Executive Summary of the Cell Decisions in Response to DNA Damage: Survival vs. Programmed Cell Death Think Tank

 

Current cancer therapy relies heavily on DNA damaging agents (radiation, DNA alkylating agents, etc.) to induce programmed cell death in cancer cells. The proven success of this therapy, albeit only in some patients, implies that cancer cells are more sensitive to killing by DNA damaging agents than normal cells. This increased sensitivity is believed to be due largely to defects in DNA damage response pathways within the cancer cell. Compelling experimental evidence suggests that it is possible to dramatically modulate the sensitivity of cells to DNA damage. Similarly it may be possible to modulate the cell-specific and stimulus-specific responses to DNA damage leading to cell death.  These responses vary dramatically between cell and tissue type, metabolic state and genetic background.  If the sensitivity of cancer cells to DNA damaging agents and the cell death response to them could be specifically increased (or the sensitivity of normal cells to these agents be specifically decreased) by just one order of magnitude this would lead to a significant increase in cancer cure rates.

 

The obvious benefits of such an outcome are:

• reduced collateral tissue damage from the use of lower doses of radiation or chemical agents that would be required to kill cancer cells,
• reduced risk of second cancers from cells irradiated at the edges of the therapeutic radiation field, 
• improved tumor targeting by combining the focusing power of radiation therapy with pharmacologic enhancement of tumor susceptibility to DNA damage.

 

A major goal of the workshop was to identify the knowledge and resources needed to optimize the DNA damage response leading to programmed cell death in human cancer.  The Think Tank participants put forth a number of recommendations that can be summarized into 4 broad areas:

 

• Support systems biology analyses of the human DNA Damage Response (DDR) networks including the complete mapping of the biochemical and regulatory circuitries that link the cell-cycle checkpoints, apoptotic, and DNA repair pathways with the DDR.
• Support further identification of molecular targets for enhancing programmed cell death in response to DNA damage, particularly by investigating p53-independent pathways of DDR-induced cell death and by investigating strategies to modulate activity of DDR sensor and mediator proteins to amplify cell death signals.
• Develop reagents to study the DDR in vivo, particularly a resource library of phosphospecific antibodies and DDR read-out reagents for quantitative and dynamic measurements.
• Convene a conference focused on enhancing molecular pathology approaches for the analysis of DDR in human tissues and undertaking comparative studies of the DDR in normal and tumor cells/tissues to elucidate novel anti-apoptotic components altered in cancer cells.

 

 

 

 

 

Introduction

 

The introduction of DNA damage is a major therapeutic strategy for killing cancer cells.  However, cell death is not the sole option for cells in response to DNA damage. In brief, the damaged cancer cell has two options: To die by regulated programmed cell death (PCD), or to survive by preventing cell division until DNA repair can be completed.  In their initial stages, both PCD and survival/DNA repair processes initiated by DNA damage share the same signaling cascades based on high-level protein kinases (e.g., ATM, ATR) and secondary kinases (e.g., Chk1 and 2) along with a number of other proteins involved in signal detection and Amediation@ of signaling and repair.  What is less clear, however, is how, at the molecular/mechanistic level, human cancer (and also normal) cells that have sustained DNA damage assess its severity and make the ultimate cellular decision between death and survival.  The aim of this Think Tank was to advance understanding of the mechanisms involved, with the ultimate goal of finding strategies that will select or enhance cell death. 

 

Think Tank Program

Session I: DNA Damage and Repair

 

Discussion topics included:

 

¨      How do human cells (normal and cancer) detect primary DNA damage?

¨      How are signals of primary DNA damage amplified by the cell?

¨      What are common and distinct responses to different types of DNA lesions?

 

The most lethal type of DNA damage is DNA double strand breaks (DSBs). DSBs can be caused directly by radiation or indirectly after DNA modified by chemotherapeutic drugs is processed by cellular enzymes. Both radiation and DNA-modifying chemotherapeutic drugs are currently used to treat human cancer.  

