Proficient Cells Research Articles

C-Myc Quadruplex-forming Sequence Pu-27 Induces Extensive Damage in Both Telomeric and Nontelomeric Regions of DNA

Quadruplex-forming DNA sequences are present throughout the eukaryotic genome, including in telomeric DNA. We have shown that the c-Myc promoter quadruplex-forming sequence Pu-27 selectively kills transformed cells (Sedoris, K. C., Thomas, S. D., Clarkson, C. R., Muench, D., Islam, A., Singh, R., and Miller, D. M. (2012) Genomic c-Myc quadruplex DNA selectively kills leukemia. Mol. Cancer Ther. 11, 66-76). In this study, we show that Pu-27 induces profound DNA damage, resulting in striking chromosomal abnormalities in the form of chromatid or chromosomal breaks, radial formation, and telomeric DNA loss, which induces γ-H2AX in U937 cells. Pu-27 down-regulates telomeric shelterin proteins, DNA damage response mediators (RAD17 and RAD50), double-stranded break repair molecule 53BP1, G2 checkpoint regulators (CHK1 and CHK2), and anti-apoptosis gene survivin. Interestingly, there are no changes of DNA repair molecules H2AX, BRCA1, and the telomere maintenance gene, hTERT. ΔB-U937, where U937 cells stably transfected with deleted basic domain of TRF2 is partially sensitive to Pu-27 but exhibits no changes in expression of shelterin proteins. However, there is an up-regulation of CHK1, CHK2, H2AX, BRCA1, and survivin. Telomere dysfunction-induced foci assay revealed co-association of TRF1with γ-H2AX in ATM deficient cells, which are differentially sensitive to Pu-27 than ATM proficient cells. Alt (alternating lengthening of telomere) cells are relatively resistant to Pu-27, but there are no significant changes of telomerase activity in both Alt and non-Alt cells. Lastly, we show that this Pu-27-mediated sensitivity is p53-independent. The data therefore support two conclusions. First, Pu-27 induces DNA damage within both telomeric and nontelomeric regions of the genome. Second, Pu-27-mediated telomeric damage is due, at least in part, to compromise of the telomeric shelterin protein complex.

Reduced migration of MLH1 deficient colon cancer cells depends on SPTAN1

IntroductionDefects in the DNA mismatch repair (MMR) protein MLH1 are frequently observed in sporadic and hereditary colorectal cancers (CRC). Affected tumors generate much less metastatic potential than the MLH1 proficient forms. Although MLH1 has been shown to be not only involved in postreplicative MMR but also in several MMR independent processes like cytoskeletal organization, the connection between MLH1 and metastasis remains unclear. We recently identified non-erythroid spectrin αII (SPTAN1), a scaffolding protein involved in cell adhesion and motility, to interact with MLH1. In the current study, the interaction of MLH1 and SPTAN1 and its potential consequences for CRC metastasis was evaluated.MethodsNine cancer cell lines as well as fresh and paraffin embedded colon cancer tissue from 12 patients were used in gene expression studies of SPTAN1 and MLH1. Co-expression of SPTAN1 and MLH1 was analyzed by siRNA knock down of MLH1 in HeLa, HEK293, MLH1 positive HCT116, SW480 and LoVo cells. Effects on cellular motility were determined in MLH1 deficient HCT116 and MLH1 deficient HEK293T compared to their MLH1 proficient sister cells, respectively.ResultsMLH1 deficiency is clearly associated with SPTAN1 reduction. Moreover, siRNA knock down of MLH1 decreased the mRNA level of SPTAN1 in HeLa, HEK293 as well as in MLH1 positive HCT116 cells, which indicates a co-expression of SPTAN1 by MLH1. In addition, cellular motility of MLH1 deficient HCT116 and MLH1 deficient HEK293T cells was impaired compared to the MLH1 proficient sister clones. Consequently, overexpression of SPTAN1 increased migration of MLH1 deficient cells while knock down of SPTAN1 decreased cellular mobility of MLH1 proficient cells, indicating SPTAN1-dependent migration ability.ConclusionsThese data suggest that SPTAN1 levels decreased in concordance with MLH1 reduction and impaired cellular mobility in MLH1 deficient colon cancer cells. Therefore, aggressiveness of MLH1-positive CRC might be related to SPTAN1.

Atorvastatin represses the angiotensin 2-induced oxidative stress and inflammatory response in dendritic cells via the PI3K/Akt/Nrf 2 pathway.

