Proficient Cells Research Articles

UCHL1 Is a New Therapeutic Target in Lymphoid Malignancies, Independent of ATM and TP53 status

Functional defects in principal DNA damage response genes ATM or TP53 are frequent events in pathogenesis of lymphoid malignancies and lead to unfavorable clinical course coupled with accumulation of unrepaired DNA damage and acquisition of genomic instability. Options for management of these tumours are currently limited mostly due to their inability to activate ATM/p53 apoptotic pathway in response to chemotherapy. Consequently, novel therapies that can overcome ATM/p53 apoptotic defect are needed.Deubiquitylation proteases (DUBs) are emerging as key regulators of the DNA damage responses (DDR). Through removal of ubiquitin, DUBs can modify protein stability, cellular localisation and activity of components of the DDR.Ubiquitin carboxy-terminal esterase L1 (UCHL1) is a DUB enzyme recently implicated in regulation of p53 stability, pathogenesis of lymphoid tumours and development of metastasis. Here we addressed whether UCHL1 is a valid therapeutic target in lymphoid malignancies with frequent ATM or p53 loss such as chronic lymphocytic leukaemia (CLL) and multiple myeloma (MM).Microarray data from 226 CLL tumour samples showed that 30% of primary CLL tumours exhibit a high expression of UCHL1 mRNA, particularly in response to DNA double strand break inducing agents. Furthermore, six of nine Multiple myeloma cell lines showed high levels of expression, whereas UCHL1 protein could not be detected in PBMC obtained from healthy controls. We determined the cytotoxic effect of the UCHL1 inhibitor, MTX082554, provided by 'MISSION Therapeutics'' in a panel of CLL and MM cell lines as well as primary CLL tumours with different ATM and TP53 status. We observed a strong cytotoxic effect of UCHL1 inhibition in all tested cell lines and primary CLL tumours. EC50 varied for CLL cell lines between 0.1 and 0.2µM, for MM cell lines between 0.05 and 0.1 µM and for primary CLL tumours between 0.2 and 3.1µM. Viability of primary CLL tumours were reduced upon exposure to MTX082554 irrespective of whether the cells were induced to cycle or not, and both ATM/TP53 proficient and mutant cells showed a dramatic loss of viability following exposure to micro molar concentrations of MTX082554. Finally we determined a gene expression signature associated with the high sensitivity of primary CLL cells to UCHL1 inhibition.Western blot analysis of CLL primaries treated with MTX082554 revealed a rise in p53 and caspase cleavage. However given the absence of further indicators of p53 activation - minimal p53 phosphorylation and p21 induction - and comparable sensitivity of p53 mutant and wild type tumours this caspase activation is believed to represent p53-independent apoptosis.Comet assays performed on CLL primaries treated with MTX082554 showed an increase in DNA strand breaks. This finding was supported by Western blotting showing an increase in the marker of double strand breaks, γH2AX. Cell cycle profiling suggested the presence of G2/M arrest. We conclude that the potent cytotoxic effect of UCHL1 inhibition observed in CLL and MM cells is caused by the combined effects of DDR inhibition and p53-independent apoptosis.In summary, we suggest that UCHL1 is a novel therapeutic target in lymphoid malignancies and that inhibition of this molecule represent a new strategy for tumours with a defect in ATM/p53 pathways. DisclosuresHarrigan:Mission Therapeutics: Employment. Jacq:Mission Therapeutics: Employment. Off Label Use: UCHL1 inhibitor, MTX082554.

Suppression of Homologous Recombination by insulin-like growth factor-1 inhibition sensitizes cancer cells to PARP inhibitors.

BackgroundImpairment of homologous recombination (HR) is found in close to 50 % of ovarian and breast cancer. Tumors with BRCA1 mutations show increased expression of the Insulin-like growth factor type 1 receptor (IGF-1R). We previously have shown that inhibition of IGF-1R results in growth inhibition and apoptosis of ovarian tumor cells. In the current study, we aimed to investigate the correlation between HR and sensitivity to IGF-1R inhibition. Further, we hypothesized that IGF-1R inhibition might sensitize HR proficient cancers to Poly ADP ribose polymerase (PARP) inhibitors.MethodsUsing ovarian and breast cancer cellular models with known BRCA1 status, we evaluated their HR functionality by RAD51 foci formation assay. The 50 % lethal concentration (LC50) of Insulin-like growth factor type 1 receptor kinase inhibitor (IGF-1Rki) in these cells was assessed, and western immunoblotting was performed to determine the expression of proteins involved in the IGF-1R pathway. Moreover, IGF-1R inhibitors were added on HR proficient cell lines to assess mRNA and protein expression of RAD51 by qPCR and western blot. Also, we explored the interaction between RAD51 and Insulin receptor substance 1 (IRS-1) by immunoprecipitation. Next, combination effect of IGF-1R and PARP inhibitors was evaluated by clonogenic assay.ResultsCells with mutated/methylated BRCA1 showed an impaired HR function, and had an overactivation of the IGF-1R pathway. These cells were more sensitive to IGF-1R inhibition compared to HR proficient cells. In addition, the IGF-IR inhibitor reduced RAD51 expression at mRNA and protein levels in HR proficient cells, and sensitized these cells to PARP inhibitor.ConclusionTargeting IGF-1R might lead to improved personalized therapeutic approaches in cancer patients with HR deficiency. Targeting both PARP and IGF-1R might increase the clinical efficacy in HR deficient patients and increase the population of patients who may benefit from PARP inhibitors.

