DNA-PK Activity Research Articles

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.

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.

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.

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.

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

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 < .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, KU-57788, in mice with a recurrent PDX line resulted in prolonged survival (P < .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.

A Novel PI3K γ/δ and DNA-PK Triple Inhibitor, BR101801, for r/r PTCL: A Phase 1a/b, Multi-Center, Open-Label Clinical Trial

Background BR101801, a triple inhibitor of PI3Kγ/δ and DNA-PK, inhibits not only the signal affecting cell growth caused by PI3Kγ/δ but also efficiently induces cell cycle arrest and apoptosis through inhibition of DNA-PK activation, with this triple target inhibition it decreases the stability of oncogenic protein, c-Myc (AACR2020 abstract #655). BR101801 is currently being evaluated in a phase 1a/b study in advanced hematologic malignancies. In phase 1a study, BR101801 exhibited evidence of clinical activity and was established at 200mg orally once daily 28-day cycle (RP2D) in patients with r/r PTCL (ASCO2023 abstract #7551). Methods This is a phase 1, open-label, multi-center, dose escalation (1a), and expansion (1b) study of BR101801 in adult patients with advanced hematologic malignancies (NCT04018248). In the phase 1a study, Dose was escalated at 50, 100, 200 and 325 mg QD. PTCL-NOS, AITL, FTCL and Nodal PTCL with TFH phenotype were determined as target indications for phase 1b with the RP2D. Results As of Jul 19, 2023, a total of 26 patients were enrolled in dose-escalation phase (n=12) and dose-expansion phase (n=14). The median age was 66 years (range of 30-83). Histological subtypes include PTCL-NOS (n=11, 42%), AITL (n=11, 42%), DLBCL (n=2, 8%), MZL (n=1, 4%) and MF (n=1, 4%). The majority of patients had stage III or IV (92%). In the phase 1a study, Dose limiting toxicity (DLT) occurred at 325 mg; thus, 200 mg QD was determined as the maximum tolerated dose (MTD) and RP2D. Of 19 evaluable patients with r/r PTCL (PTCL-NOS and AITL) in the phase 1 study. Patients' stage was III or IV (100%) with 47.4 % of patients having received three or more prior systemic therapies. The ORR was 31.6% (95% CI, 12.6-56.6) with 4 CRs (21.1%) and 2 PRs (10.5%), CBR was 47.4% (95% CI, 24.5-71.1). In median follow-up duration of 12.9 months, the median PFS was 7.5 months (95% CI, 1.7-NC), median OS and DoR (range of 1.9+-31.8+) has not reached. Currently, 5 patients still remain on treatment. Safety analysis was conducted for the 26 patients; 23 patients (88.5%) experienced adverse events (AEs). The most common (≥ 20%) AEs were Rash (23.1%), AST increased (23.1%), ALT increased (23.1%) and Cough (23.1%). Grade 3/4 ADRs occurred in 12 patients (46.2%) and those were (≥ 10%, frequency) AST increased (19.2%), ALT increased (15.4%), Neutropenia (15.4%). There were no treatment related mortality observed. Conclusion BR101801, the triple inhibitor of PI3K γ/δ and DNA-PK, demonstrated as a promising therapeutic option for r/r PTCLs patients. Preliminary results showed the ORR of 31.6% and a CR rate of 21.1%. Phase 2 study is warranted further investigate the safety and efficacy of BR101801 in r/r PTCL and Nodal TFH cell lymphoma at 200 mg QD orally.

A phase 1a/b study of BR101801, a PI3Kγδ and DNA PK triple inhibitor, in adult patients with advanced hematologic malignancies.

