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PDF file - 869K, Effect of DNA-PKcs or ATM inhibition on p-T2609 in NSCLCs. h pretreatment with 10 muM NU7441 or 10 muM KU55933. Square boxes represent cropped images shown in Figure 6C

PDF file - 900K, Radiation-induced T2609 phosphorylation in NSCLCs. Immuno-fluorescence detection of DNA-PKcs phosphorylation in indicated NSCLCs showing p-T2609 immunofluorescence (green) in nuclei (blue). Cropped images shown in Figure 6B

PDF file - 286K, Alterations in ATM or DNA-PKcs do not affect IR-induced nuclear import. Deficiency or inhibition of either ATM or DNA-PKcs is associated with an absence of EGFR-DNA-PKcs binding. The data in S1A eliminate the possibility that this is due to a potential abrogation of IR-induced nuclear translocation of EGFR which could prevent access to the predominantly nuclear DNA-PKcs. (A) WB of cytosolic and nuclear fractions at indicated time-points following 4 Gy IR from V3, V3-WT DNA-PKCS and V3-7A cells(A) or AT5 and 1BR3 fibroblast cells (B) expressing wild type, L858R or gammaE746-E750 EGFR showing EGFR, cytoplasmic marker, calnexin, and nuclear marker, lamin B. With exposure to 4 Gy, wild type but not L858R or gammaE746-E750 EGFR is seen in nuclear fractions of cells that lack DNA-PKcs or ATM. (C) DNA-PKcs or ATM inhibition does not affect EGFR nuclear import. WB of cytosolic and nuclear fractions at indicated time-points following 4 Gy IR from HBEC cells expressing wild type EGFR that were pretreated for 2 hours with vehicle or DNA-PKcs inhibitor, NU7441, (top panel) or ATM inhibitor, KU55933, (bottom panel). (D) Mutant forms of EGFR are also defective in binding DNA-PKcs. The data in S1A and S1B show that IR-induced nuclear translocation is completely abrogated in L858R or gammaE746-E750 forms of EGFR, which could prevent interactions with the predominantly nuclear DNA-PKcs. Here we use in vitro binding assay to demonstrate that relative to wild type EGFR, the L858R or gammaE746-E750 are also intrinsically defective in physical association with purified DNA-PKcs. In vitro binding assay: wild type, L858R or gammaE746-E750 forms of EGFR were immuno-precipitated with anti-V5 antibody from whole cell lysates from un-irradiated HBEC cells. Lane 1 from left shows no non-specific binding to beads. Antibody-bound EGFR was thoroughly washed. Lane 2 from left shows no evidence of endogenous bound protein. Antibody bound EGFR was incubated with purified human DNA-PKcs in the presence or absence of calf thymus DNA and immuno-precipitated. DNA-PKcs was detected by western blot assay. Lane 3 and 4 show that purified DNA-PKcs physically bound wild type but not the L858R or gammaE746-E750 forms of EGFR and the binding was slightly enhanced with presence of sheared calf thymus DNA.

The EGF receptor (EGFR) contributes to tumor radioresistance, in part, through interactions with the catalytic subunit of DNA-dependent protein kinase (DNA-PKc), a key enzyme in the nonhomologous end joining DNA repair pathway. We previously showed that EGFR-DNA-PKcs interactions are significantly compromised in the context of activating mutations in EGFR in non–small cell lung carcinoma (NSCLC) and human bronchial epithelial cells. Here, we investigate the reciprocal relationship between phosphorylation status of DNA-PKcs and EGFR-mediated radiation response. The data reveal that both the kinase activity of DNA-PKcs and radiation-induced phosphorylation of DNA-PKcs by the ataxia telangiectasia–mutated (ATM) kinase are critical prerequisites for EGFR-mediated radioresponse. Alanine substitutions at seven key serine/threonine residues in DNA-PKcs or inhibition of DNA-PKcs by NU7441 completely abrogated EGFR-mediated radioresponse and blocked EGFR binding. ATM deficiency or ATM inhibition with KU55933 produced a similar effect. Importantly, alanine substitution at an ATM-dependent DNA-PKcs phosphorylation site, T2609, was sufficient to block binding or radioresponse of EGFR. However, mutation of a DNA-PKcs autophosphorylation site, S2056 had no such effect indicating that DNA-PKcs autophosphorylation is not necessary for EGFR-mediated radioresponse. Our data reveal that in both NSCLCs and human bronchial epithelial cells, activating mutations in EGFR specifically abolished the DNA-PKcs phosphorylation at T2609, but not S2056. Our study underscores the critical importance of a reciprocal relationship between DNA-PKcs phosphorylation and EGFR-mediated radiation response and elucidates mechanisms underlying mutant EGFR-associated radiosensitivity in NSCLCs. Mol Cancer Res; 10(10); 1359–68. ©2012 AACR.

