2010 Progress Report – Executive Summary
When a cell is infected or becomes cancerous, the signaling and regulatory pathways in its cyto-network move away from homeostasis and the network enters an aberrant state. Molecular complexes that drive a specific disease have multiple, often disconnected functions and knowledge of these functions are imperfect. We reasoned that many therapeutic strategies fail because of a lack of network interaction information on what actually controls a disease, which would be essential to pick a potentially efficacious therapeutic strategy. The CCC strategy to achieve the NDC vision is to precisely interrogate and affect cell functions with combinatorial drugs aimed at regulating the cyto-network for desired cell phenotypes, to ultimately advance therapeutic goals. We aim to identify key cellular components that are responsible for the aberrant state of the cyto- network of an infected or diseased cell and use this information to develop successful therapies.
Using this integrative approach, synergistic or antagonistic interactions between molecular complexes are deciphered by quantifying multiple phosphorylation nodes in the signaling network. This strategy has already been demonstrated and linked specific protein machineries to translational medicine. Successful examples include in-vitro experiments to inhibit DNA virus infections, to eradicate cancer cells, and to maintain hES cell pluripotency and self-renewal under defined culturing conditions. The CCC overall vision and research goals are illustrated in the following schematic diagram.
Over the past few years, our center developed Feedback System Control (FSC) technology that coupled mathematical modeling algorithms from gaming theory and a rapid iterative experimental approach to quickly and reproducibly drive specific, desirable biological outcomes by optimizing the combination of input agonists and antagonists. We have used the FSC technique to interrogate aberrant cellular network activities in various biological systems including HSV-1 infection, non-small-cell lung cancer, and leukemia. With the addition of PhosphoFlow technology, we are in a unique position to identify key molecular components in these biological systems to advance therapeutic goals. For HSV-1 infection, PhosphoFlow analysis was able to identify the exceptionally high activity of an infected cell’s aberrant ribosome function. As pointed out by the 2009 Nobel Laureate in Medicine or Physiology, Ada Yonath, the ribosome is a cellular nanomachine in the genetic translation process [Ref]. With results from our study, a regulated mammalian ribosome complex, a logical but previously unsuspected nanoscale machine in infection, could now be useful as a selective target in blocking HSV-1 infection and spread. Indeed, our preliminary studies show that the direct blockade of specific units of the ribosome can produce successful results similar to optimized drug cocktail therapy in blocking HSV-1 Page 46 infection. This HSV-1 infection result serves as a roadmap for identifying essential cellular protein complex for defeating cancer.
In our cancer studies, we have used FSC to identify synergistic drug cocktails that were able to selectively eradication cancer cells from in vitro models of non-small cell lung cancer and leukemia. From these studies we revealed that the cellular responses of normal and cancer cells to the same set of drug combinations are significantly different. Furthermore, in lung cancer, we found that a single-drug resistant cell line exhibited a signaling response profile that was similar to a non-resistant cell line under treatment, suggesting that drug combinations can be effective in treating patients who develop single-drug resistance or to prevent the emergence of drug-resistance at the initiation of therapy. With unique and enabling technologies working together (FSC and PhosphoFlow), CCC labs are currently identifying key molecular components and aberrant nanomachines in lung cancer and leukemia for the development of directed therapeutic systems. Furthermore, we are able to maintain and expand hES cells in animal product-free culture media that eliminates a major hurdle for clinical applications of hES cells. Licensing agreements with industry partners are currently underway to bring this culture system to clinical applications.
The CCC continues to develop approaches to provide real-time monitoring of cellular signaling events to complement the PhosphoFlow technology already in place. TNPR technology was a high-risk project that provides the promise of unprecedented real-time intracellular monitoring of signaling pathways. We were able to overcome the first hurdle of mass fabrication of TNPRs by using nano-imprint lithography. Since then, we have performed feasibility tests exploring cellular injection techniques, toxicities and signal-to-noise ratios inside and outside the cell. We also developed OET coupled to femto-second laser pulsing for the introduction of nanoparticles, and TNPRs, into the cellular cytoplasm. Currently, we have demonstrated the feasible of this technique by selectively delivery propidium iodide (PI) dye into the cell and the ability to trap and concentrate fluorescently conjugated Au nanoparticles. We began extracellular interrogation using TNPRs to map and quantify the IL-2 secretion pattern from a single Jurkat T cell in culture. Results show that TNPRs were able to successful map the gradient pattern of IL-2 from the T cell and that TNPR was essential to achieving this measurement with quantifiable signal-to-noise ratios.
As one main focus of the CCC is translational medicine, we are working towards implementing effective combination therapies for HSV-1 infection, lung cancer, and leukemia in mouse model systems. To address the need of treating genital infections, we have used a mouse model of vaginal infection of HSV. Using intravaginal infections as well as direct vaginal treatments with drug combinations we have worked to show improvement in the efficacy of drug combinations versus drugs alone. Preliminary experiments in mouse lung cancer models using Celecoxib and Erlotinib alone and their combinations showed that the combination was able to induce lower tumor proliferation compared to single drugs but without a resolution of tumor. We have since selected a 3-drug mixture of Pioglitazone, Celecoxib, and Erlotinib to be evaluated in vivo. For leukemia, we have adopted a model of acute myeloid leukemia as our test platform. This tumor model has the following advantages 1) the molecular lesions are physiologically relevant to aggressive human AML; 2) effective therapies for this form of AML have not been identified, providing a preclinical and clinical need; 3) tumor generation time is relatively short and reproducible; 4) interesting questions in clonal evolution can be addressed with serial transfer and FSC-optimized drug combinations to determine what additional lesions defeat initially successful therapies in a well-controlled genetic system. Our approach will incorporate in vitro data to identify optimized drug combinations against harvested primary and secondary tumor explants, along with comparative analysis using in vivo combinatorial therapy initially guided by in vitro results.
Ref: Yonath, A., Large facilities and the evolving ribosome, the cellular machine for genetic-code translation. J R Soc Interface, 2009. 6 Suppl 5: p. S575-85.
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