J. Chang1, Z. Roberts1, H. Ding2, L. Stafman1 1University Of Alabama at Birmingham, Department Of Surgery/Heersink School Of Medicine, Birmingham, Alabama, USA 2University Of Alabama at Birmingham, Department Of Pathology/Heersink School Of Medicine, Birmingham, Alabama, USA
Introduction:
Neuroblastoma (NB) is the most common pediatric extracranial solid tumor and accounts for a disproportionate number of deaths. Only 50% of high risk patients survive and in those who relapse or recur, only 20% survive. Novel therapeutics are desperately needed. Lipid metabolism has been a promising target in other cancers and has been associated with differentiation, energy production, and drug resistance. Proviral Integration in Moloney murine leukemia (PIM) kinases have been shown to play a role in lipid metabolism in non-cancer cells and inhibiting PIM kinases induces differentiation and decreases proliferation in NB. Therefore, we hypothesize that PIM kinases exert their effect by altering lipid metabolism in NB and may offer an opportunity as novel therapeutics.
Methods:
Two NB cell lines, SK-N-AS (AS, MYCN non-amplified) and BE(2)-C (BE, MYCN amplified), were treated with small molecule PIM inhibitors, AZD1208 and SGI-1776. Cells were fixed and neutral lipids in lipid droplets (LDs) were stained with Oil Red O before brightfield microscopy imaging. For additional LD evaluation, cells were fixed and imaged using transmission electron microscopy. Images were analyzed using ImageJ. Student’s t-test was used with mean ± standard error of the mean reported and p<0.05 significant.
Results:
The quantity of stored lipids decreased following treatment with PIM inhibitors in NB cells. The amount of Oil Red O staining decreased by 61% (p<0.001) and 35% (p<0.001) with AZD1208 25µM and by 80% (p<0.001) and 52% (p<0.001) with AZD1208 50µM in BE and AS cells, respectively. SGI-1776 yielded a more modest decrease by 30% (p=0.04) and 35% (p=0.01) with 1µM and 35% (p=0.02) and 41% (p=0.01) with 5µM in BE and AS cells, respectively. Electron microscopy also revealed that lipid droplet quantity and size decreased with PIM inhibition.
Conclusion:
Inhibition of PIM kinases decreased the quantity of stored lipids in NB. This finding is crucial in the context of potential cancer drug targets since tumor cells with decreased lipid storage have limited access to lipids needed for proliferation, membrane biogenesis, signaling, and other cellular functions required for survival. While this finding was present in both cell lines, it was more pronounced in BE cells. This suggests that the effect of PIM kinases on lipid metabolism may be related to MYCN expression in NB. We plan to further explore the role of PIM kinases in other aspects of lipid metabolism and their effect on differentiation, energy production, and drug resistance. This line of investigation may lead to novel therapeutics for NB.