Hilal S. Khalil

Discovery and development of Seliciclib. How systems biology approaches can lead to better drug performance

Seliciclib (R-Roscovitine) was identified as an inhibitor of CDKs and has undergone drug development and clinical testing as an anticancer agent. In this review, the authors describe the discovery of Seliciclib and give a brief summary of the biology of the CDKs Seliciclib inhibits. An overview of the published in vitro and in vivo work supporting the development as an anti-cancer agent, from in vitro experiments to animal model studies ending with a summary of the clinical trial results and trials underway is presented. In addition some potential non-oncology applications are explored and the potential mode of action of Seliciclib in these areas is described. Finally the authors argue that optimisation of the therapeutic effects of kinase inhibitors such as Seliciclib could be enhanced using a systems biology approach involving mathematical modelling of the molecular pathways regulating cell growth and division.

Pharmacological inhibition of ATM by KU55933 stimulates ATM transcription

Ataxia-telangiectasia mutated (ATM) kinase is a component of a signalling mechanism that determines the process of decision-making in response to DNA damage and involves the participation of multiple proteins. ATM is activated by DNA double-strand breaks (DSBs) through the Mre11–Rad50–Nbs1 (MRN) DNA repair complex, and orchestrates signalling cascades that initiate the DNA damage response. Cells lacking ATM are hypersensitive to insults, particularly genotoxic stress, induced through radiation or radiomimetic drugs. Here, we investigate the degree of ATM activation during time-dependent treatment with genotoxic agents and the effects of ATM on phospho-induction and localization of its downstream substrates. Additionally, we have demonstrated a new cell-cycle-independent mechanism of ATM gene regulation following ATM kinase inhibition with KU5593. Inhibition of ATM activity causes induction of ATM protein followed by oscillation and this mechanism is governed at the transcriptional level. Furthermore, this autoregulatory induction of ATM is also accompanied by a transient upregulation of p53, pATR and E2F1 levels. Since ATM inhibition is believed to sensitize cancer cells to genotoxic agents, this novel insight into the mechanism of ATM regulation might be useful for designing more precise strategies for modulation of ATM activity in cancer therapy.

Systems analysis of drug-induced receptor tyrosine kinase reprogramming following targeted mono- and combination anti-cancer therapy

The receptor tyrosine kinases (RTKs) are key drivers of cancer progression and targets for drug therapy. A major challenge in anti-RTK treatment is the dependence of drug effectiveness on co-expression of multiple RTKs which defines resistance to single drug therapy. Reprogramming of the RTK network leading to alteration in RTK co-expression in response to drug intervention is a dynamic mechanism of acquired resistance to single drug therapy in many cancers. One route to overcome this resistance is combination therapy. We describe the results of a joint in silico, in vitro, and in vivo investigations on the efficacy of trastuzumab, pertuzumab and their combination to target the HER2 receptors. Computational modelling revealed that these two drugs alone and in combination differentially suppressed RTK network activation depending on RTK co-expression. Analyses of mRNA expression in SKOV3 ovarian tumour xenograft showed up-regulation of HER3 following treatment. Considering this in a computational model revealed that HER3 up-regulation reprograms RTK kinetics from HER2 homodimerisation to HER3/HER2 heterodimerisation. The results showed synergy of the trastuzumab and pertuzumab combination treatment of the HER2 overexpressing tumour can be due to an independence of the combination effect on HER3/HER2 composition when it changes due to drug-induced RTK reprogramming.

Quantitative analysis of NRF2 pathway reveals key elements of the regulatory circuits underlying antioxidant response and proliferation of ovarian cancer cells.

