DNA-dependent Protein Kinase Inhibitor Research Articles

A multicenter phase Ib/II study of DNA-PK inhibitor peposertib (M3814) in combination with capecitabine and radiotherapy in patients with locally advanced rectal cancer.

TPS4117 Background: Preoperative chemo-radiotherapy with or without sequential chemotherapy, followed by surgical intervention, is standard of care for patients with locally advanced rectal cancer (LARC). However, 1/3 of these patients still develop distant metastases, indicating the need for more effective therapies. DNA-dependent protein kinase (DNA-PK) regulates a key DNA damage repair pathway for double-strand break repair. Peposertib (M3814), a potent, selective, orally administered DNA-PK inhibitor, has been shown to potentiate the effect of ionizing radiation in a human colon cancer xenograft model and several colon cancer cell lines. Peposertib is being investigated in several different trials across multiple indications. This Phase Ib/II study (NCT03770689) aims to evaluate the safety, tolerability, pharmacokinetics (PK), and efficacy of the neoadjuvant treatment combination of peposertib, capecitabine, and radiotherapy (RT) in patients with LARC. Methods: Patients aged ≥18 years with histologically confirmed and resectable Stage II/III rectal adenocarcinoma are eligible. Induction chemotherapy is permitted, but residual disease must first be documented by MRI, digital rectal examination and endoscopy. Patients who received other anticancer therapies or those with prior pelvic RT are excluded. During open-label Phase Ib (open), 18–30 patients (n = 3 per cohort) are due to receive peposertib + capecitabine (orally, 825 mg/m2 twice daily [BID]) + RT (45–50 Gy), 5 days/week. Peposertib 50 mg once daily (QD) is the starting dose. Additional dose levels will be between 100─800 mg QD. Dose escalation is determined by the safety monitoring committee and guided by a Bayesian 2-parameter logistic regression model. At Phase II (planned), 150 patients will be randomized (1:1) to receive oral capecitabine (825 mg/m2 BID) + RT (45–50 Gy), with either oral peposertib (recommended phase II dose [RP2D]) or placebo, QD for 5 days/week. Primary objectives are to define a maximum tolerated dose and RP2D (Phase Ib), and to evaluate the efficacy of peposertib + capecitabine + RT in terms of pathological/clinical complete response (Phase II). Secondary objectives include assessment of antitumor activity (Phase Ib), quality of life outcomes (Phase II), and PK of peposertib, and the safety and tolerability of the combination therapy (both phases). One patient has received peposertib 50 mg QD and six patients have received peposertib 100 mg QD. Patients are currently receiving peposertib 150 mg QD. Clinical trial information: NCT03770689 .

M3814, a DNA-PK Inhibitor, Modulates ABCG2-Mediated Multidrug Resistance in Lung Cancer Cells

M3814, also known as nedisertib, is a potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor under phase 2 clinical trials. ABCG2 is a member of the ATP-binding cassette (ABC) transporter family that is closely related to multidrug resistance (MDR) in cancer treatment. In this study, we demonstrated that M3814 can modulate the function of ABCG2 and overcome ABCG2-mediated MDR. Mechanistic studies showed that M3814 can attenuate the efflux activity of ABCG2 transporter, leading to increased ABCG2 substrate drugs accumulation. Furthermore, M3814 can stimulate the ABCG2 ATPase activity in a concentration-dependent manner without affecting the ABCG2 protein expression or cell surface localization of ABCG2. Moreover, the molecular docking analysis indicated a high affinity between M3814 and ABCG2 transporter at the drug-binding cavity. Taken together, our work reveals M3814 as an ABCG2 modulator and provides a potential combination of co-administering M3814 with ABCG2 substrate-drugs to overcome MDR.

Pharmacologic Inhibitor of DNA-PK, M3814, Potentiates Radiotherapy and Regresses Human Tumors in Mouse Models.

