Claire W. Hardy

Pronecrotic mixed lineage kinase domain-like protein expression is a prognostic biomarker in patients with early-stage resected pancreatic adenocarcinoma

Mixed lineage kinase domain‐like protein (MLKL) is a necrosome component mediating programmed necrosis that may be an important determinant of cancer cell death. The goal of the current study was to evaluate the prognostic value of MLKL expression in patients with pancreatic adenocarcinoma (PAC).

SIRT2 directs the replication stress response through CDK9 deacetylation

Sirtuin 2 (SIRT2) is a sirtuin family deacetylase that directs acetylome signaling, protects genome integrity, and is a murine tumor suppressor. We show that SIRT2 directs replication stress responses by regulating the activity of cyclin-dependent kinase 9 (CDK9), a protein required for recovery from replication arrest. SIRT2 deficiency results in replication stress sensitivity, impairment in recovery from replication arrest, spontaneous accumulation of replication protein A to foci and chromatin, and a G2/M checkpoint deficit. SIRT2 interacts with and deacetylates CDK9 at lysine 48 in response to replication stress in a manner that is partially dependent on ataxia telangiectasia and Rad3 related (ATR) but not cyclin T or K, thereby stimulating CDK9 kinase activity and promoting recovery from replication arrest. Moreover, wild-type, but not acetylated CDK9, alleviates the replication stress response impairment of SIRT2 deficiency. Collectively, our results define a function for SIRT2 in regulating checkpoint pathways that respond to replication stress through deacetylation of CDK9, providing insight into how SIRT2 maintains genome integrity and a unique mechanism by which SIRT2 may function, at least in part, as a tumor suppressor protein.

Indocyanine green loaded liposome nanocarriers for photodynamic therapy using human triple negative breast cancer cells

BACKGROUNDnThe goal of the current research is to evaluate the potential of photodynamic therapy (PDT) in the treatment of triple negative breast cancer (TNBC) with the development of a theranostic thermosensitive liposome platform to deliver indocyanine green (ICG) as the near-infrared (NIR) photosensitizer excited by an 808 nm diode laser.nnnMETHODSnIn the PDT protocol, an optimized thermosensitive liposome formulation is investigated to formulate ICG as the photosensitizer, which is exited by laser light at the wavelength of 808 nm delivered by a fiber-coupled laser system. ICG in both free solution and thermosensitive liposomal formulation were evaluated as the NIR photosensitizer and compared in the PDT treatment on a panel of triple negative breast cancer cell lines along with the nontumorigenic mammary epithelial cell line MCF-10A. In addition to cytotoxicity, and clonogenic survival assessment, the role of DNA double strand break damage was evaluated.nnnRESULTSnBoth MTT and clonogenic assays revealed that PDT using ICG inhibited the growth of several TNBC cell lines as well as the non-tumorigenic human breast epithelial cell line MCF-10A; and the liposomal formulation of ICG did not compromise the in vitro treatment potency, though free ICG performed slightly more effective in certain cell lines, but was not statistically significant. Cell viability was dose dependent in regards to ICG concentration and irradiation energy. Interestingly, PDT using the described protocol was more potent to inhibit the growth of MDA-MB-468 and HCC-1806 cells, coinciding with the observation that these cells are more sensitive toward DNA damaging agents. In comparison, cell lines HCC-70, BT-549, and MCF-10A were found to have less of an inhibitory effect. Furthermore, substantial DNA double strand breaks (DSBs) were observed 30 min after the PDT treatment via a γ-H2AX staining assay. PDT induced DNA damage has the potential to lead to mutagenicity, which may have various responses depending on the repair capabilities of the cells.nnnCONCLUSIONnOur results suggest that PDT using indocyanine green loaded liposomes were effective in inhibiting tumor cell growth to varying extents with higher responses observed for MDA-MB-468 and HCC-1806 cells.

A gemcitabine sensitivity screen identifies a role for NEK9 in the replication stress response

The Replication Stress Response (RSR) is a signaling network that recognizes challenges to DNA replication and coordinates diverse DNA repair and cell-cycle checkpoint pathways. Gemcitabine is a nucleoside analogue that causes cytotoxicity by inducing DNA replication blocks. Using a synthetic lethal screen of a RNAi library of nuclear enzymes to identify genes that when silenced cause gemcitabine sensitization or resistance in human triple-negative breast cancer cells, we identified NIMA (never in mitosis gene A)-related kinase 9 (NEK9) as a key component of the RSR. NEK9 depletion in cells leads to replication stress hypersensitivity, spontaneous accumulation of DNA damage and RPA70 foci, and an impairment in recovery from replication arrest. NEK9 protein levels also increase in response to replication stress. NEK9 complexes with CHK1, and moreover, NEK9 depletion impairs CHK1 autophosphorylation and kinase activity in response to replication stress. Thus, NEK9 is a critical component of the RSR that promotes CHK1 activity, maintaining genome integrity following challenges to DNA replication.

