Summary: Robert Darnell studies degenerative brain disorders that are provoked by an immune response to certain cancers. This has led to the discovery of brain-specific systems for regulating RNA that may be co-opted by cancer cells, and the development of new methods to study RNA regulation in living tissues.
The study of disease allows the pursuit of mechanism while maintaining a narrow focus on questions directly relevant to human biology. The main effort of my laboratory is directed at developing a basic understanding of a group of rare brain diseases, the paraneoplastic neurologic syndromes (PNDs). These studies are producing insights into tumor immunology, autoimmunity, and neuronal cell biology.
PNDs arise when tumors outside of the nervous system express proteins that are normally made only in the brain (termed onconeural antigens). The serum and cerebrospinal fluid of PND patients harbor high titers of antibodies to specific onconeural antigens. This immune response is associated with clinically effective tumor suppression and neuronal cell death. Immunologic questions under study include the following: How are these proteins perceived as foreign by the immune system in tumor cells? What is the relationship between neuronal degeneration and the "immune-privileged" status of neurons? Why do some patients react with tumors expressing PND antigens, while others do not?
A major focus of our study is an exploration of the nature of the onconeural antigens: Why do tumors express these brain proteins, and what is their normal brain-specific function? Our laboratory pioneered the use of PND antibodies to identify cDNAs encoding the onconeural antigens. We have focused much of our work on onconeural antigens that are neuronal RNA-binding proteins (n-RBPs). Two such families of proteins, Nova and Hu, are providing a platform for gaining insight into how RNA regulation in the brain generates cellular complexity. Nova is a target antigen in a motor degeneration associated with breast and lung cancer, and is related to FMRP (the fragile X mental retardation protein), while the Hu proteins are homologs of a Drosophila n-RBP termed ELAV.
By combining biochemical, structural, and genetic approaches, ranging from RNA selection to protein:RNA crystallization to the generation of mouse knockouts, we determined optimal RNA targets for Nova and FMRP and discovered that Nova regulates neuron-specific alternative splicing. Genome-wide analysis revealed an unanticipated map of genome-wide rules by which Nova regulates alternative splicing (which have turned out to be common to other splicing factors), and led to the discovery that Nova regulates a highly coherent subset of brain transcripts. As a group, Nova regulates RNAs encoding synaptic functions, indicating that Nova, in regulating their alternative exon usage, can help shape the quality of the proteins that are present in the synapse. This work is beginning to provide new links between the regulation of gene expression and the modulation of synaptic function.
To distinguish between direct and indirect targets of n-RBP function in the brain, we have developed a new platform methodology: high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). The platform produces genome-wide "footprints" of RBP-binding sites on RNA with great accuracy, and can be applied directly to human or mouse tissues, including brain and tumor. HITS-CLIP is proving highly flexible in the development and overlay of maps for RNA-protein interactions, including precise mapping of sites of microRNA regulation, offering a general means to assess RNA regulation in living tissues.
Immunologic studies of PND patient material are a second area of focus. This work has provided the first insight into how killer T cells mediate tumor immunity in humans. We have found that tumor cell suicide (apoptotic death) serves as a potent trigger to initiate this killer T cell immune response in PND patients. In small-scale clinical research studies performed at the Rockefeller University Hospital, we are working to recapitulate this process in cancer patients. The goal of these clinical studies is to apply our understanding of the effective tumor immunity observed in PND to the general population of cancer patients.
This work is also supported by grants from the National Institutes of Health, the Starr Foundation, and the Dextra Baldwin McGonagle Foundation.
As of October 28, 2010