Physical and chemical DNA-damaging agents are used widely in the treatment of cancer. Double-strand break (DSB) lesions in DNA are the most deleterious form of damage and, if left unrepaired, can effectively kill cancer cells. DNA-dependent protein kinase (DNA-PK) is a critical component of nonhomologous end joining (NHEJ), one of the two major pathways for DSB repair. Although DNA-PK has been considered an attractive target for cancer therapy, the development of pharmacologic DNA-PK inhibitors for clinical use has been lagging. Here, we report the discovery and characterization of a potent, selective, and orally bioavailable DNA-PK inhibitor, M3814 (peposertib), and provide in vivo proof of principle for DNA-PK inhibition as a novel approach to combination radiotherapy. M3814 potently inhibits DNA-PK catalytic activity and sensitizes multiple cancer cell lines to ionizing radiation (IR) and DSB-inducing agents. Inhibition of DNA-PK autophosphorylation in cancer cells or xenograft tumors led to an increased number of persistent DSBs. Oral administration of M3814 to two xenograft models of human cancer, using a clinically established 6-week fractionated radiation schedule, strongly potentiated the antitumor activity of IR and led to complete tumor regression at nontoxic doses. Our results strongly support DNA-PK inhibition as a novel approach for the combination radiotherapy of cancer. M3814 is currently under investigation in combination with radiotherapy in clinical trials.

PARP Inhibition Synergizes with Melphalan but Does not Reverse Resistance Completely.

High-dose melphalan (MEL) and autologous stem cell transplantation (ASCT) is the standard of care in the treatment of multiple myeloma (MM). Resistance to MEL has been linked to increased DNA repair. Here we sought to identify whether inhibition of poly(ADP-ribose) polymerase (PARP) synergizes with MEL and can overcome resistance. We tested the synergistic cytotoxicity of 3 inhibitors of PARP (PARPi)-veliparib (VEL), olaparib (OLA), and niraparib (NIRA)-combined with MEL in RPMI8226 and U266 MM cell lines, as well as in their MEL resistance counterparts, RPMI8226-LR5 (LR5) and U266-LR6 (LR6). The addition of VEL, OLA, and NIRA to MEL reduced the half maximal inhibitory concentration (IC50) in RPMI8226 cells from 27.8 µM to 23.1 µM, 22.5 µM, and 18.0 µM, respectively. Similarly, the IC50 of MEL in U266 cells was decreased from 6.2 µM to 3.2 µM, 3.3 µM, and 3.0 µM, respectively. In LR5 and LR6 cells, PARPi did not reverse MEL resistance. We confirmed this in a NOD/SCID/gamma null xenograft mouse model with either MEL-sensitive (RPMI8226) or MEL-resistant (LR5) MM. Treatment with a MEL-VEL combination prolonged survival compared with MEL alone in RPMI8226 mice (107 days versus 67.5 days; P=.0009), but not in LR5 mice (41 versus 39 days; P=.09). We next tested whether 2 double-stranded DNA repair mechanisms, homologous recombination (HR) and nonhomologous end-joining (NHEJ), cause MEL resistance in LR5 and LR6 cells. In an HR assay, LR6 cells had a 4.5-fold greater HR capability than parent U226 cells (P=.05); however, LR5 cells had an equivalent HR ability as parent RPMI8226 cells. We hypothesized that NHEJ may be a mediator of MEL resistance in LR5 cells. Given that DNA-PK is integral to NHEJ and may be a therapeutic target, we treated LR5 cells with the DNA-PK inhibitor NU7026 in combination with MEL. Although NU7026 alone at 2.5 µM had no cytotoxicity, in combination it completely reversed resistance to MEL (MEL IC50, 46.4 µM versus 14.4 µM). We examined the clinical implications of our findings in a dataset of 414 patients treated with tandem ASCT. High PARP1 expressers had lower survival compared with patients with low expression (median 42.7 months versus median not reached; P=.003). We hypothesized that combined expression of the HR gene BRCA1, the NHEJ gene PRKDC (DNA-PK), and PARP1 may predict survival and found that overexpression of 0 (n=101), 1 or 2 (n=287), or all 3 (n=26) genes had a negative impact on median survival (undefined versus 57.8 months versus 14.8 months; P < .0001). Here we demonstrate that PARPi synergized with MEL, but that resistance (which may be due to HR and NHEJ pathways) is not completely reversed by PARPi. In addition, we observed that a 3-gene analysis may be tested to identify patients resistant or sensitive to high-dose MEL.

PRKDC: new biomarker and drug target for checkpoint blockade immunotherapy

BackgroundImmunological checkpoint blockade is effective in treating various malignancies. Identifying predictive biomarkers to assist patient selection for immunotherapy has become a priority in both clinical and research settings.MethodsMutations in patients...

DNA-PK Inhibitor, M3814, as a New Combination Partner of Mylotarg in the Treatment of Acute Myeloid Leukemia.