PDF file - 607K, Effect of EGFR mutation status on DNA-Pkcs phosphorylation. Immuno-fluorescence detection of DNA-PKcs phosphorylation in HBEC cells stably expressing wild type, L858R or gammaE746-E750 forms of EGFR at various time-points following 4 Gy IR. Top panel: Representative images with anti-p-T2609 and anti-p-S2056 DNA-PKcs antibodies showing nuclei (blue) and phosphorylated DNA-PKcs (green). Cropped images shown in Figure 6A.

PDF file - 544K, Proximity ligation assay (PLA): Effect of DNA-Pkcs and ATM inhibition on EGFR-DNA-PKcs binding.NSCLCs or HBEC-3KT cells expressing wild type or gammaE746-E750 forms of EGFR following a 2 h pretreatment with vehicle, 10 muM NU7441 or 10 muM KU55933 were mock irradiated or exposed to 4 Gy IR and fixed at 60 minutes. Representative images of nuclei (blue) showing EGFR-DNA-PKcs complexes (intense red dots). Cropped images shown in Figure 4C

PDF file - 774K, Proximity ligation assay (PLA): To detect radiation-induced PP2A-DNA-PKcs binding. NSCLCs and HBEC cells expressing WT or gammaE746-E750 EGFR fixed 1 h following 0 Gy or 4 Gy IR. Left panel: representative images of nuclei (blue) and EGFR-DNA-PKcs complexes (red: PLA fluorescence). Square boxes represent cropped images shown in Figure 7C

PDF file - 729K, Proximity ligation assay (PLA): Effect of T2609A and S2056A on EGFR-DNA-PKcs binding. V3-WT DNA-PKCS, V3-S2056A and V3-T2609A CHO cells expressing wild type EGFR which were either mock-irradiated or exposed to 4 Gy IR. Left panel: representative images of nuclei (blue) and EGFR-DNA-PKcs complexes (red PLA fluorescence dots) after processing for PLA. Square boxes represent cropped images shown in Figure 5C.

PDF file - 210K, API inhibits ionizing radiation-induced Akt phosphorylation/activation

Supplementary Figure 5 from Suppression of Nonhomologous End Joining Repair by Overexpression of HMGA2

A crucial step in human breast cancer progression is the acquisition of invasiveness. There is a distinct lack of human cell culture models to study the transition from preinvasive to invasive phenotype as it may occur “spontaneously” in vivo. To delineate molecular alterations important for this transition, we isolated human breast epithelial cell lines that showed partial loss of tissue polarity in three-dimensional reconstituted basement membrane cultures. These cells remained noninvasive; however, unlike their nonmalignant counterparts, they exhibited a high propensity to acquire invasiveness through basement membrane in culture. The genomic aberrations and gene expression profiles of the cells in this model showed a high degree of similarity to primary breast tumor profiles. The xenograft tumors formed by the cell lines in three different microenvironments in nude mice displayed metaplastic phenotypes, including squamous and basal characteristics, with invasive cells exhibiting features of higher-grade tumors. To find functionally significant changes in transition from preinvasive to invasive phenotype, we performed attribute profile clustering analysis on the list of genes differentially expressed between preinvasive and invasive cells. We found integral membrane proteins, transcription factors, kinases, transport molecules, and chemokines to be highly represented. In addition, expression of matrix metalloproteinases MMP9, MMP13, MMP15, and MMP17 was up-regulated in the invasive cells. Using small interfering RNA–based approaches, we found these MMPs to be required for the invasive phenotype. This model provides a new tool for dissection of mechanisms by which preinvasive breast cells could acquire invasiveness in a metaplastic context. [Cancer Res 2008;68(5):1378–87]

