Defining and manipulating quiescence associated DNA damage resistance in single cells The goal of this proposal is to study the phenomena of quiescence as it exists in tumors and relate this state to chemoresistance. All solid tumors are a heterogenous mix of proliferating and quiescent cells and many studies on different tumors have determined the quiescent population to be therapy resistant. Defining which quiescent states contribute to this resistance and identifying tool compounds to modulate the quiescent state is therefore of great interest. I will use a novel panel of live cell proliferation reporters derived from classical immunohistochemical markers of proliferation to study the nature of quiescent states and directly relate these quiescent states to DNA damage responses at the single cell level. This study will be carried out in 2- and 3D culture (Aim 1), where we have the genetic and chemical control to precisely relate DNA damage responses to quiescent states, and later taken in vivo (Aim 3) to mouse tumor models. Complementing these more observational aims, I will establish a novel screening tool to identify molecules that specifically modify the chemoresistance of quiescent cells using a fate tracing approach (Aim 2). This project will describe the diversity of quiescent states and their inter-relationships, directly relate quiescence to chemoresistance, and identify tool compounds to manipulate this phenotype. This proposal draws on my analytical skills developed as a graduate student working on yeast stress signaling and as a postdoc over the last two years studying determinates of p53 regulation in normal tissues and tumors. I will apply the single cell techniques and computational approaches I have learned to study the basic biology underlying quiescence and chemoresistance in tumors and to identify small molecules that perturb this resistance. In the mentored phase of this grant I will work under the supervision of my co-mentors Dr. Lahav and Dr. Weissleder, who are experts on the core focus of my proposal: single cell responses to genotoxic therapy. During this phase, I will gain the technical skills in cell culture, imaging, small molecule screening, and mouse work and the professional skills and profile to obtain an independent academic research position. For the initial phase, I will be embedded in the department of Systems Biology at Harvard Medical School, a world class research center for the study of complex quantitative phenotypes and surrounded by clinicians and academics working on cancer, among other problems. This proposal will complete my transition into a cancer biologist and provided me with a bridge to an independent research career studying cancer.
Tumors contain non-proliferating (quiescent) cells that are resistant to many standard therapies. This study will provide a quantitative understanding of when and what types of quiescent cells evade therapy and chemical tools to reduce this resistance.