Despite significant advances in the treatment of acute myeloid leukemia (AML) the long-term prognosis remains relatively poor and there is an urgent need for improved therapies with increased potency and tumor selectivity. Mylotarg is the first AML-targeting drug from a new generation of antibody drug conjugate (ADC) therapies aiming at the acute leukemia cell compartment with increased specificity. This agent targets leukemia cells for apoptosis with a cytotoxic payload, calicheamicin, carried by a CD33-specific antibody. Calicheamicin induces DNA double strand breaks (DSB) which, if left unrepaired, lead to cell cycle arrest and apoptosis in cancer cells. However, repair of DSB by the non-homologous end joining pathway driven by DNA-dependent protein kinase (DNA-PK) can reduce the efficacy of calicheamicin. M3814 is a novel, potent and selective inhibitor of DNA-PK. This compound effectively blocks DSB repair, strongly potentiates the antitumor activity of ionizing radiation and DSB-inducing chemotherapeutics and is currently under clinical investigation. Suppressing DSB repair with M3814 synergistically enhanced the apoptotic activity of calicheamicin in cultured AML cells. Combination of M3814 with Mylotarg in two AML xenograft models, MV4-11 and HL-60, demonstrated increased efficacy and significantly improved survival benefit without elevated body weight loss. Our results support a new application for pharmacological DNA-PK inhibitors as enhancers of Mylotarg and a potential new combination treatment option for AML patients.

Slowly Repaired Bulky DNA Damages Modulate Cellular Redox Environment Leading to Premature Senescence.

Treatments on neoplastic diseases and cancer using genotoxic drugs often cause long-term health problems related to premature aging. The underlying mechanism is poorly understood. Based on the study of a long-lasting senescence-like growth arrest (10-12 weeks) of human dermal fibroblasts induced by psoralen plus UVA (PUVA) treatment, we here revealed that slowly repaired bulky DNA damages can serve as a “molecular scar” leading to reduced cell proliferation through persistent endogenous production of reactive oxygen species (ROS) that caused accelerated telomere erosion. The elevated levels of ROS were the results of mitochondrial dysfunction and the activation of NADPH oxidase (NOX). A combined inhibition of DNA-PK and PARP1 could suppress the level of ROS. Together with a reduced expression level of BRCA1 as well as the upregulation of PP2A and 53BP1, these data suggest that the NHEJ repair of DNA double-strand breaks may be the initial trigger of metabolic changes leading to ROS production. Further study showed that stimulation of the pentose phosphate pathway played an important role for NOX activation, and ROS could be efficiently suppressed by modulating the NADP/NADPH ratio. Interestingly, feeding cells with ribose-5-phosphate, a precursor for nucleotide biosynthesis that produced through the PPP, could evidently suppress the ROS level and prevent the cell enlargement related to mitochondrial biogenesis. Taken together, these results revealed an important signaling pathway between DNA damage repair and the cell metabolism, which contributed to the premature aging effects of PUVA, and may be generally applicable for a large category of chemotherapeutic reagents including many cancer drugs.

Overcoming Melphalan Resistance By Targeting Crucial DNA Repair Pathways in Multiple Myeloma