Supplementary Information from Downregulation of Human DAB2IP Gene Expression in Prostate Cancer Cells Results in Resistance to Ionizing Radiation

Supplementary Figure 5 from A Human Breast Cell Model of Preinvasive to Invasive Transition

Supplementary Figure 1 from Suppression of Nonhomologous End Joining Repair by Overexpression of HMGA2

Supplementary Table 1 from Downregulation of Human DAB2IP Gene Expression in Prostate Cancer Cells Results in Resistance to Ionizing Radiation

The epidermal growth factor receptor (EGFR) is an important determinant of radioresponse, whose elevated expression and activity frequently correlates with radioresistance in several cancers, including non–small cell lung carcinoma (NSCLC). We reported recently that NSCLC cell lines harboring somatic, activating mutations in the tyrosine kinase domain (TKD) of the EGFR exhibit significant delays in the repair of DNA double-strand breaks (DSB) and poor clonogenic survival in response to radiation. Here, we explore the mechanisms underlying mutant EGFR-associated radiosensitivity. In three representative NSCLC cell lines, we show that, unlike wild-type (WT) EGFR, receptors with common oncogenic TKD mutations, L858R or ΔE746-E750, are defective in radiation-induced translocation to the nucleus and fail to bind the catalytic and regulatory subunits of the DNA-dependent protein kinase (DNA-PK), a key enzyme in the nonhomologous end-joining repair pathway. Moreover, despite the presence of WT EGFR, stable exogenous expression of either the L858R or the ΔE746-E750 mutant forms of EGFR in human bronchial epithelial cells significantly delays repair of ionizing radiation (IR)–induced DSBs, blocks the resolution of frank or microhomologous DNA ends, and abrogates IR-induced nuclear EGFR translocation or binding to DNA-PK catalytic subunit. Our study has identified a subset of naturally occurring EGFR mutations that lack a critical radioprotective function of EGFR, providing valuable insights on how the EGFR mediates cell survival in response to radiation in NSCLC cell lines. [Cancer Res 2007;67(11):5267–74]

Supplementary Figure 2 from A Human Breast Cell Model of Preinvasive to Invasive Transition

Supplementary Figure 4 from A Human Breast Cell Model of Preinvasive to Invasive Transition

Supplementary Table 2 from Downregulation of Human DAB2IP Gene Expression in Prostate Cancer Cells Results in Resistance to Ionizing Radiation

Supplementary Methods and Materials from A Human Breast Cell Model of Preinvasive to Invasive Transition

Despite a clear link between ataxia-telangiectasia mutated (ATM)–dependent phosphorylation of p53 and cell cycle checkpoint control, the intracellular biology and subcellular localization of p53 phosphoforms during the initial sensing of DNA damage is poorly understood. Using G0-G1 confluent primary human diploid fibroblast cultures, we show that endogenous p53, phosphorylated at Ser15 (p53Ser15), accumulates as discrete, dose-dependent and chromatin-bound foci within 30 minutes following induction of DNA breaks or DNA base damage. This biologically distinct subpool of p53Ser15 is ATM dependent and resistant to 26S-proteasomal degradation. p53Ser15 colocalizes and coimmunoprecipitates with γ-H2AX with kinetics similar to that of biochemical DNA double-strand break (DNA-dsb) rejoining. Subnuclear microbeam irradiation studies confirm p53Ser15 is recruited to sites of DNA damage containing γ-H2AX, ATMSer1981, and DNA-PKcsThr2609 in vivo. Furthermore, studies using isogenic human and murine cells, which express Ser15 or Ser18 phosphomutant proteins, respectively, show defective nuclear foci formation, decreased induction of p21WAF, decreased γ-H2AX association, and altered DNA-dsb kinetics following DNA damage. Our results suggest a unique biology for this p53 phosphoform in the initial steps of DNA damage signaling and implicates ATM-p53 chromatin-based interactions as mediators of cell cycle checkpoint control and DNA repair to prevent carcinogenesis.