Role of mismatch repair proteins in the processing of cisplatin interstrand cross-links

Mismatch repair (MMR) deficiency gives rise to cisplatin resistance and can lead to poor prognosis in cancers. Various models have been proposed to explain this low level of resistance caused due to loss of MMR proteins. We have shown that MMR proteins are required to maintain cisplatin interstrand cross-links (ICLs) on the DNA leading to increased cellular sensitivity. In our previous studies, we have shown that BER processing of the cisplatin ICLs is mutagenic. Polymerase β (Polβ) can generate mismatches which leads to the activation and the recruitment of mismatch repair proteins. In this paper, we distinguished between the requirement of different downstream MMR proteins for maintaining cisplatin sensitivity. We show that the MutSα (MSH2–MSH6) heterocomplex is required to maintain cisplatin sensitivity, whereas the Mutsβ complex has no effect. These results can be correlated with the increased repair of cisplatin ICLs and ICL induced DNA double strand breaks (DSBs) in the resistant cells. Moreover, we show that MLH1 proficient cells displayed a cisplatin sensitive phenotype when compared with the MLH1 deficient cells and the ATPase activity of MLH1 is essential to mediate this effect. Based on these results, we propose that MutSα as well as the downstream MMR pathway proteins are essential to maintain a cisplatin sensitive phenotype as a consequence of processing Polβ induced mismatches at sites flanking cisplatin ICLs.

Identification of novel, in vivo active Chk1 inhibitors utilizing structure guided drug design.

Chk1 kinase is a critical component of the DNA damage response checkpoint especially in cancer cells and targeting Chk1 is a potential therapeutic opportunity for potentiating the anti-tumor activity of DNA damaging chemotherapy drugs. Fragment elaboration by structure guided design was utilized to identify and develop a novel series of Chk1 inhibitors culminating in the identification of V158411, a potent ATP-competitive inhibitor of the Chk1 and Chk2 kinases. V158411 abrogated gemcitabine and camptothecin induced cell cycle checkpoints, resulting in the expected modulation of cell cycle proteins and increased cell death in cancer cells. V158411 potentiated the cytotoxicity of gemcitabine, cisplatin, SN38 and camptothecin in a variety of p53 deficient human tumor cell lines in vitro, p53 proficient cells were unaffected. In nude mice, V158411 showed minimal toxicity as a single agent and in combination with irinotecan. In tumor bearing animals, V158411 was detected at high levels in the tumor with a long elimination half-life; no pharmacologically significant in vivo drug-drug interactions with irinotecan were identified through analysis of the pharmacokinetic profiles. V158411 potentiated the anti-tumor activity of irinotecan in a variety of human colon tumor xenograft models without additional systemic toxicity. These results demonstrate the opportunity for combining V158411 with standard of care chemotherapeutic agents to potentiate the therapeutic efficacy of these agents without increasing their toxicity to normal cells. Thus, V158411 would warrant further clinical evaluation.

The in vitro immunogenic potential of caspase-3 proficient breast cancer cells with basal low immunogenicity is increased by hypofractionated irradiation.

BackgroundRadiotherapy is an integral part of breast cancer treatment. Immune activating properties of especially hypofractionated irradiation are in the spotlight of clinicians, besides the well-known effects of radiotherapy on cell cycle and the reduction of the clonogenic potential of tumor cells. Especially combination of radiotherapy with further immune stimulation induces immune-mediated anti-tumor responses. We therefore examined whether hypofractionated irradiation alone or in combination with hyperthermia as immune stimulants is capable of inducing breast cancer cells with immunogenic potential.MethodsClonogenic assay, AnnexinA5-FITC/Propidium iodide assay and ELISA analyses of heat shock protein 70 and high mobility group box 1 protein were applied to characterize colony forming capability, cell death induction, cell death forms and release of danger signals by breast cancer cells in response to hypofractionated radiation (4x4Gy, 6x3Gy) alone and in combination with hyperthermia (41.5 °C for 1 h). Caspase-3 deficient, hormone receptor positive, p53 wild type MCF-7 and caspase-3 intact, hormone receptor negative, p53 mutated MDA-MB231 breast cancer cells, the latter in absence or presence of the pan-caspase inhibitor zVAD-fmk, were used. Supernatants of the treated tumor cells were analyzed for their potential to alter the surface expression of activation markers on human-monocyte-derived dendritic cells.ResultsIrradiation reduced the clonogenicity of caspase deficient MCF-7 cells more than of MDA-B231 cells. In contrast, higher amounts of apoptotic and necrotic cells were induced in MDA-B231 cells after single irradiation with 4Gy, 10Gy, or 20Gy or after hypofractionated irradiation with 4x4Gy or 6x3Gy. MDA-B231 cells consecutively released higher amounts of Hsp70 and HMGB1 after hypofractionated irradiation. However, only the release of Hsp70 was further increased by hyperthermia. Both, apoptosis induction and release of the danger signals, was dependent on caspase-3. Only supernatants of MDA-B231 cells after hypofractionated irradiation resulted in slight changes of activation markers on dendritic cells; especially that of CD86 was upregulated and HT did not further impact on it.ConclusionsHypofractionated irradiation is the main stimulus for cell death induction and consecutive dendritic cell activation in caspase proficient breast cancer cells. For the assessment of radiosensitivity and immunological effects of radio- and immunotherapies the readout system is crucial.Electronic supplementary materialThe online version of this article (doi:10.1186/s13014-015-0506-5) contains supplementary material, which is available to authorized users.

