compared with empty vector controls. DNA-PK inhibition combined with inhibition of KIT signaling using the kinase inhibitors dasatinib or ibrutinib, or the protein phosphatase 2A activators FTY720 or AAL(S), led to synergistic cell death. Global phosphoproteomic analysis of KIT-D816V cells revealed that dasatinib and M3814 single-agent treatments inhibited extracellular signal–regulated kinase and AKT (RAC-alpha serine/threonine-protein kinase)/MTOR (serine/threonine-protein kinase mTOR) activity, with greater inhibition of both pathways when used in combination. Combined dasatinib and M3814 treatment also synergistically inhibited phosphorylation of the transcriptional regulators MYC and MYB. This study provides insight into the oncogenic pathways regulated by DNA-PK beyond its canonical role in DNA repair and demonstrates that DNA-PK is a promising therapeutic target for KIT mutant cancers.

Human DNA-dependent protein kinase activation mechanism

DNA-dependent protein kinase (DNA-PK), a multicomponent complex including the DNA-PK catalytic subunit and Ku70/80 heterodimer together with DNA, is central to human DNA damage response and repair. Using a DNA-PK-selective inhibitor (M3814), we identified from one dataset two cryo-EM structures of the human DNA-PK complex in different states, the intermediate state and the active state. Here we show that activation of the kinase is regulated through conformational changes caused by the binding ligand and the string region (residues 802–846) of the DNA-PK catalytic subunit, particularly the helix-hairpin-helix motif (residues 816–836) that interacts with DNA. These observations demonstrate the regulatory role of the ligand and explain why DNA-PK is DNA dependent. Cooperation and coordination among binding partners, disordered flexible regions and mechanically flexible HEAT repeats modulate the activation of the kinase. Together with previous findings, these results provide a better molecular understanding of DNA-PK catalysis.

DNA-Dependent Protein Kinase Mediates YB-1 (Y-Box Binding Protein)-Induced Double Strand Break Repair.

DNA-PK (DNA-dependent protein kinase) is a stress-activated serine/threonine kinase that plays a central role in vascular smooth muscle cell proliferation and vascular proliferative disease processes such as neointimal formation. In this study, we link the activation of DNA-PK to the function of the transcription factor YB-1 (Y-box binding protein). To identify YB-1 phosphorylation by DNA-PK, we generated different YB-1-expressing vectors. YB-1 nuclear translocation was investigated using immunoblotting and immunofluorescence staining. For YB-1 activity, luciferase assays were performed. We show by mutational analysis and kinase assay that the transcriptional regulator YB-1 is a substrate of DNA-PK. Blockade of DNA-PK by specific inhibitors revealed its critical involvement in YB-1phosphorylation as demonstrated by inhibition of an overexpressed YB-1 reporter construct. Using DNA-PK-deficient cells, we demonstrate that the shuttling of YB-1 from the cytoplasm to the nucleus is dependent on DNA-PK and that the N-terminal domain of YB-1 is phosphorylated at threonine 89. Point mutation of YB-1 at this residue abrogated the translocation of YB-1 into the nucleus. The phosphorylation of YB-1 by DNA-PK increased cellular DNA repair after exposure to ionizing radiation. Atherosclerotic tissue specimens were analyzed by immunohistochemistry. The DNA-PK subunits and YB-1 phosphorylated at T89 were found colocalized suggesting their in vivo interaction. In mice, the local application of the specific DNA-PK inhibitor NU7026 via thermosensitive Pluronic F-127 gel around dilated arteries significantly reduced the phosphorylation of YB-1. DNA-PK directly phosphorylates YB-1 and, this way, modulates YB-1 function. This interaction could be demonstrated in vivo, and colocalization in human atherosclerotic plaques suggests clinical relevance of our finding. Phosphorylation of YB-1 by DNA-PK may represent a novel mechanism governing atherosclerotic plaque progression.

Regulation of DNA-PK activity promotes the progression of TNBC via enhancing the immunosuppressive function of myeloid-derived suppressor cells.