 

The response to a DNA DSB includes DNA repair and DNA damage signaling components. DNA repair pathways are clearly important; however, DNA repair genes are not frequently mutated in human cancer and it is not clear that inhibiting these pathways will lead to cancer-specific killing. DNA damage signaling pathways (also known as DNA damage checkpoint pathways) include sensors, which detect the presence of DNA DSBs, transducers, which produce a DNA damage signal, and effectors, which induce cell death or cell cycle arrest (transient or permanent). Mutations that inactivate DNA damage checkpoint genes are extremely prevalent in human cancer, resulting in rewiring of the DNA damage signaling pathways and differences in the response of cells to DNA damage.

 

In the last ten years there has been tremendous progress in our understanding of DNA damage checkpoint pathways. Most of the known DNA damage checkpoint genes were cloned within the last ten years. Their function is just beginning to be understood and key research discoveries, especially with regard to the DNA DSB sensors, were published in the last year or are still in the process of being published. The emerging theme is that there is a small number of DNA DSB sensors and the response of the cell to DNA damage (cell cycle arrest or cell death) depends on which sensor is being used to recognize the damage. Crystallography analysis suggests that recruitment of at least one sensor (53BP1) to DNA DSBs is amenable to pharmacologic intervention. Inhibition of 53BP1 recruitment to sites of DNA DSBs would result in increased recruitment of other sensors (specifically, the NFBD1/MDC1-Mre11 complex), which is thought to be more potent in promoting cell death in response to DNA DSBs.

 

Cells also have at least two transducers of the DNA damage signal in the form of the protein kinases ATM and ATR. How these kinases are activated by the DNA DSB sensors is not yet clear. The pathway downstream of ATM and ATR is somewhat better understood, although there are still gaps in our knowledge there as well.

 

In conclusion, how cells detect the presence of DNA DSBs and transduce the DNA damage signal represents a major gap in our knowledge. The recent progress in this field suggests that we are at the verge of making considerable progress. Characterization of the DNA DSB sensors and transducers is expected to identify promising and pharmacologically amenable targets for development of cancer therapeutics.

 

Session II: Checkpoints and Apoptosis

 

 

Discussion topics included:

 

¨      How do cells assess DNA damage information to decide between survival and death?

¨      Is DNA repair linked to either checkpoint or apoptosis induction?

¨      Are DNA lesions sufficient to trigger apoptosis?

 

The p53-dependent pathway which induces apoptotic cell death in response to DNA damage is clearly relevant in terms of cancer therapy toxicity, since normal tissues exposed to DNA damage can undergo apoptosis. Stabilization of p53 following DNA damage results in its accumulation in both nuclear and cytosolic compartments.  Cytosolic p53 can directly activate the proapoptotic activity of Bax to permeabilize the mitochondrial outer membrane.   It remains to be determined to what extent this cytosolic effect, independent of transcription, contributes to p53-mediated death.  If cytosolic p53 is apoptotic, the transcriptional upregulation of PUMA may provide another block to the anti-apoptotic functions of Bcl-2/Bcl-xl.  However, in most solid tumors these pathways are unlikely to contribute to DNA damage-induced death, because in most tumors p53 is inactivated by mutations. Therefore it is anticipated that inhibiting p53-dependent apoptosis may enhance the therapeutic index of cancer therapy, although there are still reservations regarding whether such an approach would be beneficial in humans.