Cells are constantly exposed to Reactive Oxygen Species (ROS) produced both endogenously to meet physiological requirements and from exogenous sources. While endogenous ROS are considered as important signalling molecules, high uncontrollable ROS are detrimental. It is unclear how cells can achieve a balance between maintaining physiological redox homeostasis and robustly activate the antioxidant system to remove exogenous ROS. We have utilised a Systems Biology approach to understand how this robust adaptive system fulfils homeostatic requirements of maintaining steady-state ROS and growth rate, while undergoing rapid readjustment under challenged conditions. Using a panel of human ovarian and normal cell lines, we experimentally quantified and established interrelationships between key elements of ROS homeostasis. The basal levels of NRF2 and KEAP1 were cell line specific and maintained in tight correlation with their growth rates and ROS. Furthermore, perturbation of this balance triggered cell specific kinetics of NRF2 nuclear-cytoplasmic relocalisation and sequestration of exogenous ROS. Our experimental data were employed to parameterise a mathematical model of the NRF2 pathway that elucidated key response mechanisms of redox regulation and showed that the dynamics of NRF2-H2O2 regulation defines a relationship between half-life, total and nuclear NRF2 level and endogenous H2O2 that is cell line specific.

A novel mechanism of action of HER2 targeted immunotherapy is explained by inhibition of NRF2 function in ovarian cancer cells

Nuclear erythroid related factor-2 (NRF2) is known to promote cancer therapeutic detoxification and crosstalk with growth promoting pathways. HER2 receptor tyrosine kinase is frequently overexpressed in cancers leading to uncontrolled receptor activation and signaling. A combination of HER2 targeting monoclonal antibodies shows greater anticancer efficacy than the single targeting antibodies, however, its mechanism of action is largely unclear. Here we report novel actions of anti-HER2 drugs, Trastuzumab and Pertuzumab, involving NRF2. HER2 targeting by antibodies inhibited growth in association with persistent generation of reactive oxygen species (ROS), glutathione (GSH) depletion, reduction in NRF2 levels and inhibition of NRF2 function in ovarian cancer cell lines. The combination of antibodies produced more potent effects than single antibody alone; downregulated NRF2 substrates by repressing the Antioxidant Response (AR) pathway with concomitant transcriptional inhibition of NRF2. We showed the antibody combination produced increased methylation at the NRF2 promoter consistent with repression of NRF2 antioxidant function, as HDAC and methylation inhibitors reversed such produced transcriptional effects. These findings demonstrate a novel mechanism and role for NRF2 in mediating the response of cancer cells to the combination of Trastuzumab and Pertuzumab and reinforce the importance of NRF2 in drug resistance and as a key anticancer target.

NRF2 Regulates HER2 and HER3 Signaling Pathway to Modulate Sensitivity to Targeted Immunotherapies

NF-E2 related factor-2 (NRF2) is an essential transcription factor for multiple genes encoding antioxidants and detoxification enzymes. NRF2 is implicated in promoting cancer therapeutic resistance by its detoxification function and crosstalk with proproliferative pathways. However, the exact mechanism of this intricate connectivity between NRF2 and growth factor induced proliferative pathway remains elusive. Here, we have demonstrated that pharmacological activation of NRF2 by tert-butylhydroquinone (tBHQ) upregulates the HER family receptors, HER2 and HER3 expression, elevates pAKT levels, and enhances the proliferation of ovarian cancer cells. Preactivation of NRF2 also attenuates the combined growth inhibitory effects of HER2 targeting monoclonal antibodies, Pertuzumab and Trastuzumab. Further, tBHQ caused transcriptional induction of HER2 and HER3, while SiRNA-mediated knockdown of NRF2 prevented this and further caused transcriptional repression and enhanced cytotoxicity of the HER2 inhibitors. Hence, NRF2 regulates both HER2 and HER3 receptors to influence cellular responses to HER2 targeting monoclonal antibodies. This deciphered crosstalk mechanism reinforces the role of NRF2 in drug resistance and as a relevant anticancer target.