DNA-dependent protein kinase (DNA-PK) is engaged in DNA damage repair and is significantly expressed in triple negative breast cancer (TNBC). Inhibiting DNA-PK to reduce DNA damage repair provides a possibility of tumor treatment. NU7441, a DNA-PK inhibitor, can regulate the function and differentiation of CD4+ T cells and effectively enhance immunogenicity of monocyte-derived dendritic cells. However, the effect of NU7441 on the tumor progression activity of immunosuppressive myeloid-derived suppressor cells (MDSCs) in TNBC remains unclear. In this study, we found that NU7441 alone significantly increased tumor growth in 4T1 (a mouse TNBC cell line) tumor-bearing mice. Bioinformatics analysis showed that DNA-PK and functional markers of MDSCs (iNOS, Arg1, and IDO) tended to coexist in breast cancer patients. The mutations of these genes were significantly correlated with lower survival in breast cancer patients. Moreover, NU7441 significantly decreased the percentage of MDSCs in peripheral blood mononuclear cells (PBMCs), spleen and tumor, but enhanced the immunosuppressive function of splenic MDSCs. Furthermore, NU7441 increased MDSCs' DNA-PK and pDNA-PK protein levels in PBMCs and in the spleen and increased DNA-PK mRNA expression and expression of MDSCs functional markers in splenic MDSCs from tumor-bearing mice. NU7441 combined with gemcitabine reduced tumor volume, which may be because gemcitabine eliminated the remaining MDSCs with enhanced immunosuppressive ability. These findings highlight that the regulation of DNA-PK activity by NU7441 promotes TNBC progression via enhancing the immunosuppressive function of MDSCs. Moreover, NU7441 combined with gemcitabine offers an efficient therapeutic approach for TNBC and merits deeper investigation.

Abstract 10202: DNA-PKcs: A Novel Therapeutic Target in Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is a progressive disease characterized by vascular obliteration of small pulmonary arteries (PAs), notably due to excessive proliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs). Accumulating evidence indicates that PAH-PASMCs have acquired an over-efficient DNA damage response (DDR) ensuring sensing, signaling, and repair of DNA damages, accounting for their enhanced capacity to survive and proliferate under stressful conditions. DNA-dependent protein kinase (DNA-PK) is a pivotal component of the DNA repair machinery which plays an instrumental role in the overall survival and proliferation of cancer cells. Based on similarities between PAH and cancer cells, we thus hypothesized that increased expression of DNA-PK contributes to vascular remodeling in PAH. Methods and Results: Using Western blot (WB) and immunohistochemistry, we found that expression and activity of DNA-PK were increased in PAH-PASMCs (n=6) compared to controls (n=6, p<0.01). Similarly, DNA-PK was significantly up-regulated in distal PAs from PAH animal models (i.e. MCT rats and Su/Hx rats and mice, n=5/group, p<0.01). In vitro, the pharmacological inhibition of DNA-PK using AZD-7648 or Nedisertib attenuates PAH-PASMC proliferation (Ki67 and EdU labeling, MCM2, PLK1, and PNCA by WB; p<0.05) and resistance to apoptosis (AnnexinV labeling, Survivin by WB)(n=5 cell lines; p<0.05). These effects were associated with reduced expression of gH2AX. As assessed by echocardiography and right heart catheterization, treatment with AZD7648 improved pulmonary hemodynamics (RVSP, mPAP, and TPR) and right ventricular function (CO, PAT/PET, TAPSE) in Su/Hx mice with established PAH (n=10/group; all p<0.01). Accordingly, pulmonary vascular remodeling (EVG) and the proportion of PCNA-positive PASMCs were reduced in AZD7648-treated Su/Hx mice when compared to vehicle-treated Su/Hx mice (p<0.001). Conclusions: We showed for the first time that DNA-PK is overexpressed in human and experimental PAH and contributes to the pro-proliferative and anti-apoptotic phenotype of PAH-PASMCs.

Hyperactive Akt1 Signaling Increases Tumor Progression and DNA Repair in Embryonal Rhabdomyosarcoma RD Line and Confers Susceptibility to Glycolysis and Mevalonate Pathway Inhibitors.