Dendritic cells (DCs), which are highly proficient antigen-presenting cells, play a complex role in both the initiation and progression of atherosclerosis. We tested the hypothesis that the anti-inflammatory and antioxidant effects of atorvastatin may be partly mediated by the phosphatidylinositol 3-kinase/protein kinase B/transcription factor nuclear factor-erythroid 2-related factor 2 (PI3K/Akt/Nrf 2) pathway via the attenuation of DC maturation, thus reducing the inflammatory and oxidative stress responses. This study showed that angiotensin 2 (Ang 2) induced the maturation of DCs, stimulated CD83, CD40, CD80, and CD86 expression, and increased the secretion of IL-12p70, IL-6, and TNF-α. These effects were suppressed by atorvastatin. Atorvastatin also lowered the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), counteracting their initial increases in response to Ang 2 stimulation. Atorvastatin activated Nrf 2 via the PI3K/Akt pathway and thereby promoted Nrf 2 translocation from the cytoplasm to the nucleus in bone marrow-derived dendritic cells (BMDCs), a process that was reversed by the PI3K inhibitor LY294002. Therefore, the regulation of Nrf 2 expression by the PI3K/Akt pathway plays an important role in the regulation of the statin-mediated antioxidant and anti-inflammatory responses in DCs.

Abstract P3-04-08: A proteo-probe for nucleotide excision repair in breast cancers

Abstract The response of tumor cells to DNA damaging agents is often compromised, a weakness readily exploited for therapeutic purposes. Despite the general use of genotoxic treatments, the nature and extent of DNA damage in tumor cells is generally under-investigated, or remains unknown. We postulate DNA repair deficient breast tumor cells exhibit significantly more DNA damage after genotoxic treatments, compared to repair proficient cells. We present a technology to detect specific DNA damage and monitor repair as a basis to categorize cancers into homogenous groups of malignancies. We rely on the use of biochemically purified DNA damage recognition complexes as ready-to-use reagents, or “proteo-probes” for DNA damage. We developed a proteo-probe based on a purified DDB2 protein complex. DDB2 is a DNA damage recognition protein part of the nucleotide excision repair (NER) that recognizes and repairs bulky DNA adducts caused for example by UV irradiation or cisplatin. We show the DDB2 proteo-probe binds to nuclei of UV-irradiated cells. We followed the DDB2 proteo-probe signal over time, and observed a decrease concordant with the completion of NER. We then successfully distinguished repair proficient cells from genetically characterized NER-deficient cells obtained from patients. We conclude the DDB2 proteo-probe can be used to detect activation of NER by UV-induced DNA damage, and subsequent repair in situ. It has been proposed that some breast cancers carry NER deficiencies. We screened a panel of cancer cell lines using the DDB2 proteo-probe and discovered obvious NER deficient (NER-D) cell lines. In addition, we correlated NER activity with cisplatin sensitivity. We further determined whether NER deficiency in breast cancer cell lines was caused by loss-of-function of NER genes through complementation. The classification of breast cancer cell lines based on NER activity presented here suggests more investigation is needed about NER in breast cancers. Ultimately, we envision NER deficiencies could be used to select breast cancer patients for DNA-repair targeted therapy. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-04-08.

Synergistic Cytotoxic Effect Of The PARP Inhibitor ABT888 and Busulfan In Acute Leukemia Cell Lines

The alkylator Busulfan (Bu) is used in conditioning regimens for acute leukemia prior to allogeneic stem cell transplant. Multiple DNA repair mechanisms including mismatch repair and base excision repair have been implicated in resistance to Bu. The enzyme PARP is central to base excision repair. We hypothesized that treatment of acute leukemia cell lines with both ABT888 (Veliparib), an inhibitor of PARP 1 and 2, and Bu would lead to synergistic cell kill and that this effect is maximal in mismatch repair deficient cells.Two mismatch repair proficient cell lines (K562 and HL60) and 2 mismatch repair deficient cell lines (NB4 and REH) were treated with ABT888 alone, Bu alone or a combination of both. In single drug experiments, doses of drug treatment ranged from 0-400mcg/ml. In combination experiments a fixed dose of ABT888 of 1.25mcg/ml was utilized with Bu doses varying from 0-200mcg/ml. This dose of ABT888 was chosen as it approximated to patient blood levels in clinical trials. After 24 hours of treatment, cells were washed and resuspended in fresh medium. Proliferation of cells was measured by standard 3H-thymidine uptake assay at 48 hours. Sigmoidal dose response curves and GI50 values were then calculated. In addition, cells were tested for apoptosis by flow cytometry using activated caspase 3 and annexin/ PI staining at 24 and 48 hours after treatment.All 4 cell lines were found to be resistant to single agent ABT888. Despite mismatch repair deficiency in REH cells, therapeutic doses of ABT888 did not cause significant decreases in proliferation. The effect of ABT888 was, as expected, much less evident in the mismatch repair proficient K562 cells. These cells were also relatively resistant to single agent Bu in vitro. The combination of Bu and ABT888 was synergistic in all cell lines with GI50 (micromoles/ml) for Bu decreasing from 67.8 to 45.7 in K562, from 23.3 to 8.0 in HL60, from 46.6 to 36.1 in NB4 and from 34.4 to 17.0 in REH cells. The Combination Index was <1 in all cell lines indicating synergy. Dose Reduction Index, indicating the factor by which the dose of Bu can be decreased to achieve the same treatment effect size, ranged from 1.45 to 3.1. The synergistic effect was greatest in the mismatch repair deficient cell line REH (Combination Index 0.53, Dose Reduction Index 3.1). As expected, the synergistic effect observed did not correlate with increased apoptotic death of leukemic cells implying an alternative mechanism for the derease in proliferation.To our knowledge, this is the first study to show synergy of a clinically available PARP inhibitor with Bu. We believe this data warrants further study with the potential clinical application of increasing the antileukemic effect of stem cell transplantation conditioned with Bu containing preparative regimens. Disclosures:No relevant conflicts of interest to declare.

