Project 1: Dr Darran O’Connor – BET inhibition as a ... · for about 10-15% of all new breast cancer cases diagnosed. Since it is ER+, it is treated the same way as all other

Project 1: Dr Darran O’Connor – BET inhibition as a rational therapeutic strategy for Invasive Lobular Breast Cancer (ILBC)

What’s the project about?

Invasive lobular breast cancer (ILBC) is a form of hormone receptor-positive (ER+) breast cancer that accounts for about 10-15% of all new breast cancer cases diagnosed. Since it is ER+, it is treated the same way as all other ER+ breast cancer, with surgery, radiotherapy, anti-hormone therapy (and in many cases, chemotherapy). However, patients with ILBC do not have the same clinical course as other ER+ patients. Their cancer is more likely to (i) spread to the ovaries and the digestive system, (ii) occur in both breasts, (iii) come back in the other breast (iv) be unresponsive to additional chemotherapy (as well as having the same problems with hormone therapy-resistance as other forms of ER+ breast cancer). In addition to a different clinical course, the tests used to determine treatment options for ER+ patients (such as OncotypeDx), give very different results for ILBC patients, making it difficult to determine the most appropriate treatment plan. As such, the lack of tailored options for ILBC patients represents an unmet clinical need and it is time we start to consider ILBC as a distinct type of ER+ breast cancer and devise new treatment and diagnosis options specifically for these patients. Our research suggests that some ILBC patients who do not respond to anti-hormone therapy would benefit from using a BET inhibitor, and the remaining patients from a combination with an anti-BCL2 drug. This project will confirm whether BET inhibitors are a useful treatment option for ILBC patients and which drugs we need to combine them with to reach the best outcome for ILBC patients.

What will I learn during this project?

The specific aims of this project will be to test the use of BET inhibitors in a pre-clinical model of ILBC, as well as the combination with anti-BCL2 drugs, using BET sensitive and resistant ILBC cell lines. The student will receive training in cell culture, in vitro growth assays, apoptosis assays, RNAi technology, Western blotting, qRT-PCR, and determination of drug synergy using Compusyn software.

The Molecular Oncology Laboratory at the Dept. of Molecular & Cellular Therapeutics (https://www.researchgate.net/profile/Darran_Oconnor), is a young, vibrant and well-funded research group focused on the identification and mechanistic anchoring of novel cancer biomarkers and therapeutic targets.

Example recent outputs:

Li B, Ni Chonghaile T, Fan Y, Madden S, Klinger R, O'Connor AE, O’Hurley G, Mallya G, Joseph J, Tarrant F, Conroy E, Gaber A, Chin SF, Bardwell HA, Provenzano E, Dubois T, Linn S, Jirstrom K, Caldas C, O'Connor DP* & Gallagher WM*. Therapeutic rationale to target highly expressed CDK7 conferring poor outcomes in triple-negative breast cancer. Cancer Res 2017 Jul 15;77(14):3834-3845 *Shared Senior Authorship

Mulrane L, Madden SF, Brennan DJ, Gremel G, McGee SF, McNally S, Martin F, Crown JP, Jirström K, Higgins DG, Gallagher WM & O’Connor DP. miR-187 is an independent prognostic factor in breast cancer and confers increased invasive potential in vitro. Clin Cancer Res 2012 Dec 15;18(24):6702-13.

Brennan DJ*, O’Connor DP*, Rexhepaj E, Ponten F & Gallagher WM. Antibody-based proteomics: Fast-tracking molecular diagnostics in oncology. Nature Reviews Cancer, 2010 Sep;10(9):605-17. *Equal Contribution

https://www.researchgate.net/profile/Darran_Oconnor

Project 2: Prof Tracy Robson - A key role for FKBPL in the regulation of cancer stem cell signalling and the microenvironment; therapeutic implications for tumour growth and metastasis.


