**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]{.underline}

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]{.underline}:

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]{.underline}

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]{.underline}

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)*:**

    -   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.

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

    -   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:***

    -   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.

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

    -   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.

    -   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:***

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

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

    -   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**.*

    -   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.

    -   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.

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

    -   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.

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

    -   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.