Drug Discovery & Development 2020

Whether you are performing killing assays, characterizing the tumor microenvironment, or investigating immunogenicity, primary cells are essential for the discovery of new therapeutics. Conclusive and relevant assay results depend on reliable sample preparation, including Ficoll-free cell separation and efficient tumor dissociation. We have developed cell isolation solutions from various starting materials to ensure the best fit for every drug discovery program - join us to learn about the best methods and processes for your in vitro assays.

Learning Objectives:

1. Understand the fundamentals of MACS technology and magnetic cell separation

2. Discuss how the automated isolation of primary cells leads to more efficient and reproducible  in vitro assays

3. Use of primary cells in drug discovery assays

Digital spatial profiling (DSP) on the NanoString GeoMx platform enables high plex molecular profiling of clinical tissue samples. This new profiling strategy enables deep insights into the heterogenous nature of gene and protein expression as it is driven by the distribution and colocalization of cells within a tissue. Recently, DSP was featured in 2 back to back Nature publications elucidating the important role of B cells in mediated immune response to immunotherapy in melanoma. These studies, from the laboratories of Dr. Jennifer Wargo at the MD Anderson Cancer Center and Dr. Goran Jonsson at Lund University, leverage DSP as well as other traditional and next generation profiling strategies to deeply characterize the molecular expression patterns and spatial architecture of the key immune populations within tertiary lymphoid structures in the tumor. Dr. Sarah Warren will speak about both of these publications, as well as how the work was enabled by prior DSP profiling of melanoma samples from Dr. Wargo’s lab. This webinar will present an overview of the GeoMx platform as well as demonstrate its application to current clinically impactful questions in immuno-oncology.

Learning Objectives:
1. Understand the biology around B cells and tertiary lymphoid structures in cancer
2. Understand how spatial high plex profiling was used to characterize immune responses within melanoma

QIAGEN CLC Genomics Workbench is a powerful solution to analyze and visualize next generation sequencing (NGS) data. Powered by cutting-edge technology and accelerated algorithms, it supports a comprehensive set of NGS data analysis applications.

In this live webinar, Leif Schauser, Ph.D., Director Product Management Genome Analysis, will review a host of new features in the latest release of the QIAGEN CLC Genomics Workbench 20 that help you scale your research, and allow you to ramp up your productivity by taking your multi-sample analyses to the next level. Learn about: Working with reads from various platforms (Illumina, IonTorrent, Oxford Nanopore, Pacific Biosciences, BGI/MGI); Tailored solutions for RNA-seq, DNA-seq and methylation; Efficient algorithms for read trimming, mapping, de novo assembly and variant calling; Effective management of reference data; Scalable processing of many samples, with advanced workflow and reporting capabilities; Easy installation on Windows, Mac and Linux.

Learning Objectives:
1. Learn to work with reads from various platforms (Illumina, IonTorrent, Oxford Nanopore, Pacific Biosciences, BGI/MGI)
2. Understand management of reference data
3. Use algorithms for read trimming, mapping, de novo assembly and variant calling

CRISPR revolutionized gene editing, but multi-target screening remains a complex goal.  In addition, the fast pace of CRISPR technology development has brought sophisticated options for library screens including new delivery methods for CRISPR gene knockout and the possibility of genome-wide, epigenetic target gene modulation.  In this talk, we will explore CRISPR screening technologies for gene knockout, activation, and repression in projects ranging in size from use of small gene panels to whole genome pools and arrayed libraries.

Learning Objectives:

1. Describe general CRISPR screening considerations and workflows

2. Identify nuclease-independent applications of CRISPR

Recent advances in DNA sequencing and omics-based capabilities are revealing incredible therapeutic opportunities and quickly transforming drug discovery. Molecularly targeted drugs aim to exploit key tumor-specific vulnerabilities such as oncogenic or non-oncogenic addiction and synthetic lethality. The non-oncogenic addiction to tamoxifen creates the dependency on ribonucleotide reductase enzymes in Estrogen Receptor positive breast cancer cells. Moreover, targeting the host immune system with immunotherapies is proving to be another promising and complementary approach. Because of substantial genomic heterogeneity and immunologic complexities, combination strategies are required to interrupt intricate molecular signaling networks to achieve durable clinical responses in patients. The drug combinations have the potential to improve treatment response, minimize the development of resistance or minimize adverse events. Currently, designing combination trials relies mainly on clinical and experimental experience. While empirical experience has indeed crafted efficacious combination therapy clinical trials (combination trials), however, garnering experience with patients can take a lifetime. Therefore, to enhance the therapeutic success of combination therapies, we discovered that Ribonucleotide reductase inhibitors such as Didox, when combined with tamoxifen, created a novel combination therapeutics. Therefore, we proposed this most effective use of this combination should be directed towards eliminating polyclonal and heterogeneous cancer cells. Our results call to action a proactive paradigm aimed at preventing resistance rather than the current reactive paradigm of intercepting and treating drug resistance incrementally. Indeed, a paradigm shift is required from the traditional “drug-centric” model of clinical development to a “strategy-centric” model to provide personalized treatments in molecularly stratified subsets of patients or even in individual patients. Importantly, to battle the numerous challenges in combination drug development—including limited understanding of tumor biology, methodological and informatics limitations, regulatory challenges and ever-increasing monetary costs. This requires aligned goals and multidisciplinary collaboration to collectively harness knowledge and fuel continual innovation.
 
Learning Objectives:
1.     Defining the effective combination therapy
2.     Identifying various challenges in designing the rational combination therapy
3.     Current approaches to combination therapy in the clinic

Centhaquine (previously used names, centhaquin and PMZ-2010; International Non-proprietary Name (INN) recently approved by WHO is centhaquine) is being developed by us as a first-in-class resuscitative agent for treating hypovolemic shock. It acts on the venous α2B-adrenergic receptors and enhances venous return to the heart, in addition, it produces arterial dilatation by acting on central α2A-adrenergic receptors to reduce sympathetic activity and systemic vascular resistance. Enhanced cardiac output and blood flow to the vital organs, prevents organ failure and improves survival and recovery of hypovolemic shock patients. Toxicological studies showed high safety margin in preclinical studies. Its safety and tolerability have been demonstrated in a human phase I study in 25 subjects (NCT02408731). Phase II human studies showed extraordinary efficacy in patients with hypovolemic shock. A prospective, multi-centric, randomized, double-blind, parallel, saline controlled phase II study was carried out with centhaquine as a resuscitative agent for hypovolemic shock (NCT04056065). A total of 50 subjects met inclusion and exclusion criteria and were included in the study. Systolic and diastolic blood pressure in control group improved but in centhaquine group a much more significant increase in systolic and diastolic blood pressure was observed 48 hours after resuscitation. Blood lactate levels decreased from 4.30 ± 0.96 at baseline to 3.19 ± 1.07 (p=0.7729) at day 3 in control and from 4.34 ± 0.78 at baseline to 1.44 ± 0.13 (p=0.0012) at day 3 in centhaquine cohort. Two patients died in control and none in centhaquine cohort. No incidence of drug related adverse event occurred. A phase III multi-centric, randomized, double-blind, parallel, controlled phase-III efficacy clinical study of centhaquine therapy in patients with hypovolemic shock (NCT04045327) has been completed in 105 patients with positive results. Centhaquine is an innovative drug product that is likely to provide significant benefit to an unmet life-threatening medical condition of hypovolemic shock.

3D liver microtissues (also known as 3D spheroids) have the potential to positively impact drug development workflows, particularly when applied to safety assessment and drug discovery processes. These 3D in vitro liver models, which can be engineered to mimic healthy and diseased biology in human or model organisms (e.g., monkey, dog, and rat),  have been proven to be powerful tools for fulfilling the demands of modern toxicology programs, both in terms of providing translational safety insights and pushing the bench closer to the patient bedside.
 