CHD7 Expression Predicts Survival Outcomes in Patients with Resected Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with poor outcomes with current therapies. Gemcitabine is the primary adjuvant drug used clinically, but its effectiveness is limited. In this study, our objective was to use a rationale-driven approach to identify novel biomarkers for outcome in patients with early-stage resected PDAC treated with adjuvant gemcitabine. Using a synthetic lethal screen in human PDAC cells, we identified 93 genes, including 55 genes linked to DNA damage responses (DDR), that demonstrated gemcitabine sensitization when silenced, including CHD7, which functions in chromatin remodeling. CHD7 depletion sensitized PDAC cells to gemcitabine and delayed their growth in tumor xenografts. Moreover, CHD7 silencing impaired ATR-dependent phosphorylation of CHK1 and increased DNA damage induced by gemcitabine. CHD7 was dysregulated, ranking above the 90th percentile in differential expression in a panel of PDAC clinical specimens, highlighting its potential as a biomarker. Immunohistochemical analysis of specimens from 59 patients with resected PDAC receiving adjuvant gemcitabine revealed that low CHD7 expression was associated with increased recurrence-free survival (RFS) and overall survival (OS), in univariate and multivariate analyses. Notably, CHD7 expression was not associated with RFS or OS for patients not receiving gemcitabine. Thus, low CHD7 expression was correlated selectively with gemcitabine sensitivity in this patient population. These results supported our rationale-driven strategy to exploit dysregulated DDR pathways in PDAC to identify genetic determinants of gemcitabine sensitivity, identifying CHD7 as a novel biomarker candidate to evaluate further for individualizing PDAC treatment.

Improved hippocampal dose with reduced margin radiotherapy for glioblastoma multiforme

BackgroundTo dosimetrically evaluate the effect of reduced margin radiotherapy on hippocampal dose for glioblastoma multiforme (GBM) patients.MethodsGBM patients enrolled on the Radiation Therapy Oncology Group (RTOG) 0825 trial at our institution were identified. Standard RTOG 0825 expansions were 2xa0cmu2009+u20093-5xa0mm from the gross tumor volume (GTV) to the clinical tumor volume (CTV) and from the CTV to the planning tumor volume (PTV), respectively. These same patients also had reduced margin tumor volumes generated with 8xa0mm (GTV to CTV)u2009+u20093xa0mm (CTV to PTV) expansions. Individual plans were created for both standard and reduced margin structures. The dose-volume histograms were statistically compared with a paired, two-tailed Student’s t-test with a significance level of pu2009<u20090.05.ResultsA total of 16 patients were enrolled on RTOG 0825. The reduced margins resulted in statistically significant reductions in hippocampal dose at all evaluated endpoints. The hippocampal Dmax was reduced from a mean of 61.4xa0Gy to 56.1xa0Gy (8.7%), D40% was reduced from 49.9xa0Gy to 36.5xa0Gy (26.9%), D60% was reduced from 32.7xa0Gy to 18.7xa0Gy (42.9%) and the D80% was reduced from 27.3xa0Gy to 15.3xa0Gy (44%).ConclusionsThe use of reduced margin PTV expansions in the treatment of GBM patients results in significant reductions in hippocampal dose. Though the exact clinical benefit of this reduction is currently unclear, this study does provide support for a future prospective trial evaluating the neurocognitive benefits of reduced margin tumor volumes in the treatment of GBM patients.

The influence of adjuvant radiotherapy dose on overall survival in patients with resected pancreatic adenocarcinoma

Adjuvant radiotherapy (A‐RT) for patients with resected pancreatic adenocarcinoma (PAC) is controversial. In the current study, the authors aim to determine whether there is an association between overall survival (OS) and A‐RT dose.

Low CHD5 expression activates the DNA damage response and predicts poor outcome in patients undergoing adjuvant therapy for resected pancreatic cancer

The DNA damage response (DDR) promotes genome integrity and serves as a cancer barrier in precancerous lesions but paradoxically may promote cancer survival. Genes that activate the DDR when dysregulated could function as useful biomarkers for outcome in cancer patients. Using a siRNA screen in human pancreatic cancer cells, we identified the CHD5 tumor suppressor as a gene, which, when silenced, activates the DDR. We evaluated the relationship of CHD5 expression with DDR activation in human pancreatic cancer cells and the association of CHD5 expression in 80 patients with resected pancreatic adenocarcinoma (PAC) by immunohistochemical analysis with clinical outcome. CHD5 depletion and low CHD5 expression in human pancreatic cancer cells lead to increased H2AX-Ser139 and CHK2-Thr68 phosphorylation and accumulation into nuclear foci. On Kaplan–Meier log-rank survival analysis, patients with low CHD5 expression had a median recurrence-free survival (RFS) of 5.3 vs 15.4 months for patients with high CHD5 expression (P=0.03). In 59 patients receiving adjuvant chemotherapy, low CHD5 expression was associated with decreased RFS (4.5 vs 16.3 months; P=0.001) and overall survival (OS) (7.2 vs 21.6 months; P=0.003). On multivariate Cox regression analysis, low CHD5 expression remained associated with worse OS (HR: 3.187 (95% CI: 1.49–6.81); P=0.003) in patients undergoing adjuvant chemotherapy. Thus, low CHD5 expression activates the DDR and predicts for worse OS in patients with resected PAC receiving adjuvant chemotherapy. Our findings support a model in which dysregulated expression of tumor suppressor genes that induce DDR activation can be utilized as biomarkers for poor outcome.