Supplementary Figure 3 from A Human Breast Cell Model of Preinvasive to Invasive Transition

Supplementary Figure 7 from A Human Breast Cell Model of Preinvasive to Invasive Transition

Supplementary Figures 1-3 from Evidence for the Direct Binding of Phosphorylated p53 to Sites of DNA Breaks In vivo

Supplementary Figure 2 from Suppression of Nonhomologous End Joining Repair by Overexpression of HMGA2

Supplementary Figure 4 from Suppression of Nonhomologous End Joining Repair by Overexpression of HMGA2

Supplementary Figure 3 from Suppression of Nonhomologous End Joining Repair by Overexpression of HMGA2

Supplementary Figure 1 from A Human Breast Cell Model of Preinvasive to Invasive Transition

Supplementary Figures S1-S2 from Somatic Mutations in the Tyrosine Kinase Domain of Epidermal Growth Factor Receptor (EGFR) Abrogate EGFR-Mediated Radioprotection in Non–Small Cell Lung Carcinoma

Supplementary Figure Legends from Evidence for the Direct Binding of Phosphorylated p53 to Sites of DNA Breaks In vivo

Motivated by the news that Car2go replaced all of its electric vehicle fleet with gasoline-powered cars starting from 1 May 2016, we examine whether it is optimal to use electric vehicles (EVs) in the car sharing market and investigate the environmental impact of pulling the EVs from the market. We develop a model consisting of a profit-maximizing car sharing company (CSC) and a population of utility-maximizing customers. The CSC sets the number of EVs, the number of fuel vehicles (FVs), and the rental price jointly to maximize its profit. Customers decide whether to use EVs, FVs, or public transportation to complete their trips considering the rental price. We show that it is optimal to use EVs only if the charging speed, the number of charging stations, and the range of EVs are high enough. Among these three conditions, the recharging speed is the most important and the number of charging stations is more important than the range of EVs. We also find that including EVs in the car sharing market may lead to a higher total emission (due to a lower rental price that results in a higher usage rate). Moreover, we consider the problem with the objective of maximizing social welfare and find that when considering the environmental impact, the government should tax the CSC to induce a higher rental price and when ignoring this impact, the government should subsidize the CSC to reduce this price. We demonstrate our results with the case study of Car2go in San Diego and find that Car2go replaced EVs with FVs likely due to the slow recharging speed.

Joint disorders can be detrimental to quality of life. There is an unmet need for precise functional reconstruction of native-like cartilage and bone tissues in the craniofacial space and particularly for the temporomandibular joint (TMJ). Current surgical methods suffer from lack of precision and comorbidities and frequently involve multiple operations. Studies have sought to improve craniofacial bone grafts without addressing the cartilage, which is essential to TMJ function. For the human-sized TMJ in the Yucatan minipig model, we engineered autologous, biologically, and anatomically matched cartilage-bone grafts for repairing the ramus-condyle unit (RCU), a geometrically intricate structure subjected to complex loading forces. Using image-guided micromilling, anatomically precise scaffolds were created from decellularized bone matrix and infused with autologous adipose-derived chondrogenic and osteogenic progenitor cells. The resulting constructs were cultured in a dual perfusion bioreactor for 5 weeks before implantation. Six months after implantation, the bioengineered RCUs maintained their predefined anatomical structure and regenerated full-thickness, stratified, and mechanically robust cartilage over the underlying bone, to a greater extent than either autologous bone-only engineered grafts or acellular scaffolds. Tracking of implanted cells and parallel bioreactor studies enabled additional insights into the progression of cartilage and bone regeneration. This study demonstrates the feasibility of TMJ regeneration using anatomically precise, autologous, living cartilage-bone grafts for functional, personalized total joint replacement. Inclusion of the adjacent tissues such as soft connective tissues and the TMJ disc could further extend the functional integration of engineered RCUs with the host.