Cancer stem cells (CSCs) are a special type of cell found within tumors that are able to undergo unlimited self-renewal and are highly resistant to therapy. Indeed, these cells are left behind and go on to divide rapidly, leading to tumor re-growth. Even more worrying, this population of cells have special features allowing them to move through the body, invading vital organs; a process known as metastasis. We have identified a novel protein, called FKBPL, that occurs naturally in the body and which inhibits tumor blood vessel development, thereby stopping tumor growth. A therapeutic drug derived from the protein and designed to harness its therapeutic effects, has successfully completed phase I cancer clinical trials and was recently granted Orphan Drug status in ovarian cancer by the FDA. However, we have acquired data that suggests that this protein also targets breast and ovarian CSCs by transforming them into a more ‘normal’ cancer cell, which can be easily killed by chemotherapy. This project will assess the impact of FKBPL on other cells within the ovarian tumor microenvironment that are known to support the growth and survival of CSCs cells in the primary tumor and at distant sites. We will evaluate exactly how FKBPL controls these cells and the implications on the ability of CSCs to become metastatic. Understanding how this protein works will allow us to design future clinical trials that are more likely to demonstrate better response rates in cancer patients.

Valentine A, O’Rourke M, Yakkundi A, Worthington J, Hookham M, Bicknell R, McCarthy H, McClelland K, McCallum L, Dyer H, McKeen H, Waugh D, Roberts J, McGregor J, Cotton G, James I, Harrison T, Hirst D, Robson T FKBPL and peptide derivatives: novel biological agents that inhibit angiogenesis by a CD44-dependent mechanism. Clin Cancer Res. 2011 Mar 1;17(5):1044-56. PMID: 21364036.

McClements L, Yakkundi A, Papaspyropoulos A, Harrison H, Ablett MP, Jithesh PV, McKeen HD, Bennett R, Donley C, Kissenpfennig A, McIntosh S, McCarthy HO, O’Neill E, Clarke RB, Robson T. Targeting treatment resistant breast cancer stem cells with FKBPL and its peptide derivative, AD-01, via the CD44 pathway. Clin Cancer Res. 2013 Jul 15;19(14):3881-93. PMID: 2374106.


The student will undergo training in basic laboratory health and safety. Once this has been completed training in basic laboratory skills will include tissue culture of tumor cells and macrophages and isolation of BMDMs from mice; cell-based assays of stemness, including tumorsphere assays, flow cytometry, cell migration and invasion assays; measurement of RNA and protein using ELISA, qRT-PCR and western blot analysis; gene expression manipulation using siRNA knockdown. This training will be carried out within MCT laboratories, where two highly experienced post-doctoral researchers are already actively involved in FKBPL research and are competent in the above techniques. The proposed supervisor for this study, Professor Tracy Robson, has an established research laboratory focusing on the role of FKBPL/AD-01 in stemness, metastasis and inflammation and has all of the available tools in place to ensure the success of this project. In addition to the laboratory skills and techniques the student will learn basic data handling and data analysis and use basic statistical methodology. They will also become familiar with critical analysis of the literature, research ethics, research integrity, research communication & presentation; with respect to the latter the student will present their data and findings at weekly group meetings.

Project 3: Dr Annie Curtis - Time Matters. Uncovering the Mechanisms by which the Body Clock controls Dendritic Cell Function and Immunity.


Within our cells we have a timekeeping system called “the molecular clock” or “body clock”, which regulates daily (or circadian) patterns of our sleep/wake cycle and energy metabolism throughout the day. Dendritic cells are cells of the immune system and are one of the first line responders to infection or damage. This is the cell that responds to and propagates the protective effects of vaccinations. Remarkably we have shown that the function of dendritic cells is controlled by the molecular clock. The ability of dendritic cells to process external signals is altered over the course of the day and is entirely dependent on proteins of the molecular clock. Mitochondria are organelles within cells that produce energy and we have shown that these changes in processing external signals by dendritic cells correlate with activity of the mitochondria. In this project, we will investigate what proteins within the dendritic cell are driving this time of day response to external signals. Our studies will provide new opportunities for using clock dependent or time-of-day approaches for modulating DCs function in disease conditions or to improve vaccination efficiency.