Improved Efficacy of Acylfulvene in Colon Cancer Cells When Combined with a Nuclear Excision Repair Inhibitor

The efficacy of DNA-damaging anticancer drugs is highly influenced by cellular DNA repair capacity, and by inhibiting the relevant DNA repair pathway, efficacy of alkylating agents may be increased. Therefore, combining DNA repair inhibitors with anticancer agents that selectively target tumor tissue should improve cancer treatment. The objective of this study was to test the hypothesis that cotreatment of cancer cells with acylfulvene (AF, alkylating agent) and UCN-01 (DNA repair inhibitor) would improve drug efficacy and promote the persistence of DNA adducts. Previous data regarding the relative susceptibility of repair proficient versus deficient cells toward an AF analogue suggests that corresponding adducts are repaired by nuclear excision repair (NER), a cellular process that has been shown to be prevented with UCN-01. In this study, cells were cotreated with nontoxic levels of UCN-01 together with increasing doses of AF. The efficacy of AF was assessed by measuring cytotoxicity and DNA adducts. In addition, cells were cotreated with nontoxic levels of methoxyamine, a known base excision repair (BER) inhibitor, to determine if inhibiting BER also promotes cytotoxicity of AF. DNA-adducts were measured in a sensitive and precise manner by using stable isotope-labeled mass spectrometry analysis. The data obtained in this study demonstrate for the first time that pharmacological inhibition of the NER pathway of DNA repair leads to the persistence of AF-specific adducts and promotes AF cytotoxicity.

Abstract B210: Novel inhibitors of nicotinamide phosphoribosyl transferase and their evaluation in combination with bortezomib.

Abstract Nicotinamide phosphoribosyl transferase (NAMPT) is the enzyme that catalyzes the rate limiting step in the salvage pathway of Nicotinamide Adenine Dinucleotide (NAD) biosynthesis. NAMPT is reported to be overexpressed in a number of cancer and inflammatory indications. Because of the requirement of NAD for a number of key biochemical pathways, inhibition of NAMPT has been shown to result in antitumor efficacy in preclinical models. NAMPT inhibitors have also been reported to enhance sensitivity to a number of targeted agents and overcome resistance to available therapies such as bortezomib resistance in multiple myeloma. In view of the sub-optimal profile of the reported first generation NAMPT inhibitors with respect to pharmacokinetics and drug-drug interaction, we sought to develop NAMPT inhibitors with the "best-in-class" profile for overcoming mechanism-based toxicities and/or resistance to current therapies. Utilizing structure-guided drug design and SAR-based approaches, we have optimized two chemical series of inhibitors of NAMPT. Determination of co-crystal structures with several de novo designed hits greatly aided in the identification of lead compounds that exhibited potent inhibition of NAMPT. Lead compounds were highly active in inhibiting proliferation that correlated well with cellular NAD depletion of cell lines derived from multiple myeloma, prostate and breast cancers. The anti-proliferative activities were rescued in NAPRT- proficient cell lines with the addition of NA due to the NAMPT independent salvage pathway for biosynthesis of NAD, confirming the mechanism of action through NAD depletion. Lead compounds exhibited desirable drug-like properties including solubility, permeability, metabolic stability, lack of CYP inhibition and pharmacokinetic exposure. In a xenograft model of pancreatic cancer, treatment with lead compounds resulted in regression of tumors with no signs of toxicity. Recent reports demonstrating the overexpression of NAMPT in bortezomib-resistant cells and a synergistic efficacy with a combination of NAMPT inhibitor and bortezomib against the resistant cells prompted us to evaluate the combination with our optimized leads. The combination of lead NAMPT inhibitors with bortezomib showed synergistic killing of cultured multiple myeloma cell lines. Evaluation of efficacy of the lead compounds as a single agent or in combination with bortezomib in xenograft models of multiple myeloma is currently underway. Modulation of NAD levels at lower doses of NAMPT inhibitors in combination with bortezomib could overcome the limitations such as mechanism-based toxicity and/or resistance of both these therapies and provide an effective treatment option for multiple myeloma. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B210. Citation Format: Dinesh Chikkanna, Anirudha Lakshminarasimhan, Vinayak Khairnar, Sunil Panigrahi, Anuradha Ramanathan, Aparna Satyanandan, Narasimha Rao, S Karthikeyan, Kishore Narayanan, Sreevalsam Gopinath, Raghuveer Ramachandra, Hosahalli Subramanya, Chetan Pandit, Murali Ramachandra. Novel inhibitors of nicotinamide phosphoribosyl transferase and their evaluation in combination with bortezomib. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B210.