In pediatric rhabdomyosarcoma (RMS), elevated Akt signaling is associated with increased malignancy. Here, we report that expression of a constitutively active, myristoylated form of Akt1 (myrAkt1) in human RMS RD cells led to hyperactivation of the mammalian target of rapamycin (mTOR)/70-kDa ribosomal protein S6 kinase (p70S6K) pathway, resulting in the loss of both MyoD and myogenic capacity, and an increase of Ki67 expression due to high cell mitosis. MyrAkt1 signaling increased migratory and invasive cell traits, as detected by wound healing, zymography, and xenograft zebrafish assays, and promoted repair of DNA damage after radiotherapy and doxorubicin treatments, as revealed by nuclear detection of phosphorylated H2A histone family member X (γH2AX) through activation of DNA-dependent protein kinase (DNA-PK). Treatment with synthetic inhibitors of phosphatidylinositol-3-kinase (PI3K) and Akt was sufficient to completely revert the aggressive cell phenotype, while the mTOR inhibitor rapamycin failed to block cell dissemination. Furthermore, we found that pronounced Akt1 signaling increased the susceptibility to cell apoptosis after treatments with 2-deoxy-D-glucose (2-DG) and lovastatin, enzymatic inhibitors of hexokinase, and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), especially in combination with radiotherapy and doxorubicin. In conclusion, these data suggest that restriction of glucose metabolism and the mevalonate pathway, in combination with standard therapy, may increase therapy success in RMS tumors characterized by a dysregulated Akt signaling.

Abstract 2596: Impact of novel Ku-DNA binding inhibitors on the DNA DSBs-induced DNA damage response

Abstract The DNA-dependent protein kinase (DNA-PK) plays a critical role in the non-homologous end joining (NHEJ) double-strand break (DSB) repair pathway and the DNA damage response (DDR). Consequently, blocking DNA-PK kinase activity is a novel anti-cancer therapeutic strategy in combination with ionizing radiation (IR). Towards developing a new class of DNA-PK inhibitors, our laboratory has exploited the mechanism of DNA-PK activation which requires binding to DNA termini via the Ku 70/80 heterodimer. We have previously reported the development of Ku-DNA binding inhibitors (Ku-DBi’s) that act via this novel mechanism of action to inhibit DNA-PK catalytic kinase activity. Ku-DBi’s display nanomolar activity in vitro, possess cellular DNA-PK and NHEJ inhibitory activity and sensitize non-small cell lung cancer (NSCLC) cells to DSB generating chemotherapeutics bleomycin and etoposide. In this study, we demonstrate that pre-incubation of our novel Ku-DBi’s can potentiate the cellular effects of bleomycin and IR and also induce p53 phosphorylation through the activation of the ATM pathway, which is concomitant with a decrease in DNA-PKcs autophosphorylation events at the S2056 (pS2056) cluster. Using a combination of Western blot and immunofluorescence assays in the NSCLC NCI-H460 and A549 cell lines, Ku-DBi’s treatment in combination with DNA DSBs-inducing agents such as bleomycin and IR, showed a significant reduction of autophosphorylation events of DNA-PKcs at the S2056 cluster compared to DNA DSBs-inducing agent alone. In addition, analysis of phospho-ATM and phospho-p53 protein levels in these NSCLC cell lines, suggested activation of the ATM pathway as a function of Ku-DBi’s treatment followed by bleomycin, evidenced by an increase of phosphorylation of ATM at Ser1981, and a modest increase of p53 phosphorylation at Ser15. Our findings demonstrate that Ku-DBi’s block DNA-DSB dependent DNA-PKcs autophosphorylation, resulting in potentiating cellular sensitivity to bleomycin and IR, and a likely effect on the ATM-dependent signaling pathway. These data are consistent with Ku-DBi’s possessing a novel mechanism of action that abrogates autophosphorylation of DNA-PKcs to impact DSB repair and potentially DDR signaling, becoming in a promising approach as part of an anticancer therapeutic strategy in combination with DNA DSBs-inducing agents. Citation Format: Pamela L. Mendoza-Munoz, Navnath S. Gavande, Pamela S. VanderVere-Carozza, Katherine S. Pawelczak, Joseph R. Dynlacht, Joy E. Garrett, John J. Turchi. Impact of novel Ku-DNA binding inhibitors on the DNA DSBs-induced DNA damage response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2596.