High dose melphalan (MEL) and autologous stem cell transplant (ASCT) is the standard of care in the treatment of multiple myeloma (MM). Resistance to MEL has been linked to increased DNA repair. Here we sought to identify which DNA repair pathways lead to MEL resistance. As inhibitors of the single strand DNA repair enzyme PARP1 are clinically available, we first examined whether PARP1 gene expression affected clinical outcomes. In a dataset of 414 patients treated with tandem ASCT, high PARP1 expression lead to reduced survival compared to patients with low expression (42.7 months vs. median not reached, p=0.003). We therefore tested in vitro the cytotoxicity of PARPi with veliparib (VEL), olaparib (OLA) or niraparib (NIRA) in RPMI8226 and U266 MM cell lines as well as their MEL resistance counterparts, RPMI8226-LR5 (LR5) and U266-LR6 (LR6). At physiologic doses PARPi alone was not cytotoxic. However, the MEL IC50 in RPMI8226 cells decreased from 27.8 to 23.1, 22.5 and 18.0 µM with the addition of VEL, OLA and NIRA respectively. Similarly, MEL IC50 decreased in U266 cells from 6.2 to 3.2, 3.3 and 3.0 µM. However, in LR5 and LR6 cells, PARPi did not completely reverse MEL resistance. We confirmed this in a NOD/SCID/gamma null xenograft mouse model with either MEL sensitive (RPMI8226) or resistant (LR5) MM. Treatment with a MEL-VEL combination prolonged survival in RPMI8226 mice compared to MEL alone (107 vs. 67.5 days, p=0.0009), but not in LR5 mice (41 vs. 39 days, p=0.09). Next we tested whether the double strand DNA repair mechanisms, homologous recombination (HR) and non-homologous end joining (NHEJ), cause MEL resistance which is not overcome by PARPi. Using an HR assay, LR6 cells had 4.5 fold higher HR ability than parent U226 cells (p = 0.05) showing this to be a possible mechanism of resistance. However, LR5 cells had equivalent HR ability to parent RPMI8226 cells. We therefore hypothesized that NHEJ may be a mediator of MEL resistance in LR5 cells. As DNA-PK is integral to NHEJ and may be a therapeutic target, we treated LR5 cells with the DNA-PK inhibitor NU7026 in combination with MEL. Although NU7026 alone at 2.5 µM had no cytotoxicity, in combination it completely reversed resistance to MEL resulting in a lower IC50 than even parent RPMI8226 cells (46.4 vs. 14.4 µM). Based on our findings, we hypothesized that combined expression of the HR gene BRCA1, the NHEJ gene DNA-PK, and PARP1 may predict survival in patients undergoing ASCT. Using the same clinical dataset, we found that overexpression of 0 (n=101), 1-2 (n=287), or all 3 genes (n=26) strongly predicted median survival (undefined vs. 57.8 vs. 14.8 months, p Here we demonstrated that MEL resistance is not completely reversed by PARPi, while it may be overcome by targeting HR and NHEJ pathways. In addition, we observed that a 3 gene analysis pre-ASCT may help predict patients that are inherently resistant or sensitive to high dose MEL.

APOBEC3B reporter myeloma cell lines identify DNA damage response pathways leading to APOBEC3B expression.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) DNA cytosine deaminase 3B (A3B) is a DNA editing enzyme which induces genomic DNA mutations in multiple myeloma and in various other cancers. APOBEC family proteins are highly homologous so it is especially difficult to investigate the biology of specifically A3B in cancer cells. To easily and comprehensively investigate A3B function in myeloma cells, we used CRISPR/Cas9 to generate A3B reporter cells that contain 3×FLAG tag and IRES-EGFP sequences integrated at the end of the A3B gene. These reporter cells stably express 3xFLAG tagged A3B and the reporter EGFP and this expression is enhanced by known stimuli, such as PMA. Conversely, shRNA knockdown of A3B decreased EGFP fluorescence and 3xFLAG tagged A3B protein levels. We screened a series of anticancer treatments using these cell lines and identified that most conventional therapies, such as antimetabolites or radiation, exacerbated endogenous A3B expression, but recent molecular targeted therapeutics, including bortezomib, lenalidomide and elotuzumab, did not. Furthermore, chemical inhibition of ATM, ATR and DNA-PK suppressed EGFP expression upon treatment with antimetabolites. These results suggest that DNA damage triggers A3B expression through ATM, ATR and DNA-PK signaling.

CHAF1B induces radioresistance by promoting DNA damage repair in nasopharyngeal carcinoma

BackgroundRadiotherapy is the main treatment for nasopharyngeal carcinoma (NPC); however radioresistance restricts its efficacy. Therefore, new molecular regulators are required to improve the radiosensitivity of NPC. Chromatin assembly factor 1 subunit B (CHAF1B) plays a role in DNA synthesis and repair, and participates in the progression of various malignancies. However, the expression and function of CHAF1B in NPC is unclear. MethodsThe expression of CHAF1B was determined using real-time PCR and western blotting. CHAF1B expression in 160 human NPC tissue samples was evaluated using immunochemistry (IHC). The correlations between CHAF1B expression and NPC clinicopathological features were determined. The effect of CHAF1B on the radiosensitivity of NPC cells was detected using 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and colony formation assays. Apoptosis rates were analyzed using flow cytometry. A nude mouse subcutaneous xenograft model and living fluorescence imaging were applied to evaluate tumor regression in vivo. The molecular mechanisms of radioresistance were confirmed by bioinformatics analysis and detection of phosphorylated H2A histone family member X (γH2AX) foci. ResultsSignificantly increased CHAF1B levels were observed in NPC tissues, which correlated positively with radioresistance and poor prognosis. In addition, CHAF1B was upregulated in radioresistant NPC cell lines. Overexpression of CHAF1B reduced, while silencing of CHAF1B enhanced, the radiosensitivity of NPC cells in vitro and in vivo. Mechanistically, CHAF1B inhibited NPC cell apoptosis by promoting DNA damage repair. Finally, the DNA-dependent protein kinase (DNA-PK) pathway was observed to be essential for CHAF1B promotion of DNA damage repair-mediated radioresistance. ConclusionThe results suggested CHAF1B enhances radioresistance by promoting DNA damage repair and inhibiting cell apoptosis, in a DNA-PK pathway-dependent manner. CHAF1B may serve as a novel factor for predicting radiorsensitivity. Besides, DNA-dependent protein kinase inhibitor could serve as a radiosensitizer for patients with NPC and high CHAF1B expression.

