DNA Damage Response Markers Research Articles

Abstract 308: Role of Snf1-related Kinase as a Regulator of Chromatin Modifications and Dna-damage Response in Heart Injury

Introduction: Altered DNA damage response (DDR) can result in phenotypic changes that share features with age-associated pathological conditions, including metabolic and cardiovascular abnormalities. Snf1-related kinase (SNRK) is a serine/threonine kinase that belongs to the AMPK family. Our recent data demonstrates that SNRK expression is upregulated in heart failure (HF) and low energy states and that SNRK improves cardiac mitochondrial efficiency and cell survival. Here, we assessed the hypothesis that in response to stress, SNRK translocates to the nucleus where it modulates nuclear processes to enhance DNA repair and ultimately reduces cardiomyocyte death. Results: We first showed that the nuclear distribution of endogenous SNRK in cardiomyoblast is enhanced under low nutrient and oxidative stress. Next, we performed global phosphoproteomics analysis on cells with overexpression of wild type SNRK and mutant kinase-dead SNRK (D158A). We found significant increase of several phosphopeptides involved in nuclear processes, in particular in the regulation of chromatin remodeling and DDR. SNRK downregulation resulted in an increase in nucleus volume and heterochromatin content, with increased nuclear dysmorphia when subjected to oxidative stress. Despite having lower expression of DDR signal effectors p53 and Mdm2, these cells accumulated higher levels of pH2AX (DDR marker) and displayed higher cellular death after oxidative stress, consistent with increased DNA damage. Etoposide treatment in SNRK knockdown cells failed to induce p53 levels despite pH2AX accumulation. Additionally, pharmacological stabilization of p53 protein through nutlin-3A treatment did not increase p53 in SNRK knockdown cells. These findings suggest that SNRK regulates p53 expression, thereby acting as a new modulator of DDR. Conclusions: Our results demonstrate increased nuclear localization of SNRK under stress conditions, and that SNRK regulates phosphorylation of nuclear proteins involved in chromatin remodelling and DDR. Thus, SNRK may induce its pro-survival cardiac functions through regulation of chromatin architecture and enhancing DDR. Those novel findings can contribute to development of new therapies against HF.

Abstract CT099: DNA damage response and therapeutic activity following once-daily administration of the Wee 1 inhibitor AZD1775 (adavosertib)

Abstract Background: Wee1 tyrosine kinase phosphorylates and inactivates cyclin dependent kinase (Cdk) 1 as part of DNA damage response (DDR) signaling, resulting in G2 cell cycle arrest to enable DNA repair. We are conducting a Phase I study of oral Wee1 inhibitor AZD1775 in patients (pts) with advanced solid tumors using twice-daily (Arm A) and once-daily (Arm B) dosing. Results for Arm A were published previously. Here, we report updated pharmacodynamic (PD), pharmacokinetic (PK), and clinical response results for Arm B. Methods: In Arm B, AZD1775 was administered once daily on days 1-5 and 8-12 of each 21-day cycle in a 3+3 design. Primary objectives were to establish safety, tolerability, PK, and MTD. Secondary objectives included determining PD effects on DDR markers in tumor tissue and circulating tumor cells (CTCs), and tumor response (RECIST 1.1). Post-treatment PD effects were established in Arm A. Given the importance of sustained molecular target control in maximizing targeted therapy efficacy, paired tumor biopsies in Arm B were obtained at baseline and day 8 (pre-dose) to assess durability of Wee1 inhibition and downstream PD effects. Immunofluorescence assays were used to measure target inhibition (pY15-Cdk1/2) and DDR markers (e.g., γH2AX). Results: Arm B included 42 pts; of the 34 evaluable for response, 6 (18%) had a partial response (PR; 4 ovarian, 2 endometrial), and 20 (59%) had stable disease (SD; 2-26 cycles, mean 7). Of 10 evaluable BRCA-deficient pts, 2 had a PR (20%) and 6 had SD (60%; mean: 7.17 cycles). AZD1775 was well-tolerated, as previously reported. Three of 11 paired biopsies had elevated baseline pY15-Cdk (8-23% nuclear area positive [NAP]) and showed persistence of target suppression following the dosing break during days 6-8. Of the 8 pts with low baseline pY15-Cdk (0-3% NAP), 5 had elevated pY15-Cdk levels at day 8 (range: 192-1094%), suggesting a target rebound effect. Of the 8 pts with elevated pY15-CdK at baseline or day 8, 4 (50%) achieved a PR. Day 8 γH2AX activation was detected for 3 pts, all who progressed within 2 cycles. Preliminary PK data revealed low plasma drug concentrations at day 8 pre-dose (55 ± 50 nM; n = 4) relative to peak levels at day 5 (1697 ± 1062 nM; n = 9). Conclusions: Daily AZD1775 exhibits antitumor activity in pts with both BRCA-proficient and -deficient ovarian and endometrial cancer. Responses amongst pts with elevated pY15-Cdk levels at baseline or day 8 provide additional clinical/PD evidence for the presumed mechanism of action of AZ1775 on the cell cycle and suggest that levels of pY15-Cdk may be an important correlate of response. In contrast, γH2AX activation on day 8 demonstrates the presence of drug-induced DNA double strand breaks that are not associated with clinical drug activity. PK data confirmed substantially reduced plasma AZD1775 concentrations at day 8. Ongoing genomic and CTC analyses may uncover additional molecular determinants of response. NCT01748825. Supported in part by NCI Contract HHSN261200800001E. Citation Format: Arjun Mittra, Geraldine O'Sullivan Coyne, Shivaani Kummar, Khanh Do, Ashley Bruns, Lamin Juwara, Richard Piekarz, Larry Rubinstein, Sheila Prindiville, Elad Sharon, Howard Streicher, Tiffaney Hsia, Ganesh Mugundu, Jiuping Ji, Deborah Wilsker, Angie B. Dull, Brandon Miller, Robert J. Kinders, Ralph Parchment, James H. Doroshow, Alice P. Chen, Naoko Takebe. DNA damage response and therapeutic activity following once-daily administration of the Wee 1 inhibitor AZD1775 (adavosertib) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT099.