In this lecture, you will learn:
 
1. Practical liver toxicology applications for 3D human liver microtissues, from safety testing to complex disease modeling, using non-alcoholic steatohepatitis (NASH) as an example.  
2. Considerations for adapting existing protocols to leverage 3D liver microtissues and upgrade your tox toolbox for investigating specific scientific questions related to drug induced liver injury (DILI) and drug efficacy testing of new drug candidates, such as promising therapies currently under development for NASH —all with a high degree of flexibility.
3. Guidelines for adopting 3D human liver microtissues for use in high-throughput applications, which require uniform spheroid quality, functional robustness, assay scalability, and reproducibility of results.
4. How 3D cell technology is evolving into an investigative tool that will meet the challenges of the next generation of therapeutic modalities.

Non-alcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease in developed countries, and it affects over 25% of the population worldwide. Within the next five years, the more advanced form of this disease, Non-alcoholic steatohepatitis (NASH), is expected to become the leading cause for liver transplants in the US. There are currently no approved pharmacologic therapies for NAFLD or NASH.  Several drug pipelines with diverse mechanisms of action are in clinical development to address this unmet medical need.  While improvement in liver histology confirmed through biopsy is still a required endpoint for regulatory approval, non-invasive biomarkers may be used in early-stage trials to assess treatment effect. Serum Cytokeratin 18 (CK18) levels are reflective of hepatocyte apoptosis and necrosis, which are thought to be important processes involved in the progression of NAFLD. Monitoring serum CK18 levels, therefore, provides mechanistic information on the effect of drug treatment. Furthermore, changes in Serum Cytokeratin 18 Levels have been shown to correlate with changes in liver histology.

The aims of this talk are to:
1. Introduce the audience to the growing epidemic of NAFLD
2. Elucidate challenges in developing therapies for the treatment of NASH
3. Present data that support the value of CK18 as a biomarker for assessing the efficacy of drugs in NASH clinical trials.

The use of primary cells in vitro is compromised by the limited quantity of cells that can be isolated from one donor, a lack of or very restricted proliferation capacity (e.g. hepatocytes) and/or the change from a quiescent to an activated state (e.g. hepatic stellate cells). The use of immortalized cell lines, on the other hand, is compromised by their transformed, cancerous phenotype. Using the upcyte® technology, primary cells are driven into proliferation using a viral gene transfer system, thus allowing controlled and reversible bypass of cell cycle control mechanisms without inducing immortalization, uncontrolled cell growth or changing the typical phenotype. In this talk liver-specific cells such as upcyte hepatocytes and liver sinusoidal endothelial cells will be presented.

Learning Objectives:

1. Identifying different cell types of the liver and their roles

2. advantages and disadvantages of genetic modification of primary cells

The OrganoPlate is a microtiterplate based Organ-on-a-Chip platform for high throughput drug safety and efficacy screening. It contains up to 96 cell culture  chambers that allow co-culture of 3D tissues under continuous perfusion. Highly physiologically relevant tissue and disease models are constructed by (co-)culturing human cells in 3D under continuous perfusion whilst maintaining ease of use and compatibility with standard equipment. This presentation will highlight several applications of the OrganoPlate including models of vasculature, gut, kidney, liver, brain and tumor physiology including iPS and organoid based tissues. The models are interrogated using a variety of assays including (Confocal) Immunocytochemistry, calcium imaging, barrier integrity assessment, qPCR and TEER measurements.

Learning Objectives:

1. Introduce the audience to the fields of organ-on-a-chip and micro physiological systems for preclinical research

2. Explain current tissue culture modalities and (high throughput) assays available in organs on chips

Biomarkers are critical tools for all stages of cancer research, from drug development through clinical applications.  Cancer is fundamentally a disease of unregulated cell growth and circumvented cell death; therefore biomarkers focused on these two cellular processes are valuable in the various stages of drug development for many cancer types.  Few commonly used biomarkers offer the sensitivity and specificity needed to efficiently take drugs through all stages of development and clinical uses.  Thymidine Kinase 1 (TK1) is an old biomarker with a new use in solid tumor studies, while Keratin 18 is a useful biomarker for assessing and distinguishing between necrotic vs apoptotic cell death.  

This talk aims to introduce these two biomarkers as superior options to use over other biomarkers and present data to support their value in developing drugs to target solid tumors.