Inhibition of Ciliogenesis Enhances the Cellular Sensitivity to Temozolomide and Ionizing Radiation in Human Glioblastoma Cells

ObjectiveTo investigate the function of primary cilia in regulating the cellular response to temozolomide (TMZ) and ionizing radiation (IR) in glioblastoma (GBM). MethodsGBM cells were treated with TMZ or X-ray/carbon ion. The primary cilia were examined by immunostaining with Arl13b and γ-tubulin, and the cellular resistance ability was measured by cell viability assay or survival fraction assay. Combining with cilia ablation by IFT88 depletion or chloral hydrate and induction by lithium chloride, the autophagy was measured by acridine orange staining assay. The DNA damage repair ability was estimated by the kinetic curve of γH2AX foci, and the DNA-dependent protein kinase (DNA-PK) activation was detected by immunostaining assay. ResultsPrimary cilia were frequently preserved in GBM, and the induction of ciliogenesis decreased cell proliferation. TMZ and IR promoted ciliogenesis in dose- and time-dependent manners, and the suppression of ciliogenesis significantly enhanced the cellular sensitivity to TMZ and IR. The inhibition of ciliogenesis elevated the lethal effects of TMZ and IR via the impairment of autophagy and DNA damage repair. The interference of ciliogenesis reduced DNA-PK activation, and the knockdown of DNA-PK led to cilium formation and elongation. ConclusionPrimary cilia play a vital role in regulating the cellular sensitivity to TMZ and IR in GBM cells through mediating autophagy and DNA damage repair.

Inhibitory effect of tanshinone IIA, resveratrol and silibinin on enterovirus 68 production through inhibiting ATM and DNA-PK pathway

BackgroundHuman enterovirus 68 (EV68) is a primary etiological agent for respiratory illnesses, while no effective drug has yet used in clinics largely because the pathogenesis of EV68 is not clear. DNA damage response (DDR) responds to cellular DNA breaks and is also involved in viral replication. Three DDR pathways includes ataxia telangiectasia mutated (ATM), ATM and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PK). Natural products proved to be an excellent source for the discovery and isolation of novel antivirals. Among them, tanshinone IIA, resveratrol, silibinin, rutin and quercetin are reported to target DDR, therefore their roles in anti-EV68 are investigated in this study. PurposeThis study investigated the anti-EV68 ability of various natural compounds related to DDR. Study design and methodsThe methods include cell counting, flow cytometry, western blot, Immunofluorescence staining, comet assays, quantitative real-time RT PCR and short interfering RNAs (siRNAs) for analysis of cell number, cell cycle, protein expression, protein location, DNA damage, mRNA level and knock down target gene, respectively. ResultsEV68 infection induced DDR. Down-regulation or inhibition of ATM or DNA-PK lowered DDR in EV68-infected cells and mitigated viral protein expression, however, down-regulation or inhibition of ATR unexpectedly up-regulated DDR, and promoted viral protein expression. Meanwhile tanshinone IIA, resveratrol, and silibinin inhibited ATM and/or DNA-PK activation and decreased viral proliferation, while rutin and quercetin inhibited ATR activation and promoted viral production. The role of them in ATM, DNA-PK and ATR activation was consistent with previous reports. ConclusionTanshinone IIA, resveratrol and silibinin inhibited EV68 proliferation through inhibiting ATM and/or DNA-PK activation, and they were effective anti-EV68 candidates.

The DNA-PK Inhibitor AZD7648 Sensitizes Patient-Derived Ovarian Cancer Xenografts to Pegylated Liposomal Doxorubicin and Olaparib Preventing Abdominal Metastases.