HPV infection associated DNA damage correlated with cervical precancerous lesions and cancer in the highest area of cervical cancer mortality, Longnan, China.

ObjectivesThis study was to assess whether human papillomavirus (HPV) resulting in genetic instability is one reason for the high incidence and mortality of cervical cancer in Longnan.MethodsBetween 2012 and 2016, a total of 346 samples from Longnan were collected and divided into four groups: cervicitis group (n=57), cervical intraepithelial neoplasia I group (CIN I, n=63), CIN II/III group (n=79) and invasive squamous cell carcinoma group (SCC, n=147). HPV E6/E7 mRNA was detected by Quantivirus® HPV E6/E7 RNA 3.0 assay (bDNA). The markers of DNA damage response (DDR) – ataxia telangiectasia mutated (ATM) pSer1981, H2AX pSer139 (γH2AX), Chk2 pThr68 and P53 – were analyzed by immunohistochemistry.ResultsThe activation of ATM, γH2AX, Chk2 and P53 was increased with increasing severity of cervical lesion. A significant difference of ATM expression in simple infection was also shown accompanied by the cervical lesion. The expression of γH2AX between HPV16+ and HPV16- specimens, γH2AX and P53 between HPV58+ and HPV58- groups had statistical significance. The expression and copy number of HPV E6/7 mRNA increases with the cervical lesion severity. A significant difference was shown for P53 expression between HPV E6/7 mRNA+ and mRNA- specimens. A close correlation with CHK2 expression for HPV E6/7 mRNA+ and HPV16 E6/7 mRNA+ specimens and γH2AX and CHK2 expression for SCC specimens was shown between low and high viral load groups.ConclusionsDDR, HPV genotypes and HPV E6/E7 oncogene expression correlated with the level of dysplasia of cervical lesions. HPV infection resulted in genetic instability may be one reason for the high incidence and mortality in Longnan.

Preclinical Combination Studies of an FGFR2 Targeted Thorium-227 Conjugate and the ATR Inhibitor BAY 1895344

Fibroblast growth factor receptor 2 (FGFR2) has been previously reported to be overexpressed in several types of cancer, whereas the expression in normal tissue is considered to be moderate to low. Thus, FGFR2 is regarded as an attractive tumor antigen for targeted alpha therapy. This study reports the evaluation of an FGFR2-targeted thorium-227 conjugate (FGFR2-TTC, BAY 2304058) comprising an anti-FGFR2 antibody, a chelator moiety covalently conjugated to the antibody, and the alpha particle-emitting radionuclide thorium-227. FGFR2-TTC was assessed as a monotherapy and in combination with the DNA damage response inhibitor ATRi BAY1895344. The invitro cytotoxicity and mechanism of action were evaluated by determining cell viability, the DNA damage response marker γH2A.X, and cell cycle analyses. The invivo efficacy was determined using human tumor xenograft models in nude mice. Invitro mechanistic assays demonstrated upregulation of γH2A.X and induction of cell cycle arrest in several FGFR2-expressing cancer cell lines after treatment with FGFR2-TTC. Invivo, FGFR2-TTC significantly inhibited tumor growth at a dose of 500 kBq/kg in the xenograft models NCI-H716, SNU-16, and MFM-223. By combining FGFR2-TTC with the ATR inhibitor BAY 1895344, an increased potency was observed invitro, as were elevated levels of γH2A.X and inhibition of FGFR2-TTC-mediated cell cycle arrest. In the MFM-223 tumor xenograft model, combination of the ATRi BAY 1895344 with FGFR2-TTC resulted in significant tumor growth inhibition at doses at which the single agents had no effect. The data provide a mechanism-based rationale for combining the FGFR2-TTC with the ATRi BAY 1895344 as a new therapeutic approach for treatment of FGFR2-positive tumors from different cancer indications.

