Leaders in Pharmaceutical Business Intelligence (LPBI) Group

Immunotherapy in Cancer: A Series of Twelve Articles in the Frontier of Oncology by Larry H Bernstein, MD, FCAP

Reporter: Aviva Lev-Ari, PhD, RN

Introduction

This project has been the most intense search for answers in my career as a pathologist.  It was perhaps made somewhat necessary after my retirement from a career in clinical pathology with a feeling of not having finished my life work, which began in my childhood reading of Paul De Kruif’s Microbe Hunters, and continued with my readings in collegiate course in scientific German. I began serious work on the crystallins of the eye lens and also on the ontogeny of the lactic dehydrogenase isoenzymes in the laboratory of Prof. Harry Maisel after my sophomore year in medical school, and when I graduated in 1968, I had chosen a career in pathology, but a career limited to service in anatomic pathology would not be a good fit.  I was mentored in residency by a superb pathologist and biochemist who suggested after a year that I work in the laboratory of Nathan O. Kaplan, at University of California, San Diego, where I completed my training in biochemistry and enzymology, in particular, and also completed my residency in pathology under Averill Liebow.  It was an intense experience. Unappreciated by me at the time, NO Kaplan never used the terminology for the pyridine nucleotide coenzymes – NAD and NADP – but stuck to the terms used by Otto Warburg, DPN and DPNH.  I was still not prepared for the half century old hypothesis by Warburg that cancer involves a dysmetabolism of the mitochondrion, and he had referred to the work 60 years earlier by Louis Pasteur to identify in the proliferation of cancer cells, a reliance on glycolysis (as in fermentation), even in the presence of oxygen.  However, in my first academic appointment I continued studies of the mitochondrial and cytoplasmic malate dehydrogenases. I developed a simple assay to determine the ratio of the two activities based on the work done earlier on differences in their inhibition by a ternary complex formed between oxaloacetate and the NAD⁺ formed during the forward reaction with the reduced coenzyme. I also obtained fast growing and minimal deviation hepatocellular carcinoma tissue from my Chairman, Herschel Sidransky, and found interesting differences between cytoplasmic malate dehydrogenases from benign liver, minimal deviation and fast growing cancer that I could not continue long term.

The Warburg investigations have been reexplored in a new light in the 21^st century. The scientific instrumentation and the computational tools available today have brought a greater depth to biological and medical sciences, which has given real promise to a reconstruction of pharmaceutical sciences. This is the case for infectious diseases, autoimmune disease, diabetes, genetic diseases, cardiovascular disease, endocrinology, and also cancer.

The difficulty with cancer has been the variety of presentations and courses of development for carcinogenesis by types of tissue, age, and sex, as well as differences within types. This has been made clear by the significant number of mutations that have been uncovered associated with cancers in humans and animal models. The first generations of cancer therapies were directed at DNA and replication, and they have carried toxicities to nonmalignant cells. However, we have unlocked an inner dimension of cells in which there are networks of interacting pathways that are involved in cellular regulation. We also have a much better grasp of the processes of cell replication, cellular remodeling, proliferation, and cell death, which ranges from some degree of autophagia involving mitochondria, the endoplasmic reticulum, and the lysosome, and cell death (apoptosis). The proteins, enzymes and pathways have been evolved for thousands of years, and are of primordial descent.

This chapter is concerned with the possibilities for pharmaceutical developments in cancer for the near future.  It will cover the outlined subchapters. It is based on extensive searches for articles using a combination of sources. The basic theme of these presentations will be in more than one direction as follows:

• The encapsulation of a drug on conjugates so embedded that the action is locally directed to the site of the tissue disrupted.
• The targeting of specific pathways related to cancer oncogenes, or more specifically, having a key role in cell proliferation, cell adhesion, and metastatic potential.
• The effect of overexpression of identified pathways, and the effect of suppression of the same pathways, and the interaction between other key pathways that are upregulated or downregulated.
• The investigation of these mechanisms brings one to some conclusion about the amazing intricacy of how we age, and how we interact with a stressful environment.

This is what makes it difficult to design a treatment that is the so thought of “magic bullet”, and it necessitates more than one course of treatment, and combination treatments will probably not go away. However, there may still be many opportunities for treatments that are optimum for the patient and the condition. In addition, there is another dimension that was not so possible even a decade ago.

The clear knowledge of how these pathways are related to the particular cancer, and the ability to measure the substrates and small molecules involved has introduced a better way to follow the effectiveness of treatment, and at an early stage.

