(P 173) DYSREGULATION OF MITOCHONDRIAL DYNAMICS IN WELL-DIFFERENTIATED/DEDIFFERENTIATED LIPOSARCOMA PROVIDES A UNIQUE SUSCEPTABILITY TO NOVEL THERAPY

Objective: Mitochondria display a high degree of plasticity and subcellular distribution to meet cellular demands. This highly orchestrated property is termed mitochondrial dynamics. The dysregulation of mitochondrial dynamics has been implicated in tumor progression and metastasis among a diverse group of malignancies. Well-differentiated (WD) and dedifferentiated (DD) liposarcoma (LPS) display a wide clinical spectrum of disease with limited response to current therapies. We hypothesized dysregulated mitochondrial dynamics are important for tumor progression among WD/DD LPS, which may provide a unique susceptibility to targeted therapy.

Methods: Mitochondrial oxygen flux measurements in available WD/DD LPS cell lines were obtained using Agilent Seahorse XF Analyzer. Human tumor samples were collected immediately following surgery, and dissociated into single cells prior to Seahorse flux analysis. Super resolution fluorescent microscopy with Airyscan processing (LSM 880, Zeiss) was used to image live cells following incubation with MitoTracker (TM) Deep Red FM and Hoechst 33342. ImageJ software was used to quantify mitochondrial morphology (total area, aspect ratio, circularity). Relative sensitivity to mitochondrial inhibition was assessed using LPS2 and 93T449 cells, pre-treated for 2 hours with titrations of various mitochondrial complex I inhibitors (metformin, phenformin, rotenone, IACS-010759 (IACS)) followed by Seahorse flux analysis. Cell proliferation was assessed using an ATP luminescent assay (CellTiter Glo 2.0) following 48 hours of treatment, luminescent values normalized to vehicle (DMSO). To evaluate these effects in vivo, NOD scid gamma (NSG) mice harboring LPS2 flank xenografts were treated with IACS, tumor volume was assessed by serial caliper measurements every 2-3 days. Immunohistochemistry was performed on fixed tumor tissue for markers of mTORC1 activity (p4EBP1) and apoptosis (CC3). Cell positivity was quantified by QuPath v0.3.2 software. Unpaired T tests were used to compare means between groups, p values < 0.05 were considered significant.

Results: DD LPS exhibit low oxygen consumption rates (OCR) (Fig1A-B) and highly fragmented mitochondria compared to WD LPS (Fig1C). DD LPS display heightened sensitivity to mitochondrial targeting by complex I inhibition (Fig 2A-B). Treatment with IACS inhibits tumor growth in a DD LPS xenograft murine model (Fig3A-B). Xenograft IHC demonstrates loss of mTORC1 activity and increased apoptosis in the treatment group (Fig 3C).

Conclusion: WD and DD LPS subtypes display unique mitochondrial dynamics. Low cellular respiration and fragmented mitochondrial morphology is a hallmark of DD LPS, which appears to enhance sensitivity to complex I inhibition both in vitro and in vivo. Taken together, our data suggests dysregulated mitochondrial dynamics in DD LPS provides a unique susceptibility to novel therapy.