Impact of ATM and DNA-PK Inhibition on Gene Expression and Individual Response of Human Lymphocytes to Mixed Beams of Alpha Particles and X-Rays.

Accumulating evidence suggests a synergistic effect in cells simultaneously exposed to different types of clustered and dispersed DNA damage. We aimed to analyse the effect of mixed beams of alpha particles and X-rays (1:1 dose of each) on DNA damage response genes in human peripheral blood lymphocytes isolated from four donors. Two donors were compared upon inhibition of ATM or DNA-PK and at different sampling times. qPCR was used to measure mRNA levels of FDXR, GADD45A, BBC3, MDM2, CDKN1A, and XPC 24 h following exposure. Generally, alpha particles and mixed beams were stronger inducers of gene expression compared to X-rays, displaying saturated versus linear dose–response curves, respectively. Three out of four donors responded synergistically to mixed beams. When two donors were sampled again one year later, the former additive effect in one donor was now synergistic and no significant difference in intrinsic radiosensitivity was displayed, as determined by gamma-radiation-induced micronuclei. ATM, but not DNA-PK inhibition, reduced the radiation-induced gene expression, but differently for alpha radiation between the two donors. In conclusion, synergy was present for all donors, but the results suggest individual variability in the response to mixed beams, most likely due to lifestyle changes.

The effect of inhibitors of phosphatidylinositol 3-kinase-related kinases on dibenzo[def,p]chrysene genotoxicity measured by γH2AX levels and neutral comet assay in HepG2 human hepatocellular cancer cells

In the study the modulating effect of inhibition of phosphatidylinositol 3-kinase-related kinases (PIKK): ATM (Ataxia Telangiectasia Mutated), ATR (Ataxia Telangiectasia and Rad3 Related) and DNA-PK (DNA-dependent protein kinase) on genotoxicity of dibenzo[def,p]chrysene (DBC) in HepG2 human hepatocellular cancer cells was investigated. The cytotoxicity of DBC was determined, also in combination with PIKK inhibitors, using the MTT reduction assay. The high cytotoxicity of DBC was observed after 72 h incubation (IC50 = 0.06 μM). The PIKK inhibitors applied at non-cytotoxic concentrations: caffeine (1 mM) and KU55933 (2.5 μM) had no significant influence on the DBC cytotoxicity, however NU7026 (5 μM) caused significant increase in the cell viability by about 25%. The combinations of the inhibitors (double or triple) where NU7026 was present also caused increase in the cell viability (i.e. cytoprotective effect) compared to the effect of DBC. The level of damage to the genetic material (DNA double strand breaks, DSB) was assessed by measuring levels of phosphorylated form of H2A histone (γH2AX) and neutral comet assay. DBC induced DSB in a concentration and time-dependent manner. NU7026 considerably reduced the level of DSB level measured by γH2AX and comet assay.The obtained results confirm that DBC is cytotoxic and causes damage to the genetic material including DSB. The DNA-PK inhibitor NU7026 increases cell viability after exposure to DBC and reduces DNA damage, what indicates an important role of the sensor kinase in mediating the effect.