This internship will provide training in some of the most cutting edge molecular biology and immunology techniques. A new field is emerging called immunometabolism in which we now believe that the metabolism of the cell is directing the immune response. This projects now utilizes some of the most up to date techniques of immunometabolism such as metabolic profiling of cells by Seahorse technology

https://www.agilent.com/en/products/cell-analysis-(seahorse)/seahorse-analyzers

In addition this internship will provide training in cell culture, cell harvesting, cell differentiation, protein analysis by western blot, gene knockdown techniques by transfecting cells with siRNAs. Finally, RCSI is equipped with some of the finest microscopes in Ireland allowing us to conduct live cell imaging and machine learning analysis of the images.

https://www.agilent.com/en/products/cell-analysis-(seahorse)/seahorse-analyzers

Project 4: Prof Annette Byrne - Improving the Efficacy of Bevacizumab (Avastin) in Glioblastoma through the Development of a Novel Anti-Invasion Nanotherapeutic.


Patients diagnosed with the brain cancer glioblastoma (GBM) have a 14-month life expectancy. GBM requires significant blood flow to survive, and as such has the ability to form new blood vessels. Bevacizumab (Avastin) is a drug given to patients that targets these blood vessels and should prevent tumor growth and increase life expectancy. However, while the quality of life of GBM patients on avastin is improved, is no increase in life expectancy. In some of these patients, avastin actually makes GBM invade into healthy brain tissue. We have uncovered mechanisms to explain how this happens and have developed a new therapy to target the mechanism GBM tumors use to invade into the normal brain. In this project we seek to demonstrate efficacy of this putative new therapeutic in a clinically relevant GBM disease model. We seek to show that combining this new therapy with Avastin will increase life expectancy.


The student will receive training in a number of in vitro and in vivo techniques essential to their career as a tumor biologist. Specifically:

In vitro techniques:

• Cell culture; Western blotting; 3D Invasion assay techniques; wound healing assays; microscopy; data analysis (densitometry/wound assessment via Image J)

The student will then receive In vivo research training as follows:

• Animal handling and restraint (mice); Husbandry of animals; Sexing breeding and weaning (mice); Correct handling of syringes and needles; Subcutaneous injection (mice); Intracranial injection of tumor cells (Observation only; mice); Intravenous injection (tail vein); Suturing techniques; Gaseous anesthesia (mice); CO2 euthanasia (mice); BLI IVIS imaging (mice); necropsy; data analysis.

Other:

• The student will carry out their LAST Ireland animal handling course and exam (or relevant equivalent). HPRA Individual Authorization will then be sought.

• The student will receive in depth training in the design of statistically powered in vivo studies.

• The student will be fully trained in calculating dosing regimens for animals.

Project 5: Prof Andreas Heise - Synthesis of cell penetrating oligopeptides by automated synthesis.


The development of new drugs has offered treatment for many diseases and positively affected the well-being of patients. However, some drugs do not reach the site of action for various reasons and this has consequences for their dosage. In some instances they have to be overdosed to compensate for this but that can enhance undesired side effects. In an ideal treatment the drugs would only target the diseased area but this is not a trivial task to achieve. This project aims at the development of targeting units, signaling molecules, which can be attached to drug carriers to facilitate a local delivery of drugs where they are needed. It is using automated systems to design these molecules allowing fast synthesis of libraries to identify the most efficient structures.


The internship will provide hands on training on the new state-of-the-art Intavis peptide synthesizer. This synthesizer allows the synthesis of larger quantities of peptides by programmed and software controlled automated dosing of the reagents. The handling is straightforward and after an introduction the student will be able to operate the equipment independently. Training will also be provided on the operation of analytical HPLC and the interpretation of the chromatograms. Training in basic peptide chemistry and the fundamentals of solid-state peptide synthesis will be provided as well as some theoretical insights into alternative techniques. As part of the participation in research group activities, development of complementary skills will be provided such as scientific presentations, scientific communication, organization of project and interaction with research group members.