Ovarian cancer is the deadliest gynecologic cancer, with a 5-year survival rate of 30%, when the disease has spread throughout the peritoneal cavity. We investigated the efficacy to delay disease progression by the DNA-dependent protein kinase (DNA-PK) inhibitor AZD7648, administered in combination with two of the therapeutic options for patient management: either pegylated liposomal doxorubicin (PLD) or the PARP inhibitor olaparib. Patient-derived ovarian cancer xenografts (OC-PDX) were transplanted subcutaneously to evaluate the effect of treatment on tumor growth, or orthotopically in the peritoneal cavity to evaluate the effect on metastatic spread. AZD7648 was administered orally in combination with PLD (dosed intravenously) or with olaparib (orally). To prove the inhibition of DNA-PK in the tumors, we measured pDNA-PKcs, pRPA32, and γH2AX, biomarkers of DNA-PK activity. AZD7648 enhanced the therapeutic efficacy of PLD in all the OC-PDXs tested, regardless of their BRCA status or sensitivity to cisplatin or PLD. The treatment caused disease stabilization, which persisted despite therapy discontinuation for tumors growing subcutaneously, and significantly impaired the abdominal metastatic dissemination, prolonging the lifespan of mice implanted orthotopically. AZD7648 potentiated the efficacy of olaparib in BRCA-deficient OC-PDXs but did not sensitize BRCA-proficient OC-PDXs to olaparib, despite an equivalent inhibition of DNA-PK, suggesting the need of a preexisting olaparib activity to benefit from the addition of AZD7648. This work suggests that AZD7648, an inhibitor of DNA-PK, dosed in combination with PLD or olaparib is an exciting therapeutic option that could benefit patients with ovarian cancer and should be explored in clinical trials.

Autophosphorylation transforms DNA-PK from protecting to processing DNA ends

The DNA-dependent protein kinase (DNA-PK) initially protects broken DNA ends but then promotes their processing during non-homologous end joining (NHEJ). Before ligation by NHEJ, DNA hairpin ends generated during V(D)J recombination must be opened by the Artemis nuclease, together with autophosphorylated DNA-PK. Structures of DNA-PK bound to DNA before and after phosphorylation, and in complex with Artemis and a DNA hairpin, reveal an essential functional switch. When bound to open DNA ends in its protection mode, DNA-PK is inhibited for cis-autophosphorylation of the so-called ABCDE cluster but activated for phosphorylation of other targets. In contrast, DNA hairpin ends promote cis-autophosphorylation. Phosphorylation of four Thr residues in ABCDE leads to gross structural rearrangement of DNA-PK, widening the DNA binding groove for Artemis recruitment and hairpin cleavage. Meanwhile, Artemis locks DNA-PK into the kinase-inactive state. Kinase activity and autophosphorylation of DNA-PK are regulated by different DNA ends, feeding forward to coordinate NHEJ events.

Golgi Phosphoprotein 3 Confers Radioresistance via Stabilizing EGFR in Lung Adenocarcinoma

Radioresistance is a major cause of treatment failure in tumor radiation therapy, and the underlying mechanisms of radioresistance are still elusive. Golgi phosphoprotein 3 (GOLPH3) has been reported to associate tightly with cancer progression and chemoresistance. Herein, we explored whether GOLPH3 mediated radioresistance of lung adenocarcinoma (LUAD) and whether targeted suppression of GOLPH3 sensitized LUAD to radiation therapy. The aberrant expression of GOLPH3 was evaluated by immunohistochemistry in LUAD clinical samples. To evaluate the association between GOLPH3 and radioresistance, colony formation and apoptosis were assessed in control and GOLPH3 knockdown cells. γ-H2AX foci and level determination and micronucleus test were used to analyze DNA damage production and repair. The rescue of GOLPH3 knockdown was then performed by exogenous expression of small interfering RNA-resistant mutant GOLPH3 to confirm the role of GOLPH3 in DNA damage repair. Mechanistically, the effect of GOLPH3 on regulating stability and nuclear accumulation of epidermal growth factor receptor (EGFR) and the activation of DNA-dependent protein kinase (DNA-PK) were investigated by quantitative real-time polymerase chain reaction, western blot, immunofluorescence, and coimmunoprecipitation. The role of GOLPH3 in vivo in radioresistance was determined in a xenograft model. In tumor tissues of 33 patients with LUAD, the expression of GOLPH3 showed significant increases compared with those in matched normal tissues. Knocking down GOLPH3 reduced the clonogenic capacity, impaired double-strand break (DSB) repair, and enhanced apoptosis after irradiation. In contrast, reversal of GOLPH3 depletion rescued the impaired repair of radiation-induced DSBs. Mechanistically, loss of GOLPH3 accelerated the degradation of EGFR in lysosome, causing the reduction in EGFR levels, thereby weakening nuclear accumulation of EGFR and attenuating the activation of DNA-PK. Furthermore, adenovirus-mediated GOLPH3 knockdown could enhance the ionizing radiation response in the LUAD xenograft model. GOLPH3 conferred resistance of LUAD to ionizing radiation via stabilizing EGFR, and targeted suppression of GOLPH3 might be considered as a potential therapeutic strategy for sensitizing LUAD to radiation therapy.

