Identification of EPLIN as a novel MYCN-independent target in neuroblastoma and medulloblastoma
Neuroblastoma and medulloblastoma are the most common solid tumors in children, with MYC(N) proto-oncogene amplification serving as a key marker of aggressive disease and poor treatment outcomes. However, the complex nature of the challenging MYC(N) protein underscores the urgent need for additional targets and therapies to tackle neuroblastoma and medulloblastoma. In this study, we identified FLIX5, a small compound that exhibits broad toxicity against both neuroblastoma and medulloblastoma cells by inducing apoptosis and ferroptosis. Furthermore, FLIX5 enhances the cholesterol dependency of neuroblastoma cells under conditions where mitochondrial function is impaired. FLIX5 as well synergistically enhances the effectiveness of conventional drugs vincristine and vinorelbine against neuroblastoma cells and patient-derived xenograft organoids. Through proteome integral solubility alteration, computational molecular docking predictions, and cellular thermal shift assays for target identification and validation, FLIX5 revealed EPLIN (Epithelial Protein Lost In Neoplasm) as a previously unexplored drug target. EPLIN functions as a tumor suppressor and plays roles in various cellular processes, including cholesterol uptake and mitochondrial activity. The discovery of FLIX5 targeting EPLIN opens new avenues for addressing the significant challenges posed by neuroblastoma and medulloblastoma, and potentially offering broader applications in other types of cancer. Overall design: Neuroblastoma cell line was obtained from (ATCC, Manassas, VA, USA). Neuroblastoma cell line was maintained in 1:1 ratio of EMEM (ATCC, Cat. #30-2003) and Ham's F-12 Nutrient Mix (Thermo Fisher Scientific, Waltham, MA, USA, Cat. #11765054) supplemented with 1% penicillin streptomycin (Thermo Fisher Scientific, Cat. # 15140-122) and 10% FBS (Thermo Fisher Scientific, Cat. #10270-106). U87MG and Daoy was maintained in EMEM supplemented with 1% penicillin streptomycin and 10% FBS. HDFa and D283 was maintained in DMEM + Glutmax (Thermo Fisher Scientific, Cat. # 61965026) supplemented with 1% penicillin streptomycin and 10% FBS. GMYC126, GTML-S1, GTML2 and GTML3 were maintained in Neurobasal – A medium (Thermo fisher scientific, Cat. #10888022) supplemented with B27-A (Thermo Fisher Scientific, Cat. #12587010),1% penicillin streptomycin1, 1% L-glutamine (100x) (Thermo fisher Scientific, Cat. #25030081), 20ng/mL bFGF (Thermo Fisher Scientific, Cat. # 100-18B), 20ng/ml EGF (Sigma Aldrich, Cat. #E9644). All cells were maintained at 37°C and 5% CO2. All cell lines were authenticated by STR profiling (ATCC cell authentication service) and the MycoAlert mycoplasma detection kit (Lonza) was used to frequently test for mycoplasma infection. Spheroids was generated by plating 10,000 cells/well in 100µL medium in 96-well ultra-low attachment plates (Corning, NY, USA, Cat. #7007). Cells were settled in the wells for 20min followed by centrifugation at 1000RPM for 5min and cultured in 37°C in 5% CO2. Spheroids were cultured for 4 days before drug exposure and images was taken by Incucyte followed by calculation of the spheroids volume by: V =(4/3*p) *r3