Christine Richardson - US grants

DESCRIPTION (provided by applicant): The long-term objective of my research is to understand the influence of hematopoietic-specific developmental programs on the repair DNA damage such as double strand breaks (DSBs) and the initial molecular events that lead to translocations, which are a hallmark of leukemia, lymphoma, and soft-tissue sarcomas. DSBs are highly recombinogenic, increasing the exchange of information between two homologous DNA duplexes by several orders of magnitude; thus, mammalian cells are potentially at risk for rearrangements arising during DSB repair. Chromosomal DSBs result following exposure to irradiation, alkylating agents, and topoisomerase II (topoII) inhibitors that are common therapies in the treatment of human cancers. Treatment regimens that include the topoII inhibitor etoposide are associated with one class of therapy-related acute myeloid leukemia (t-AML) and chromosomal translocations involving the mixed lineage leukemia (MLL) gene on chromosome band 11q23. Similarity of 11q23 MLL breakpoints in t-AML and infant leukemias suggests an association between de novo infant leukemia and in utero exposure to topoII inhibitors. The list of potential topo II inhibitors is extensive, and it remains unclear which of these compounds have a direct potential to induce the chromosomal translocations observed in the clinical setting. Using a unique genetic system to determine the potential for repair of DSBs within the breakpoint cluster regions of the 11q23 MLL gene and common partner genes to result in chromosomal translocations, this proposal will (1) determine the potential for exposure to a range of topoII inhibitors to initiate chromosomal rearrangements within the breakpoint cluster region of the MLL and AF9 genes similar to those observed in the clinical setting; and (2) create a targeted mouse model to determine in vivo the potential for exposure to topoII inhibitors to initiate chromosomal rearrangements within the breakpoint cluster region of the MLL and AF9 genes as measured by the presence of MLL-AF9 genome rearrangements in bone marrow and peripheral blood. These approaches in both ex vivo cell culture and in vivo mouse models will provide significant insight into the initiation of potentially oncogenic chromosomal rearrangements and leukemogenesis. Unraveling the etiology and consequences of translocations may lead to new approaches to therapy and prevention. PUBLIC HEALTH RELEVANCE: Exposure to the topoisomerase II (topoII) inhibitors is associated with chromosomal translocations involving the mixed lineage leukemia (MLL) gene on chromosome band 11q23, one class of therapy-related acute myeloid leukemia (t-AML), and possibly de novo infant leukemias following in utero exposure to topoII inhibitors. The list of potential topo II inhibitors is extensive, and it remains unclear which of these compounds have a direct potential to induce the chromosomal translocations observed in the clinical setting. Our approaches in both ex vivo cell culture and in vivo mouse models will provide significant insight into the initiation of these oncogenic chromosomal translocations and leukemogenesis and may lead to new approaches to therapy and prevention.

This REU Site award to the University of North Carolina Charlotte, located in Charlotte,
NC, will support the training of 10 students in a 10-week program during the summers of
2015-2017. The REU program is open to all undergraduate students who are citizens,
nationals or permanent residents of the United States. Students trained in the program
will gain skills in lab research, develop their critical thinking and problem-solving skills,
understand the process of science, and be able to communicate their research results to
their peers and the general public. Students will have an opportunity to present their
results at a national conference. The REU program provides an experience to students
that is typically not available to them at their home institutions. Students from schools
with limited opportunities for research and from underrepresented groups are encouraged
to apply.

This REU Site program will focus on research in biology and biotechnology in a
traditional research experience while conveying how basic biotechnology research leads
to business creation and industrial development. Example projects include bioinformatics
and whole genome analyses to understand drought resistance of potato crops, generation
and study of transgenic soybeans for agricultural use, study of microbial populations for
biofuel development or water treatment, use of nanoparticles in bioavailability or
environmental impact studies. During the 10-week program, students will participate in
activities including workshops on Research Ethics, Applying to Graduate School, Career
Opportunities, Biotechnology Innovation and Start-Ups, and a program at NC Research
Campus and David Murdock Research Institute featuring research and employment
opportunities. At the end of the program, students will present their research in a poster
and/or oral presentation at the UNC Charlotte Summer Research Symposium.

A common web-based assessment tool used by all REU programs funded by the Division
of Biological Infrastructure (Directorate for Biological Sciences) will be used to
determine the effectiveness of the training program. Students are required to be tracked
after the program and must respond to an automatic email sent via the NSF reporting
system. More information is available by contacting the PI (Dr. Christine Richardson at
C.Richardson@uncc.edu) or co-PI (Dr. Sharon Bullock at sbulloc8@uncc.edu).