 

p53-independent cell death pathways are likely to be of greater relevance to killing of most solid tumors in response to therapy. Cell death in p53-mutant cancers may be due to apoptosis or perhaps more likely to progress through the cell cycle with unrepaired DNA DSBs, which occurs when DNA damage checkpoint genes are mutated. While in yeast and humans it is well established that defects in DNA damage checkpoint genes lead to extreme radiosensitivity (for example, in ataxia-telangiectasia patients in which the ATM transducer is mutant), a link between mutations in DNA damage checkpoint genes in human cancers and their radiosensitivity has not been systematically investigated (with the exception of the effect of p53 mutations). Recent studies have identified a p53-independent apoptotic pathway, involving p53-related p63 and p73, in mediating DNA damage induced apoptosis.  Activation of p73 by genotoxic agents has been shown to involve the nuclear c-Abl tyrosine kinase and contributes to the apoptotic response to p53-negative cells.  But in general, it is fair to say that we do not understand well how DNA DSBs kill cancer cells that bear mutant p53.

 

In addition, apoptosis is not the only way for damaged cells to die.  Excessive or persistent damage can trigger other modes of cell death, including necrosis and mitotic catastrophe.  It is also possible that these modes of cell death could offer opportunities for new therapeutic strategies.

 

Session III: DNA Damage and Cancer Therapy

 

Discussion topics included:

 

¨      How can our knowledge about DNA damage responses be best exploited to therapeutic benefit?

¨      What are the current roadblocks to translating our knowledge about DNA-damage responses to therapeutic strategies?

 

Tumor cells start out as intrinsically more sensitive to apoptosis by virtue of their obligate oncogenic lesions, which push cells closer to their apoptotic activation threshold. This is thought to explain the innate sensitivity of most tumors (at least initially) to the crude and blunt types of classical therapies we currently use. Clearly, such innate apoptotic sensitivity is eroded during tumor evolution, and especially in response to the strong selective pressure of classical cancer therapies. However, such erosion is an evolutionary process and arises through a specific and restricted repertoire of anti-apoptotic mutations that the tumor acquires. Because of this, tumor cells remain chronically dependent on their restricted repertoire of anti-apoptotic mutations for their survival. In contrast, normal cells have no pro-apoptotic oncogenic lesions and are supported by a variety of trophic signals by virtue of their residing in their correct orthotopic somatic environments. By definition, tumor cells are outside their orthotopic environment and should therefore be acutely sensitive to therapies that negate their anti-apoptotic mutations.

 

Unfortunately, we know too little as yet of the range or repertoire of anti-apoptotic mutations that operate in different types of cancer cell. Since such mutations are acquired through natural selection, and since different selective pressures operate on different tissue types as they evolve into tumors, anti-apoptotic mechanism are likely to vary between different tumor types. This provides an unparalleled wealth of possible therapeutic targets, if we could but identify them. This must remain a priority.

 

 

 

 

 

 

Conclusions:

 

Pathways

 

A substantial body of data exists on the pathways of responses to DNA damage in bacteria and yeast, and studies of pathways generally follow two main strategies: mutations in pathways that trigger cell death, and responses of cells (i.e., responses at the cellular level) to damage.  Despite extensive information on some pathways in certain organisms, many key gaps in knowledge exist in how cells detect and repair DNA lesions and orchestrate these cellular responses.  Further investigation of DNA repair mechanisms in mitochondria and of the mechanisms of target resistance and sensitivity is also warranted.  We also need to increase our knowledge of how DNA damage kills cells that lack a functional p53, and the role of metabolism and microenvironment on cell death decisions in response to DNA damage.  We have accumulated clustered knowledge in areas such as apoptosis and DNA repair, but we lack the knowledge needed to link these clusters.  The basic mechanisms of DNA repair and molecular interactions in signal transduction pathways are probably best studied in cellular models, including yeast and mammalian cells, and advances in these areas may assist in the identification of novel therapeutic interventions (e.g., IR and targets of NHEJ repair). 