Effect of zinc oxide nanomaterials-induced oxidative stress on the p53 pathway

Excessive production of reactive oxygen species (ROS) is a hallmark feature in nanomaterials (NMs) induced cellular toxicity. However, the inter-relationship between NMs induced ROS generation and the cells innate ability to regulate intracellular ROS level in effecting a particular cellular outcome is currently underexplored. Here, using a BJ fibroblast p53 knockdown system, we showed that p53 may be implicated in playing a dual regulatory role to determine cell survivability in response to oxidative stress induced by ZnO NMs. At low level of ZnO NMs induced ROS, p53 triggers expression of antioxidant genes such as SOD2, GPX1, SESN1, SESN2 and ALDH4A1 to restore oxidative homeostasis while at high concentration of ZnO NMs, the elevated level of intracellular ROS activated the apoptotic pathway through p53. The implication of our finding that p53 can function as an important regulator in determining ZnO induced cytotoxicity is highlighted by the differential action of ZnO on p53 deficient and proficient colorectal cell lines. p53 deficient cells cancer cells such as DLD-1 and SW480 are more susceptible to ZnO induced cell death compared to p53 proficient cells such as colon epithelial cells NCM460 and HCT116 cells in a ROS dependent manner. Collectively, our findings showcased a role p53 plays in the context of nanotoxicity and highlights the need to consider the interplay of physicochemical properties of NMs and cell biology.

ATM is required for the repair of Topotecan-induced replication-associated double-strand breaks

PurposeDNA replication is a promising target for anti-cancer therapies. Therefore, the understanding of replication-associated DNA repair mechanisms is of great interest. One key factor of DNA double-strand break (DSB) repair is the PIK kinase Ataxia-Telangiectasia Mutated (ATM) but it is still unclear whether ATM is involved in the repair of replication-associated DSBs. Here, we focused on the involvement of ATM in homology-directed repair (HDR) of indirect DSBs associated with replication. Material and methodsExperiments were performed using ATM-deficient and -proficient human cells. Replication-associated DSBs were induced with Topotecan (TPT) and compared with γ-irradiation (IR). Cell survival was measured by clonogenic assay. Overall DSB repair and HDR were evaluated by detecting residual γH2AX/53BP1 and Rad51 foci, respectively. Cell cycle distribution was analysed by flow cytometry and protein expression by Western blot. ResultsATM-deficiency leads to enhanced numbers of residual DSBs, resulting in a pronounced S/G2-block and decreased survival upon TPT-treatment. In common with IR, persisting Rad51 foci were detected following TPT-treatment. ConclusionsThese results demonstrate that ATM is essentially required for the completion of HR-mediated repair of TPT-induced DSBs formed indirectly at replication forks.

Biological effects of simple changes in functionality on rhodium metalloinsertors

DNA mismatch repair (MMR) is crucial to ensuring the fidelity of the genome. The inability to correct single base mismatches leads to elevated mutation rates and carcinogenesis. Using metalloinsertors-bulky metal complexes that bind with high specificity to mismatched sites in the DNA duplex-our laboratory has adopted a new chemotherapeutic strategy through the selective targeting of MMR-deficient cells, that is, those that have a propensity for cancerous transformation. Rhodium metalloinsertors display inhibitory effects selectively in cells that are deficient in the MMR machinery, consistent with this strategy. However, a highly sensitive structure-function relationship is emerging with the development of new complexes that highlights the importance of subcellular localization. We have found that small structural modifications, for example a hydroxyl versus a methyl functional group, can yield profound differences in biological function. Despite similar binding affinities and selectivities for DNA mismatches, only one metalloinsertor shows selective inhibition of cellular proliferation in MMR-deficient versus -proficient cells. Studies of whole-cell, nuclear and mitochondrial uptake reveal that this selectivity depends upon targeting DNA mismatches in the cell nucleus.