Project 6: Dr Olga Piskareva - Reconstructing tumour microenvironment in a 3D in vitro model of neuroblastoma using collagen-based scaffolds


In the native tissue, cancer cells are surrounded by three-dimensional (3D) settings that provide biological and physical support and determine disease initiation, progression, patient prognosis and response to treatment. This feature is absent in two-dimensional (2D) traditional cell culture resulting in discrepancies between in vitro and in vivo results and leading to longer drug development journey. Recently, a collaborative effort between Dr. Piskareva’s and Prof. O’Brien’s research teams led to the development of a 3D tissue-engineered tumor cell model (TumourColl) for neuroblastoma, an aggressive childhood cancer. Neuroblastoma cells were grown on collagen-made sponges to form a critical mass of cancer cells mimicking physiologically and clinically applicable scenario. Then cells were treated with cisplatin, a drug commonly used in anticancer therapy, at different doses including clinically relevant. Neuroblastoma cells growth on collagen-made sponges reduced in size in response to the clinically relevant dose of cisplatin only. This dose is more than 100 times higher than that used in traditional 2D cell models. Thus, TumourColl displayed a physiological similarity, making evident the potential of this model to serve as a tool to elucidate neuroblastoma pathogenesis and for the development of new drugs. At the next step, we aim to advance TumourColl using the “off-to-on” fluorescence response tracking system in real time. An ability to monitor the TumourColl status in real-time would contribute to better understanding of this cell system requirements and give a great advantage to this model in the identification of novel targets for the development of more effective personalized therapy.


In vitro cell analysis: the basic principles of cell culture in 2D and 3D (scaffold-based), cell viability assays;

DNA extractions and quantifications

Histology: H&E staining

Microscopy: contrast light microscopy; confocal microscopy. Fluorescent microscopy using the pH sensitive NIR lysosome-responsive fluorophore.

Software and Statistics: PowerPoint, Microsoft Excel; Fiji (ImageJ); GraphPad Prism.

Gaining knowledge in the area of cancer research through hands-on laboratory work and literature review of the subject.

Project 7: Dr Oran Kennedy - Development of a novel system to study bone -cartilage crosstalk


Osteoarthritis (OA) is traditionally defined as a disorder that is (1) age-related and (2) only relevant to cartilage tissue. In relation to the first point, it is now clear that a younger population is also affected, particularly in cases of Anterior Cruciate Ligament (ACL) rupture in the knee. This leads to a Post-Traumatic Osteoarthritis (PTOA). Based on epidemiological studies around 2.6 million children/adolescents (5-19 years old) are treated in emergency departments for knee injuries. In relation to the second point, it has also become clear recently that bone tissue directly beneath the joint (called ‘subchondral bone’) may be indirectly regulating cartilage degradation. Indeed, it was recently shown that 80% of young patients with ACL rupture, who have a diagnostic MRI scan, also show evidence of Bone Marrow Lesions (BMLs) in the subchondral bone – while the cartilage tissue appears normal. The precise makeup of these lesions is unknown, but it is widely accepted that they represent some form of mechanical damage to the bone tissue. Thus, to develop these ideas further, we propose using an explant system whereby damage is introduced to subchondral bone in an explant model , and cytokine expression and local morphological changes are quantified in cartilage tissue in relation to bone damage.


The techniques that will be used here are standard aseptic technique for maintaining explant models, alamar blue assays and significant histological and histomorphometric and imaging training for explant analyses.

Project 8: Dr Sudipto Das and Prof Celine Marmion - Assessing the functional effect and efficacy of novel histone deacetylase inhibitors in breast cancer.