Correlation of peripheral blood markers of DNA damage and immune response with chemoradiation response in patients with locally advanced rectal cancer.

e15109 Background: Approximately 60% of newly diagnosed rectal cancers are locally advanced. The standard of care for these malignancies is neoadjuvant chemoradiation therapy (nCRT) followed by surgery. Unfortunately, only approximately 20% of patients experience a complete response, but all are at risk for toxicity. There are no biomarkers to predict who will derive the greatest benefit from CRT. We hypothesized that inherent DNA damage recognition and repair (DNA-DRR) capacity contributes to CRT response in rectal cancer. Methods: We used primary peripheral blood lymphocytes (pPBLs) from rectal cancer patients to study differences in DNA-DRR capacity. The blood samples used were either drawn prior to nCRT or post a standard course of ~5.5 weeks of nCRT. We used immunofluorescence and Luminex-based multianalyte approaches to study DNA-DRR capacity in pPBLs. To determine if the tested biomarkers correlated with CRT response, we segregated patients by neoadjuvant rectal (NAR) score, a validated surrogate endpoint in rectal cancer. Results: Using pPBLs, we found that a subset of patients had elevated phosphorylated histone H2AX (γ-H2AX) foci, a well-described marker of DNA double strand breaks. We found that poor responders (PoR; NAR score > 14; n = 21) had lower γ-H2AX foci then complete responders (CR; NAR score < 1; n = 21) (P < 0.0001, Kruskal-Wallis test). We also did not observe any significant differences in γ-H2AX foci from pPBLs drawn prior to CRT versus post nCRT (p = 0.519, n = 11, Wilcoxon rank sum test). Next, by multianalyte testing, we found that a combined score derived from six DNA-DRR markers (ATR, MDM2, phosphorylated forms of H2AX (γ), p53, Chk1, Chk2) strongly correlated with CR (p < 0.001). Finally, by multianalyte testing, we also detected a serum signature of inflammatory markers that significantly correlated with CRT response (p < 0.05). Conclusions: Our data suggest that rectal cancer patients who differ in their response to CRT differ in global, inherent DNA-DRR capacity, suggesting that DNA-DRR capacity in pre-treatment pPBLs can form the basis of a predictive biomarker. This predictive capacity may reflect underlying epigenetic or genetic differences, or inflammatory state. Validation and further development of this novel assay is warranted, as it has the capacity to substantially improve patient selection in an era with growing treatment options for these patients.

Expression of DNA Damage Response Markers in Early-Onset or Familial Gastric Cancers

Background:Early-onset or familial gastric cancer (GC) is known to have clinicopathologic profiles different from those of sporadic GC. We aimed to compare DNA damage response marker expression between early-onset or familial GC and sporadic GC.Methods:GC samples were obtained from patients who underwent gastrectomy for GC at Seoul National University Hospital. Immunohistochemical analyses of various DNA damage response markers, including BRCA1, BRCA2, MRE11, RAD51C, and γH2AX, were performed using 54 early-onset GC, 59 familial GC, and 337 sporadic GC tissue microarray samples. Correlations between marker expression and clinicopathologic features were evaluated by univariate and multivariate analyses, and overall survival was analyzed.Results:The rate of γH2AX positivity was significantly higher (p < 0.001) in early-onset or familial GC than in sporadic GC. In contrast, the rates of MRE11 negativity and RAD51C negativity were significantly higher in sporadic GC than in early-onset or familial GC. BRCA1 negativity was associated with decreased overall survival in sporadic GC (p = 0.002), and MRE11 negativity was associated with decreased overall survival in sporadic GC (p = 0.012).Conclusion:Our results show significant differences in DNA damage response marker expression between early-onset or familial GC and sporadic GC.