7551 Background: BR101801, a triple inhibitor of PI3Kγ/δ and DNA PK, inhibits not only the signal affecting cell growth caused by PI3Kγ/δ but also efficiently induces cell cycle arrest and apoptosis through inhibition of DNA-PK activation, and finally decreases the stability of oncogenic protein, c-Myc(AACR2020 abstract #655). We initiated a first-in-human study to investigate BR101801 across advanced hematologic malignancies that had relapsed or was refractory to the standard therapies. Methods: This is a phase 1, open-label, multi-center, dose escalation (1a), and expansion (1b) study of BR101801 in adult patients with advanced hematologic malignancies including relapsed/refractory(R/R) B-cell lymphoma, CLL/SLL, and PTCL (NCT04018248). Dose was escalated at 50, 100, 200 and 325 mg QD. PTCL-NOS, AITL, FTCL and Nodal PTCL with TFH phenotype were included for dose expansion study at 200mg as RP2D. Results: 12 patients with advanced hematologic malignancies were enrolled in phase 1a study and treated with 50-325mg of BR101801. The median age was 63 years (range 30-76 years). Histological subtypes include AITL (n = 5, 41.7%), PTCL-NOS (n = 3, 25.0%), DLBCL (n = 2, 16.7%), MZL (n = 1, 8.3%) and MF (n = 1, 8.3%). Of 7 evaluable patients with PTCL (PTCL-NOS and AITL), ORR was 42.9% (95% CI, 9.9-81.6) and CBR was 85.7% (95% CI, 42.1-99.6). In median follow-up duration of 22.5 months, mPFS was 22.2 months (95% CI, 3.6-22.2) and mOS has not yet reached. Duration of response of the 3 responders were 9.2, 14.1+ and 20.7+ months, respectively. Safety was analyzed in 12 patients. 10 patients (83.3%) experienced ≥1 ADRs. The most common (≥ 10%) ADRs were Rash (33.3%), ALT increased (33.3%), AST increased (25.0%), and Diarrhea (16.7%). Grade 3 ADRs occurred in 6 patients (50.0%) and those were ALT increased (25.0%), AST increased (16.7%), Rash (16.7%), erythema multiforme (8.3%), and neutropenia (8.3%). All ADRs were manageable and has recovered. There were no Grade 4 or 5 ADRs and no treatment related death observed. DLTs were occurred at 325mg; thus, 200mg QD was determined as the MTD and RP2D. The pharmacokinetics data showed an ideal profile in general. The PK results did not remarkably deviate from the non-clinical studies, and a more thorough interpretation will be performed in phase Ib. In phase 1b study, 7 PTCL patients were enrolled and it is ongoing till enrollment of total 12 patients. Conclusions: The results for the phase 1a study evaluated the safety, efficacy results and pharmacokinetic characteristics, 200 mg QD was shown to provide target exposure for clinical efficacy with the tolerable and safe profile. BR101801 showed of antitumor activity, especially in R/R PTCL patients. Hence, the phase 1b study of BR101801 is warranted in R/R PTCL patients and we expect BR101801 would become a promising therapeutic option for these patients. Clinical trial information: NCT04018248 .

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.

Integrative multi-omics networks identify PKCδ and DNA-PK as master kinases of glioblastoma subtypes and guide targeted cancer therapy

Despite producing a panoply of potential cancer-specific targets, the proteogenomic characterization of human tumors has yet to demonstrate value for precision cancer medicine. Integrative multi-omics using a machine-learning network identified master kinases responsible for effecting phenotypic hallmarks of functional glioblastoma subtypes. In subtype-matched patient-derived models, we validated PKCδ and DNA-PK as master kinases of glycolytic/plurimetabolic and proliferative/progenitor subtypes, respectively, and qualified the kinases as potent and actionable glioblastoma subtype-specific therapeutic targets. Glioblastoma subtypes were associated with clinical and radiomics features, orthogonally validated by proteomics, phospho-proteomics, metabolomics, lipidomics and acetylomics analyses, and recapitulated in pediatric glioma, breast and lung squamous cell carcinoma, including subtype specificity of PKCδ and DNA-PK activity. We developed a probabilistic classification tool that performs optimally with RNA from frozen and paraffin-embedded tissues, which can be used to evaluate the association of therapeutic response with glioblastoma subtypes and to inform patient selection in prospective clinical trials.

DNA damage stress-induced translocation of mutant FUS proteins into cytosolic granules and screening for translocation inhibitors.