Activity of M3814, an Oral DNA-PK Inhibitor, In Combination with Topoisomerase II Inhibitors in Ovarian Cancer Models

DNA-dependent protein kinase (DNA-PK) has been shown to play a crucial role in repair of DNA double-strand breaks, facilitating nonhomologous end-joining. DNA-PK inhibitors have the potential to block DNA repair and therefore enhance DNA-damaging agents. M3814 is a DNA-PK inhibitor that has shown preclinical activity in combination with DNA-damaging agents, including radiotherapy and topoisomerase II inhibitors. Here we evaluated the activity of M3814 in combination with multiple topoisomerase II inhibitors, doxorubicin, etoposide, and pegylated liposomal doxorubicin (PLD) in vivo, utilizing ovarian cancer xenografts. Using cell lines representative of P53 wild-type ovarian cancer (A2780), and P53 mutant ovarian cancer (SKOV3), cells were implanted in the flank of athymic nude female mice. Mice were treated with vehicle, M3814 alone, topoisomerase II inhibitor alone, and M3814 in combination with topoisomerase II inhibitor, and change in tumor volume over time was documented. The addition of M3814 was well tolerated. We demonstrated that M3814 shows limited efficacy as a single agent in ovarian cancer models. The combination of M3814 with PLD showed enhanced activity over PLD as a single agent. Further study of this combination is warranted.

Therapeutic Implications of p53 Status on Cancer Cell Fate Following Exposure to Ionizing Radiation and the DNA-PK Inhibitor M3814.

Inhibition of DNA double-strand break (DSB) repair in cancer cells has been proposed as a new therapeutic strategy for potentiating the anticancer effects of radiotherapy. M3814 is a novel, selective pharmacologic inhibitor of the serine/threonine kinase DNA-dependent protein kinase (DNA-PK), a key driver of nonhomologous end-joining, one of the main DSB-repair pathways, currently under clinical investigation. Here, we show that M3814 effectively blocks the repair of radiation-induced DSBs and potently enhances p53 phosphorylation and activation. In p53 wild-type cells, ataxia telangiectasia-mutated (ATM) and its targets, p53 and checkpoint kinase 2 (CHK2), were more strongly activated by combination treatment with M3814 and radiation than by radiation alone, leading to a complete p53-dependent cell-cycle block and premature cell senescence. Cancer cells with dysfunctional p53 were unable to fully arrest their cell cycle and entered S and M phases with unrepaired DNA, leading to mitotic catastrophe and apoptotic cell death. Isogenic p53-null/wild-type A549 and HT-1080 cell lines were generated and used to demonstrate that p53 plays a critical role in determining the response to ionizing radiation and M3814. Time-lapse imaging of cell death and measuring apoptosis in panels of p53 wild-type and p53-null/mutant cancer lines confirmed the clear differences in cell fate, dependent on p53 status. IMPLICATIONS: Our results identify p53 as a possible biomarker for response of cancer cells to combination treatment with radiation and a DNA-PK inhibitor and suggest that p53 mutation status should be considered in the design of future clinical trials. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/17/12/2457/F1.large.jpg.

APC loss affects DNA damage repair causing doxorubicin resistance in breast cancer cells

Chemoresistance is one of the leading causes of cancer-related deaths in the United States. Triple negative breast cancer (TNBC), a subtype lacking the known breast cancer receptors used for targeted therapy, is reliant on chemotherapy as the standard of care. The Adenomatous Polyposis Coli (APC) tumor suppressor is mutated or hypermethylated in 70% of sporadic breast cancers with APC-deficient tumors resembling the TNBC subtype. Using mammary tumor cells from the ApcMin/+ mouse model crossed to the Polyoma middle T antigen (PyMT) transgenic model, we previously showed that APC loss decreased sensitivity to doxorubicin (DOX). Understanding the molecular basis for chemoresistance is essential for the advancement of novel therapeutic approaches to ultimately improve patient outcomes. Resistance can be caused via different methods, but here we focus on the DNA repair response with DOX treatment. We show that MMTV-PyMT;ApcMin/+ cells have decreased DNA damage following 24 hour DOX treatment compared to MMTV-PyMT;Apc+/+ cells. This decreased damage is first observed 24 hours post-treatment and continues throughout 24 hours of drug recovery. Activation of DNA damage response pathways (ATM, Chk1, and Chk2) are decreased at 24 hours DOX-treatment in MMTV-PyMT;ApcMin/+ cells compared to control cells, but show activation at earlier time points. Using inhibitors that target DNA damage repair kinases (ATM, ATR, and DNA-PK), we showed that ATM and DNA-PK inhibition increased DOX-induced apoptosis in the MMTV-PyMT;ApcMin/+ cells. In the current work, we demonstrated that APC loss imparts resistance through decreased DNA damage response, which can be attenuated through DNA repair inhibition, suggesting the potential clinical use of DNA repair inhibitions as combination therapy.