Age-related dysfunction of the DNA damage response in intestinal stem cells

BackgroundSenescence increases the risks of inflammatory bowel diseases and colon cancer. Intestinal stem cells (ISCs) in crypts differentiate into epithelial cells and thereby maintain intestinal homeostasis. However, the influence of aging on the functions of ISCs is largely unknown. The mutation rate is highest in the small and large intestines. Numerous types of naturally occurring DNA damage are removed by the DNA damage response (DDR). This response induces DNA repair and apoptosis; therefore, its dysregulation leads to accumulation of damaged DNA and consequently cellular dysfunctions, including tumorigenesis. This study investigated whether aging affects the DDR in mouse ISCs.MethodsYoung (2–3-month-old) and old (> 19-month-old) Lgr5-EGFP-IRES-creERT2 mice were irradiated. The DDR in Lgr5-positive ISCs was compared between these mice by immunohistochemical analyses.ResultsInduction of DDR marker proteins (phosphorylated ATR and 53BP1), inflammatory factors (phosphorylated NF-κB and interleukin-6), and a mitochondrial biogenesis-associated gene (peroxisome proliferator-activated receptor-γ coactivator 1α) was lower in old ISCs than in young ISCs in vivo.ConclusionThe competence of the DDR in ISCs declines with age in vivo.

High levels of EGFR prevent sulforaphane-induced reactive oxygen species-mediated apoptosis in non-small-cell lung cancer cells

BackgroundSulforaphane (SFN) has been shown to induce the production of reactive oxygen species (ROS) and inhibit epidermal growth factor receptor (EGFR)–mediated signaling in non-small-cell lung cancer (NSCLC). NSCLC cells harboring constitutively active EGFR mutations are more sensitive to SFN treatment than cells with wild-type EGFR, but whether NSCLC cells with high levels of EGFR expression are more resistant or sensitive to SFN treatment is not known. Study designWe employed a pair of cell lines, CL1-0 and CL1-5, which have the same genetic background but different levels of EGFR expression, to examine the effects of high EGFR level in the sensitivity to SFN. MethodsThe effect of SFN on cell viability and tumorigenicity was examined by trypan blue dye-exclusion assay, clonogenic assays, flow cytometry, and immunoblotting in vitro as well as tumorigenicity study in vivo. ROS levels in cells were assessed by flow cytometry using the ROS-reactive fluorescent indicator CM-H2DCFDA. Knockdown of EGFR in CL1-5 cells was infected with an EGFR-targeting small hairpin (interfering) RNA (shRNA)–containing lentivirus. ResultsWe present evidence that cells with high-level EGFR expression (CL1-5) are more resistant to SFN treatment than those with low-level expression (CL1-0). SFN treatment produced a similar increase in ROS and caused arrest of a cell population at S-phase accompanied by the induction of γH2AX, a DNA damage-response marker, in both cell sublines. However, SFN induced apoptosis only in the high-EGFR-expressing CL1-0 subline. Pretreatment with the antioxidant N-acetyl-L-cysteine prevented SFN-induced apoptosis in CL1-0 cells and production of γH2AX in both CL1-0 and CL1-5 cells. shRNA-mediated knockdown of EGFR in CL1-5 cells rendered the cells susceptible to SFN-induced apoptosis. ConclusionThe cellular effects produced by SFN in NSCLC cells are largely mediated by SFN-induced production of ROS. Cells with higher levels of EGFR were more resistant to SFN treatment and showed resistance to SFN-induced apoptosis, suggesting that high EGFR levels protect cells from SFN-induced apoptosis. Despite this, we found that SFN retained the ability to inhibit the growth of NSCLC tumors with high-level EGFR expression in vivo.

Novel Combination Therapy Targeting rDNA Transcription and Histone Deacetylation Provides Effective Treatment for Multiple Myeloma, and Synergises in Bortezomib-Resistant MM