Reactive oxygen species spermine metabolites generated from amine oxidases and radiation represent a therapeutic gain in cancer treatments

The most frequent interventions in cancer therapy are currently the destruction of cells by irradiation or administration of drugs both able to induce radical formation and toxic metabolites by enzyme-catalyzed reactions. The aim of this study was to determine the cell viability of cells undergoing a DNA damage threshold accomplished by ROS overproduction via both ectopic expression of murine spermine oxidase (mSMOX) and bovine serum amine oxidase (BSAO) enzymes. Low dose of X-irradiation delivers a challenging dose of damage as evaluated in proficient Chinese hamster AA8 cell line and both deficient transcription-coupled nucleotide excision repair (NER) UV61 cells and deficient base excision repair (BER) EM9 cells, at 6 and 24 h after exposure. The priming dose of ROS overexposure by mSMOX provokes an adaptive response in N18TG2, AA8 and EM9 cell lines at 24 h. Interestingly, in the UV61 cells, ROS overexposure by mSMOX delivers an earlier adaptive response to radiation. The enzymatic formation of toxic metabolites has mainly been investigated on wild-type (WT) and multidrug-resistant (MDR) cancer cell lines, using and spermine as substrate of the BSAO enzyme. MDR cells are more sensitive to the toxic polyamine metabolites than WT cells, thus indicating a new therapeutic strategy to overcome MDR tumors. Since SMOX in mammals is differentially activated in a tissue-specific manner and cancer cells can differ in terms of DNA repair and MDR capabilities, it could be of interest to simultaneously treat with very low dose of X-rays and/or to alter SMOX metabolism to generate a differential response in healthy and cancer tissues.

The Efficiency of Homologous Recombination and Non-Homologous End Joining Systems in Repairing Double-Strand Breaks during Cell Cycle Progression

This study investigated the efficiency of Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) repair systems in rejoining DNA double-strand breaks (DSB) induced in CCD-34Lu cells by different γ-ray doses. The kinetics of DNA repair was assessed by analyzing the fluorescence decrease of γ-H2AX foci measured by SOID (Sum Of Integrated Density) parameter and counting foci number in the time-interval 0.5–24 hours after irradiation. Comparison of the two methods showed that the SOID parameter was useful in determining the amount and the persistence of DNA damage signal after exposure to high or low doses of ionizing radiation. The efficiency of DSB rejoining during the cell cycle was assessed by distinguishing G1, S, and G2 phase cells on the basis of nuclear fluorescence of the CENP-F protein. Six hours after irradiation, γ-H2AX foci resolution was higher in G2 compared to G1 cells in which both NHEJ and HR can cooperate. The rejoining of γ-H2AX foci in G2 phase cells was, moreover, decreased by RI-1, the chemical inhibitor of HR, demonstrating that homologous recombination is at work early after irradiation. The relevance of HR in DSB repair was assessed in DNA-PK-deficient M059J cells and in CCD-34Lu treated with the DNA-PKcs inhibitor, NU7026. In both conditions, the kinetics of γ-H2AX demonstrated that DSBs repair was markedly affected when NHEJ was absent or impaired, even in G2 phase cells in which HR should be at work. The recruitment of RAD51 at DSB sites was, moreover, delayed in M059J and in NU7026 treated-CCD-34Lu, with respect to DNA-PKcs proficient cells and continued for 24 hours despite the decrease in DNA repair. The impairment of NHEJ affected the efficiency of the HR system and significantly decreased cell survival after ionizing radiation, confirming that DSB rejoining is strictly dependent on the integrity of the NHEJ repair system.

Characterization of Mutagenicity Test and Identification of N-Nitroso Mutagens in Some Soju Alcoholic Spirits

Excision repair after combined treatments of UV and N-acetoxy-2-acetylaminofluorene (AAAF) was studied by three different techniques in cells proficient in UV excision repair and in cells deficient in UV repair. Two patterns of repair were observed: A) in repair proficient cells total repair was additive, and B) in repair deficient cells total repair was much less than additive—usually less than observed for separate treatments—and AAAF inhibited dimer excision. We conclude that in the 1st class of cells pathways for repair of UV and AAAF lesions are not identical, and in the 2nd class the residual excision enzymes are different from those in repair proficient cells.

MSH3 Mismatch Repair Protein Regulates Sensitivity to Cytotoxic Drugs and a Histone Deacetylase Inhibitor in Human Colon Carcinoma Cells