Background: Everolimus (EVE) plus exemestane (EXE) doubled progression-free survival (PFS) while maintaining quality of life versus EXE alone in postmenopausal women with hormone receptor positive (HR+), HER2-negative metastatic breast cancer (mBC) (BOLERO-2 phase 3; NCT00863655). Pretreatment serum activin A was previously reported as a prognostic factor in first-line hormone therapy (letrozole vs tamoxifen) (Novartis P025) and anti-HER2 mBC (lapatinib vs trastzmab) (CCTG MA.31) trials. Here we investigate the prognostic and predictive ability of activin A in BOLERO-2. Methods: Activin A levels were determined on pretreatment serum samples using ELISA. Cox-proportional hazards model was used to assess the efficacy of EVE in the activin A low and high subgroups (median cut-point), and the prognostic effect of activin A on PFS and overall survival (OS). Results: Baseline activin A levels were determined in 513 patients (71% of 725 BOLERO-2 patients randomized 2:1 to EVE+EXE or EXE). Predictive and prognostic signals are shown in the table below Predictive and prognostic signalsPredictive/PrognosticEnd-pointAct-ivin ATreatmentNEventsMedian PFSHR (95% CI); p valuepredictivePFSHEXE93832.5 (1.5-2.8)-predictivePFSHEVE+EXE1631325.4 (4.1-6.8)0.46 (0.34 - 0.60); . In multivariate analysis (including sensitivity to prior hormone therapy and visceral disease), activin A remained a significant independent prognostic factor for PFS and OS [HR 0.57 (0.46-0.69) and 0.34 (0.27-0.43), respectively]. Conclusions: Higher serum activin A was strongly associated with shorter PFS and OS in HR+/HER2- mBC patients. Everolimus was efficacious regardless of serum activin A level. These results are similar to our previous studies in phase 3 trials of letrozole-tamoxifen (Novartis P025), and HER2-targeted therapy, lapatinib vs trastuzmab (CCTG MA.31): pretreatment serum activin A was prognostic for outcome, but was not a predictive factor for treatment arm selection. Citation Format: Ali SM, Chen D, Ali A, Krecko L, Leitzel K, Vasekar M, Nagabhairu V, Marks E, Polimera H, Richardson A, May M, He W, Patel P, Lavin M, Hofsess S, Sweetman R, Hortobagyi G, Baselga J, Lipton A. Serum activin A and outcomes in HR+ /HER2- metastatic breast cancer patients treated with everolimus: Results from BOLERO-2 [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-07-09.

We study service systems with parallel servers and random customer arrivals, with a focus on the total waiting cost of customers. Using a Markov decision process (MDP) modeling approach, we analytically characterize the structures of the dynamic server assignment policies for two important systems, one consisting of multiple homogeneous servers and two classes of customers; and the other consisting of two heterogeneous servers and multiple classes of customers. Based on the obtained results, we propose a threshold-type heuristic policy for the generalized system consisting of multiple heterogeneous servers and multiple classes of customers. To design such a heuristic policy, we first develop techniques for the performance evaluation of general threshold-type policies with any given threshold values. We then construct a path to search for the optimal threshold values. Finally, we compare the performance of the best threshold-type heuristic policy with that of the optimal policy, and show that our proposed heuristic policy is computationally efficient yet generates great performance. We compare the system under our threshold-type dynamic server assignment policy with other commonly seen and simple systems (including the dedicated and the work-conserving flexible priority systems), and demonstrate the importance and usefulness of dynamic server assignment control, for service systems having multiple classes of customer arrivals. We also conduct sensitivity analysis to explore the impacts of various system configuration features (including the number of servers; service rate variation; number of customer classes; and system utilization) on the performance regarding waiting-cost minimization.

DNA-dependent protein kinase (DNA-PK) is a serine/threonine kinase that plays an essential role in the repair of DNA double-strand breaks (DSBs) in the non-homologous end-joining (NHEJ) pathway. The DNA-PK holoenzyme consists of a catalytic subunit (DNA-PKcs) and DNA-binding subunit (Ku70/80, Ku). Ku is a molecular sensor for double-stranded DNA and once bound to DSB ends it recruits DNA-PKcs to the DSB site. Subsequently, DNA-PKcs is activated and heavily phosphorylated, with these phosphorylations modulating DNA-PKcs. Although phosphorylation of DNA-PKcs is well studied, other post-translational modifications of DNA-PKcs are not. In this study, we aimed to determine if acetylation of DNA-PKcs regulates DNA-PKcs-dependent DSB repair. We report that DNA-PKcs is acetylated in vivo and identified two putative acetylation sites, lysine residues 3241 and 3260. Mutating these sites to block potential acetylation results in increased radiosensitive, a slight decrease in DSB repair capacity as assessed by γH2AX resolution, and increased chromosomal aberrations, especially quadriradial chromosomes. Together, our results provide evidence that acetylation potentially regulates DNA-PKcs.

