DDDR-53. Novel indenoisoquinoline inhibitors dual function in modulating TOP1 and primary cilia expression in glioblastoma

Abstract Primary cilia are highly conserved, non-motile, transient organelles that play crucial roles in development, transduction of extracellular signals, and regulation of canonical signaling pathways. Primary cilia are also implicated in the pathogenesis of several primary brain neoplasms including medulloblastomas, glioblastomas, and ependymomas. In glioblastoma–the most common primary CNS malignancy–primary cilia are implicated in tumor invasion, angiogenesis, and chemoradiation resistance. There is a paucity of effective therapies for glioblastomas which remain uniformly fatal. LMP744 is a novel non-camptothecin indenoisoquinolone designed to target the TOP1 axis and has shown high efficacy and safety in systemic cancers. It is characterized by chemical stability, high brain penetrance, and irreversible binding providing distinct advantages over the camptothecin class of drugs. Human glioblastoma cells are exquisitely sensitive to LMP744 in vitro, and there is a significant survival benefit in mouse glioblastoma models in vivo. Despite the efficacy of the drug in vivo and in vitro, we did not observe changes in TOP1 levels. Therefore, we analyzed proteomic changes following LMP744 administration in human glioblastoma cells treated with D5W or 100nM of LMP744 by LC-mass spectrometry followed by a bioinformatics pipeline. We observed a significant decrease in DNAH2, a protein involved in primary cilia assembly and concomitant increase in AURKB, a protein implicated in primary cilia disassembly. Network analysis also revealed multiple interactions between primary cilia proteins and other differentially expressed proteins including ALB, TK1, SDHC, YIPF5, and PPIF suggesting that disruption of ciliary signaling may be central to the efficacy of LMP744. Immunocytochemical staining confirmed decreased frequency of primary cilia following LMP744 treatment. Western blots for GLI1 and Phospho-GLI1 confirmed decreased SHH signaling–a surrogate of primary cilia functional activity. These findings suggest that LMP744–and the class of non-camptothecin indenoisoquinolones–may modulate primary cilia and are promising as a ‘ciliotherapeutic’ strategy in glioblastomas.