Background MSH3 is a DNA mismatch repair (MMR) gene that undergoes frequent somatic mutation in colorectal cancers (CRCs) with MMR deficiency. MSH3, together with MSH2, forms the MutSβ heteroduplex that interacts with interstrand cross-links induced by drugs such as cisplatin. To date, the impact of MSH3 on chemosensitivity is unknown.MethodsWe utilized isogenic HCT116 (MLH1−/MSH3−) cells where MLH1 is restored by transfer of chromosome 3 (HCT116+ch3) and also MSH3 by chromosome 5 (HCT116+3+5). We generated HCT116+3+5, SW480 (MLH1+/MSH3+) and SW48 (MLH1−/MSH3+) cells with shRNA knockdown of MSH3. Cells were treated with 5-fluorouracil (5-FU), SN-38, oxaliplatin, or the histone deacetylase (HDAC) inhibitor PCI-24781 and cell viability, clonogenic survival, DNA damage and apoptosis were analyzed.Results MSH3-deficient vs proficient CRC cells showed increased sensitivity to the irinotecan metabolite SN-38 and to oxaliplatin, but not 5-FU, as shown in assays for apoptosis and clonogenic survival. In contrast, suppression of MLH1 attenuated the cytotoxic effect of 5-FU, but did not alter sensitivity to SN-38 or oxaliplatin. The impact of MSH3 knockdown on chemosensitivity to SN-38 and oxaliplatin was maintained independent of MLH1 status. In MSH3-deficient vs proficient cells, SN-38 and oxaliplatin induced higher levels of phosphorylated histone H2AX and Chk2, and similar results were found in MLH1-proficient SW480 cells. MSH3-deficient vs proficient cells showed increased 53BP1 nuclear foci after irradiation, suggesting that MSH3 can regulate DNA double strand break (DSB) repair. We then utilized PCI-24781 that interferes with homologous recombination (HR) indicated by a reduction in Rad51 expression. The addition of PCI-24781 to oxaliplatin enhanced cytotoxicity to a greater extent compared to either drug alone.ConclusionMSH3 status can regulate the DNA damage response and extent of apoptosis induced by chemotherapy. The ability of MSH3 to regulate chemosensitivity was independent of MLH1 status. PCI-24781-mediated impairment of HR enhanced oxaliplatin sensitivity, suggesting that reduced DSB repair capacity may be contributory.

Effect of PARP inhibitors on sensitivity of lung and breast cancer cells to trans-platinum as compared to cis-platinum.

e22115 Background: Poly(ADP-ribose) polymerase inhibitors (PARP-i) represent promising strategy in treatment of cisplatin resistant lung cancer and triple-negative breast cancer. We compared PARP inhibition by 3-aminobenzamide (3-AB) or NU1025, on activity of cis- and trans-platinum on NSCLC cells (A549) and breast cancer cells (MDA-MB-361), and correlated with p53 and ERCC1 status. Methods: Cell cycle distribution was determined by FACS-flow and PI-staining, while morphological changes were followed by light microscopy. ERCC1 siRNA transfection was performed by HiperFect reagent, and mRNA was analyzed by qRT-PCR. Protein expression levels were determined by Western blot. PARP-inhibition was quantified as NAD(P)H depletion using colorimetric XTT assay. Results: Trans-platinum compound with planar ammine ligands, trans-[PtCl2(4-acetylpyridine)2] showed cytotoxic activity comparable to cis-platin and exhibited enhanced action in combination with PARP-i. 3-AB enhanced the activity of trans more efficient comparing to cis-platinum in MDA-MB-361 cells. At equitoxic concentrations corresponding to IC50 values in A549 cells, cis-platin and trans-platinum compound differently affected cell survival in combination with PARP-i. XTT assay confirmed efficient inhibition of PARP activity at 200 μM NU1025 and 2 mM 3-AB in both cell lines. ERCC1 silencing in p53 proficient A549 cells, alone or in combination with NU1025, almost completely diminished resistance to trans-[PtCl2(4-acetylpyridine)2], decreasing cell survival to 50 % following 24 h action and more than 85 % following 48 h treatment, comparing to siRNA/NU1025 treated control. By contrast, sensitivity to cisplatin was much less altered. Simultaneous alteration of ERCC1 and PARP itself did not affect cell cycle progression, but prolonged sub-G1 fraction by trans-platinum as well as G2-M fraction by cis-platin. Conclusions: PARP-inhibition enhanced tumor cell sensitivity to trans-platinum drug, stronger than cis-platinum. Knockdown of ERCC1 further increases sensitivity to platinum drugs, irrelevant to p53 status. Trans-platinum complexes should be further explored as good candidates for combining with PARP-i.

Evaluation of flap endonuclease-1 (FEN1) as a prognostic, predictive, and therapeutic target in breast (BC) and ovarian cancer (OVC).