Abstract 3103: Differences in the DDR enzymes activation kinetics between normal and cancer cells could be utilized to achieve targeted cellular sensitivity towards genotoxic agents

The DNA damage response (DDR) pathway is a complex signalling network that is activated when eukaryotic cells undergo DNA damage caused by exposure to genotoxic agents. Depending upon the type and scale of DNA damage, the DDR can either cause cell cycle arrest if the DNA lesions are repairable, or trigger apoptosis if the DNA lesions are beyond the repair capacity of the cell. A variety of enzymes take part in this complex signalling network that decides cellular fate after DNA damage, and is comprised of kinases, phosphatases and glycosylases. Here we explored the kinetics of activation of these key enzymes, the rates of their product formation and the interplay between them that impact the decision making process of the cell. We exposed normal (MCF10A, HaCat) and breast cancer cell lines (MCF7, MDA MB231, MDA MB468) to different combinations of radiomimetic drugs, UV radiation and enzyme inhibitors in a time and dose dependent manner and looked at the resulting enzyme activation rates of DDR kinases such as ATM, ATR, Chk1, Chk2, and DDR phosphatases PP2A and WIP1. We studied the effects of such enzymatic activation on their substrates P53, E2F1, BRCA1 and γ-H2AX and examined its outcome on cell survival. Firstly, we have identified key differences in enzyme activation kinetics that impacted the cell fate decision between normal and cancer cell lines. In addition we established treatment regimes based on those differences that bias the cell fate decision towards apoptosis in cancer cells and cell cycle arrest and repair of DNA in their normal counterparts. Thus, we have demonstrated a link between the rate of induction of different enzyme activities in the DDR pathway, the resulting product formation and cellular sensitivity to genotoxic agents. We have successfully used such information to devise a combinatorial drug treatment regime that is selective for cancer cells. Elucidation of the enzyme activation kinetics in a time and dose dependent manner of the proteins governing cell fate decision can be used to achieve targeted cellular selectivity and sensitivity with lesser side effects on normal cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3103. doi:1538-7445.AM2012-3103

Abstract 4913: A quantitative integrated systems biology approach for modeling cell cycle pathways in normal and tumor cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL We have developed a novel biological system for quantitative analysis of biochemical pathways in normal and tumour human cells. The system is based on cells growing in tissue culture media where the concentrations of growth factors, hormones and other components of the media are precisely defined and continuously monitored. Quantitative immunoblotting and luminescence-based reporter assays are then used to measure the cellular concentration of key cell cycle regulatory proteins and their activity in real time in live cells. This technology resulted in accurate measurement of cellular concentration of a number of key cell cycle regulatory proteins such as Cyclins, CDKs and CDK inhibitors during the cell cycle. The data are being utilized in the development of a quantitative integrated systems biology approach to the cell cycle. The approach consists of a comprehensive network of the molecular interaction pathways regulating cell cycle in normal and tumor cells and a model which incorporates the activity of key molecular species in a single dynamical system which can be solved using genetic algorithms designed to match experimental data, both qualitative and quantitative, to the model kinetics. The model could be utilized for the development of new drug targets and would be capable of consistently measuring the effects of existing drugs (either single or in combination) and opens up a methodology for establishing the effectiveness of these drugs with clear implications for cost/benefit assessment. Major impact is expected on clinical research where the model can be a tool in determining intervention strategies in the molecular pathways concerned. Apart from the development of new drug targets the model will be capable of consistently measuring the effects of existing drugs (either single or in combination) and opens up a methodology for establishing the effectiveness of anti-cancer drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4913. doi:1538-7445.AM2012-4913

Human 825C > T polymorphism in GNB3 gene promotes enhanced cell migration by inducing cytosolic calcium influx and hyper phosphorylation of ERK regulated mTOR pathway