 

It is also critical in this area of investigation to understand the pathophysiology of normal versus tumor tissue and the progression of normal tissue to tumor tissue.  It is assumed that mutations in mechanisms that alter DNA repair or damage response pathways can potentiate tumor development.  However, it is not clear whether this process is (1) a stochastic phenomenon that “opens the door” to a cell progressing to transformation or to cell death; or (2) an ordered, repeated process that continues until reaching a specific threshold beyond which repair no longer occurs, ultimately generating a cancer cell or causing a cell to die.  Tracing the effect of a DNA repair defect from its initial stage to the final outcome in a highly advanced tumor cell would likely be highly informative in distinguishing between these two possibilities and in identifying different death suppression mechanisms that lead to variable sensitivity to DNA damage.  Comparisons of immediate responses to DNA damage, which appear to involve posttranslational changes, and longer term adaptive responses, would likely be informative.

 

Tumors may be considered as dysfunctional tissue.  For example, tumor cells are attracted to a type of vasculature to which normal cells are not.  In addition, specific lesions in tumors prevent terminal differentiation.  A more sophisticated analysis of signaling pathways within currently curable human tumors would be enormously valuable.  While these tumors (mostly pediatric leukemias, sarcoma, germ cell tumors, certain lymphomas) have been studied in the past, a more sophisticated approach, which compares and contrasts their signaling responses to chemotherapy (particularly DNA damaging agents) relative to incurable tumors, is likely to shed important light on key variables in treatment success. 

 

Tools:

 

Considering that the NCI Developmental Therapeutics Program is being "reshaped," the NCI could now play a direct role in providing infrastructure and support for the development of new tools.  The NCI should be looking hard at data already in hand with an eye to making it more accessible (e.g., cross-indices of chemical compounds vs. the ‘NCI60’ cell lines) or simply funding development to make needed reagents available (e.g., screening to identify panels of phospho-specific antibodies that work on tissue).  A model is found with the development of the PubMed database, where the previous MedLine was rolled over into something that is web-accessible, readily searchable and easily elaborated to add additional features at the local level.

 

In addition, improved, advanced computer and web-based systems modeling programs relevant to DNA damage signaling also are needed.  Participants noted a two-dimensional computer model developed by Kurt Kohn that incorporates the underlying biochemical mechanisms involved in the cell cycle (a similar model for apoptosis also has been developed).  Although these models can be helpful and serve as a platform for further investigation, participants generally agreed that a research model should be dynamic and should allow for contingency pathways and multiple decisions by the cell.  It was noted that other more quantitative computer models are under development.  However, because of the limited amount of data available, applications of these models currently are, in turn, also limited.

 

Sophisticated mouse models that accurately recapitulate human neoplastic processes are also needed.  Most existing transgenic and knockout models merely “mimic” cancer.  Efforts, like that of the MMHCC, to more faithfully recapitulate relevant neoplastic processes should be strongly supported.  Faithful mouse tumor models are essential since, as discussed countless times in the Think Tank, the response of somatic cells (normal and neoplastic) to DNA damage and/or other therapeutic insults is, in great part, dictated by interaction between the tumor cell and its dynamic somatic environment.    

 

A particularly attractive suggestion regarding animal models was to engineer "reporter transgenics/knockins."  Such mice would contain germline-encoded reporters (such as GFP, LacZ, Luciferase) that would respond to a variety of important signaling or transcriptional pathways (eg p53 responsive, NFkB responsive, HIF, hypoxia, and many more). Until molecular imaging becomes possible via PET (not likely for a while still), such animals could prove enormously valuable.

 

Human Tissues

 

Much of the emphasis in the past two decades had been on defining genetic differences between tumor and normal cells. This has come, quite naturally, from the emphasis on cancer as a somatic genetic disease process. The most likely way to identify truly useful differences between normal and tumor cells (i.e., those that when targeted would have the offer the largest possible therapeutic indices) is to determine which normal pathways in tumors are still functional and can be exploited in the background of the tumor to force specific endpoints, such as cell death.  This analysis will be facilitated by the ability to assay the activity of damage response networks by protein expression levels.