Background: Multiple myeloma (MM) requires combination drug therapies to delay acquired drug resistance and clinical relapse. We co-developed CX-5461, a highly-selective inhibitor of RNA polymerase I-mediated rDNA transcription(1), currently in phase I trials for relapsed haematological malignancies (Peter Mac). CX-5461 produces a targeted nucleolar DNA damage response (DDR), triggering both a p53-dependent and -independent nucleolar stress response and killing malignant cells while sparing normal cells(2,3). Single-agent CX-5461 provides an impressive survival benefit in mouse models of B-cell lymphoma, acute myeloid leukaemia and now MM(2,4,5). However, drug resistance eventually occurs, confirming the need for combination therapies.Aim: To test the efficacy of CX-5461 in combination with the histone deacetylase inhibitor panobinostat, (prioritised from a boutique high-throughput screen of anti-myeloma agents), with a focus on the setting of resistance to proteasome-inhibitors (PIs).Methods: We assessed the impact of CX-5461 and panobinostat on overall survival in mouse models of MM, then surveyed the effects on cellular response and molecular markers of DDR. We developed bortezomib-resistant cell lines and an in vivo model of bortezomib-resistance to test this combination in the setting of PI-resistance.Results: CX-5461 in combination with panobinostat provides a significant survival advantage in both the transplanted Vk*MYC and the 5T33/KaLwRij models, with minimal bone marrow toxicity.The combination showed increased anti-proliferative effects and cell death in vitro. Interestingly, experiments interrogating the downstream cellular response of this combination suggest that the mechanism(s) driving synergy are complex and cell context-dependent. Cell cycle analysis indicates that both CX-5461- and panobinostat-driven cell cycle effects, i.e. G2/M and G1/S arrest, respectively, are dominant in the combination setting in a cell line-dependent manner, suggesting that context-dependent factors such as p53 may influence the cellular response.Mechanistically, in both p53-wild type and -null cell lines we observe an increase in DDR signalling with single agent CX-5461, with only moderate further increase with the combination. Moreover, CX-5461-mediated MYC downregulation is not universally observed, with the combination promoting further downregulation only in some cell lines. Given the potential for affecting global transcription programs downstream of panobinostat, we are performing transcriptome analyses in the combination setting compared to single agent treatment.We have generated bortezomib-resistant cell lines, sequentially increasing drug exposure to establish populations growing at concentrations above the IC90 of the parental lines. The resistant 5T33 cells retain their resistance to bortezomib in vivo and we have demonstrated that CX-5461 remains effective in this model, significantly increasing survival. We are currently examining the combination of CX-5461 with panobinostat in this model of bortezomib-resistance, which will give critical information guiding patient selection for future clinical trials.Conclusion: The rDNA transcription inhibitor CX-5461 synergises in vitro and in vivo with panobinostat, and CX-5461 retains efficacy in the setting of bortezomib-resistant myeloma.ReferencesDrygin et al., Cancer Research 2011Bywater et al., Cancer Cell 2012Quin et al, Oncotarget, 2016Devlin et al., Cancer Discovery 2016Hein et al., Blood 2017 DisclosuresHarrison:Janssen-Cilag: Other: Scientific advisory board.

Nuclear PTEN Localization Contributes to DNA Damage Response in Endometrial Adenocarcinoma and Could Have a Diagnostic Benefit for Therapeutic Management of the Disease.

Endometrial adenocarcinoma (EndoCA) is the most common gynecologic cancer type in the United States, and its incidence is increasing. The majority of patients are disease-free after surgical resection of stage I tumors, which is often followed by radiotherapy, but most patients with advanced disease recur and have a poor prognosis, largely because the tumors become refractory to cytotoxic chemotherapies. PTEN, a commonly mutated tumor suppressor in EndoCAs, is well known for its ability to inhibit the AKT/mTOR signaling pathway. Nuclear functions for PTEN have been proposed as well, but whether those affect EndoCA development, progression, or outcomes is not well understood. Using immunohistochemistry, nuclear PTEN expression was observed in approximately half of EndoCA patient tumors, independent of grade and cytoplasmic PTEN expression. Higher levels of the DNA damage response (DDR) marker, γH2AX, were observed by immunohistochemistry and immunofluorescence in human EndoCA tumor sections that were PTEN-negative, in murine EndoCA tissues that were genetically modified to be PTEN-null, and in Ishikawa EndoCA cells, which do not express endogenous PTEN. Overexpression of exogenous PTEN-WT or PTEN-NLS, a modified PTEN with an added nuclear localization signal, significantly improved both DDR and G2-M transition in Ishikawa cells treated with a DNA-damaging agent. Whereas PARP inhibition with Olaparib was not as effective in Ishikawa cells expressing native or PTEN-NLS, inhibition with Talazoparib was not affected by PTEN overexpression. These results suggest that nuclear PTEN subcellular localization in human EndoCA could be diagnostic when considering DDR therapeutic intervention. Mol Cancer Ther; 17(9); 1995-2003. ©2018 AACR.