Histone Deacetylase (HDAC) inhibitors such as SAHA and Belinostat have been extensively used as treatment options across several cancer types. However, development of side effects as well as acquired resistance significantly limits their usage as a primary treatment option. This to a great extent is attributed to the reduced drug efficacy owing to the poor delivery of drug in the cell. Taking this into consideration, our collaborators in the Dept. of Chemistry, RCSI have developed novel conjugates involving conjugation of onto HDAC inhibitors including SAHA and Belinostat. However, the therapeutic efficacy of this novel conjugates remains to be tested in context of anti-cancer properties.

The overarching goal of this project is to examine the cellular and molecular phenotypes developed following treatment of breast cancer cell lines, with these novel conjugates when compared to their non-conjugated forms. The specific objectives of this project include:

- Determine the impact of the treatment of molecular markers of proliferation, metastasis and immune modulation using qRT-PCR and western blotting.

- Examine the metabolism of the novel conjugates using ELISA.

This proposed study will allow us to identify the key pathways that are impacted by the novel conjugates, which largely differ from their parent drug thus explaining the enhanced efficacy of the drug in breast cancer cells. The data generated from this project will play a critical role in identifying potential biomarkers and/or therapeutic targets which can be used to predict and/or accentuate the response of breast cancer cells to treatment by these novel conjugates.


- ELISA

- RNA and protein extraction from cells

- qRT-PCR and western blots

Project 9: Dr Brona Murphy - Increasing Treatment Efficacy in GBM


Glioblastoma (GBM) is the most aggressive and malignant tumor of the central nervous system. Despite intense effort to combat GBM with surgery, radiation and temozolomide (TMZ) chemotherapy, 90-95% of patients succumb to the disease within 5 years of diagnosis. A major contributor to the limited effectiveness of current therapies is the extreme resistance to death-inducing stimuli which GBM cells display. As a result of overexpression of survival proteins and attenuated levels of pro-death proteins, GBMs are able to resist both chemotherapy and radiation treatment.

Within our research group, we have a panel of patient-derived cell lines that display extreme resistance to treatment with the therapeutically used temozolomide. In an effort to explain such a strong resistance, we analyzed the activation of survival signaling pathways and observed strong up-regulation of the Ras/RAF/MEK/ERK survival pathway. Upon inhibition of this survival pathway, cell survival was reduced and up-regulation of pro-death proteins was observed. Our working hypothesis is that the intractable nature of GBMs to treatment can be overcome by utilizing therapeutic combinations that target pro-survival signaling and enhance the activation of pro-death pathways.


The student will initially become competent in cell culture techniques. (S)he will also learn how to treat these cell lines using various concentrations of inhibitors and drugs. (S)he will become skilled in how to analyse treatment effects on the cell lines using PI staining, MTT assays and flow cytometry. (S)he will also learn how to complement these analyses with the biochemical techniques of qPCR and Western Blot analysis.

Project 10: Dr Triona ni Chonghaile - The role of histone deacetylase 6 in non-small cell lung cancer


Despite the development of novel targeted therapies, lung cancer still remains the leading cause of cancer deaths worldwide. All too often though tumor responds poorly to chemotherapy, or relapse and resistance follow an initial response. We performed a small molecule screen to identify new therapeutic small molecules that can preferentially kill cancer cells independent of mitochondrial apoptosis. This phenotypic screen led to the discovery of a highly specific histone deacetylase 6 (HDAC6) inhibitor termed BAS-2.

HDAC6 is a complex protein it has two deacetylates domains and an ubiquitin binding domain. Therefore, HDAC6 has been shown to play a role in cell mobility through the acetylation of tubulin and through its ubiquitin binding domain traffics unfolded proteins for degradation. We aim to determine the molecular signaling events that occur following inhibition of HDAC6 in lung cancer cells. We will use a panel of cell lines with various genetic defects to determine which subtypes of NSCLC are sensitive to HDAC6. Lastly, we aim to determine the signaling pathways activated following HDAC6 to kill NSCLC.


CRISPR-Cas9 knockout

cell culture

Viability assays including annexin V/ propidium iodide staining by flow cytometry

Migration and invasion assays.