AZD7648 is a potent and selective DNA-PK inhibitor that enhances radiation, chemotherapy and olaparib activity

DNA-dependent protein kinase (DNA-PK) is a critical player in the DNA damage response (DDR) and instrumental in the non-homologous end-joining pathway (NHEJ) used to detect and repair DNA double-strand breaks (DSBs). We demonstrate that the potent and highly selective DNA-PK inhibitor, AZD7648, is an efficient sensitizer of radiation- and doxorubicin-induced DNA damage, with combinations in xenograft and patient-derived xenograft (PDX) models inducing sustained regressions. Using ATM-deficient cells, we demonstrate that AZD7648, in combination with the PARP inhibitor olaparib, increases genomic instability, resulting in cell growth inhibition and apoptosis. AZD7648 enhanced olaparib efficacy across a range of doses and schedules in xenograft and PDX models, enabling sustained tumour regression and providing a clear rationale for its clinical investigation. Through its differentiated mechanism of action as an NHEJ inhibitor, AZD7648 complements the current armamentarium of DDR-targeted agents and has potential in combination with these agents to achieve deeper responses to current therapies.

DNA-PKcs is activated under nutrient starvation and activates Akt, MST1, FoxO3a, and NDR1

BackgroundPresence of unperfused regions containing cells under hypoxia and nutrient starvation; contributes to radioresistance in solid human tumors. We have previously reported that cultured cells; under nutrient starvation show resistance to ionizing radiation compare with cells under normal; condition, and that nutrient starvation increases ATM activity, which causes cellular resistance to; ionizing radiation (Murata et al., BBRC2018). For further investigation of molecular mechanisms; underlying radioresistance of cells under nutrient starvation, effects of nutrient starvation on activity; of DNA-PKcs have been investigated because both DNA-PKcs and ATM belong to the PIKK family; and are required for DNA DSBs repair. In addition to DNA-PKcs, effects of nutrient starvation on; activities of FoxO3a and its regulators Akt, MST1 and AMPK have been investigated because FoxO3a; mediates cellular responses to stress and is activated under nutrient starvation. MethodsA human glioblastoma cell line, T98G was used to examine the effects of nutrient starvation on activities and expression of DNA-PKcs, Akt, MST1, FoxO3a, NDR1, and AMPK. To elucidate; signal transduction pathways for FoxO3a activation under nutrient starvation, we examined effects of; specific inhibitors or siRNA for DNA-PKcs or Akt on activities and expression of MST1, FoxO3, NDR1, andAMPK. ResultsUnder nutrient starvation, phosphorylations of DNA-PKcs at Ser2056, Akt at Ser473, MST at Thr183, FoxO3a at Ser413, NDR1 at Ser281 and Thr282, and AMPK at Thr172 were increased, which suggests their activation. Nutrient starvation did not affect expression of DNA-PKcs, Akt, MST1, or NDR1, with decreased expression of FoxO3a and increased expression of AMPK. Inhibition; of DNA-PK suppressed phosphorylation of Akt under nutrient starvation. Inhibition of DNA-PK or; Akt suppressed phosphorylations of MST1, FoxO3a, and NDR1 under nutrient starvation, which; suggests DNA-PKcs and Akt activate MST1, FoxO3a, and NDR1. Inhibition of DNA-PK did not; suppress phosphorylation ofAMPK under nutrient starvation. ConclusionOur data suggest that DN-PKcs is activated under nutrient starvation and activates AktMST1, FoxO3a, and NDR1.

DNA-PKcs Mediates An Epithelial-Mesenchymal Transition Process Promoting Cutaneous Squamous Cell Carcinoma Invasion And Metastasis By Targeting The TGF-β1/Smad Signaling Pathway.

PurposeDNA-dependent protein kinase catalytic subunit (DNA-PKcs) has attracted extensive attention in various types of malignant tumors. However, the role of DNA-PKcs in cutaneous squamous cell carcinoma (cSCC) development has not been elucidated. In this study, we investigated the role of DNA-PKcs in cSCC and the molecular mechanisms of TGF-β1-induced cSCC progression mediated by DNA-PKcs.MethodsWe performed bioinformatic analysis and RT-PCR to examine the DNA-PKcs expression level in cSCC. Then, we downregulated DNA-PKcs using a DNA-PK-specific inhibitor or small interfering RNA (siRNA) to explore the effects of DNA-PKcs on SCL-1 cell migration and invasion. To further investigate the mechanism by which DNA-PKcs promotes cSCC progression, TGF-β1 and the TGF-β receptor (TGF-βR) I/II dual inhibitor LY2109761 were used to examine whether DNA-PKcs participates in TGF-β1/Smad signaling.ResultsDNA-PKcs expression was upregulated in cSCC. DNA-PK inhibition or expression knockdown resulted in inhibited migration and invasion and altered epithelial-mesenchymal transition (EMT) marker expression patterns in SCL-1 cells. Importantly, TGF-β1 mediated EMT induction in cSCC cells, and DNA-PKcs was identified as a TGF-β1-responsive gene. TGF-β1 promoted DNA-PKcs transcription, and DNA-PKcs enhanced the TGF-β1-induced EMT program involved in cSCC invasion and metastasis by phosphorylating Smad3.ConclusionThis study is the first to show that DNA-PKcs mediates EMT to promote cSCC aggressiveness by targeting the TGF-β1/Smad signaling pathway, which provides insight into how DNA-PKcs impacts cSCC progression and identifies a new therapeutic target.