The liver plays a critical role in the metabolism and clearance of more than 70% of marketed drugs. Furthermore, toxicity to the liver is a major reason for preclinical and clinical drug failures. Animals are not always predictive of human liver-drug interactions due to significant species-specific differences in drug metabolism and toxicity pathways. Thus, in vitro models of the human liver are now routinely utilized in pharmaceutical practice to complement animal testing; such models enable faster and cheaper drug testing and hold the promise to prevent harm to living patients. Isolated primary human liver cells are the gold standard for fabricating human liver models, but these cells rapidly lose their liver functions, including drug metabolism capacities, in conventional 2-dimensional monocultures. In contrast, semiconductor-driven microfabrication tools and co-culture with non-parenchymal cells have been used to precisely modulate the microenvironment around hepatocytes towards enhancing and stabilizing phenotypic functions for 4-6 weeks. Such engineered liver co-cultures have significantly improved the sensitivity for the detection of drug clearance, drug metabolite identification, drug-transporter interactions, and drug-induced liver toxicity. This presentation will showcase the development and validation of engineered human liver co-cultures for the above-mentioned applications; it will then discuss emerging trends in further improving the physiological relevance of the platform using liver non-parenchymal cells and determining the role of major liver diseases (e.g., non-alcoholic fatty liver disease) in changing drug outcomes.

Learning objectives:
1. The key design features necessary for building robust in vitro human liver models and
2. The various applications within the drug development pipeline where such models can be effectively utilized.

Microphysiological systems (MPS), also known as organ-on-chips, are small scale in vitro cell cultures which mimic facets of tissue or organ level function. MPS frequently utilise primary human cells, often cultured in 3D, to obtain highly functional, physiologically relevant models. MPS can be utilized alone, but can also be connected through fluidic circuits to create advanced multi-MPS that can model the interactions between organ systems, allowing greater analysis of molecular pathways and disease mechanisms.

Most current in vitro liver models are simple mono-cultures of hepatocytes or use hepatic cell lines and do not have the complexity to analyse the interactions between different tissue types and determine how these interactions drive specific pathologies. We have developed a novel system for the in vitro culture of hepatocytes in a perfused three-dimensional format, with/without a separate co-culture allowing interactions to be studied with a second MPS. The model of the human liver, contains multiple primary cell types, which can be cultured together for several weeks and maintain their phenotype and metabolic activity. The MPS platform provides a tool to study in greater detail the key interactions between the gut, the lungs, the liver and the immune system that drive pathologies including steatohepatitis, hepatocellular carcinoma and viral hepatitis.

Learning Objectives:

1. Introduction to the field of microphysiological systems

2. Demonstrate use of MPS for efficacy, tox and ADME applications

“The poor translatability of early-stage preclinical models is a major setback in oncology drug development. Immortalized cell lines, that are extensively used in drug screens, undergo genetic drift over time due to adapting to in vitro growth, thus showing limited relevance to the human disease. Interpatient tumor heterogeneity is also a major hurdle with standard in vitro models failing to recapitulate this variability. Patient-derived xenografts are the gold standard preclinical models for their proven translatability to the human disease but can be costly and time consuming to generate and maintain. The Hubrecht Organoid Technology (HUB) has pioneered the development of tumor organoids, a revolutionary patient-derived 3D in vitro system that recapitulates the genomic, morphological, and pathophysiological characteristics of their parental tumor. HUB organoids provide highly clinically relevant 3D in vitro models for oncology drug development. To overcome the limitation imposed by sourcing patient material for organoid preclinical development PDX can be used as a source of human tumor tissue leading to a patient-relevant matched in vitro/in vivo platform for oncology drug screening.”

Learning bullets:
1. Find out why HUB organoids are considered best in class systems
2. Understand how organoids are revolutionizing oncology drug development
3. Learn about how patient-derived xenografts (PDX) can be leveraged to develop the largest organoid library for oncology drug discovery

Accurate DNA replication is essential to transmit the genetic information from one generation to another. However, replication is frequently challenged by barriers that originate from exogenous and endogenous sources. These obstacles perturb normal DNA replication by slowing or stalling replication fork progression, a process known as replication stress. High intrinsic replication stress is a hallmark of cancer. Intriguingly, cancer cells can proliferate and replicate their DNA under replication stress leading to therapy resistance, but how they do so is not understood.