Changes in DNA damage response markers with treatment in advanced ovarian cancer

Changes in DNA damage response markers with treatment in advanced ovarian cancer

Alterations in the expression of DNA damage response-related molecules in potentially preneoplastic oral epithelial lesions

The aim of this study was to evaluate the expression levels of DNA damage response (DDR) markers in potentially preneoplastic oral epithelial lesions (PPOELs). Immunohistochemical expression of DDR markers (γΗ2 ΑΧ, pChk2, 53 BP1, p53, and phosphorylated at Ser 15 p53) was assessed in 41 oral leukoplakias, ranging from hyperplasia (H) to dysplasia (D) and in comparison with oral squamous cell carcinoma (OSCC) and normal mucosa (NM). Statistical and receiver operating characteristic curve analysis were performed. γH2 AX immunoexpression demonstrated a gradual increase and upper layer extension from NM to H to higher D degrees to OSCC. pChk2 expression was minimal in NM, relatively low in PPOELs, with an increasing tendency from H to D, and higher in OSCC. 53 BP1 demonstrated higher levels in OSCC than in NM, whereas its expression in PPOELs was heterogeneous, gradually increasing according to D. p53 demonstrated progressively higher levels and upper layer extension from H to D to OSCC. Phosphorylated p53 was absent in NM and relatively low in PPOELs and OSCC. DDR markers' expression is variable in PPOELs, showing a tendency to increase along with dysplasia. Activated DDR mechanisms may play an important protective role at early stages of oral carcinogenesis, but probably suffer progressive deregulation, eventually failing to suppress malignant transformation.

Vitamin K2, a menaquinone present in dairy products targets castration-resistant prostate cancer cell-line by activating apoptosis signaling

The aim of this study was to evaluate the therapeutic effects of vitamin K2 (VK2) on castration-resistant prostate cancer (CRPC) and its anti-cancer mechanisms in a pre-clinical study using a VCaP cell line (ATCC® CRL-2876™) which was established from a vertebral bone metastasis from a patient with hormone refractory prostate cancer. Our data showed that VK2 significantly inhibited CRPC VCaP cell proliferation in a dose-dependent manner at 48 h treatment in vitro. In addition, VK2 reduced the migration potential of VCaP cells and inhibited anchorage-independent growth of these cells. Our results also showed that VK2 induces apoptosis in VCaP cells. Furthermore, VK2 enforced growth arrest in VCaP cells by activating cellular senescence. Notably, VK2 treatment elevated the levels of reactive oxygen species in VCaP cells. Western blot analysis revealed that VK2 downregulated the expression of androgen receptor, BiP, survivin, while activating caspase-3 and -7, PARP-1 cleavage, p21 and DNA damage response marker, phospho-H2AX in VCaP cells. In conclusion, our study suggests that VK2 might be a potential anti-cancer agent for CRPC by specifically targeting key anti-apoptotic, cell cycle progression and metastasis-promoting signaling molecules.

DNA damage checkpoint pathway modulates the regulation of skeletal growth and osteoblastic bone formation by parathyroid hormone-related peptide.

We previously demonstrated that parathyroid hormone-related peptide (PTHrP) 1-84 knockin (Pthrp KI) mice, which lacked a PTHrP nuclear localization sequence (NLS) and C-terminus, displayed early senescence, defective osteoblastic bone formation, and skeletal growth retardation. However, the mechanism of action of the PTHrP NLS and C-terminus in regulating development of skeleton is still unclear. In this study, we examined alterations of oxidative stress and DNA damage response-related molecules in Pthrp KI skeletal tissue. We found that ROS levels, protein expression levels of γ-H2AX, a DNA damage marker, and the DNA damage response markers p-Chk2 and p53 were up-regulated, whereas gene expression levels of anti-oxidative enzymes were down-regulated significantly. We therefore further disrupted the DNA damage response pathway by deleting the Chk2 in Pthrp KI (Chk2-/-KI) mice and did comparison with WT, Chk2-/- and Pthrp KI littermates. The Pthrp KI mice with Chk2 deletion exhibited a longer lifespan, improvement in osteoblastic bone formation and skeletal growth including width of growth plates and length of long bones, trabecular and epiphyseal bone volume, BMD, osteoblast numbers, type I collagen and ALP positive bone areas, the numbers of total colony-forming unit fibroblasts (CFU-f), ALP+ CFU-f and the expression levels of osteogenic genes. In addition, the genes associated with anti-oxidative enzymes were up-regulated significantly, whereas the tumor suppressor genes related to senescence were down-regulated in Chk2-/- KI mice compared to Pthrp KI mice. Our results suggest that Chk2 deletion in Pthrp KI mice can somewhat rescue defects in osteoblastic bone formation and skeletal growth by enhancing endochondral bone formation and osteogenesis. These studies therefore indicate that the DNA damage checkpoint pathway may be a target for the nuclear action of PTHrP to regulate skeletal development and growth.