We have selected the mediterranean shrub Euphorbia characias as an experimental model to study the complexity of plant latex chemistry. Latex is a mixture with diversified composition, that includes alkaloids, terpenoid compounds, polymeric substances such as resins and gums, starch, oil and a large number of proteins and enzymatic activities. The aim of the present study is to contribute to the knowledge of this plant product evaluating the antioxidant properties of extracts of E. characias and searching for polymeric substances as natural rubber. We analyzed different extracts from the latex of E. characias and performed a new extraction method (involving the use of trichloacetic acid, TCA) that turned out to be easier, faster and higher reproducible if compared to common extraction methods involving organic solvents like methanol, ethanol, and petroleum ether/methanol. TCA extract of E. characias latex exhibits antioxidant activities determined as total content of free-radical scavenging, polyphenols and total flavonoids. GC-MS analysis confirms the presence of several compound identified as antioxidant molecules. E. characias latex contains a natural rubber. The optimum rubber extraction is achieved with acetic acid followed by cyclohexane/ethanol treatment. The rubber content was shown to be 14% (w/v) of the E. characias latex and the gel content is 2.5% of the rubber weight. E. characias natural rubber showed a molecular weight of 93000 and Mw/Mn of 2.9. On the basis of H NMR, C NMR and FT-IR spectroscopy, the structure of this rubber can be identified as cis-1,4-polyisoprene. This study was partially supported by a grant from Regione Autonoma della Sardegna, Progetti di ricerca di base CRP2-22.Session 1—Maternal Medicine 1. INCIDENCE OF SUPPLEMENTAL POLYDRUG USE IN IRISH PREGNANT WOMEN ON METHADONE MAINTENANCE PROGRAMME Akhter P, Coulter-Smith S, Lee J, Brennan M, Clarke T, Geary M; Dublin, Ireland. 3. MAXIMAL EXERCISE TESTING CAN BE SAFELY USED TO ASSESS CARDIOVASCULAR RESERVE IN PREGNANCY Barker D, Mason G, Schlosshan D, McLoughlin H, Blackburn M, Simpson N, Tan LB; Leeds, UK 4. PREGNANCY OUTCOME IN RENAL TRANSPLANT RECIPIENTS AT ADDENBROOKES HOSPITAL: A 10 YEAR REVIEW Burrell C, Lees C, Daniels I, Somoye G; Cambridge, UK 5. HYPERTENSION IN PREGNANCY: A SURVEY OF CLINICAL PRACTICE TO DOCUMENT CURRENT PRACTICE IN THE UK AND REFINE QUESTIONS FOR A RANDOMIZED CONTROLLED TRIAL (RCT) Churchill D, Duley L, Farrell B; Wolverhampton, UK 6. THE IMPACT OF ACQUIRED THROMBOPHILIA ON MATERNAL AND FETAL WELLBEING IN A LOW-RISK POPULATION Cooley SM, Donnelly J, Walsh T, Geary MP, Gillan J, MacMahon C; Dublin, Ireland 7. FREE MATERNAL DNA IS INCREASED BEFORE 20 WEEKS GESTATION IN WOMEN WHOSE PREGNANCIES ARE SUBSEQUENTLY COMPLICATED BY IUGR BUT NOT PREECLAMPSIA Crowley A, Fitzpatrick P, Sheils O, O’Leary J, O’Herlihy C, Byrne B; Dublin, Ireland 8. IMPACT OF NATIONAL GUIDELINES ON THE MANAGEMENT OF VENOUS THROMBOEMBOLISM IN PREGNANCY Doumouchtsis SK, Tartaglia MA, Thomas GMA, Wilson MP, Thomson AJ; Paisley, UK 9. AN AUDIT ON THE MANAGEMENT OF ANAEMIA IN PREGNANCY IN A LARGE DISTRICT GENERAL HOSPITAL Dua A, Schram CMH, Karunakaran B; Blackburn, UK 10. MECHANISMS OF ACTION OF TNF-a ON ENDOTHELIUM-DEPENDENT RELAXATION IN OMENTAL ARTERIES OBTAINED FROM WOMEN WITH HEALTHY PREGNANCIES Gillham JC, Taggart MJ, Baker PN; Manchester, UK 11. THE QUEENMOTHERS HOSPITAL REVIEW OF PRE-GESTATIONAL DIABETES MELLITUS Hale J, Nicoll AE, Munaza S, Macara LM, Small M, Capaldi B, Cameron AD; Glasgow, UK 12. HEPATITIS B VACCINATION IN AT RISK DRUG USING WOMEN IN PREGNANCY Hyde J, Blunsden V, Irish C; Bristol, UK 13. ALVEOLAR O2 AND CO2 TENSIONS DURING PREGNANCY, MEASURED WITH A NOVEL NON-INVASIVE TECHNIQUE Litos M, Hadjistavrou C, Antsaklis A, Xygakis A, Jordanoglou J; Athens, Greece 14. VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) LEVELS IN DIABETIC PREGNANCY: RELATIONS TO NORMAL PREGNANCY AND GLYCAEMIC CONTROL Manderson JG, McClure N, Patterson CC, Hadden DR, Traub AI, McCance DR; Belfast, UK 15. HYPOTHYROIDISM IN PREGNANCY: INCREASE IN THYROXINE DOSE MAY BE TOO LATE USING OUR CURRENT PROTOCOL Masheshwari S, Singh A, Trinder J; Bristol, UK 16. PREGNANCY CARE IN OBESE WOMEN. AN OBSERVATIONAL STUDY IN A DISTRICT GENERAL HOSPITAL Nawar-Youssef MN, Iqbal F, Odukoya OA; Scunthorpe, UK 17. MANAGEMENT OF POST-PARTUM HYPERTENSION (PHT) Ogunnoiki O, Hirsi-Farah S, Gray G, Nelson-Piercy C; London, UK 18. STUDY ON SCREENING FOR GESTATIONAL DIABETES ParveenAS,PeploeD,Bell-ThomasS;Abergavenny,UK 19. DOES THE INTRODUCTION OF A SCREENING PROCESS FOR MENTAL HEALTH PROBLEMS IMPROVE DETECTION OF AT RISK WOMEN IN THE ANTENATAL PERIOD? Scholefield HJ, Hernon M, Topping J; Liverpool, UK 20. MATERNAL OBESITY AND ITS IMPACT ON PREGNANCY OUTCOME Singhal T, Vogiatzi M, Parmeshwaran S, Howarth ES; Leicester, UK 21. RETROSPECTIVE ANALYSIS OF THE OUTCOME OF HIV PREGNANCIES IN A LARGE DISTRICT GENERAL HOSPITAL Sivarajan S, Hooi A, Roy M, Thomas P, Modi M, Howard R, Sahoo S; London, UK 22. PLACENTAL ABCA1 PROTEIN EXPRESSION IN ANTI-PHOSPHOLIPID SYNDROME TetlowN, Albrecht C, Lakasing L, Soumian S, Patel P, Sullivan M, Nicolaides K, Williamson C; London, UK 23. VIRILIZATION IN PREGNANCY ASSOCIATED WITH OVARIAN LEIOMYOMAS Udayasankar V, Moselhi M, Fielding A; Swansea, UK Journal of Obstetrics and Gynaecology (April 2005) Vol. 25, Supplement 1, S27–S31