 

Few tools are available to study responses to DNA repair in tissues.  However, such tools are necessary to map processes in normal and tumor tissues.  To accomplish this goal ‘modern molecular pathology’ needs to be better defined in this context.  For example, what functional imaging or histopathology markers are useful to examine endpoints such as apoptosis, energy state and viability in vivo or in tissue/tumor specimens from humans or mouse models?  Methods that cut across different types of specimens: e.g., functional imaging, biopsy samples and archival tumor specimens (currently the world’s largest untapped human tumor biology resource) would be particularly valuable.  The suggestion of phosphorylation site-specific antibodies that work on tissue is a good example, as is simple staining for p53 abundance.  This would be the best place to fund some descriptive normal biology, which we desperately need to provide the ‘numerator’ for all of the tumor studies and begin to understand the tissue correlates of therapeutic index. Use of imaging as a noninvasive tool to examine molecular pathways in carcinogenesis and in response to DNA damage is still in the early stages of development but holds great promise.  Expanded use of skin (or biopsies) was suggested as a model for imaging studies and assessment of the pathology of normal versus carcinogenic cellular processes.

 

At least two types of effort are focused on mechanisms and markers of DNA repair in human tumor tissue.  They are academic-commercial laboratory collaborations developing reagents (e.g., phosphoepitope-specific antibodies) and researchers conducting tumor specimen analyses (e.g., chromatin status or modification in relation to tumor type or stage).  Forging better connections between the two would facilitate movement of new analytical reagents and techniques into the clinical setting and yield more information from human specimens.

 

Broader issues that need further investigation include gaining a better understanding of the persistence of DNA damage versus transient DNA damage systems; determining the relative rates of proliferation and apoptosis in conjunction with DNA damage signals in situ (e.g., in oncogenic mouse models); assessing processes in real time; conducting longitudinal studies in humans; and understanding the different mechanisms underlying chemotherapy and radiation.  One notable gap that warrants further investigation involves the distinct differences in the in vitro versus in vivo responses to DNA damage and therapeutic interventions (e.g., radiation).  An approach to gaining a better understanding of these differences and bridging the gap between these systems may be through the study of pathway reporters inside tumors (see Tools).  Another general challenge to the field is the standardization of technologies, assays, definitions, interventions, and outcomes across research to improve the power of between-study comparisons.

 

Targets, Strategies

 

The emerging science indicates that it will be possible to differentially modulate sensitivity to DNA damaging agents in cancer and normal cells. Practically all cancer cells have mutations targeting DNA damage response genes. Mutations in these genes are essential for cancer development and result in rewiring of the DNA damage response pathways in cancer cells. Because of this difference in rewiring, inhibiting the same molecule is expected to have different effects in normal and cancer cells. We need to support research to identify and characterize DNA double strand break sensors and transducers. These proteins are promising targets for development of drugs that could specifically enhance the efficacy of current cancer therapeutics. Research to validate which of these sensors and transducers would be suitable targets for development of cancer therapeutics should be supported, including development of prototypical small chemical inhibitors to test proof of principle. It is expected that the proposed research will utilize a multitude of experimental systems, including human cells, human cancer tissues, mouse models and lower eukaryotes, as each experimental system has its own advantages and disadvantages and knowledge gained in one system can be easily applied to the others.  In addition, tumor cells have different sensitivities to DNA damage and other apoptotic stimuli.  Sensitization models that focus on unique mutations and/or unique clinical situations with high sensitivity may help determine the role of DNA damage and repair in the carcinogenic process.

 

