The EVA-PCD™ Platform

At the recent meeting of the American Association for Cancer Research (AACR) held in Washington D.C (April 16-21, 2010), the theme remained consistent with the ground swell of interest in personalized care. Many of the sessions reflected the changing paradigm of clinical trials with a growing focus on biomarker analysis and patient selection predicated on genomic and proteomic features. Among the most compelling presentations were those that examined the manifest complexities of human signaling circuits. One presentation by Dr. Neal Rosen from Memorial Sloan Kettering in New York examined redundancy and feedback as principal determinants of clinical response to signal inhibitors. That session, chaired by Dr. Engleman from Harvard Medical School, examined the cross talk between EGFr and PI3K pathways. Using cell line systems, these investigators drilled down onto RNA and DNA expression profiles to examine how inhibitors acting for one pathway might up or down regulate parallel pathways.

This work dovetailed perfectly with our presentation on Monday, April 19, 2010 (Nagourney, R. et. al, Horizontal and vertical signal pathway inhibition in human tumor primary culture micro-spheroids. Abstract 1764, proceedings AACR 2010). In this analysis, we used small molecules tyrosine and serine/threonine kinase inhibitors to examine the points of commonality and disparity in these two crucial signaling pathways to assess how future drug combinations might provide response with these novel classes of agents.

The most exciting aspect of our work is the capacity of the human tumor micro-spheroid platform (EVA-PCD™) to capture all of the operative mechanisms of response and resistance. This, more closely than other platforms, recapitulates the complexity of human tumors and provides insight into these  complex and redundant biological pathways. No genomic or proteomic tool can approximate the clinical relevance of the EVA-PCD™ platforms’ predictions.

Forms of Cell Death

Following the description of apoptosis in the British Journal of Cancer in 1972, scientists around the world incorporated the concept of programmed cell death into their cancer research. What is less understood is the fact that apoptosis is not synonymous with programmed cell death. Programmed cell death is a fundamental feature of multicellular organism biology. Mutated cells incapable of performing their normal functions self-destruct in service of the multicellular organism as a whole. While apoptosis represents an important mechanism of programmed cell death, it is only one of several cell death pathways. Apoptotic cell death occurs with certain mutational events, DNA damage, oxidative stress and withdrawal of some growth factors particularly within the immune system. Non-apoptotic programmed cell death includes: programmed necrosis, para-apoptosis, autophagic cell death, nutrient withdrawal, and subtypes associated with mis-folded protein response, and PARP mediated cell death. While apoptotic cell death follows a recognized cascade of caspase mediated enzymatic events, non-apoptotic cell death occurs in the absence of caspase activation.

With the recognition of programmed cell death as a principal factor in carcinogenesis and cancer response to therapy, there has been a growing belief that the measurement of apoptosis alone will provide the insights needed in cancer biology. This oversimplification underestimates the complexity of cell biology and suggests that cancer cells have but one mechanisms of response to injury. It has previously been shown that cancer cells that suffer lethal injury and initiate the process of apoptosis can be treated with caspase inhibitors to prevent caspase-mediated apoptosis. Of interest, these cells are not rescued from death. Instead, these cells committed to death, undergo a form of non-apoptotic programmed cell death more consistent with necrosis. Thus, commitment to death overrides mechanism of death.

Labs that focus on measurements of caspase activation can only measure apoptotic cell death. While apoptotic cell death is of importance in hematologic cancers and some solid tumors, it does not represent the mechanism of cell death in all tumors. This is why we measure all cell death events by characterizing metabolic viability at the level of cell membrane integrity, ATP content, or mitochondrial function. While caspase activation is of interest, comparably easy to measure and useful in many leukemias and lymphomas, it does not represent cancer cell death in all circumstances and can be an unreliable parameter in many solid tumors.

Probing Human Biology

Functional Analyses Unravel The Complexities of Signal Transduction

The application of functional analyses in human tumors is the topic of our upcoming presentation at the American Association of Cancer Research to be held in Washington D.C, in April 2010 (Nagourney, R. et. al, Horizontal and vertical signal pathway inhibition in human tumor primary culture micro-spheroids. Abstract 1764, to be presented Monday, April 19, 2010).

Scientists now realize that cancer biology, indeed all biology, is driven by signaling pathways. Cells speak to each other and the messages they send are interpreted via intracellular pathways known as signal transduction. Many of these pathways are activated or deactivated by phosphorylations on select cellular proteins. Tyrosine kinases, and serine/threonine kinases are among the most important classes of enzymes responsible for these chemical cascades inside the cell. In recent years small molecules have been developed to inhibit these chemical reactions. Hundreds of such compounds are in development for cancer today. While most scientists use genomic or proteomic platforms to detect mutations in these pathways that might result in response to these chemicals, we have taken a different tack. By applying functional analysis, to measure the end result of pathway activation or deactivation, we can predict whether patients will actually respond. Our results in lung cancer patients to date have exceeded the best outcomes using DNA profiles, clearly supporting the predictive accuracy use of functional analyses in this and related areas.

Focusing upon two fundamental pathways, the EGFR and the insulin-like growth factor pathway, we have explored how small molecule inhibitors influence these important survival signaling pathways. This is but one of many applications of functional profiling in the study of human tumor biology.