NRF2 Regulates HER1 Signaling Pathway to Modulate the Sensitivity of Ovarian Cancer Cells to Lapatinib and Erlotinib

NF-E2-related factor 2 (NRF2) regulates the transcription of a battery of metabolic and cytoprotective genes. NRF2 and epidermal growth factor receptors (EGFRs/HERs) are regulators of cellular proliferation and determinants of cancer initiation and progression. NRF2 and HERs confer cancers with resistance to several therapeutic agents. Nevertheless, there is limited understanding of the regulation of HER expression and activation and the link between NRF2 and HER signalling pathways. We show that NRF2 regulates both basal and inducible expression of HER1, as treatment of ovarian cancer cells (PEO1, OVCAR3, and SKOV3) with NRF2 activator tBHQ inducing HER1, while inhibition of NRF2 by siRNA knockdown or with retinoid represses HER1. Furthermore, treatment of cells with tBHQ increased total and phosphorylated NRF2, HER1, and AKT levels and compromised the cytotoxic effect of lapatinib or erlotinib. Treatment with siRNA or retinoid antagonised the effect of tBHQ on NRF2 and HER1 levels and enhanced the sensitivity of ovarian cancer cells to lapatinib or erlotinib. Pharmacological or genetic inhibition of NRF2 and/or treatment with lapatinib or erlotinib elevated cellular ROS and depleted glutathione. This extends the understanding of NRF2 and its regulation of HER family receptors and opens a strategic target for improving cancer therapy.