Radiation-Induced DNA Damage in Operators Performing Endovascular Aortic Repair.

Radiation exposure during fluoroscopically guided interventions such as endovascular aortic repair (EVAR) is a growing concern for operators. This study aimed to measure DNA damage/repair markers in operators perfoming EVAR. Expression of the DNA damage/repair marker, γ-H2AX and DNA damage response marker, phosphorylated ataxia telangiectasia mutated (pATM), were quantified in circulating lymphocytes in operators during the peri-operative period of endovascular (infrarenal, branched, and fenestrated) and open aortic repair using flow cytometry. These markers were separately measured in the same operators but this time wearing leg lead shielding in addition to upper body protection and compared with those operating with unprotected legs. Susceptibility to radiation damage was determined by irradiating operators' blood in vitro. γ-H2AX and pATM levels increased significantly in operators immediately after branched endovascular aortic repair/fenestrated endovascular aortic repair (P<0.0003 for both). Only pATM levels increased after infrarenal endovascular aortic repair (P<0.04). Expression of both markers fell to baseline in operators after 24 hours (P<0.003 for both). There was no change in γ-H2AX or pATM expression after open repair. Leg protection abrogated γ-H2AX and pATM response after branched endovascular aortic repair/fenestrated endovascular aortic repair. The expression of γ-H2AX varied significantly when operators' blood was exposed to the same radiation dose in vitro (P<0.0001). This is the first study to detect an acute DNA damage response in operators performing fluoroscopically guided aortic procedures and highlights the protective effect of leg shielding. Defining the relationship between this response and cancer risk may better inform safe levels of chronic low-dose radiation exposure.

γ-H2AX formation in the urinary bladder of rats treated with two norharman derivatives obtained from o-toluidine and aniline.

Aminomethylphenylnorharman (AMPNH) and aminophenylnorharman (APNH) are mutagenic norharman derivatives obtained from o-toluidine and aniline, respectively. APNH is carcinogenic to the urinary bladder of rats and present in urine samples of healthy volunteers, indicating that norharman derivatives may be associated with cancer development in the urinary bladder of humans. To evaluate the possible role of AMPNH and APNH in bladder carcinogenesis, we examined the formation of γ-H2AX, a DNA damage response marker, in the urinary bladder of rats. Seven-week-old male F344 rats were treated with 400ppm AMPNH or 40ppm APNH in the diet for 4weeks. Animals were killed at the end of administration or after 2weeks of recovery, and immunohistochemistry for γ-H2AX and Ki67, a cell proliferation marker, was performed. At week 4, γ-H2AX formation in bladder epithelial cells was significantly increased by APNH treatment as compared with that in controls. AMPNH also induced upregulation of γ-H2AX formation, although there was no statistical significance. After the recovery period, γ-H2AX-positive cells were reduced but remained significantly higher in AMPNH and APNH groups than in the control group. Ki67-positive cells were significantly increased by AMPNH and APNH at week 4 and reduced to the same level as the control after 2weeks of recovery. Expression of KRT14, a bladder stem cell marker, was also increased in the basal layer by the two norharman derivatives. Thus, AMPNH and APNH showed in vivo genotoxicity in the bladder epithelium of rats, and APNH may be a potent causative agent of bladder carcinogenesis.

Systematic Kinase Inhibitor Profiling Identifies CDK9 as a Synthetic Lethal Target in NUT Midline Carcinoma

SummaryKinase inhibitors represent the backbone of targeted cancer therapy, yet only a limited number of oncogenic drivers are directly druggable. By interrogating the activity of 1,505 kinase inhibitors, we found that BRD4-NUT-rearranged NUT midline carcinoma (NMC) cells are specifically killed by CDK9 inhibition (CDK9i) and depend on CDK9 and Cyclin-T1 expression. We show that CDK9i leads to robust induction of apoptosis and of markers of DNA damage response in NMC cells. While both CDK9i and bromodomain inhibition over time result in reduced Myc protein expression, only bromodomain inhibition induces cell differentiation and a p21-induced cell-cycle arrest in these cells. Finally, RNA-seq and ChIP-based analyses reveal a BRD4-NUT-specific CDK9i-induced perturbation of transcriptional elongation. Thus, our data provide a mechanistic basis for the genotype-dependent vulnerability of NMC cells to CDK9i that may be of relevance for the development of targeted therapies for NMC patients.