The influence of radiation therapy dose escalation on overall survival in unresectable pancreatic adenocarcinoma

PURPOSEnRadiation therapy (RT) dose escalation in unresectable pancreatic adenocarcinoma (PAC) remains investigational. We examined the association between total RT dose and overall survival (OS) in patients with unresectable PAC.nnnMETHODS AND MATERIALSnNational cancer data base (NCDB) data were obtained for patients who underwent definitive chemotherapy and RT (chemo-RT) for unresectable PAC. Univariate (UV) and multivariate (MV) survival analysis were performed along with Kaplan-Meier (KM) estimates for incremental RT dose levels.nnnRESULTSnA total of 977 analyzable patients met inclusion criteria. Median tumor size was 4.0 cm (0.3-40 cm) and median RT dose was 45 Gy. Median OS was 10 months (95% CI, 9-10 months). On MV analysis RT dose <30 Gy [HR, 2.38 (95% CI, 1.85-3.07); P<0.001] and RT dose ≥30 to <40 Gy [HR, 1.41 (95% CI, 1.04-1.91); P=0.026] were associated with lower OS when compared with dose ≥55 Gy. Patients receiving RT doses from 40 to <45, 45 to <50, 50 to <55, and ≥55 Gy did not differ in OS.nnnCONCLUSIONSnLack of benefit to OS with conventionally delivered RT above 40 Gy is shown. Optimal RT dose escalation methods in unresectable PAC remain an important subject for investigation in prospective clinical trials.

Abstract 1767: A synthetic lethal screen identifies determinants of gemcitabine sensitivity in pancreatic cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnPurpose/Objectives:nnPancreatic cancer is notorious for its devastating disease course and prognosis. Gemcitabine is a widely used regimen for pancreatic cancer treatment but lack of response and resistance often limits its effectiveness. Thus, a better understanding of which patients are likely to respond to gemcitabine treatment would allow the personalization of therapies that are most effective for a patient while potentially reducing toxicity. The objective of this study was to identify the genes and mechanisms involved in determining gemcitabine sensitivity in pancreatic cancer.nnMaterials/Methods:nnWe completed a loss of function genetic screen to identify genes, which when silenced cause sensitization of resistance to a low dose of gemcitabine in human pancreatic cancer cells. We optimized a high-throughput assay using ATR or CHK1 siRNA as positive controls and ATM or non-targeting (NT) siRNA as negative controls. Our siRNA library included 4,474 siRNAs corresponding to 1,006 unique human genes arrayed in a one-gene:one-well format in 96-well plates. Genes chosen for our library consisted predominantly of nuclear enzymes, which we reasoned were more likely to function directly in DNA repair processes and be targetable. Mia PaCa-2 cells were transfected with 25 nM siRNA and treated 48 hours later with or without 13 nM gemcitabine for 72 hours prior to assaying for cell proliferation using WST-1 reagent. Candidate genes were deconvoluted with individual siRNAs to eliminate off-target effects and validated by secondary screens for cell cycle recovery after a challenge of hydroxyurea (HU) and γH2AX phosphorylation in the absence of exogenous damage following gene silencing. A rigorous statistical algorithm was used to determine positive hits. Genes with variable expression in pancreatic cancer tissue samples were identified by mining through published data sets.nnResults:nnWe identified 49 genes in which at least 2 unique siRNAs yielded gemcitabine sensitization and 17 genes in which at least 2 unique siRNAs yielded gemcitabine resistance. Positive hits included 25 known genome maintenance genes, including well characterized ATR signaling pathway genes CHK1, RAD9, HUS1, and CDC25A, 27 putative ATM/ATR substrates, and 26 genes identified in previously published DNA damage sensitivity screens. Eight of our genes are above the 90th percentile in variability of expression amongst a panel of pancreatic cancer tissue samples.nnConclusion:nnWe identified gemcitabine sensitization and resistance genes that are variably expressed in pancreatic cancer, which may function as novel targets or biomarkers for individualizing treatment for patients with pancreatic cancer.nnCitation Format: Matthew D. Warren, Claire W. Hardy, Brooke G. Pantazides, Khanjan Gandhi, Jerome C. Landry, Joseph W. Shelton, Shishir K. Maithel, Bassel El-Rayes, Jeanne Kowalski, David S. Yu. A synthetic lethal screen identifies determinants of gemcitabine sensitivity in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1767. doi:10.1158/1538-7445.AM2013-1767