Fused in sarcoma/translated in liposarcoma (FUS) is an RNA-binding protein, and its mutations are associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), through the DNA damage stress response, aberrant stress granule (SG) formation, etc. We previously reported that translocation of endogenous FUS into SGs was achieved by cotreatment with a DNA double-strand break inducer and an inhibitor of DNA-PK activity. In the present study, we investigated cytoplasmic SG formation using various fluorescent protein-tagged mutant FUS proteins in a human astrocytoma cell (U251) model. While the synergistic enhancement of the migration of fluorescent protein-tagged wild-type FUS to cytoplasmic SGs upon DNA damage induction was observed when DNA-PK activity was suppressed, the fluorescent protein-tagged FUSP525L mutant showed cytoplasmic localization. It migrated to cytoplasmic SGs upon DNA damage induction alone, and DNA-PK inhibition also showed a synergistic effect. Furthermore, analysis of 12 sites of DNA-PK-regulated phosphorylation in the N-terminal LC region of FUS revealed that hyperphosphorylation of FUS mitigated the mislocalization of FUS into cytoplasmic SGs. By using this cell model, we performed screening of a compound library to identify compounds that inhibit the migration of FUS to cytoplasmic SGs but do not affect the localization of the SG marker molecule G3BP1 to cytoplasmic SGs. Finally, we successfully identified 23 compounds that inhibit FUS-containing SG formation without changing normal SG formation. Highlights Characterization of DNA-PK-dependent FUS stress granule localization.A compound library was screened to identify compounds that inhibit the formation of FUS-containing stress granules.

EXTH-21. DEVELOPMENT OF THERAPEUTIC STRATEGIES BY PATHWAY-BASED MULTI-OMICS APPROACH AND MASTER KINASE ANALYSIS IN GLIOBLASTOMA MULTIFORME

Abstract Current transcriptomic classification of Glioblastoma Multiforme (GBM) has been ineffective to predict survival and therapeutic vulnerabilities. Recently, we proposed a four-group functional classification of GBM that included proliferative/progenitor, neuronal, mitochondrial and glycolytic/plurimetabolic subtypes with prognostic and therapeutic implications as the mitochondrial subtype carries the best survival and exhibits distinct sensitivity to mitochondrial OXPHOS inhibitors. To uncover novel therapeutic targets for each functional GBM subtype, we focused on protein kinases for their attractive features as both drivers and drug targets, with current availability of 62 FDA-approved inhibitors available for cancer precision therapeutics. We designed an unbiased integrative, machine learning-based proteomics/phosphoproteomics network for the identification of Master Kinases (MKs) responsible for effecting key phenotypic hallmarks of each of the four GBM subtypes. Here we report the identification and validation of protein kinase C delta (PRKCd) and DNA-PKcs as MKs that sustain the glycolytic/plurimetabolic and proliferative/progenitor GBM subtypes, respectively. Genetic and pharmacologic inactivation of PKCd in GBM patient-derived organoids of the glycolytic/plurimetabolic subtype blocked glucose uptake and lipid accumulation, resulting in marked anti-tumor effects. We also validated the role of PKCd in oncometabolic processes at the intersection between insulin, IGF, and lipid metabolism. DNA-PKcs was qualified as MK for the proliferative/progenitor GBM subtype, which is characterized by high basal level of replication stress. Biochemical experiments showed activation of DNA-PK in GBM patient-derived organoids of the proliferative/progenitor subgroup. Targeting DNA-PK in proliferative/progenitor GBM organoids with the clinically tested DNA-PKcs inhibitor nedisertib in association with gamma irradiation, the essential component of the standard of care for patients with GBM, led to an unsustainable level of DNA damage and massive GBM cell death selectively in this GBM subtype. As DNA-PKcs inhibitors have been introduced into clinical trials, our findings suggest that pre-selection of patient with PPR tumors is likely to enhance therapeutic success.

Inhibition of DNA Repair by Inappropriate Activation of ATM, PARP, and DNA-PK with the Drug Agonist AsiDNA.

