Tuesday, April 1, 2025
4/1/2025
The Role of SMARCAL1 Depletion on Increased Immunogenicity in ALT+ Gliomas - PROJECT SUMMARY/ABSTRACT. Approximately 10 to 15% of all cancers utilize the alternative lengthening of telomeres (ALT) mechanism to maintain telomere length and achieve replicative immortality. ALT occurs frequently in high-grade gliomas (HGGs), including IDH-mutant astrocytomas, pediatric HGGs with H3G34R mutations, and a subset of adult primary GBMs. The overarching goal of this F32 Fellowship Award is to investigate the role of SMARCAL1, an annealing helicase that catalyzes replication fork reversal, in promoting glioma cell proliferation and suppressing tumor-intrinsic inflammation. HGGs are notoriously immunologically cold with large suppressive myeloid compartments. Current therapies have struggled to generate an immune response. Our preliminary data in pre-clinical glioma models shows that therapeutic targeting of SMARCAL1 represents a path to tumor- cell specific DNA damage and inflammation, and depletion of SMARCAL1 increases replication stress, DNA damage, and aberrant DNA and RNA species. This project will investigate the intrinsic and extrinsic inflammatory response following SMARCAL1 depletion in ALT+ HGG cell lines and orthotopic syngeneic murine glioma models. SMARCAL1 will be depleted in human ALT+ HGGs and syngeneic murine ALT+ cell lines utilizing RNAi lentiviral particles containing two distinct doxycycline-inducible SMARCAL1-targeting shRNAs. In vitro studies will involve investigation of pattern recognition receptor (PRR) signaling within tumor cells and the resulting effects on the secretome, measured using western blot and LegendPlex. The specific mechanism of PRR derived inflammation will be elucidated using CRISPR/Cas9 gRNA knockdown studies and visualized using IF-FISH with confocal microscopy. The effect of inflammation on phagocytic myeloid cells will be monitored using living cell imaging and flow cytometry. In vivo studies will investigate the inflammatory response in orthotopic murine glioma models in C57Bl/6 mice with syngeneic tumors. These studies will identify alterations to the inflammatory profile of ALT+ tumors within tissue context and provide information on the role of the immune system following SMARCAL1 depletion using both in vivo and ex vivo models. Alterations in the inflammatory response and T cell mediated cytotoxic killing will be investigated using flow cytometry and transcriptomics. Both mentors are experts in the field with well-respected publication records in the fields of HGG, ALT, and SMARCAL1 and will provide the appropriate mentorship as Dr. Erman transitions her immunology expertise from nephrology to oncology, as well as facilitating her mentorship of more junior members. The resources and facilities at Duke University are exemplary and all studies will be performed using the most modern techniques with the guidance of experienced and respected collaborators. The proposed studies include techniques Dr. Erman has extensive experience with including flow cytometry and transcriptomics yet allowing her to apply them to a new field, and areas for growth including murine glioma models, cell culture, and IF-FISH with confocal microscopy.
HHS’ Tracking Accountability in Government Grants System (TAGGS) website provides access to detailed descriptions of grants, loans, aggregated direct payments and other types of financial assistance awarded by the HHS Operating Divisions (OPDIVs) and the Office of the Secretary Staff Divisions (STAFFDIVs).