-By Bevin P. Engelward
On April 22, 2021, Dr. Leona D. Samson was elected a member of the American Academy of Arts and Sciences. Dr. Samson is exceptionally deserving of this recognition. Her innovative research has truly been groundbreaking, with a consistent string of pioneering work that has not only moved her research field forward, but that has also opened doors to personalized medicine and a deeper understanding of cancer etiology. Her work has consistently focused on aspects of DNA damage and repair. With the knowledge that DNA damage has the potential to lead to mutations, and that accumulated mutations can drive cancer, she set out to discover genes that modulate susceptibility to DNA damage and to uncover their molecular mechanisms. Importantly, and somewhat ironically, DNA damaging agents are also used to treat cancer when they are delivered at a high concentration. As such, her work impacts both our understanding of the causes of cancer and our ability to effectively treat cancer. Here, a subset of her remarkable contributions are summarized.
Dr. Samson started her career working with Dr. John Cairns. As a graduate student, she was tasked with the goal of using E. coli as a way to mimic some aspects of somatic cell behavior. In particular, she set out to study accumulation of mutations in E. coli that were exposed to a DNA damaging agent under long-term low-dose conditions. The idea was that somatic stem cells can persist in the body and accumulate mutations, and that fundamental aspects of this process could be studied in E. coli. For these studies, they used a type of DNA damaging agent that adds methyl groups to the DNA (in other words, carbons are added where they do not belong). It was known at the time that methylated bases could be mutagenic (and toxic). Unexpectedly, Dr. Samson observed that E. coli were resistant to DNA damage-induced mutations. After struggling for many months thinking that something was wrong with the experiment, John Cairns and Dr. Samson had an “aha” moment when they came up with the idea that perhaps the cells were adapting during the long low-dose exposure. Indeed, when cells were exposed to a low dose of a DNA damaging agent prior to receiving a much higher dose, they became remarkably resistant to DNA damage-induced mutations and toxicity. This led to the discovery of several genes that play fundamental roles in sensing, responding to, and repairing DNA damage. Published in Nature, this remarkable discovery both uncovered an important biological process (adaptation) and paved the way for cloning and characterization of the genes responsible for adaptation.
One of Dr. Samson’s breakthrough ideas was that DNA repair could be functional across kingdoms. This is because DNA repair proteins recognize structural changes to DNA, and such changes are present in virtually all cell types. In other words, if a protein can recognize a particular methylated base, such as an adenine with an extra carbon on the N3 position (3-methyladenine), it doesn’t matter which cell type the protein is in. This paradigm-shifting idea led Dr. Samson to clone the first eukaryotic gene that repairs methylation damage. To do this study, her team fragmented DNA from yeast and subcloned it into an E. coli expression vector, thus creating a yeast genomic library. The library was then put into E. coli cells that lacked key DNA repair genes responsible for the repair of toxic 3-methyladenine lesions. After creating plates with thousands of colonies, she recruited the lab to test the colonies one by one for their ability to grow in the presence of a methylating agent. Persistence paid off! One colony out of 15,000 showed resistance to the methylating agent. This led to the discovery of the first eukaryotic gene that defends against methylation damage-induced toxicity. She then extended this approach and used a human cDNA library to clone the first human gene able to defend against methylation damage, and she called this gene the alkyl adenine DNA glycosylase (AAG).
Wanting to understand better what Aag does in mammals, she next set out to knock out the Aag gene in mice. This was at a time when there were very few knockout mice in existence, and there was only one knockout mouse that lacked a DNA repair activity. Boldly moving forward, she teamed up with Jan Hoeijmakers to make the Aag KO mouse. These mice turned out to have fascinating phenotypes some of which are described below. She also went on to create a different knockout mouse, one that is lacking a gene called 06-methylguanine DNA methyltransferase (MGMT) that functions to remove methyl groups from the 06 position of guanine by attaching them to its active site. Through studies of cells from these mice, it became clear that the Mgmt gene protects against not only methylating agents, but also other chemicals that create DNA adducts, including BCNU, a chemotherapeutic agent.
Dr. Samson always has had an eye on the utility of understanding the mechanisms of DNA repair in the context of treating cancer. One of the problems with chemotherapy is that it is not only toxic to the tumor, but it is also toxic to normal healthy cells in the body. In fact, the dose of BCNU that can be used to treat a patient is limited by the fact that it can destroy bone marrow cells, which can be lethal to the patient. After presenting her data showing that DNA methyltransferases can rescue human cells from being annihilated by BCNU, she was approached by a clinician in the audience about ways that they could leverage this new understanding. This led to experiments in an animal model in which the bone marrow was removed and a gene for the DNA repair methyltransferase was inserted into the genome of the bone marrow cells. The cells were then returned to the animal, which rendered them resistant to BCNU toxicity. This was the first example of gene therapy using a DNA repair enzyme and it paved the way for clinical trials where the gene for a DNA methyltransferase is being inserted into the bone marrow of children with childhood cancers.