AsiDNA is a DNA repair inhibitor mimicking DNA double-strand breaks (DSB) that was designed to disorganize DSB repair pathways to sensitize tumors to DNA damaging therapies such as radiotherapy and chemotherapy. We used the property of AsiDNA of triggering artificial DNA damage signaling to examine the activation of DSB repair pathways and to study the main steps of inhibition of DNA repair foci after irradiation. We show that, upon AsiDNA cellular uptake, cytoplasmic ATM and PARP are rapidly activated (within one hour) even in the absence of irradiation. ATM activation by AsiDNA leads to its transient autophosphorylation and sequestration in the cytoplasm, preventing the formation of ATM nuclear foci on irradiation-induced damage. In contrast, the activation of PARP did not seem to alter its ability to form DNA repair foci, but prevented 53BP1 and XRCC4 recruitment at the damage sites. In the nucleus, AsiDNA is essentially associated with DNA-PK, which triggers its activation leading to phosphorylation of H2AX all over chromatin. This pan-nuclear phosphorylation of H2AX correlates with the massive inhibition, at damage sites induced by irradiation, of the recruitment of repair enzymes involved in DSB repair by homologous recombination and nonhomologous end joining. These results highlight the interest in a new generation of DNA repair inhibitors targeting DNA damage signaling.

Abstract 2600: AsiDNA® treatment protects healthy cells from anticancer treatment toxicity

Abstract Purpose: Conventional cytotoxic therapies are still the standard of care for the treatment of many cancers. However, the associated severe side effects, especially damage to normal proliferating cells like stem and progenitor cells, often lead to drug dose reduction, which limits treatment success. Indeed, chemotherapy-induced myelosuppression is manifested by neutropenia, lymphopenia, anemia, and thrombocytopenia. Although growth factors ameliorate this myelosuppression, their efficacy is still suboptimal and lineage specific. Pre-clinical and clinical studies have shown that AsiDNA, a double-stranded (DS) DNA molecule that mimics DS DNA breaks to interfere with DNA repair by over-activating a false DNA damage signaling through DNA-PK and PARP enzymes (decoy agonist), is extremely well tolerated in standalone in mammals. These observations led us to assess the potential of AsiDNA to protect healthy cells from toxicities of several anti-cancer treatments. Experimental design: In vivo, we analyzed the safety profile of AsiDNA during a recent clinical trial (DRIIV-1b/NCT03579628) in combination with platinum-based chemotherapy. In vitro, we used isolated blood cells from healthy donors, and epithelial and fibroblast cells as models to study if AsiDNA could protect healthy cells to chemo- and radiotherapy-induced toxicity. We monitored cell survival, DNA damage (comet assays) and repair (53BP1 and Rad51 foci), DNA-PK (HSP90 and H2AX phosphorylation) and PARP (PARylation) activation, cell cycle modulation and p53 dependency to identify mechanisms underlying the effects of AsiDNA on healthy cells. Results: In vivo, long-term treatment of several patients with carboplatine+/-paclitaxel + AsiDNA showed no increase of chemotherapies toxicities allowing longer periods of disease control and suggesting a protective effect of AsiDNA. In the in vitro models, we showed that AsiDNA enters non dividing and dividing healthy cells as revealed by intracellular PARylation but induces its nuclear target engagement (H2AX and HSP90 phosphorylation) only in dividing cells. Association of AsiDNA to antitumor treatments increased survival of healthy proliferative cells. Interestingly, AsiDNA displayed two distinct mechanisms of protection depending on the origin of the cells: p53-dependent G1/S cell cycle arrest in fibroblasts and epithelial cells, and DNA repair “doping” in hematological cells revealed by higher recruitment of Rad51 and 53BP1 at damage sites in those cells. Enzymatic inhibition and gene editing revealed that hyperactivation of DNA-PK/p53 pathway by AsiDNA might be required for healthy cells preservation. Conclusion: These findings suggest that the combination of AsiDNA with anticancer treatments should provide a means to attenuate therapy-induced toxicity, while showing the well-documented synergy in tumor cells, thus providing an opportunity to increase the therapeutic window. Citation Format: Wael Jdey, Anouk Sesink, Agathe Cohendet, Juliette Rieu, Chloe Doizelet, Vincent Hayes, Pierre-Marie Girard, Marie Dutreix, Judith Greciet. AsiDNA® treatment protects healthy cells from anticancer treatment toxicity [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 2600.