Abstract 673: Vulnerability of LKB1 deficient NSCLC to C8-modified adenosine analogs is associated with diminished autophagy induction

Abstract The C8-modified adenosine analogs, 8-chloroadenosine (8-Cl-Ado) and 8-aminoadenosine (8-NH2-Ado) are cytotoxic to a number of liquid and solid tumor cells. Compared with nucleoside analogs established for cancer therapeutics, these analogs are unique as they possess a ribose sugar. These distinctive analogs deplete cellular ATP and are RNA directed and 8-Cl-Ado, is currently in a phase I clinical trial for hematological malignancies. Previously, using a screen of non-small cell lung cancer (NSCLC) cell lines for growth sensitivity against a panel of therapeutic agents, we established that LKB1 mutant NSCLC cells were highly resistant to standard chemotherapy agents but showed high sensitivity to 8-Cl-Ado. This sensitivity was diminished in cells made LKB1-proficient [Cancer Res 74s19:5480, 2014]. LKB1 promotes the maintenance of the cellular energy balance by enhancing the activation of AMP activating protein kinase (AMPK) under conditions of reduced ATP levels. Consequently, LKB1 deficient tumors lack proper energy regulation, thus are vulnerable to ATP depletion. To examine the tumoricidal activity of 8-Cl-Ado in NSCLC, we assessed isogenic A549 LKB1 proficient and deficient cell lines for 8-Cl-Ado-induced death by flow cytometry. 8-Cl-Ado was tumoricidal to these cells with greater sensitivity in the LKB1 deficient cells, (38 versus 23% annexin V/propidium iodide positivity with 20 μM 8-Cl-Ado for 4d, p = 0.001). HPLC analysis indicated that the endogenous ATP levels are lower in LKB1 deficient cell lines than LKB1 proficient cells (2800 versus 3400 μM, p = 0.011, respectively in the H460 cells and 2556 versus 2812 μM, p = 0.38, respectively in the A549 cells). This was further perturbed by 8-Cl-Ado treatment as the ATP levels were depleted to a greater extent in the deficient cells compared with the proficient cells (60 versus 40% reduction, p = 0.002, respectively in the H460 cells and 67 versus 52% reduction, p = 0.023, respectively in the A549 cells). In concert, 8-Cl-Ado-induced AMPK phosphorylation was detected in LKB1 proficient cells. Due to AMPK's ability to promote autophagy for energy restoration and the role of autophagy in chemoresistance, we examined the NSCLC cells for 8-Cl-Ado-induced changes in autophagy and demonstrated significantly greater LC3B-lipidation and acridine orange staining (30% higher, p = 0.014) in the LKB1 proficient cells. We also initiated studies with 8-NH2-Ado and demonstrated there was higher metabolism of the analog to its cytotoxic form in the NSCLC cell lines as compared with 8-Cl-Ado. This was associated with enhanced tumoricidal activity in the LKB1 deficient cells with cell death occurring earlier after 1 d of treatment. Our results indicate that LKB1 mutant NSCLC cells are vulnerable to energy altering effects of C8-modified adenosine analogs and that the protective role LKB1 plays during treatment of these cells is due in part to greater induction of autophagy. Citation Format: Christine M. Stellrecht, Lisa S. Chen, Mary L. Ayres, John V. Heymach, Varsha Gandhi. Vulnerability of LKB1 deficient NSCLC to C8-modified adenosine analogs is associated with diminished autophagy induction. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 673. doi:10.1158/1538-7445.AM2015-673

Abstract 1656: MK-1775 (WEE1 inhibition) lacks efficacy against DNA repair deficient pancreatic cancer cells