Abstract PO-023: Impact of a novel Ku-DNA binding inhibitor on the IR-induced DNA damage response

Abstract The DNA-dependent protein kinase (DNA-PK) plays a critical role in the non-homologous end joining (NHEJ) double stand break (DSB) repair pathway and the DNA damage response (DDR). Consequently, blocking DNA-PK kinase activity is a novel anti-cancer therapeutic strategy in combination with ionizing radiation (IR). Towards developing a series of a new class of DNA-PK inhibitors, our laboratory has exploited the mechanism of DNA-PK activation which requires binding to DNA termini via the Ku 70/80 heterodimer. We recently reported the development of Ku-DNA binding inhibitors (Ku-DBi’s) that act via this novel mechanism of action to inhibit DNA-PK catalytic kinase activity. Ku-DBi’s display nanomolar activity in vitro, possess cellular DNA-PK and NHEJ inhibitory activity and sensitize non-small cell lung cancer (NSCLC) cells to chemotherapeutics bleomycin and etoposide. In this study, we demonstrate that pre-incubation of our novel Ku-DBi’s can potentiate the cellular effects of IR. We demonstrate that the increase in IR sensitivity is preceded by a decrease in DNA-PKcs autophosphorylation events at the S2056 (pS2056) cluster. Using a combination of Western blot and immunofluorescence assays in the NSCLC NCI-H460 cell line, Ku-DBi 245 treatment in combination with IR showed a significant reduction of phosphorylation of DNA-PKcs at S2056 compared to IR alone. Analysis of gH2A.X also showed an inhibition of the DDR as a function of 245 treatment, evidenced by reduction of S139 phosphorylation. These results demonstrate that Ku-DBi 245 blocks DNA-break dependent DNA-PKcs autophosphorylation, resulting in an increased in radiation sensitivity. These data are consistent with Ku-DBi’s possessing a novel mechanism of action that abrogates autophosphorylation of DNA-PKcs and DDR signaling as part of an anticancer therapeutic strategy in combination with IR. Citation Format: Pamela Mendoza-Munoz, Navnath S. Gavande, Pamela S. VanderVere-Carozza, Katherine S. Pawelczak, Joseph R. Dynlacht, Joy Garrett, John J. Turchi. Impact of a novel Ku-DNA binding inhibitor on the IR-induced DNA damage response [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-023.

Abstract PO-018: Hypoxia activated prodrugs of DNA-dependent protein kinase inhibitors as radiosensitisers of head and neck squamous cell carcinomas

Abstract Inhibition of the repair of radiation-induced DNA double strand breaks offers the potential to sensitize tumors to radiation therapy. DNA-dependent protein kinase (DNA-PK) is a well validated target for radiosensitisation, but inhibition also has the potential to radiosensitize normal tissues within the radiation field, and to target normal tissue functions outside of DNA-PK’s canonical role in DNA repair. Exploiting hypoxia to selectively activate hypoxia-activated prodrugs of DNA-PK inhibitors (DNA-PKi) in tumors may enhance tumor selectivity, with the advantage of sensitizing the most radioresistant (hypoxic) subpopulation, as well as sensitizing adjacent oxic cells through a local bystander effect. We recently conducted a drug discovery campaign, scaffold-hopping from the pan-PIKK inhibitor dactolisib to identify new DNA-PKi. We screened compounds against three signal targets DNA-PK, PI3Kα and mTOR, and determined in vitro sensitization of UT-SCC-54C head and neck squamous cell carcinoma (HNSCC) cells using a growth inhibition endpoint. We identified a highly selective lead DNA-PKi, SN39536, and a corresponding hypoxia-activated prodrug, SN39884. SN39536 is an effective inhibitor of DNA-PK activity, both biochemically and in cells, with substantial selectivity compared to phosphoinositide-3-kinase (PI3K) enzymes and other PI3K-related kinase enzymes. SN39536 provided radiosensitization of DNA-PK proficient HAP1 cells, but was inactive against isogenic cells with knockout of the DNA-PK catalytic subunit (PRKDC), demonstrating on-mechanism activity. SN39536 also provided robust radiosensitization of oxic UT-SCC-54C HNSCC cells using a clonogenic endpoint. Prodrug SN39884 was deactivated as a DNA-PKi and released SN39536 under reductive conditions. Incubation of the prodrug with HCT116 cells overexpressing cytochrome P450 oxidoreductase or UT-SCC-54C cells led to hypoxia-dependent metabolism of the prodrug and efficient release of SN39536. SN39536 effectively sensitized UT-SCC-54C cells to radiation under either aerobic or anoxic conditions, while prodrug SN39884 only sensitized cells under anoxia. SN39536 and its prodrug also demonstrated radiosensitization of hypoxic cells in UT-SCC-54C tumor xenografts using an ex vivo clonogenic survival endpoint. We have identified a new class of potent and selective DNA-PK inhibitors and demonstrated the ability of a hypoxia-activated prodrug to selectively release the inhibitor under hypoxia in vitro and in vivo. Citation Format: Michael P. Hay, Way W. Wong, Benjamin D. Dickson, Gary J. Cheng, Liew P. Liew, Cho R. Hong, William R. Wilson, Stephen M. Jamieson. Hypoxia activated prodrugs of DNA-dependent protein kinase inhibitors as radiosensitisers of head and neck squamous cell carcinomas [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-018.