Abstract 2594: Discovery and characterization of ZL-2201, a potent, highly-selective, and orally bioavailable small-molecule DNA-PK inhibitor

Abstract DNA-damaging poisons and replicative stress commonly result in increased double-strand breaks (DSBs) in tumor cells. Non-homologous end joining (NHEJ), driven by the enzymatic function of DNA-PK, is one of the critical pathways employed to repair such DSBs. Inhibition of DNA-PK kinase activity impairs NHEJ, potentiates the activity of DNA-damaging agents, and inhibits survival of tumor cells incapable of repairing DSBs via other means, including homologous recombination (HR). We have developed an orally bioavailable, highly potent, and selective pharmacological inhibitor of DNA-PK activity, ZL-2201, for the treatment of cancer. ZL-2201 demonstrates enzymatic inhibition of DNA-PK in the single-digit nM range, with nearly 500-fold or greater selectivity against other phosphatidylinositol 3-kinase-related kinases (PIKKs). In addition, ZL-2201 showed high selectivity for DNA-PK kinase activity in a kinase panel screening study and a clean off-target profile with no appreciable inhibition against a panel of diverse, safety-relevant targets. We demonstrate that ZL-2201 effectively inhibits DNA-PK autophosphorylation and its downstream target phospho-RPA in multiple cell lines in a concentration- and time-dependent manner. DNA-PK has been shown previously to have synthetically lethal interactions with ATM. Therefore, we profiled the impact of ATM expression on the antiproliferative effects of ZL-2201. Cancer cell lines possessing defects in ATM displayed greater sensitivity to ZL-2201 compared to ATM-WT cancer cell lines. These observations were recapitulated in two ATM knockout isogenic cell lines. Moreover, ZL-2201 displayed minimal effects in a DNA-PK deficient model or in normal human and canine colon and kidney cell lines. Independent of ATM status, ZL-2201 showed strong synergy in vitro with topoisomerase II inhibitors. Following oral administration of ZL-2201 in vivo, ZL-2201 demonstrated dose-dependent and exposure-dependent antitumor activity against multiple models, with the greatest activity against ATM deficient human cancer xenografts. Antitumor activity correlated with duration of ZL-2201 exposure and level of target engagement. In addition, ZL-2201 significantly enhanced the activity of DNA-damaging agents in vivo, even in human cancer xenograft models with limited monotherapy antitumor activity for ZL-2201. The projected human PK properties, based on the ADME properties across species and PK properties in preclinical species of ZL-2201, suggest that ZL-2201 is suitable for oral administration. In conclusion, ZL-2201 demonstrates potent and selective inhibition of DNA-PK enzymatic activity, strong synergy with DNA damaging agents in vitro and in vivo, antitumor activity against multiple human xenograft models, and a favorable projected human PK profile. ZL-2201 is expected to enter clinical development in 2022. Citation Format: Shruti Lal, Bee-Chun Sun, Yuping Chen, Tom Huang, Neil Bhola, Vivian Morton, Kevin Chen, Shanghua Xia, Haoyu Zhang, Qiuping Ye, Petter Veiby, David I. Bellovin, Yuhua Ji. Discovery and characterization of ZL-2201, a potent, highly-selective, and orally bioavailable small-molecule DNA-PK inhibitor [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 2594.

DNA repair protein DNA-PK protects PC12 cells from oxidative stress-induced apoptosis involving AKT phosphorylation

BackgroundEmerging evidence suggest that DNA-PK complex plays a role in the cellular response to oxidative stress, in addition to its function of double strand break (DSB) repair. In this study we evaluated whether DNA-PK participates in oxidative stress response and whether this role is independent of its function in DNA repair.Methods and resultsWe used a model of H2O2-induced DNA damage in PC12 cells (rat pheochromocytoma), a well-known neuronal tumor cell line. We found that H2O2 treatment of PC12 cells induces an increase in DNA-PK protein complex levels, along with an elevation of DNA damage, measured both by the formation of γΗ2ΑX foci, detected by immunofluorescence, and γH2AX levels detected by western blot analysis. After 24 h of cell recovery, γΗ2ΑX foci are repaired both in the absence and presence of DNA-PK kinase inhibitor NU7026, while an increase of apoptotic cells is observed when DNA-PK activity is inhibited, as revealed by counting pycnotic nuclei and confirmed by FACS analysis. Our results suggest a role of DNA-PK as an anti-apoptotic factor in proliferating PC12 cells under oxidative stress conditions. The anti-apoptotic role of DNA-PK is associated with AKT phosphorylation in Ser473. On the contrary, in differentiated PC12 cells, were the main pathway to repair DSBs is DNA-PK-mediated, the inhibition of DNA-PK activity causes an accumulation of DNA damage.ConclusionsTaken together, our results show that DNA-PK can protect cells from oxidative stress induced-apoptosis independently from its function of DSB repair enzyme.Graphical