The reader will find that not all of the treatments are necessarily by use of a monoclonal antibody. All of the reactions do rely on an intermolecular reaction like a lock-and-key that binds with a critical small molecule that is critically engaged in a regulatory process.  Whether it is sufficient is a matter to be discovered.  We are now familiar with a library of terms, such as, conformational change, linkers, promoters, inhibitors, upregulation, downregulation, baggage-carriers, heteromer, dimer, trimer, etc.  These all are players in this process.

Growth Factors, Suppressors and Receptors in Tumorigenesis

Larry H Bernstein, MD, FCAP

http://pharmaceuticalintelligence.com/2015/04/07/growth-factors-suppressors-and-receptors-in-tumorigenesis-7-1/

7.1 Quantum dots

Larry H Bernstein, MD, FCAP

http://pharmaceuticalintelligence.com/2015/04/13/quantum-dots-7-1/

7.2 Liposomal encapsulated drug

Larry H Bernstein, MD, FCAP

http://pharmaceuticalintelligence.com/2015/04/13/liposomal-encapsulated-drug-7-2/

7.3 Protein-binding, Protein-Protein interactions & Therapeutic Implications

Larry H Bernstein, MD, FCAP

http://pharmaceuticalintelligence.com/2015/04/14/protein-binding-protein-protein-interactions-therapeutic-implications-7-3/

7.4       EpCAM

Larry H. Bernstein, MD, FCAP

http://pharmaceuticalintelligence.com/2015/04/07/epcam-7-4/

7.5       Upregulate Tumor Suppressor Pathways

Larry H Bernstein, MD, FCAP

http://pharmaceuticalintelligence.com/2015/04/08/upregulate-tumor-suppressor-pathways-7-5/

7.6       Manipulate Signaling Pathways

Larry H Bernstein, MD, FCAP

http://pharmaceuticalintelligence.com/2015/04/08/manipulate-signaling-pathways-7-6/

7.7       Pathway Specific Targeting in Anticancer Therapies

Larry H. Bernstein, MD, FCAP

http://pharmaceuticalintelligence.com/2015/04/09/pathway-specific-targeting-in-anticancer-therapies-7-7/

7.8     Sirtuins

Larry H. Bernstein, MD, FCAP

http://pharmaceuticalintelligence.com/2015/04/10/sirtuins-7-8/

7.9     Hypoxia Inducible Factor 1 (HIF-1)

Larry H Bernstein, MD, FCAP

http://pharmaceuticalintelligence.com/2015/04/10/hypoxia-inducible-factor-1-hif-17-9/

7.10     Wnt/β-catenin Signaling

Larry H. Bernstein, MD, FCAP

http://pharmaceuticalintelligence.com/2015/04/10/wnt%CE%B2-catenin-signaling-7-10/

7.11       Targeting the Wnt Pathway

Larry H Bernstein, MD, FCAP

http://pharmaceuticalintelligence.com/2015/04/10/targeting-the-wnt-pathway-7-11/

7.12       Novel Approaches to Cancer Therapy

Larry H. Bernstein, MD, FCAP

http://pharmaceuticalintelligence.com/2015/04/11/novel-approaches-to-cancer-therapy-7-12/

 Summary

I introduced these presentations with a message of high complexity and intricate networks that govern our cells.  We have had a spectacular development in the research on genomics, but the picture was incomplete. There are repeated discoveries of new oncogenes that are related to cancer discoveries. In some cases they have had a large impact on diagnosis and relationship to an at risk population, most notably in breast cancer. Then there is the long history of PSA in prostate cancer.  These are biomarkers, and biomarkers of another generation are coming into play.  These biomarkers are the signaling molecules that are directly involved in the tumorigenesis progression.

While we know about the large number of genomes that can develop mutations over a lifetime, we cannot necessarily make a determination of cause and effect.  In addition, the study of genomics is not necessarily related to phenotypic observables. The genome is the script, and it is not directly engaged in the dynamic processes of the living cell.  We now have a host of molecular agents that interact with genetic chromatin, or interact with the cytoskeleton, which can have an impact in the immediate cell structure and function. There are small RNAs, large RNAs, and peptides and proteins. The cell is engaged with other cells by an intercellular matrix, by transport proteins, by pore structures that convey electrolytes, and the cell is sensitive to hydrostatic pressure and to temperature.

We also see that there are protein substrate, and protein-protein interactions that occur in close proximity and affect protein conformation. These many reactions occur in milliseconds to maybe a hundredth of a millisecond. This is the architecture needed to survive in a stressful environment, which might arise intracellularly as well as extracellulary.

This chapter has gone into much detail about the relationship of signaling cascades and key regulatory targets involved in cell stemness, cell proliferation, cell aggregation, and cell metastasis. The reactions discussed don’t bring into the discussion of a dimension of the metabolic process that also needs consideration. Some reactions have been unknown until the development of mass spectroscopy. If there is a reaction, we have to consider either a cationic cofactor, or a nucleotide catalyst.  Briton Chance pioneered the study of in vivo cell monitoring for changes in the ratio of NADH/NAD⁺.  This is a dynamic viewing of cellular processes.