Abstract Introduction: In this study, we explored the possibility of developing a personalized approach of using MK-1775, a potent WEE1 inhibitor, as mono- or combination therapy to treat pancreatic ductal adenocarcinoma (PDA) cells with DNA repair deficiency. Experimental Methods: We treated PDA cells harboring diverse genetic backgrounds with IC50 doses of clinically-relevant DNA damaging agents: mitomycin C (MMC) and the WEE1 inhibitor MK-1775. Drug sensitivity assays were performed in isogenic PDA cells that are either proficient or deficient in Fanconi Anemia (FA)/BRCA2 pathway. In addition, proficient cell lines were treated with siRNA oligos targeted against FANCD2 and BRCA2 genes. Mechanistic studies such as Annexin V staining were performed to measure apoptotic cells upon MMC and MK-1775 treatments. The degree of DNA damage was measured by immunofluorescence (IF) assay using γH2AX antibody. Mitotic entry was analyzed by both IF and flow cytometry analyses using pH3 antibody. Results: Drug sensitivity assays demonstrated that FA/BRCA2-pathway proficient PDA cell lines (MiaPaCa2 and Panc1) are sensitive to the cytotoxic effect of MK-1775 compared to DNA repair-deficient cell lines (Capan1:BRCA2 deficient and Hs766T:FANCG deficient) which showed acute resistance to MK-1775. Immunoblotting showed that MK-1775 efficiently reduces the expression of WEE1 and accordingly phosphorylation of CDK1 in all cell lines. Annexin V staining showed a higher percentage of cell death in the FA/BRCA2-pathway proficient cell lines compared to deficient cell lines when exposed to MK-1775. Furthermore, IF experiments demonstrated that MMC treatment induces γH2AX foci in all cell lines, however, the FA/BRCA2 proficient cell lines showed a higher degree of nuclear abnormality and multi-nucleation after MK-1775 treatment compared to deficient cell lines. In addition, the FA/BRCA2 proficient cell lines showed significantly higher phospho histone 3 (pH3) staining, a marker of mitotic cells. FACS analyses validated that proficient cell lines showed a higher percentage of cells in the mitotic phase when exposed to MK-1775. In addition, we evaluated the cytotoxic effect of MK-1775 in combination with MMC and showed that FA/BRCA2 proficient cells are more sensitive to dual treatment than deficient cells. In addition, immnobloting detected cleaved caspase 3, a marker of apoptosis, after MK-1775 treatment alone or in combination with MMC, demonstrating cells undergoing mitotic catastrophe. We validated the results in the FA/BRCA2-pathway proficient cell lines pre-treated with siFANCD2 or siBRCA2 oligos as compared to the proficient control lines. Conclusions: These results support a paradigm in which identified high risk FA/BRCA2-mutated patients would not benefit from WEE1 inhibitor monotherapy; and thus, would most likely respond better to conventional DNA damaging agent-based therapies (e.g., oxaliplatin or MMC). Citation Format: Shruti Lal, Saswati N. Chand, Emanuela Dylgjeri, Charles J. Yeo, Jordan M. Winter, Jonathan R. Brody. MK-1775 (WEE1 inhibition) lacks efficacy against DNA repair deficient pancreatic cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1656. doi:10.1158/1538-7445.AM2015-1656

Abstract 3624: A novel, selective DNA-PK inhibitor sensitizes poor prognosis chronic lymphocytic leukemia (CLL) cells to mitoxantrone

Abstract Approximately 20% of Chronic Lymphocytic Leukaemia (CLL) cases carry 11q deletions and 10% carry 17p deletions (associated with loss of p53 function), both of which are poor prognostic markers. Ataxia telangiectasia mutated kinase (ATM) is located on chromosome 11q23 and 30% of del11q patients lack ATM function. Previous work has shown that the DNA-PK inhibitor NU7441 chemosensitises CLL cells to fludarabine ex vivo (Willmore E et al 2008), and monotherapy with KU60648, a dual inhibitor of PI3kδ and DNA-PK has been shown to be effective in ATM deficient cells (Riabinska A et al 2013). Furthermore, a dual DNA-PK/mTOR inhibitor, CC-115, is now in clinical trials (NCT01353625). Here, we aimed to validate the selective inhibition of DNA-PK over related kinases as a therapeutic approach in CLL, using a novel, more selective compound (NDD0004, cell-free IC50 10nM). Both primary CLL cells and an isogenic pair of DNA-PK-deficient and proficient HCT116 cells were used as models. Primary CLL cells were cultured ex vivo and treated with the topoisomerase II poison mitoxantrone in the presence and absence of NU7441 and NDD0004. Cell viability was determined after 48h by XTT assay. Apoptosis and DNA damage (yH2AX foci) were measured by caspase 3/7GloAssay and immunofluorescence, respectively. ATM function was determined by the cells ability to autophosphorylate ATM on serine 1981 after ionising radiation. Chemosensitisation to mitoxantrone in primary CLL cells was substantial and it was higher with NDD0004 than with NU7441 (mean 202- vs. 69-fold, ranges 1-1000 vs. 1-543-fold). Intriguingly, cells lacking ATM or p53 function were also sensitised by DNA-PK inhibition (mean 15- and 21-fold, respectively). HCT116 cells lacking DNA-PK were not chemosensitised by NDD0004 but DNA-PK proficient cells were sensitised 5-fold. Mitoxantrone-induced DSBs (as measured by yH2AX foci) were repaired by 24h, but persisted in the presence of DNA-PK inhibitors. In keeping with this observation, apoptosis as measured by caspase 3/7 activity was potentiated by DNA-PK inhibitors. NDD0004 and KU60648 were also evaluated in a panel of ATM deficient (HT144, H1395) and ATM proficient (A549, A375) cell lines. ATM status had no impact on (a) the level of chemosensitisation to mitoxantrone by both inhibitors and (b) single agent activity for NDD0004 and KU60648. In conclusion, our data show that selective inhibition of DNA-PK chemosensitises primary CLL cells even when p53 is mutated or ATM function is lost. Current work is focused on identifying biomarkers which predict good response to DNA-PK inhibitors. Citation Format: Gesa Junge. A novel, selective DNA-PK inhibitor sensitizes poor prognosis chronic lymphocytic leukemia (CLL) cells to mitoxantrone. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3624. doi:10.1158/1538-7445.AM2015-3624