Project 11: Prof Sally Ann Cryan – Development of inhalable anti-tubercular therapies


Mycobacterium tuberculosis (TB) is the primary infectious disease killer in the world. In 2016 alone 1.7million people died from TB and 10 million people fell ill and drug-resistant TB is a growing problem worldwide. Therefore new therapies and treatment modalities are urgently needed. As TB is primarily a pulmonary pathogen by localizing new and existing TB therapies to the lungs via aerosol instead of using oral or parenteral routes of delivery it is possible to target the site of TB infection in the alveolar macrophage (AM).This project seeks to encapsulate emerging therapies targeting tuberculosis into inhalable delivery platforms designed for cell-specific targeting through additive manufacturing (3D PRINT). Previous work by our group has investigated the key parameters required for targeting the macrophage using inhalable particle technology. Working alongside a clinical research group in St James Hospital Dublin a range of novel host-directed and peptide-based therapeutics that have emerged from research within our teams over the last number of years will be first assessed for their efficacy using a well-established in vitro TB infection mode. In the second stage of the project these lead therapeutics will be encapsulated into inhalable polymeric particles using additive manufacturing (3D PRINT) technology being developed within the Drug Delivery and Advanced Materials team in RCSI. These drug-loaded particles will be characterized pharmaceutically and their cellular targeting and efficacy assessed using well established advanced cellular imaging and infection models respectively.


Prof. Cryan’s Drug Delivery & Advanced Materials Team is part of the RCSI Tissue Engineering Research Group (TERG) which is one of the largest bioengineering research groups in Ireland. TERG is an exceptionally well resourced group with a multidisciplinary PI-base and access to labs and facilities across RCSI, as well as the Trinity Centre for Bioengineering (TCBE). All necessary equipment and facilities required to complete the project as described are in place in our labs already and extensive support from senior researchers will be provide

Techniques will include:

• TB infection model: Cell culture, microbiology, bacillary viability assay, apoptosis & multiplex cytokine assays, high content cell imaging (HCS) & confocal laser scanning microscopy (CLSM)

• Particle manufacture: spray-drying, additive manufacture/3D PRINT processes

• Pharmaceutical characterization: particle sizing, stability testing, drug loading & release, scanning electron microscopy (SEM)

• Aerosol testing: aerodynamic particle sizing, respirable dose determination, patient breath-simulation studies

• Statistics: Formal in-house biostatistics training and training in Graphpad Prism software

The student will have an opportunity to participate in weekly TERG meetings including journal clubs, scientific updates and guest speakers/seminars.

Project 12: Prof Jochen Prehn – Systems modeling of colorectal cancer to inform better patient stratification.


Colorectal cancer (CRC) has one of the highest worldwide incidences (>1.3 million new cases/year) and mortality rates (~610,000 deaths/year). Chemotherapy benefits only 5% of stage II patients and 15-20% of stage III patients, and there are limited predictive markers to inform therapy selection. Our central hypothesis is that systems-based modeling of apoptotic cell death at a single-cell level, in combination with CRC subtype and tumor heterogeneity measures (including stromal, metabolism, and immune markers), will provide unprecedented insights into tumor cell behavior, chemotherapy responsiveness and prognosis in stage II and III CRC patients.


Single cell apoptosis protein data, will be used to model apoptosis competency in every cell and tumor heterogeneity metrics will be calculated for all patients. Multivariate statistics will be used to model and predict tumor responsiveness to chemotherapy in stage II and III patients. Finally, rationally selected apoptosis-targeted therapies will be tested in cell lines, organoids, and co-culture systems derived from stage II and III patients with high risk signatures. Such a multi-parameter modeling approach, comprising tumor heterogeneity, has not been previously implemented for predicting patient response to chemotherapy.

Documents

Project 1: Dr Darran O’Connor – BET inhibition as a ... · for about 10-15% of all new breast cancer cases diagnosed. Since it is ER+, it is treated the same way as all other