Phosphoproteomics Uncovers Synergy between DNA-PK and FLT3 Inhibitors in Acute Myeloid Leukaemia

*These authors contributed equally to this work Background:Acute Myeloid Leukaemia (AML) is the most common and aggressive form of acute leukaemia, with a 5-year survival rate of just 24%. Activating mutations in the receptor tyrosine kinase FLT3 are the most common driver mutations in AML (25-30% of patients). Inhibiting the FLT3 receptor as a mono-therapeutic strategy in AML has proven difficult however, due to the development of treatment resistance and relapse. In order to identify improved therapeutic targets, the oncogenic signalling pathways downstream of mutant FLT3 require characterisation. Methods:Quantitative, label-based phosphoproteomics was performed on primary blasts from 7 AML patients (4 mutant-FLT3, 3 wildtype-FLT3). Differentially phosphorylated pathways were identified using Ingenuity Pathway Analysis, and kinase activation was assessed by kinase substrate enrichment analysis. Validation of results was performed using targeted mass spectrometry. Proliferation, apoptosis, and cell cycle assays were used to assess drug toxicity; drug synergy was evaluated using Chou-Talalay and Webb analyses. Results:Analysis of differentially expressed phosphoproteins in mutant-FLT3 compared to wildtype-FLT3 AML patient blasts revealed dysregulation of DNA repair pathways. Specifically, mutant-FLT3 samples displayed increased phosphorylation of proteins within the error-prone Non-Homologous End Joining (NHEJ) repair pathway, indicating NHEJ pathway activation. Kinase enrichment analysis predicted increased activity of the NHEJ core kinase, DNA-dependent protein kinase (DNA-PK), in mutant-FLT3 samples. Accordingly, proliferation assays revealed that mutant-FLT3 cell lines were sensitive to inhibition of DNA-PK. FLT3-inhibitor treatment reduced DNA-PK phosphorylation in mutant-FLT3 cells, suggesting that activation of DNA-PK is downstream of FLT3 activation. Inhibition of DNA-PK kinase activity combined with FLT3 inhibitors led to synergistic induction of cell death, selectively in mutant-FLT3 cell lines. DNA-PK inhibitors combined with FLT3 inhibitors also co-operatively induced cell death in mutant-FLT3 primary AML patient samplesex vivo, and significantly prolonged survival compared to either monotherapy in a human AML xenograft mouse model. Conclusions:Mutant-FLT3 AML is associated with activation of the error-prone NHEJ repair pathway, which may contribute to genomic instability. Targeting the NHEJ kinase, DNA-PK, in combination with FLT3 inhibitors has the potential to improve outcomes for this poor-prognosis AML subtype. Disclosures Enjeti: Bayer:Speakers Bureau;AbbVie:Membership on an entity's Board of Directors or advisory committees;Alexion:Speakers Bureau;Novartis:Membership on an entity's Board of Directors or advisory committees;Astellas:Membership on an entity's Board of Directors or advisory committees;Sanofi:Speakers Bureau.