Another key to Dr. Samson’s remarkable impacts is that she has consistently jumped at the opportunity to use whatever cutting-edge technology was available in order to delve deeper into the mechanisms by which DNA damaging agents cause mutations and toxicity. A great example of this is her work studying the transcriptome. At a time when Affymetrix was still in the development phase in the creation of microarrays used to study the level of expression of thousands of genes at a time, Dr. Samson was able to obtain chips even before they were commercially available. Using the Affymetrix chips, her team uncovered hundreds of genes that are differentially expressed upon exposure to a methylating agent. As such, she pioneered studies of the transcriptome in response to DNA damage. One of her key discoveries was that while any particular gene might not be informative regarding cell behavior, analysis of several genes from the same pathway was effective in discerning the impact of gene expression on cell behavior. In other words, she helped to break open the field of pathway analysis in human cells.
One of the fascinating things about the Aag gene is that it is the first step in a repair pathway. Aag is a glycosylase that removes the damaged base, creating an abasic site that then needs to be repaired by downstream enzymes. An essential step is to cut the backbone so that new DNA can be created to replace the damaged piece. Dr. Samson discovered that if Aag is expressed at a high level in mammalian cells, it can actually be detrimental. And she uncovered the mechanism, which was the toxic accumulation of abasic sites. It later became clear that Aag can not only remove toxic DNA damage, but it can also remove benign DNA damage and even undamaged bases. As such, having extra Aag activity puts pressure on the pathway, and if downstream enzymes cannot keep up, repair intermediates can be highly toxic. Having observed these effects, Dr. Samson next asked what the consequences are for Aag activity in animals. She hypothesized that under some circumstances Aag might be detrimental rather than helpful. Remarkably, she discovered that this is indeed the case. For example, when mice are exposed to a methylating agent, they develop retinal degeneration. If Aag is knocked out, the mice actually become resistant to the methylating agent, showing definitively that under conditions of high levels of DNA damage, a DNA repair activity can do more harm than good. Taking this line of thinking even further, Dr. Samson hypothesized that Aag might actually modulate responses to inflammation, because Aag not only removes methylation damage, but it also removes certain DNA damages that are formed by oxidative stress. To test this, Dr. Samson leveraged models of ischemia reperfusion in mice and she demonstrated that indeed, Aag activity greatly increases ischemia-induced tissue damage in the liver, kidney and brain. In other words, mice without Aag were relatively resistant to ischemia reperfusion. This brought Aag into the spotlight as a key enzyme with broad implications for many physiologically important conditions.
Another aspect of Dr. Samson’s work that is remarkable was her work on studies of the transcriptome in response to environmental chemicals, and one of the questions that she set out to answer was the extent to which a baby in utero might be impacted by exposure to the mother. By obtaining cord blood from mothers who had been exposed to arsenic in their drinking water, she was able to analyze the transcriptional responses of the baby. This work led to the discovery that numerous stress responses are induced in the fetus when mothers were exposed to arsenic, and from these results her team was able to identify a set of just 11 genes that serve as an effective biomarker for arsenic exposure for babies in utero.
Dr. Samson has always had an eye on the possibility that differences in DNA repair capacity among people might affect their susceptibility to cancer and their sensitivity to chemotherapy. For some of her most recent work, she focused on developing high-throughput tools to evaluate DNA repair capacity in human cells. To do this, she took an old-fashioned, rarely used assay for the presence of DNA lesions in transcribed genes and modernized it by exploiting site-specific lesion technology and the ability to rapidly assess the efficiency of transcription using fluorescent proteins. She won a prestigious Pioneer Award to pursue the idea of a modernized “host-cell reactivation assay” wherein DNA repair either turns on or off expression of genes for fluorescent proteins. Through a number of clever tricks, she was able to devise methods that enable parallel analyses of multiple DNA repair pathways in human lymphocytes. Her new assay opens doors to both studies of interindividual variation repair and its relationship to disease susceptibility, as well as applications to personalized medicine to evaluate tumors for their ability to use DNA repair as a way to resist the toxic effects of chemotherapeutic agents.
These are merely highlights from an astounding number of discoveries. Her work has made a significant and lasting impact not only in the field of DNA damage and repair, but more broadly in our ability to understand how cells respond to exposures and how we can leverage our understanding to gain insights that help us to better predict, prevent, and treat disease.