An Early and Robust Activation of Caspases Heads Cells for a Regulated Form of Necrotic-like Cell Death

Apoptosis is triggered by the activation of caspases and characterized by chromatin condensation and nuclear fragmentation (type II nuclear morphology). Necrosis is depicted by a gain in cell volume (oncosis), swelling of organelles, plasma membrane leakage, and subsequent loss of intracellular contents. Although considered as different cell death entities, there is an overlap between apoptosis and necrosis. In this sense, mounting evidence suggests that both processes can be morphological expressions of a common biochemical network known as "apoptosis-necrosis continuum." To gain insight into the events driving the apoptosis-necrosis continuum, apoptotically proficient cells were screened facing several apoptotic inducers for the absence of type II apoptotic nuclear morphologies. Chelerythrine was selected for further studies based on its cytotoxicity and the lack of apoptotic nuclear alterations. Chelerythrine triggered an early plasma membrane leakage without condensed chromatin aggregates. Ultrastructural analysis revealed that chelerythrine-mediated cytotoxicity was compatible with a necrotic-like type of cell death. Biochemically, chelerythrine induced the activation of caspases. Moreover, the inhibition of caspases prevented chelerythrine-triggered necrotic-like cell death. Compared with staurosporine, chelerythrine induced stronger caspase activation detectable at earlier times. After using a battery of chemicals, we found that high concentrations of thiolic antioxidants fully prevented chelerythrine-driven caspase activation and necrotic-like cell death. Lower amounts of thiolic antioxidants partially prevented chelerythrine-mediated cytotoxicity and allowed cells to display type II apoptotic nuclear morphology correlating with a delay in caspase-3 activation. Altogether, these data support that an early and pronounced activation of caspases can drive cells to undergo a form of necrotic-like regulated cell death.

Abstract 2556: Trifluridine induces p53-dependent sustained G2 phase arrest with its massive misincorporation into DNA and few DNA strand breaks

Abstract Background: TAS-102 is a combination of an antineoplastic thymidine-based nucleoside analogue, trifluridine (FTD), and a thymidine phosphorylase inhibitor, tipiracil (TPI), at a molar ratio of 1:0.5. FTD is the active antitumor component of TAS-102, and its triphosphate form is incorporated into the DNA in tumor cells. TPI, a potent inhibitor of the FTD-degrading enzyme thymidine phosphorylase, maintains adequate plasma concentrations of orally administered FTD, thereby potentiating its antitumor activity. In an international multicenter randomized double blind Phase III study (RECOURSE), TAS-102 administration resulted in a significant improvement in overall survival and progression-free survival and a favorable safety profile compared to placebo treatment in patients with metastatic colorectal cancer refractory to standard chemotherapies. In this study, we investigated the mechanism of FTD-induced cytotoxicity in colorectal cancer and other cell lines. Method: The colorectal cancer cell lines RKO and HCT-116, the subline HCT-116 p53−/−, and the lung adenocarcinoma cell line A549 were used. Cells were treated with FTD or FdUrd using the IC50 values for 12 to 72 h. Molecules involved in DNA damage and the cell cycle were evaluated by western blot and RT-PCR. Cell cycle analysis and DNA strand-breaks were evaluated by flow cytometry and alkaline- and neutral-comet assays. Results: FTD and FdUrd activated similar DNA damage response pathways via transient phosphorylation of Chk1 at Ser345, followed by accumulation of p53 and p21 in HCT-116 cells. A similar biochemical response was observed using the IC50 concentration of FdUrd or FTD in RKO and A549 cells. FTD repressed G2 to M phase progression and the cells accumulated in G2 phase 72 h after treatment, with a 35% and 29% decrease in the CDK1 and CCNB1 mRNA levels compared to the control. The G2 phase arrest by FTD was commonly induced in each p53 proficient cell lines (HCT-116, RKO, and A549). In contrast, FTD-induced G2 phase arrest was not observed when p53 expression was suppressed. Furthermore, the p53-dependent G2 phase arrest by FTD was associated with the downregulation of Cyclin B1, both through its transcriptional repression and proteasomal degradation. Although FdUrd induced numerous single- and double-strand DNA breaks, FTD did not induce detectable DNA strand breaks. FTD was massively incorporated into DNA (41.4 pmol/μg DNA), whereas less FdUrd was incorporated (0.6 pmol/μg DNA) 72 h after treatment. Conclusion: Our analyses revealed that FTD induced sustained G2 phase arrest and few DNA strand breaks, in spite of massive incorporation into DNA. These results suggest that the mechanism of FTD action is unique at the cellular level, resulting in the clinical efficacy of TAS-102. Citation Format: Kazuaki Matsuoka, Makoto Iimori, Shinichiro Niimi, Hiroshi Tsukihara, Sugiko Watanabe, Shinichi Kiyonari, Mamoru Kiniwa, Koji Ando, Eriko Tokunaga, Hiroshi Saeki, Eiji Oki, Hiroyuki Kitao, Yoshihiko Maehara. Trifluridine induces p53-dependent sustained G2 phase arrest with its massive misincorporation into DNA and few DNA strand breaks. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2556. doi:10.1158/1538-7445.AM2015-2556

Centrosome aberrations in human mammary epithelial cells driven by cooperative interactions between p16INK4a deficiency and telomere-dependent genotoxic stress.