A Phase 1 Dose Escalation Study of Dual PI3K and DNA PK Inhibitor, BR101801 in Adult Patients with Advanced Hematologic Malignancies

Background: BR101801 not only blocks the signaling responsible for cell growth caused by PI3K, but also efficiently induces cell cycle arrest and apoptosis through inhibition of DNA-PK activation and stimulates decreasing stability of the oncogenic protein, c-Myc(AACR2020 abstract #655). This phase I study evaluated safety, tolerability, pharmacokinetics and preliminary activity of the BR101801 (PI3Kγ/δ and DNA PK inhibitor) in patients with advanced hematologic malignancies.Method: This is a Phase I, multi-center, open-label, first-in-human study. The Phase Ia (dose escalation) part of the study was designed to determine the maximum tolerated dose (MTD)/recommended dose for expansion (RDE) of BR101801. BR101801 was administered orally once daily in 28-day cycles. The dose escalation part was initiated with a dose titration in the initial cohort, followed by a 3 + 3 design.Results: 11 patients were enrolled and have been treated at 4 dose levels: 50mg, 100mg, 200mg, 325mg and expanded 200mg through fifth cohort escalation. Pathological subtypes include 7 PTCL, 2 DLBCL, 1 MZBL and 1 composite CTCL/MF. 3 females and 8 males have been treated to date. Median age is 58 (range 30-71) and ECOG PS is in the range of 0-1. All patients had taken at least one prior chemotherapy. 10 of total patients have completed at least one cycle except 1 premature drop-out case due to disease progression. First interim analysis after completion of cycle 3 of the last patient of 200mg QD cohort had been conducted, which was to include 5 patients (1 DLBCL and 4 PTCLs). No DLT had been identified in Cohorts 1-3, and 2 patients discontinued the study treatment due to adverse event (G4 thrombocytopenia, not related to IP) and disease progression, respectively. The PK values from multiple dosing range of 50mg to 200mg cohort resulted in an approximate 2.92-fold and 4.97 fold increase in exposure based on Cmax and AUCtau, respectively. BR101801 PK profile showed that the exposure of concentration increased in a dose dependent manner and there was no accumulation observed in the dose range of 50mg to 200mg. 2 DLTs was observed at 325mg QD cohort. The dose was de-escalated to the previous lower dose level (200mg QD) and was expanded to 3 additional patients. The expansion cohort is ongoing at present. 2 of 11 patients had G3 skin reaction and 3 had G3 hepatotoxicites. All adverse effects were manageable and recovered to grade 0-1 upon BR1010801 discontinuation. Total 5 patients have been currently ongoing. For overall tumor response assessment, 4 SDs and 2 PRs have been obsereved.Summary/Conclusion: 200 mg QD of BR101801 was shown to provide target exposure for clinical efficacy with the tolerable and safe profiles. BR101801 was well tolerated and showed preliminary signs of activity in patients with relapsed or refractory hematologic malignancies. The phase Ib/II study of BR101801 is warranted in relapsed/refractory NHL. This study is registered at clinicaltrials.gov identifier NCT04018248. DisclosuresKim: AstraZeneca-KHIDI: Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; GI CELL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Hanmi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Boryung: Consultancy, Membership on an entity's Board of Directors or advisory committees; BeyondBIO: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca/MedImmune: Consultancy, Membership on an entity's Board of Directors or advisory committees. Curran: Servier pharmaceuticals and Amgen: Consultancy. Kim: Boryung pharmaceuticals: Current Employment.