ABSTRACT Chromosomal double-strand breaks (DSBs) are formed during normal metabolic processes, following exposure to DNA damaging agents including irradiation, alkylating agents, and topoisomerase II poisons. DSBs are also implicated as forming as a result of exposure to a growing list of dietary compounds, supplements including bioflavonoids, and environmental toxins. DSBs are highly recombinogenic, increasing the exchange of information between two homologous DNA duplexes by several orders of magnitude, and cultured mammalian cells do utilize this mechanism to faithfully restore sequence following DSBs. Although the role of HR is well appreciated in meiosis of prokaryotes, yeast, and metazoans, the role of interchromosomal HR to occur in vivo in somatic cells of mammals is only minimally understood although it has the potential to promote genome stability, and also genetic diversity, chromosomal rearrangements, and drive evolution. Our laboratory established unique mouse models to determine the potential of DSBs to promote DSB induced recombination in vivo. These models were the first to demonstrate that interchromosomal HR occurs in vivo in multiple organ systems, and provide an ideal platform to further elucidate which cells at specific developmental stages of development or differentiation may be most likely to undergo this type of DSB repair. Further we will determine if altered altered expression of one protein central to DSB repair and recombination?Rad51--is sufficient to promote promiscuous interchromosomal HR. This work will lead to an understanding of the fundamental mechanisms of DSB rejoining at the chromosomal level, and also provide insight on genome stability and genetic evolution. Further, there is a growing list of dietary supplements and environmental toxins that promote or stabilize chromosomal DSBs, and thus our findings may have implications to the susceptibility of differentiating somatic cell types to mutagenic DSB repair and genome rearrangements that may result from exposure to them.

ABSTRACT The University of North Carolina Charlotte will partner with Gaston College and Rowan- Cabarrus Community College to implement the proposed NIH R25 Bridges to Baccalaureate Program to promote UR student success in obtaining a biomedical sciences degree and being competitive post-graduation. Low retention and graduation rates at the community colleges indicate that few students can begin their college careers at community colleges and earn a B.S. in biomedical sciences. Overall, the three institutions will identify and remediate multiple attrition points and work to increase the viability of the community college-to-UNC Charlotte biomedical sciences B.S. degree transfer pathway. The Program will aid a total of 50 student participants from community college partners Gaston College and Rowan-Cabarrus Community College in obtaining an A.A. or A.S. degree and then to transfer into, and graduate from, UNC Charlotte with a B.S. in the biomedical sciences. This Program will address the needs of academically qualified students to foster success. First, to promote preparedness and achievement in academic curricula, students will receive intense academic advising and ?degree maps?, individualized mentorship, cohort learning, and embedded course tutoring at the community college and university institutions. Second, to promote preparedness and achievement for biomedical sciences research careers, students will conduct independent research projects with faculty at the community college and later at UNC Charlotte. Research at UNC Charlotte will include both full-time summer and academic term independent research in a laboratory conducting cutting- edge biomedical sciences research. These experiences will culminate in poster and oral presentations both on campus and at national scientific meetings as well as opportunities to publish in peer-reviewed journals. Third, to promote a sense of inclusion and understanding of how biomedical sciences fit in the broader community context, students will be paired with senior student mentors at the university level, attend regional networking events for biomedical professionals, participate in professional development workshops, and take courses in bioethics. !

Abstract Genomic instability is a major driving force for cancer and age-related diseases. While DNA damage responses were long thought to regulate genome integrity and cell fates, evidence is accumulating that genomic instability also triggers inflammatory responses. Recent studies have provided insight into the mechanisms underlying such responses with the demonstration that the cytosolic DNA sensor, cyclic GMP-AMP synthase (cGAS), can play a key role in linking DNA damage to innate immunity. Interestingly, our research team has recently described the ability of human microglia and astrocytes to respond to foreign cytosolic double-stranded DNA and we demonstrated that human glia show robust levels of cGAS protein expression at rest and following activation. Furthermore, we showed these cell types constitutively express the critical downstream cGAS adaptor protein, stimulator of interferon genes (STING). In this R03 pilot study, we will begin to test the hypothesis that the cGAS-STING pathway detects cytoplasmic DNA in microglia and/or astrocytes after genotoxic stress and initiates glial auto- inflammatory responses. This project stems from a new collaboration between two experienced investigators with complementary expertise in the study of DNA damage repair mechanisms and glial innate immune sensor molecules. In these preliminary studies, we will determine whether genomic DNA damage elicits micronuclei formation and an elevation in the level of the cGAS product cGAMP in cultured glia, and we will correlate such responses with the production of auto-inflammatory mediators or potentially anti-tumor factors such as type I interferon. Furthermore, we will directly assess the relative importance of the cGAS-STING pathway in glial responses to DNA damage following pharmacological inhibition and/or CRISPR/cas9 genome editing. The proposed pilot R03 studies are an important first step in this new research direction and will provide a solid rationale for a more comprehensive investigation into the role of the cGAS-STING pathway in CNS cellular senescence and cancer for which future R01 mechanism support will be sought.