Virtually all human cancers display chromosome instability (CIN), a condition in which chromosomes are gained or lost at a high rate. CIN occurs early in cancer development where it may undermine the advance of the neoplastic disease. With the aim of establishing the mechanisms underlying CIN in cancer, we investigated possible links between telomere-dysfunction and centrosome defects, which were seen to coincide in early in breast carcinogenesis using human mammary epithelial cells (HMECs). In this study, we show that TP53 proficient vHMECs cells develop centrosome aberrations when telomere-dysfunction genotoxic stress is produced in the presence of a defective p16INK4a setting and in parallel with an activation of the DNA damage checkpoint response. These aberrations consist of the accumulation of centrosomes in polyploid vHMECs, plus centriole overduplication in both diploid and polyploid cells, thus reflecting that distinct mechanisms underlie the generation of centrosome aberrations in vHMECs. Transduction of vHMEC with hTERT, which rescued the telomere dysfunction phenotype and consequently reduced DNA damage checkpoint activation, led to a progressive reduction of centrosome aberrations with cell culture, both in diploid and in polyploid vHMECs. Radiation-induced DNA damage also raised centrosome aberrations in vHMEC-hTERT. Collectively, our results, using vHMECs define a model where p16INK4a deficiency along with short dysfunctional telomeres cooperatively engenders centrosome abnormalities before p53 function is compromised.

SUB1 Plays a Negative Role during Starvation Induced Sporulation Program in Saccharomyces cerevisiae.

Saccharomyces cerevisiae Sub1 is involved in several cellular processes such as, transcription initiation, elongation, mRNA processing and DNA repair. It has also been reported to provide cellular resistance during conditions of oxidative DNA damage and osmotic stress. Here, we report a novel role of SUB1 during starvation stress-induced sporulation, which leads to meiosis and spore formation in diploid yeast cells. Deletion of SUB1 gene significantly increased sporulation efficiency as compared to the wild-type cells in S288c genetic background. Whereas, the sporulation functions of the sub1(Y66A) missense mutant were similar to Sub1. SUB1 transcript and protein levels are downregulated during sporulation, in highly synchronized and sporulation proficient wild-type SK1 cells. The changes in Sub1 levels during sporulation cascade correlate with the induction of middle sporulation gene expression. Deletion of SUB1 increased middle sporulation gene transcript levels with no effect on their induction kinetics. In wild-type cells, Sub1 associates with chromatin at these loci in a temporal pattern that correlates with their enhanced gene expression seen in sub1Δ cells. We show that SUB1 genetically interacts with HOS2, which led us to speculate that Sub1 might function with Set3 repressor complex during sporulation. Positive Cofactor 4, human homolog of Sub1, complemented the sub1Δ sporulation phenotype, suggesting conservation of function. Taken together, our results suggest that SUB1 acts as a negative regulator of sporulation.

Lipid metabolism regulator, Acetyl-coA carboxylase: a possible therapeutic target for dendritic cell-based immunotherapy (IRC10P.414)

Abstract Dendritic cells (DCs) are proficient antigen-presenting cells that regulate the activation and suppression of the innate and adaptive immunity. There has been a growing interest in the regulation of DC function by tailoring metabolic requirements in DC-based immunotherapy of cancer. Here we report that targeting acetyl-CoA carboxylase, a key regulator of de novo fatty acid synthesis and lipid oxidation, facilitates the down-modulation of DC functions during the maturation process. DCs treated with ACC blockade, CP-640186, dramatically down-regulated the expression of co-stimulatory molecules including CD80, CD86 and CD40 together with MHC II(major histocompatibility complex II) during LPS-mediated maturation by a dose-dependent manner. Coincidently, the secretion of pro-inflammatory cytokines (IFN-r, IL-6, IL-12) was decreased as well. Blocking fatty acid synthesis by targeting ACC down-regulated the expression of CCR7, a homing receptor of DCs, but up-regulated the expression of CCR5, a chemokine receptor largely expressed in immature DCs. Our data elucidate the role of fatty-acid synthesis in DC maturation and have implications for the development of metabolism based-approaches in DC-mediated immunotherapies.

Role for Artemis nuclease in the repair of radiation-induced DNA double strand breaks by alternative end joining.

Exposure of cells to ionizing radiation or radiomimetic drugs generates DNA double-strand breaks that are processed either by homologous recombination repair (HRR), or by canonical, DNA-PKcs-dependent non-homologous end-joining (C-NHEJ). Chemical or genetic inactivation of factors involved in C-NHEJ or HRR, but also their local failure in repair proficient cells, promotes an alternative, error-prone end-joining pathway that serves as backup (A-EJ). There is evidence for the involvement of Artemis endonuclease, a protein deficient in a human radiosensitivity syndrome associated with severe immunodeficiency (RS-SCID), in the processing of subsets of DSBs by HRR or C-NHEJ. It is thought that within HRR or C-NHEJ Artemis processes DNA termini at complex DSBs. Whether Artemis has a role in A-EJ remains unknown. Here, we analyze using pulsed-field gel electrophoresis (PFGE) and specialized reporter assays, DSB repair in wild-type pre-B NALM-6 lymphocytes, as well as in their Artemis(-/-), DNA ligase 4(-/-) (LIG4(-/-)), and LIG4(-/-)/Artemis(-/-) double mutant counterparts, under conditions allowing evaluation of A-EJ. Our results substantiate the suggested roles of Artemis in C-NHEJ and HRR, but also demonstrate a role for the protein in A-EJ that is confirmed in Artemis deficient normal human fibroblasts. We conclude that Artemis is a nuclease participating in DSB repair by all major repair pathways.