1016 Background: FEN1 is a multifunctional protein with essential roles in long patch base excision repair (LP-BER), Okazaki fragment maturation during replication, resolution of tri-nucleotide repeat sequence-derived secondary structures, rescue of stalled replication forks, maintenance of telomere stability and apoptotic fragmentation of DNA. In the current study we have evaluated FEN1 as a prognostic, predictive and therapeutic target in BC and OVC. Methods: Clinico-pathological significance of FEN1 mRNA expression was evaluated in a BC training cohort [n=128], test cohort (n=249) and validated in a large cohort of 1,980 BC (METABRIC). Neural network analysis (NNA) was conducted to identify FEN1 interaction genes. FEN1 protein expression was investigated in three consecutive series of BC and OVC: 568 ER negative (ER- BC), 894 ER positive (ER+ BC) and 195 OVC cohorts. Pre-clinically, FEN1 deficient and proficient HeLa cell lines were investigated for chemotherapy sensitization. A high throughput screening (HTS) strategy was developed to identify FEN1inhibitors for therapeutic application. Results: FEN1mRNA overexpression is associated with adverse clinicopathological features such as high grade, high mitotic index, and triple negative (ps&lt;0.0001). High FEN1 mRNA expression was highly significantly associated with PAM50 Her2, PAM50 Basal and PAM50 LumB (ps&lt;0.0001) BC. FEN1 mRNA overexpression is associated with resistance to chemotherapy (p=0.019), endocrine therapy (p&lt;0.0001) and independently with poor survival (p&lt;0.0001). NNA revealed novel interaction genes with predominant roles in proliferation, cell growth, DNA repair, differentiation, invasion, migration, metabolism and apoptosis. FEN1 protein overexpression is significantly associated with aggressive clinico-pathological features and independently with poor survivals in ER+ BC, ER- BC and in OVC (ps&lt;0.001). HTS assay has identified novel FEN1 inhibitors for therapeutic development. Conclusions: This is the first study to investigate FEN1 in BC and OVC. We provide confirmatory evidence that FEN1 is a promising biomarker as well as a therapeutic anti-cancer drug target for clinical application.

Can microsatellite status of colorectal cancer (CRC) be reliably assessed after neoadjuvant therapy?

1525 Background: Universal testing of resected CRC for Microsatellite Instability (MSI) status has been widely advocated. While PCR-based MSI analysis is the current gold standard test, immunohistochemistry (IHC) of mismatch repair (MMR) proteins is more commonly performed. At present, it remains un-established whether PCR-based MSI analysis remains reliable when performed on post-neoadjuvant treated specimens. Our goal was to examine the effect of neoadjuvant therapy on MSI analysis using both in vitro and clinical data. Methods: MSI analysis was performed using the standard panel of 7 markers. In vitro, MMR deficient (HCT116) and proficient (HCT116+Ch3) isogenic cell lines were exposed to 3 cycles of single agent 5-Fluorouracil, Oxaliplatin and Irinotecan and MSI analysis was tested after each cycle. To evaluate the induction of genomic instability, 3 additional coding microsatellite tracts in ATR, BLM and CHK1 were assessed by fragment analysis. In parallel, clinical data from 238 CRCs that were resected after neoadjuvant chemoradiation (n=191) or chemotherapy (n=47) between 9/2009 and 8/2011 were queried for MSI results. MSI analysis performed on paired pre-treatment and post-treatment resection tissues was compared. Results: No changes in MSI status were detected and there was no increase in genomic instability post-treatment in cell line models. In the clinical setting, pre-neoadjuvant MSI testing was limited by the availability of pre-treatment biopsy tissue. 3 patients found to be MMR proficient based on MSI analysis performed on pre-treatment biopsy remained microsatellite stable when analyzed on post-treatment resection tissue. 7 patients were MMR deficient based on MSI analysis performed on post-treatment resection tissue but most had strong clinical evidence to expect MMR deficiency. Conclusions: Based on both preclinical in vitro and clinical data, results of the MSI analysis does not appear to be affected by neoadjuvant therapy for both MMR proficient and deficient cases. In addition, preclinical results do not suggest that neoadjuvant chemoradiation induces an increase in genomic instability. Therefore, MSI analysis remains a reliable gold standard test on post-treatment specimens.

Abstract 5389: Novel inhibitors of nicotinamide phosphoribosyl transferase (NAMPT).