Transcriptional-mediated effects of radiation on the expression of immune susceptibility markers in melanoma

Background and purposeWe recently reported a time-sensitive, cooperative, anti-tumor effect elicited by radiation (RT) and intra-tumoral-immunocytokine injection in vivo. We hypothesized that RT triggers transcriptional-mediated changes in tumor expression of immune susceptibility markers at delayed time points, which may explain these previously observed time-dependent effects. Materials and methodsWe examined the time course of changes in expression of immune susceptibility markers following in vitro or in vivo RT in B78 murine melanoma and A375 human melanoma using flow cytometry, immunoblotting, and qPCR. ResultsFlow cytometry and immunoblot revealed time-dependent increases in expression of death receptors and T cell co-stimulatory/co-inhibitory ligands following RT in murine and human melanoma. Using high-throughput qPCR, we observed comparable time courses of RT-induced transcriptional upregulation for multiple immune susceptibility markers. We confirmed analogous changes in B78 tumors irradiated in vivo. We observed upregulated expression of DNA damage response markers days prior to changes in immune markers, whereas phosphorylation of the STAT1 transcription factor occurred concurrently with changes following RT. ConclusionThis study highlights time-dependent, transcription-mediated changes in tumor immune susceptibility marker expression following RT. These findings may help in the design of strategies to optimize sequencing of RT and immunotherapy in translational and clinical studies.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract 119: Vitamin K2 targets castration-resistant prostate cancer VCaP cells by reactive oxygen species mediated apoptotic cell death

Abstract Prostate Cancer (PCa) is the second most common cancer in western countries especially in US population, in which castration-resistant prostate cancer (CRPC) is the major cause for patient mortality. Current treatment options available for CRPC are not efficient and have undesirable side effects. Hence there is an urgent need to develop non-toxic and effective treatment strategies for CRPC. Vitamin K2 (VK2), a natural menaquinone has several medicinal values including anti-cancer activity and anti-osteoporosis effect. The aim of this study is to evaluate the therapeutic effects of Vitamin K2 (VK2) and its anti-cancer mechanism against CRPC. In this study, we have used VCaP cell line (ATCC) which is established from a patient with hormone refractory prostate cancer. VCaP cells were treated with various concentrations of VK2 to evaluate its effects on cell viability by MTT assay, anchorage independent growth by soft agar assay, cellular senescence by beta-galactosidase staining assay and cancer cell migration by wound healing assay. We have also assessed the VK2-induced production of intracellular reactive oxygen species (ROS) using DCF (2′,7′-dichlorofluorescein) probe based fluorescence assay. VK2 induced apoptosis was detected by Annexin-V FITC and TUNEL assays. Western blot analysis was utilized to uncover the anti-proliferative and anti-metastatic mechanisms of VK2 against CRPC. Our results showed that VK2 significantly inhibits the proliferation of VCaP cells in a dose dependent manner at 48 hrs treatment in vitro. MTT data also showed that anti-proliferative effects of VK2 were significantly abrogated in the presence of anti-oxidant N-acetyl cysteine (NAC) and caspase inhibitor Z-VAD-FMK suggesting that ROS and caspase activation as the underlying anti-cancer mechanisms of VK2 in CRPC cells. In addition, VK2 reduced the migration potential of VCaP cells in wound healing assay and inhibited anchorage independent growth of these cells when compared to untreated cells. Annexin-V and TUNEL assays confirmed that VK2 induces apoptosis in VCaP cells. Our results also suggested that the VK2 has the ability to enforce growth arrest in CRPC cells by activating cellular senescence. Western blot analysis revealed that VK2 downregulated the expression of BiP, survivin, MMP-2, and PCNA while activating PARP-1, p21 and DNA damage response marker, phospho-H2AX in VCaP cells. Furthermore, VCaP cells treated with VK2 resulted in the activation of Caspase-3 and-7 apoptotic mediators. These results correlated with translocation of Bax and Cytochrome C to cytoplasm following VK2 treatment in VCaP cells as determined by confocal immunofluorescence analysis. In conclusion our study suggests that VK2 might be an effective anti-proliferative and anti-metastatic agent for CRPC by specifically targeting key anti-apoptotic, cell cycle progression and metastasis promoting signaling molecules. Citation Format: Subramanyam Dasari, Maarten C. Bosland, Andre Kajdacsy- Balla, Gnanasekar Munirathinam. Vitamin K2 targets castration-resistant prostate cancer VCaP cells by reactive oxygen species mediated apoptotic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 119. doi:10.1158/1538-7445.AM2017-119