SG-04 * THE eEF2 KINASE, A MEDIATOR OF MEDULLOBLASTOMA ADAPTATION TO METABOLIC STRESS, SUPPORTS FATTY ACID OXIDATION

During tumour progression, brain tumour cells are exposed to nutrient deprivation due to defective tumour vasculature. The ability of brain tumour cells to adapt to reduced nutrient availability favours selection for aggressive clones. The molecular pathways supporting such metabolic adaptation are still poorly defined. We previously reported that the translation elongation factor 2 kinase (eEF2K), which controls mRNA translation at the step of elongation, is an evolutionarily conserved mediator of the response to nutrient deprivation. Gene expression analysis show that eEF2K expression is upregulated in the most aggressive subgroup of medulloblastoma (MB), namely group 3, and that high eEF2K expression is strongly associated with poor survival in both MB. In addition, genetic targeting of eEF2K significantly increases sensitivity of MB cells to nutrient deprivation, supporting a role for eEF2K in facilitating adaptation of MB to nutrient stress. To decipher the basis for eEF2K protective functions under nutrient deprivation, we performed gene expression analysis of eEF2K proficient versus deficient cells under nutrient-deprived conditions. Gene set enrichment analysis (GSEA) highlighted fatty acid oxidation (FAO) as one of the biological pathways most dramatically deregulated upon eEF2K loss. Remarkably, many genes involved in FAO are downregulated in eEF2K deficient cells under starvation, including genes encoding acyl-coA synthetases and acyl-coA dehydrogenases. Furthermore, cells lacking eEF2K are deficient in metabolizing exogenous palmitate to produce ATP, strongly supporting a role for eEF2K in stimulating FAO activity. Finally, our data show that pharmacological induction of FAO in eEF2K deficient cells using bezafibrate was sufficient to rescue cell death occurring under nutrient depleted conditions. The molecular basis for eEF2K-mediated regulation of the FAO transcriptional program is under investigation. Together, our work supports a critical role for eEF2K in driving FAO activity under nutrient deprivation, which could profoundly impact medulloblastoma adaptation to nutrient stress and therefore emergence of aggressive phenotypes.

294 Higher Prevalence of Cystic Lesions of the Pancreas in First Degree Relatives of Familial Pancreatic Cancer Cases Than in Carriers of Pancreatic Cancer-Prone Gene Mutations

G A A b st ra ct s disease, and current in vitro systems do not lend to studying metastasis either. Hence, in this abstract, we demonstrate the development of a new model, called Tumor in a Dish or TiD. Methods: Epithelial cells and fibroblasts from the colon and lung, as well as human vascular endothelial cells were used to develop a colon and lung organoid, respectively. For mismatch repair [MMR] proficient and defective cells, we used HCT-116 and DLD-1, and SW480 and LS174T cells, respectively. In addition, we used freshly resected colon cancer samples. For therapeutic interventions, we used 5-fluouracil (5FU), oxaloplatin (OX), irinotecan (IRI) and SN38, the active metabolite of irinotecan. Results: In the multi-cell type organoid system, cancer cells were grown in a 3-dimensional (3D) culture containing normal epithelial cells, fibroblasts and endothelial cells, which took approximately 10 days to form. The model resembles in vivo tumor microenvironments, including cell-cell contact, tumor architecture, and the influence of different cell types. To study colon cancer, we developed both primary TiD (priTiD) and metastatic TiD (metTiD). In the priTiD, we used normal colon epithelial cells and fibroblasts to develop a primary colon organoid and included colon cancer cells to develop a colon tumor. For metTiD, we similarly developed a lung organoid in which we added colon cancer cells and demonstrate colonization. We next determined the efficacy of standard colon cancer treatment agents. We observed differential activity of 5FU in cells with MMR defects in both priand metTiD system when compared to standard 2D cultures. Another surprising result we found was with freshly resected patient samples. Without knowing the genetic characteristics of the cancer tissue, the TiD method was able to identify cells that were resistant to oxaloplatin. Moreover, using this method, we have developed novel drugs that target cancer cells while not affecting the normal tissue. Conclusion: We have developed a novel method for identifying the appropriate therapeutic agent that specifically target tumor tissues, especially with mismatch repair defects. More importantly, the technique has significant utility in precision/personalized medicine, wherein this phenotypic screen can be coupled with current DNA pharmacogenetics to identify the ideal therapeutic agent, thereby increasing the probability of response to treatment while reducing unnecessary side effects.

The link between cell-cycle dependent radiosensitivity and repair pathways: a model based on the local, sister-chromatid conformation dependent switch between NHEJ and HR.