Abstract Nicotinamide phosphoribosyl transferase (NAMPT) is the enzyme that catalyzes the rate limiting step in the salvage pathway of Nicotinamide Adenine Dinucleotide (NAD) biosynthesis. NAMPT is reported to be overexpressed in a number of cancer and inflammatory indications. Because of the requirement of NAD as a co-factor or substrate for a number of key biochemical pathways including those catalyzed by PARP1, Sirtuins and ADP-ribosyl cyclase, inhibition of NAMPT has been shown to result in anti-tumor efficacy in preclinical models. Two NAMPT Inhibitors FK866/APO866 and GMX1778 are currently in clinical trials for oncology indications. In the presence of these clinical agents, cultured cell lines show development of resistance due to mutations underscoring the potential need for inhibitors from distinct chemical series. Here, we report a structure-guided drug design based approach for identification of lead compounds from two chemical series selectively targeting NAMPT. Determination of co-crystal structures with several de novo designed hits greatly aided in the identification of lead compounds that exhibited potent inhibition of NAMPT against both wild type and resistance mutants (G217R and H191R) Lead compounds were highly active in inhibiting proliferation that correlated well with cellular NAD depletion in several cancer cell lines. Normal cells and selected cancer cell lines have an NAMPT independent salvage pathway for biosynthesis of NAD, which is dependent on nicotinic acid phosphoribosyltransferase (NAPRT) and Nicotinic acid (NA). The anti-proliferative activities were fully rescued in NAPRT- proficient cell lines with the addition of NA, confirming the mechanism of action through specific NAD depletion. Lead compounds from both series exhibited excellent drug-like properties including solubility, metabolic stability and permeability, and desired exposure in pharmacokinetic studies. Anti-tumor activities of these compounds including NA rescue in NAPRT-proficient tumor models are currently being evaluated in preclinical models. Citation Format: Murali Ramachandra, Chetan Pandit, Hosahalli Subramanya, Dinesh Chikkanna, Anirudha Lakshminarasimhan, Vinayak Khairnar, Sunil Panigrahi, Anuradha Ramanathan, Aparna Satyanandan, Narasimha Rao, Arnab Bera, Kishore Narayanan, Sreevalsam Gopinath, Raghuveer Ramachandra. Novel inhibitors of nicotinamide phosphoribosyl transferase (NAMPT). [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 5389. doi:10.1158/1538-7445.AM2013-5389

Targeting XRCC1 Deficiency in Breast Cancer for Personalized Therapy

XRCC1 is a key component of DNA base excision repair, single strand break repair, and backup nonhomologous end-joining pathway. XRCC1 (X-ray repair cross-complementing gene 1) deficiency promotes genomic instability, increases cancer risk, and may have clinical application in breast cancer. We investigated XRCC1 expression in early breast cancers (n = 1,297) and validated in an independent cohort of estrogen receptor (ER)-α-negative breast cancers (n = 281). Preclinically, we evaluated XRCC1-deficient and -proficient Chinese hamster and human cancer cells for synthetic lethality application using double-strand break (DSB) repair inhibitors [KU55933 (ataxia telangectasia-mutated; ATM inhibitor) and NU7441 (DNA-PKcs inhibitor)]. In breast cancer, loss of XRCC1 (16%) was associated with high grade (P < 0.0001), loss of hormone receptors (P < 0.0001), triple-negative (P < 0.0001), and basal-like phenotypes (P = 0.001). Loss of XRCC1 was associated with a two-fold increase in risk of death (P < 0.0001) and independently with poor outcome (P < 0.0001). Preclinically, KU55933 [2-(4-Morpholinyl)-6-(1-thianthrenyl)-4H-pyran-4-one] and NU7441 [8-(4-Dibenzothienyl)-2-(4-morpholinyl)-4H-1-benzopyran-4-one] were synthetically lethal in XRCC1-deficient compared with proficient cells as evidenced by hypersensitivity to DSB repair inhibitors, accumulation of DNA DSBs, G2-M cell-cycle arrest, and induction of apoptosis. This is the first study to show that XRCC1 deficiency in breast cancer results in an aggressive phenotype and that XRCC1 deficiency could also be exploited for a novel synthetic lethality application using DSB repair inhibitors. Cancer Res; 73(5); 1621-34. ©2012 AACR.

Ataxia telangiectasia mutated and Rad3 related (ATR) protein kinase inhibition is synthetically lethal in XRCC1 deficient ovarian cancer cells.

IntroductionAtaxia telangiectasia mutated and Rad3 Related (ATR) protein kinase is a key sensor of single-stranded DNA associated with stalled replication forks and repair intermediates generated during DNA repair. XRCC1 is a critical enzyme in single strand break repair and base excision repair. XRCC1-LIG3 complex is also an important contributor to the ligation step of the nucleotide excision repair response.MethodsIn the current study, we investigated synthetic lethality in XRCC1 deficient and XRCC1 proficient Chinese Hamster ovary (CHO) and human ovarian cancer cells using ATR inhibitors (NU6027). In addition, we also investigated the ability of ATR inhibitors to potentiate cisplatin cytotoxicity in XRCC1 deficient and XRCC1 proficient CHO and human cancer cells. Clonogenic assays, alkaline COMET assays, γH2AX immunocytochemistry, FACS for cell cycle as well as FITC-annexin V flow cytometric analysis were performed.ResultsATR inhibition is synthetically lethal in XRCC1 deficient cells as evidenced by increased cytotoxicity, accumulation of double strand DNA breaks, G2/M cell cycle arrest and increased apoptosis. Compared to cisplatin alone, combination of cisplatin and ATR inhibitor results in enhanced cytotoxicity in XRCC1 deficient cells compared to XRCC1 proficient cells.ConclusionsOur data provides evidence that ATR inhibition is suitable for synthetic lethality application and cisplatin chemopotentiation in XRCC1 deficient ovarian cancer cells.