It is critically important to explore ways in which sub-lethal pro-apoptotic signals can be combined to overcome the apoptotic-buffering threshold in cells.  Merely refining, exacerbating or increasing the persistence of DNA damage alone may not accomplish selective tumor cell death.  No amount of damage signaling is going to provide a therapeutically discriminate pro-apoptotic signal if, as in many tumors, DNA damage-induced apoptosis has become compromised by the mutation of p53. Exacerbating DNA damage also carries with it the future problem of inducing further oncogenic mutations.  While it is reasonable to argue that therapy-induced mutagenic/carcinogenic risk is secondary to curing the immediate neoplastic disease in patients, it would be better to avoid the problem completely if a better way of triggering apoptosis in tumor cells can be found.  No tumor cell has ever been identified that lacks the apoptotic machinery if hit hard enough.  This reflects our understanding that the programmed cell death (PCD) machinery is highly redundant and mechanistically defocused to employ several overlapping processes, including mitochondrial dysfunction, pro-phagocytic signaling, and caspase activation, each of which is alone sufficient to ensure death of the affected cell.  Therefore, it appears that PCD cannot be lost by progressive mutation. Rather, tumors acquire refractoriness to apoptosis through corruption of upstream signaling pathways and through lesions that raise the threshold at which apoptosis is triggered.  Thus, adroit stimulation of multiple disparate apoptotic pathways, each at a sub-lethal level, might together trigger activation of the apoptotic program

 

Other research questions of interest focus on the role of the cell cycle in DNA repair and cell responses to DNA damage.  We must find out whether blocking the cell cycle is a good clinical strategy.

Specific Recommendations for the NCI:

 

¨       Analyses of the human DNA-Damage Response networks (DDR): 

 

o       Complete the mapping of biochemical and regulatory circuitries that link the cell-cycle checkpoints, apoptotic and DNA-repair pathways into the hDDR with special emphasis on putative regulatory nodes.

o       Develop a quantitative, computer-driven, systems-biology model for the hDDR network in human cells.

o       Complete (or initiate) the integration of hDDR proteomics, gene-regulatory and gene expression databases with more dynamic cellular metabolite and energy profiles as functions of DNA damage.

 

¨      Identification of Molecular Targets & Translational Strategies for Enhancing Programmed Cell Death over Cell Survival in Response to DNA damage:

 

o       Support more research on ways to modulate the activity/expression of early-time (“upstream”) mediator and sensor proteins to activate the pathways of programmed cell death.

o       Support more research on the underlying mechanisms of  p53-independent programmed cell death, because in most tumors p53 is inactivated by mutations. 

o       Validate, as targets, mechanisms other than apoptosis that can induce cell death in human tissue, such as those linked to metabolic pathways that are altered in specific tumors.

o       Place greater research emphasis on mechanisms that determine whether dual-function (“downstream”) signaling proteins (e.g., p53, E2F1) signal survival or death responses.

 

¨      Normal versus Cancer Tissues:

 

o       Conduct comparative proteomic studies of tumor cells/tissues, progenitor cells, and normal cells/tissues to elucidate additional anti-apoptotic components altered in cancer.

o       Characterize and catalogue molecules from the signaling, checkpoint and apoptotic parts of the hDDR network as possible therapeutic targets.

o       Develop predictive profiles for radiation sensitive/resistant individuals; tumors with or without functional p53, ATM or other key signaling proteins, in order to tailor DNA-damage-based therapy to the patient.

 

¨      Reagents to Study and Technology to Study the DDR in vivo. 

 

o       Develop a resource library of phospho-specific antibodies or other “readout” reagents that can provide quantitative measures of DDR signaling and output pathways (e.g., caspase induction and apoptosis; focus formation and DNA repair).  This should include phospho-specific antibodies that can examine the activities of the DNA-damage signaling pathways in cultured cells and primary human tumors, before and after treatment and at different stages in tumor progression, etc.

o       Develop small molecule inhibitors of specific targets implicated in the response of cells to radiation, particularly where the target protein three-dimensional structure is known.

o       Promote dynamic imaging in the study of mechanisms of DNA repair in in vivo and in vitro studies.

o       Develop programs or incentives to allow for research access to early-phase clinical drugs belonging to pharmaceutical companies and studies of pharmaceutical reagents in mouse models of human cancer.

o       Support application of molecular imaging capabilities to monitor the dynamics and cellular compartmentalization of the hDDR.

o       Convene a conference or think tank focused on new tissue markers and modern pathology to improve connections between investigators in basic science and clinical research.