DNA-PK phosphorylation at Ser2056 during adenovirus E4 mutant infection is promoted by viral DNA replication and independent of the MRN complex

Adenovirus (Ad) early region 4 (E4) mutants activate cellular DNA damage responses (DDRs) that include non-homologous end joining (NHEJ) pathways mediated by the DNA repair kinase DNA-PK and its associated factors Ku70/Ku86. NHEJ results in concatenation of the viral linear double-stranded DNA genome and inhibits a productive infection. E4 proteins normally prevent activation of cellular DDRs in wild-type Ad type 5 (Ad5) infections, thereby promoting efficient viral growth. The purpose of this study was to evaluate the factors that govern DNA-PK activation during adenovirus infection. Our data indicate that viral DNA replication promotes DNA-PK activation, which is required for genome concatenation by NHEJ. Although the Mre11/Rad50/Nbs1 (MRN) DDR sensor complex is not required for DNA-PK activation, Mre11 is important for recruitment of the NHEJ factor Ku86 to viral replication centers. Our study addresses the interplay between the DNA-PK and MRN complexes during viral genome concatenation by NHEJ.

Identification of hub molecules of FUS-ALS by Bayesian gene regulatory network analysis of iPSC model: iBRN

Fused in sarcoma/translated in liposarcoma (FUS) is a causative gene of amyotrophic lateral sclerosis (ALS). Mutated FUS causes accumulation of DNA damage and cytosolic stress granule (SG) formation, thereby motor neuron (MN) death. However, key molecular aetiology remains unclear. Here, we applied a novel platform technology, iBRN, "Non- biased" Bayesian gene regulatory network analysis based on induced pluripotent stem cell (iPSC)-derived cell model, to elucidate the molecular aetiology using transcriptome of iPSC-derived MNs harboring FUSH517D. iBRN revealed "hub molecules", which strongly influenced transcriptome network, such as miR-125b-5p-TIMELESS axis and PRKDC for the molecular aetiology. Next, we confirmed miR-125b-5p-TIMELESS axis in FUSH517D MNs such that miR-125b-5p regulated several DNA repair-related genes including TIMELESS. In addition, we validated both introduction of miR-125b-5p and knocking down of TIMELESS caused DNA damage in the cell culture model. Furthermore, PRKDC was strongly associated with FUS mis-localization into SGs by DNA damage under impaired DNA-PK activity. Collectively, our iBRN strategy provides the first compelling evidence to elucidate molecular aetiology in neurodegenerative diseases.

Caveolin-1 promotes radioresistance in rhabdomyosarcoma through increased oxidative stress protection and DNA repair.

The aim of this work was to investigate whether Caveolin-1 (Cav-1), a membrane scaffolding protein widely implicated in cancer, may play a role in radiation response in rhabdomyosarcoma (RMS), a pediatric soft tissue tumor. For this purpose, we employed human RD cells in which Cav-1 expression was stably increased via gene transfection. After radiation treatment, we observed that Cav-1 limited cell cycle arrest in the G2/M phase and enhanced resistance to cell senescence and apoptosis via reduction of p21Cip1/Waf1, p16INK4a and Caspase-3 cleavage. After radiotherapy, Cav-1-mediated cell radioresistance was characterized by low accumulation of H2AX foci, as confirmed by Comet assay, marked neutralization of reactive oxygen species (ROS) and enhanced DNA repair via activation of ATM, Ku70/80 complex and DNA-PK. We found that Cav-1-overexpressing RD cells, already under basal conditions, had higher glutathione (GSH) content and greater catalase expression, which conferred protection against acute treatment with hydrogen peroxide. Furthermore, pre-treatment of Cav-1-overexpressing cells with PP2 or LY294002 compounds restored the sensitivity to radiation treatment, indicating a role for Src-kinases and Akt pathways in Cav-1-mediated radioresistance. These findings were confirmed using radioresistant RD and RH30 lines generated by hypofractionated radiotherapy protocol, which showed marked increase of Cav-1, catalase and Akt, and sensitivity to PP2 and LY294002 treatment. In conclusion, these data suggest that concerted activity of Cav-1 and catalase, in cooperation with activation of Src-kinase and Akt pathways, may represent a network of vital mechanisms that allow irradiated RMS cells to evade cell death induced by oxidative stress and DNA damage.

The plié by DNA-PK: dancing on DNA

In this issue of Molecular Cell, Chen etal. (2020) report the structural transition during DNA-dependent activation of DNA-PK, shedding light on the mechanism by which kinase inhibitors and auto-phosphorylation-deficient DNA-PKcs compromise non-homologous end-joining (Chen etal., 2020).