Background: The Y537S and D538G mutations in ESR1 are recurrent alterations in metastatic breast cancer (MBC) that promote ligand-independent receptor activation and resistance to estrogen deprivation therapy in laboratory models. The clinical prevalence of these mutations is not well established, but has been reported to be ∼10% by tumor sequencing. Cell free DNA (cfDNA) analysis typically reflects tumor-derived genetic alterations and can be used to characterize a population of patients at a common time point. We hypothesized that cfDNA could be used to detect ESR1 mutation at entry and its clinical impact in a large subset from BOLERO-2 that randomized patients with MBC to Exemestane (EXE) or EXE plus Everolimus (EVE). Methods: Patients with ER+/HER2– MBC with prior exposure to at least 1 non-steroidal aromatase inhibitor (NSAI) were enrolled in BOLERO-2. cfDNA was extracted from 560 baseline plasma samples using QIAamp Circulating DNA kit or QIAsymphony DSP Virus/Pathogen kit. Samples were analyzed by droplet digital PCR for Y537S and D538G. Cox-proportional hazards model was used to assess progression free survival (PFS) in patient subgroups defined by each ESR1 mutation, and the prognostic effect of each ESR1 mutation on overall survival (OS). Results: Of 541 evaluable patients (74.7% of study population), 156 (28.8%) had mutation in ESR1 in D538G (21.1%) and/or Y537S (13.3%) with 30 samples having both mutations. Sequencing of 302 archival tumor specimens (244 primary and 57 metastases) from this study only yielded 4 instances of D538G (1.3%) and 1 Y537S (0.3%). In the overall population, both mutations were poor prognostic factors associated with shorter OS (Table 1). Table 1AlterationNEventsMedian OS (95%CI)HR (95%CI)WT38521732.1 (28.1-36.4) D538G835726.0 (19.2-32.4)1.25 (1.02-1.54)Y537S423020.0 (13.0-29.3)2.31 (1.34-3.97)Double mt302415.2 (10.9-27.4)1.77 (1.31-2.39) PFS results were different for the two mutations. D538G but not Y537S mutation was associated with a shorter PFS with EXE compared to wild type (WT), (hazard ratios, D538G: 1.44 [95%CIs, 1.04-1.99] and Y537S: 0.92 [95%CIs, 0.44-1.93]). The D538G mutant group derived a similar benefit as WT from the addition of EVE to EXE, whereas the Y537S group did not (Table 2). Table 2AlterationGroupNEventsMedian PFS (95%CI)HR (95%CI)WTEXE1281163.9 (2.8-4.2)0.4 (0.31-0.51) EXE/EVE2571728.5 (6.9-9.9) D538GEXE24222.7 (1.4-2.8)0.34 (0.2-0.57) EXE/EVE59455.8 (4.2-8.4) Y537SEXE21164.1 (1.4-6.7)0.98 (0.49-1.94) EXE/EVE21194.2 (1.4-5.4) Conclusions: cfDNA analysis identifies a high rate of the Y537S and D538G ESR1 mutations in ER+ NSAI-treated MBC. As the two mutations may only represent 50-60% of all activating ESR1 mutations, the clinical prevalence of ESR1 mutations in ER+ MBC may be much higher than previously reported. Both mutations appear to be associated with a more aggressive disease biology. Interestingly, these two activating mutations appear to have differential effects on EXE and EVE sensitivity, highlighting new areas for research in ER biology. Citation Format: Chandarlapaty S, Sung P, Chen D, He W, Samoila A, You D, Bhatt T, Patel P, Voi M, Gnant M, Hortobagyi G, Baselga J, Moynahan ME. cfDNA analysis from BOLERO-2 plasma samples identifies a high rate of ESR1 mutations: Exploratory analysis for prognostic and predictive correlation of mutations reveals different efficacy outcomes of endocrine therapy–based regimens. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S2-07.