One of the mechanisms that helps cells to deal with replication stress and restart DNA replication is fork reversal, a pathway that can facilitate replication stress relief. This mechanism involves replication fork remodelers, among them the DNA translocase HLTF. Inactivation of several DNA translocases leads to defective fork restart, slowed fork progression, hypersensitivity to genotoxic agents, and increased double-strand break formation. However, we showed that HLTF-deficient cells display several phenotypes unique among known remodelers, including unrestrained replication fork progression and resistance to replication stress. Using a novel single-cell imaging technique, we have shown that these cells move faster through S-phase under replication stress. To further investigate this phenotype, we took advantage of a single molecule technique called “DNA spreading”. This amazing technique allows us to directly visualize replication fork progression in vivo at a single molecule level through labeling the replicating DNA using different nucleotide analogues. By combining this technique to genetic manipulations of our cells (CRISPR knock out), we have shown that all these phenotypes were dependent on the primase/polymerase PrimPol, mediating discontinuous DNA replication in HLTF-deficient cells. We hypothesize that, in the absence of HLTF, PrimPol mediates active fork repriming downstream of the block leaving gaps behind the fork.

Altogether, our data suggest that the molecular composition of the replication fork machinery is changed in HLTF-deficient cells, allowing the recruitment of specialized polymerases (i.e. PrimPol) that help the cells to bypass replication obstacles, progress unrestrained under replication stress and proliferate. As HLTF is silenced in many cancers, this phenotype could explain how cancer cells sustain the stress-resistant replication that drives their proliferation and resistance to therapy. Therefore, understanding how HLTF-deficient cells deal with replication stress at the molecular and cellular levels is paramount to improve cancer therapy and DNA spreading will largely contribute to shed the light on our understanding of replication under stress conditions.

Learning Objectives:

1. Understand how replication stress tolerance can lead to cancer therapy resistance.

2. Explain an elegant single-molecule approach to study replication stress in vivo in cells.

With an increasing push to improve safety, efficacy, and efficiency throughout the drug development pipeline, researchers are evermore looking to improve the predictive capacity of their in vitro models. By employing 3D cell culture models (i.e. spheroids, organoids), which better recapitulate the native tissue environment in comparison to traditional 2D cultures, disease states can be mimicked more effectively, and thus therapeutics can be evaluated in a more meaningful context. Yet the use of these more complex 3D cell culture models stipulates a more sophisticated method of analysis in order to extract the full potential of these models. In this webinar, Erin Edwards of Visikol will discuss 1) how 3D cell culture models can be designed and applied to evaluate new therapeutics, and 2) how tissue clearing and 3D high content confocal imaging can be used to extract the most meaningful conclusions from studies involving 3D cell culture models. Examples will be given from toxicity evaluation (neural, cardiac, liver) and traditional and immuno-oncology.

Learning Objectives:
1. Understand the differences, pros, and cons of each 2D and 3D cell culture models
2. Learn about specific applications in drug discovery where 3D cell culture models can afford advantages over traditional 2D cell culture models.

In the United States, vancomycin is the single most commonly prescribed antibiotic in the hospital setting and is a well-known nephrotoxin. Despite over 60 years of clinical use, there is still debate on the drivers, modifiers, and time-course of the kidney injury associated with vancomycin. Drug-induced kidney injury results in significant long term patient morbidity and mortality. Meanwhile, clinical trials are difficult and expensive, with an estimated median cost of $40,000 USD per enrolled patient. Thus, human trials are less frequently performed. Preclinical models and newer urinary biomarkers provide a feasible pathway to creating safer vancomycin treatment courses for patients. Positive findings can be carried forward to create Precision Dosing for vancomycin (i.e. drug optimization based on individual patient exposures and biomarkers that indicate sub-clinical toxicity).

Learning Objectives:

1. Describe the exposure-response relationships with vancomycin and acute kidney injury
2. Define biomarkers that may be useful to improve Precision Dosing