The different DNA damage repair pathways like homologous recombination (HR) and non-homologous end joining (NHEJ) have been linked to the variation of radiosensitivity throughout the cell cycle. However, no attempts have been made to test the various hypotheses derived from these studies in a quantitative way e.g. by using modeling approaches. Here we present the first modeling approach that allows predicting the cell cycle dependent radiosensitivity of repair proficient as well as of repair deficient cell lines after photon irradiation based on a small set of parameters and assumptions. A key element of the model is the classification of DNA damage according to its complexity on the level of chromatin loops of about 2Mbp size. Isolated DSB (iDSB), characterized by a single DSB within a chromatin loop, are distinguished from clustered DSB (cDSB), characterized by two or more DSB within a chromatin loop. The class of iDSB is further subdivided into two sub-classes, characterized by the replication status of the corresponding chromatin loop. For iDSB in replicated loops that are in close contact, error-free homologous recombination is assumed to be effective; in unreplicated loops or in replicated loops that have already been separated, iDSB are assumed to be repaired by error-prone non-homologous end joining. cDSB are assumed not to be repairable effectively by neither HR nor NHEJ. Assigning empirically derived lethalities to these three damage classes and pathways, we demonstrate that the model is able to accurately reproduce cell cycle dependent survival probabilities. Notably, the relevant parameters are derived solely from two survival curves for normal, repair proficient cells in G1 and late-S phase. Based on a comparison of model predictions with a large data set reported in the literature, we show that the lethality values for wild type cells are simultaneously predictive for the cell cycle dependent variation of sensitivity observed for HR-deficient and NHEJ-deficient cells.

Discovery of 1-substituted benzyl-quinazoline-2,4(1H,3H)-dione derivatives as novel poly(ADP-ribose)polymerase-1 inhibitors

Poly(ADP-ribose)polymerase-1 (PARP-1) has emerged as a promising anticancer drug target due to its key role in the DNA repair process. In this work, a novel series of 1-benzyl-quinazoline-2,4(1H,3H)-dione derivatives were designed and synthesized as human PARP-1 inhibitors, structure–activity relationships were conducted and led to a number of potent PARP-1 inhibitors having IC50 values of single or double digit nanomolar level. Compound 7j was a potent PARP-1 and PARP-2 inhibitor and it could selectively kill the breast cancer cells MX-1 and MDA-MB-468 with mutated BRCA1/2 and PTEN, respectively, in comparison with homologous recombination proficient cell types such as breast cancer cells MDA-MB-231. In addition, compound 7j displayed the strongest potentiation effect on temozolomide in MX-1 cells (PF50=3.77) in this series of PARP-1 inhibitors.

Dendritic cells: The warriors upfront-turned defunct in chronic hepatitis C infection.

Hepatitis C virus (HCV) infection causes tremendous morbidity and mortality with over 170 million people infected worldwide. HCV gives rise to a sustained, chronic disease in the majority of infected individuals owing to a failure of the host immune system to clear the virus. In general, an adequate immune response is elicited by an efficient antigen presentation by dendritic cells (DCs), the cells that connect innate and adaptive immune system to generate a specific immune response against a pathogen. However, HCV seems to dysregulate the activity of DCs, making them less proficient antigen presenting cells for the optimal stimulation of virus-specific T cells, hence interfering with an optimal anti-viral immune response. There are discordant reports on the functional status of DCs in chronic HCV infection (CHC), from no phenotypic or functional defects to abnormal functions of DCs. Furthermore, the molecular mechanisms behind the impairment of DC function are even so not completely elucidated during CHC. Understanding the mechanisms of immune dysfunction would help in devising strategies for better management of the disease at the immunological level and help to predict the prognosis of the disease in the patients receiving antiviral therapy. In this review, we have discussed the outcomes of the interaction of DCs with HCV and the mechanisms of DC impairment during HCV infection with its adverse effects on the immune response in the infected host.

SRT1720 induces lysosomal-dependent cell death of breast cancer cells.

SRT1720 is an activator of SIRT1, a NAD(+)-dependent protein and histone deacetylase that plays an important role in numerous biologic processes. Several studies have illustrated that SRT1720 treatment could improve metabolic conditions in mouse models and in a study in cancer SRT1720 caused increased apoptosis of myeloma cells. However, the effect of SRT1720 on cancer may be complex, as some recent studies have demonstrated that SRT1720 may not directly activate SIRT1 and another study showed that SRT1720 treatment could promote lung metastasis. To further investigate the role of SRT1720 in breast cancer, we treated SIRT1 knockdown and control breast cancer cell lines with SRT1720 both in vitro and in vivo. We showed that SRT1720 more effectively decreased the viability of basal-type MDA-MB-231 and BT20 cells as compared with luminal-type MCF-7 breast cancer cells or nontumorigenic MCF-10A cells. We demonstrated that SRT1720 induced lysosomal membrane permeabilization and necrosis, which could be blocked by lysosomal inhibitors. In contrast, SRT1720-induced cell death occurred in vitro irrespective of SIRT1 status, whereas in nude mice, SRT1720 exhibited a more profound effect in inhibiting the growth of allograft tumors of SIRT1 proficient cells as compared with tumors of SIRT1-deficient cells. Thus, SRT1720 causes lysosomal-dependent necrosis and may be used as a therapeutic agent for breast cancer treatment.