The Chalfant Laboratory has historically focused on two major but diverse areas of basic science Biochemistry, Biophysics, and Molecular Biology. Specifically, the Chalfant Laboratory studies mechanisms of cell signaling associated with both bioactive lipids and RNA splicing with a focus on both basic science mechanisms related to cancer, wound healing, and inflammation, in many cases, to clinical translation. Regarding our bioactive lipid research, these studies have recently exploded into numerous sectors with clinical applications. For example, the Chalfant Laboratory initially described the lipid: protein interaction of the sphingolipid, ceramide-1-phosphate (C1P), with the phospholipase, cPLA2a. This initial basic research finding has now advanced to a possible new therapeutic target to treat sepsis and a therapeutic target to drive rapid wound healing for both acute and chronic wounds. The Chalfant Laboratory began a global lipidomics initiative through this research area to rapidly analyze the downstream bioactive lipids associated with this lipid: protein interaction and various human disease states. Through this initiative, clinicians were brought together with basic scientists to determine new biomarkers for placental and wound healing dysfunction as bioactive lipids are most proximal to clinical phenome (Fig.1). We also continue our strong basic research in this area by exploring additional lipid: protein interactions as well as a new temporal paradigm for eicosanoid biosynthesis.
Regarding RNA splicing research, the Chalfant Laboratory has identified key RNA splicing events and signaling mechanisms mediating the tumor maintenance of non-small cell lung cancer (NSCLC) cells. The Chalfant laboratory further merges these findings with novel lipid signaling events linked to cell survival. More recently, we have also examined the function of specific splice variants upregulated in NSCLC to determine the reason for our observed dysregulation of RNA splicing cascades linked to specific oncogenotypes. For example, we examined the downstream signaling function of modulations in caspase 9 RNA splicing induced by EGFR mutations. Indeed, two splice variants of caspase 9, caspase 9a and caspase 9b (C9b), are derived from alternative mRNA processing via the inclusion of the exon 3,4,5,6 cassette. C9b, the anti-apoptotic isoform lacking the large subunit encoded by the exon 3,4,5,6 cassette, has been reported to augment the anchorage-independent growth (AIG) and tumorigenic capacity of cancer cells, specifically non-small cell lung cancer (NSCLC) cells. Originally, our preliminary investigations suggested that caspase 9b activated key survival pathways via GSK3b and Stat3 (Fig.2). Still, in-depth investigations over the last two years have also shown a key role for C9b in the activation of the canonical arm of the nuclear factor κB (NF-kB) pathway, a major pathway linked to the tumorigenesis in NSCLC.
Further mechanistic studies demonstrated that C9b activates this pathway via direct interaction with a cellular inhibitor of apoptosis 1 (cIAP1) and subsequent induction of the E3 ligase activity of this IAP family member. The C9b:cIAP1 interaction occurred via the BIR3 domain of cIAP1 and the IAP-binding motif of C9b but did not require the proteolytic cleavage of C9b. Lastly, this protein interaction was required for C9b to promote the viability, AIG, and tumorigenicity of NSCLC cells and broadly translate to diverse NSCLC genotypes. Our current studies show that caspase 9b can drive in vitro transformation of human bronchial epithelial cells and cooperate with oncogenic KRAS to induce full transformation and induce tumorigenesis in vivo. Furthermore, we are finding links between the expression of caspase 9b, inflammation, and the tumor microenvironment. Thus, small molecule inhibitors of this interaction may lead to new and effective therapeutics to treat NSCLC, possibly in conjunction with newer immunotherapies.
The Chalfant Laboratory has also identified key RNA splicing events and signaling mechanisms mediating the tumor maintenance of NSCLC cells (Fig.3). These findings have been further expanded using deep RNA sequencing to identify specific RNA splicing signatures for individual NSCLC patients, which are linked to therapeutic sensitivity. Even more exciting from a basic science point of view, our mechanistic research has now validated the “unmasking hypothesis.” For years, researchers in the RNA splicing field overlooked the presence of multiple RNA binding domains (RBDs) in RNA trans-factors. Essentially, SELEX was used to identify high-affinity binding sites for a specific RNA trans-factor. Then a dogmatic scientific view was founded: One RNA trans-factor, one optimal binding preference. We asked, “Why is the RNA trans-factor, hnRNP L, binding to a non-standard RNA cis-element in NSCLC cells?” The asking of this straightforward question led to the identification of a specific phosphorylation site that “unmasks” C-terminal RBDs in hnRNP L to bind a separate RNA sequence with high affinity. The RNA trans-factor retains its constitutive RNA splicing function but has additional regulatory abilities for a separate set of RNA splicing events important in cell survival and tumor maintenance. These findings are expected to drive a complete paradigm shift in cell signaling and RNA splicing once we publish next year. Importantly, we have extended our RNA splicing research into different cancers (e.g., breast cancer and anoikis resistance) and type 1 diabetes. More recently, we identified cytoplasmic splice variants of an uncharacterized lncRNA associated with CDK2NA mutations, which we are examining for biological functions.
Overall, both research areas described above for the Chalfant Laboratory have proven fruitful in obtaining national funding for the laboratory (20 years continually funded for our cancer research). Furthermore, these research areas and the proven productivity of the Chalfant Laboratory have led to the appointments of Dr. Charles Chalfant to several Editorial Boards and National Committees, which include: the Editorial Boards of the Journal of Lipid Research and the Journal of Biological Chemistry, formal membership on the Cancer and Molecular Pathobiology Study Section of the National Institutes of Health, and formal membership on the Oncology A Study Section of the Veterans Administration. These research areas also led to the communication of the 2011 ASBMB Avanti Junior Investigator Award for Lipid Research to Dr. Chalfant. The applicant, Dr. Chalfant, has also been very productive with over 120peer-reviewed publications (see CV). His current research is supported by multiple extramural grants. He has trained more than 40 trainees at various levels, with a number of them being faculty members at various institutions with funded research programs.