Drug Sensitivity Prediction Models Reveal a Link between DNA Repair Defects and Poor Prognosis in HNSCC.

Head and neck squamous cell carcinoma (HNSCC) is characterized by the frequent manifestation of DNA crosslink repair defects. We established novel expression-based DNA repair defect markers to determine the clinical impact of such repair defects. Using hypersensitivity to the DNA crosslinking agents, mitomycin C and olaparib, as proxies for functional DNA repair defects in a panel of 25 HNSCC cell lines, we applied machine learning to define gene expression models that predict repair defects. The expression profiles established predicted hypersensitivity to DNA-damaging agents and were associated with mutations in crosslink repair genes, as well as downregulation of DNA damage response and repair genes, in two independent datasets. The prognostic value of the repair defect prediction profiles was assessed in two retrospective cohorts with a total of 180 patients with advanced HPV-negative HNSCC, who were treated with cisplatin-based chemoradiotherapy. DNA repair defects, as predicted by the profiles, were associated with poor outcome in both patient cohorts. The poor prognosis association was particularly strong in normoxic tumor samples and was linked to an increased risk of distant metastasis. In vitro, only crosslink repair-defective HNSCC cell lines are highly migratory and invasive. This phenotype could also be induced in cells by inhibiting rad51 in repair competent and reduced by DNA-PK inhibition. In conclusion, DNA crosslink repair prediction expression profiles reveal a poor prognosis association in HNSCC. SIGNIFICANCE: This study uses innovative machine learning-based approaches to derive models that predict the effect of DNA repair defects on treatment outcome in HNSCC.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/21/5597/F1.large.jpg.

Inhibition of Ku70 in a high-glucose environment aggravates bupivacaine-induced dorsal root ganglion neurotoxicity

BackgroundBupivacaine (BP) is commonly used as a local anaesthetic(LA) in the clinic, but it can also cause neurotoxicity, especially in patients with diabetes. Previous studies have found that high-glucose environments can aggravate BP-induced DNA damage in nerve cells. Ku70 is subunit of the DNA damage repair enzyme DNA-PK. This study was designed to determine whether high-glucose conditions enhance BP neurotoxicity and DNA damage by inhibiting Ku70 expression. MethodsWe examined the effect of BP on apoptosis and DNA damage in murine dorsal root ganglion (DRG) neurons under hyperglycaemic conditions. Untreated DRG cells and DRG cells pretreated with NU7441, a DNA-PK inhibitor, were cultured for 3 days under normal culture conditions or with 50 mM glucose, and the cells were then treated with BP for 3 h. DNA damage was investigated via comet assays, the ratio of early to late apoptotic cells was assessed by Annexin V-FITC/PI staining, and cell viability was measured by CCK-8 assays. The protein expression levels of DNA-PK, Ku70, Bax, Bcl-2 and γH2ax were measured by immunofluorescence or Western blotting. ResultsCompared to its effect under normal culture conditions, BP treatment led to decreased cell viability and increased DNA damage in DRG cells grown under high-glucose conditions. The rate of DRG cell apoptosis and the expression of γH2ax, the ratio of Bax to Bcl-2 also increased under the high-glucose conditions. Furthermore, Ku70 expression was inhibited. The DNA-PK inhibitor, NU7441, could significantly inhibit DNA-PK and Ku70 expression, simultaneously further aggravating BP-induced apoptosis and DNA damage under high-glucose conditions. ConclusionThese data indicate that hyperglycaemia may enhance BP-induced neurotoxicity and DNA damage by inhibiting the DNA repair protein Ku70.