Session: 604. Molecular Pharmacology and Drug Resistance: Myeloid Neoplasms: Poster II
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Metabolism, Biological Processes, Technology and Procedures, Molecular biology
6ME suppressed clonogenic growth of patient-derived AML cells (IC50: 2.025 ± 0.187 μM) while having no effect on normal hematopoietic cells. In mouse engraftment studies, where patient-derived AML cells are engrafted into the mouse bone, 6ME (5 mg/kg, three times/week for 4 weeks) was given after a 3-week post-transplantation of patient cells into immunocompromised mice, once bone marrow aspirate engraftment was confirmed. The subsequent 4-week treatment with 6ME significantly reduced patient-derived AML cell engraftment in mouse bone marrow by about 45% (p<0.0001) without imparting toxicity.
Using published computational methods, the family of PPAR transcription factors were identified as potential targets of 6ME. To confirm this potential mechanism, cell-based luciferase reporter assays, immunoblotting and co-immunoprecipitation (co-IP) coupled with ultra-high performance liquid chromatography (UHPLC) assay were performed to confirm that 6ME bound to PPARδ and inhibited its activity. To further study this interaction, we quantified downstream targets of PPARδ-transcriptional activity in 6ME-treated AML cells by immunoblotting; this showed that PPARδ-related genes including CD36, CPT2, UCP3, and PPARδ itself were significantly reduced by 6ME. Finally, shRNA-mediated PPARδ knockdown (KD) AML cells were created to probe the functional impact of PPARδ in AML cells. As expected, PPARδ KD AML cells were much less sensitive to 6ME, and those cells with reduced PPARδ resulted in a similar pattern of protein changes to that of 6ME-treated cells.
As 6ME reduced levels of CD36, CPT2, UCP3, and PPARδ, which are all essential proteins involved in fatty acid oxidation (FAO), we measured FAO in AML cells using high-resolution respirometry. When provided with an FAO substrate (palmitate-BSA), 6ME significantly hindered oxygen flux of intact AML cells, indicating that 6ME impairs FAO-supported mitochondrial respiration. Similarly, treatment with a PPARδ antagonist (i.e., GSK0660) but not an agonist (i.e., GW0742), also resulted in the inhibition of FAO-supported intact AML cell respiration, suggesting 6ME targets PPARδ to impair mitochondrial FAO.
Taken together, our 6ME targets the PPARδ-FAO pathway and may open a new avenue for therapeutic targeting in the treatment of AML.
Disclosures: Spagnuolo: SP Nutraceuticals: Consultancy.
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