There are three articles in the Apr 15 issue of Genetic Engineering News (Genengnews.com) that are specifically related to this discussion.

1. Vicki Glasser – Diverse Pathways to Drug Targets.

Protein paths through the gene-expression undergrowth have been well trodden, but RNA paths want wear too.

More than 90 percent of the genome that is transcribed into RNA is not translated into protein, and the growing numbers of naturally occurring microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) being identified and characterized, the important role that they have in normal biological processes and diseases is becoming ever more clear. An example to illustrate this point is the case of a Phase II clinical study of micravirsen, antisense oligonucleotide, in patients with hepatitis C (hep C), published in 2013 in the New England Journal of Medicine that describes its effectiveness as dependent on the miR-122 binding hepC for stability, and inhibition of miR-122 in HCV infected patients was associated with decreased levels of HCV that continued beyond the treatment period.
However, does this also apply to cancer. According to George Calin and coworkers at MD Anderson Cancer Center, Houston, Tx, regulatory RNAs – both miRNAs and other ncRNAs are being investigated to identify miRNAs of about 21-22 nucleotides length that can serve as reliable biomarkers for cancer diagnosis and to guide treatment. These miRNAs are stably expressed in tumor cells and  the exosomes are present in body fluids, where they act like hormones and signaling molecules. The work was described in 2014 in CA: A Cancer Journal for Clinicians (“MicroRNAome genome: a treasure for cancer diagnosis and therapy”, and was also presented in Feb 2014 at the Molecular Med Tri Conference in San Francisco. Nevertheless, finding a miRNA target is difficult because an individual miRNA could have a role in regulating tens, hundreds, and even thousands of protein-coding genes. The message is to identify mRNAs that affect a single pathway of interest to help limit off-target effects. The solution depends on identifying which metabolic and/or signaling pathways are activated of inhibited.

2. Lisa Heiden – Precision Tuning GPCR Pathways
   G protein coupled receptors (GPCRs) are essential drug targets for therapeutic intervention due to their integral roles in a plathora of fundamental signal transduction pathways. But discovering, designing, and synthesizing GPCR-targeted compounds for modulating signaling has been difficult, mainly because the pathways are so complex. Consequently, of 800 proteins that have been classified as GPCRs only drugs have been developed against only 50.
3. Kate Marusina – RNA Constructs: Thread Translational Needle

Noncoding RNA plays a major role in gene expression and gene regulation, and its malfunction often results in abnormal cellulat activity. This understanding led to the development of treatment strategies that use RNA both as therapeutics targets and treatment agents. This years’ Gordon Conference on Nanotechnology is dedicated to RNA nanotechnology research. Cancer pathways and miRNAs (that regulate the expression of more than 90 percent of the human genome) are linked, according to Carlo Croce at Ohio State University Institute of Genetics.  miRNA is often the downstream target of an initial tumorigenic event. Dr. Croce’s team identified a cause of chronic lymphocytic leukemia (CLL) in a chromosomal region that is lost in 70 percent of CLL, which contains two miRNA genes, miRNA-15 and mi-RNA-16. They demonstrated that these are negative regulators of another gene in the cascade, BCL-2. In May 2014 AbbVie presented results from a Phase Ib clinical trial of ABT-199, a BCL-2 selective inhibitor. The response rate was 84% in patients with relapsed/refractory CLL.  Furthermore, Peixuan Guo’s team at University of Kentucky Nanobiotechnology Center introduced the phi29 motor pRNA nanotechnology that is a hexameric pRNA nanoparticle with a ribozyme, a receptor-binding aptamer, a siRNA, an image reporter molecule, a bound drug site, and a component for endosome disruption.   The key to success will be sustaining tumor suppression using the scaffold, targeting lignads and therapeutic modules that can be composed entirely of RNA. The prototype was achieved by packaginging bacteriophage phi29 (pRNA) fragments designed to form multimeric RNA nanoparticles with defined size and structure.

This chapter focused on the following key molecular targets:

EpCAM	Gene silencing
Wnt/β-catenin	nanoparticles
Erp49	Quantum dots
IGFBP2/PTEN	Liposome encapsulation
EGF/EGFR
Argonaute and Triman
GPCR
N-terminal modifications
HIF-1
Tumor suppressors
–         NR4A
–         NLRX1
–         Mre11
–         SCDF1 and CXCR4
–         DLC1
–         Smad 7
IQGAPs
Cathepsin B
Raf-Mek-Erk
shh
NF-kB