Abstract Topic: 3. Acute myeloid leukemia - Biology & Translational Research
Background: KIT mutation coexisted with t(8;21) translocation is frequently found in acute myeloid leukemia (AML) and drives leukemogenesis. Previously, we revealed that cabozantinib had therapeutic potential in Kasumi-1 cell line harboring t(8;21) and KIT mutations, but not in SKNO-1 with a similar cytogenetic backgrounds. The same went for other PI3K/mTOR dual inhibitors, suggesting the urgency of investigating the molecular mechanism of refractory and exploring the therapeutic strategies. To elucidate the refractory of SKNO-1 cells, transcriptome profiles of SKNO-1 and Kasumi-1 from DepMap Portal (https://depmap.org/portal/) with ID of ACH-001656 and ACH-000263, respectively, were used. We identified a total of 395 differential expressed genes (DEGs) with the selection criteria of p ≤ 0.05 and log2 (fold change) ≥1 or ≤1 between the Kasumi-1 and SKNO-1 cells. KEGG Mapper analysis explored the most significant pathways were “metabolic pathway” and “pathways in cancer” in SKNO-1 cells. Metascape analysis highlighted “carbon metabolism”, “biosynthesis of amino acids” and “MYC active pathway” were the top three active pathways in SKNO-1 cells. GSEA coupled with Molecular Signatures Database v7.4 indicated that gene sets associated with “MYC targets”, “mTORC1 signaling” and “IL2-STAT5 signaling” were enriched in SKNO-1 cells. Finally, we noticed that the gene with the greatest changes in these pathways between SKNO-1 and Kasumi-1 cells was PIM1.
Aims: To comprehensively explore the therapeutic potential and strategies of PIM447, a pan-PIM inhibitor, in refractory myeloid leukemia.
Methods: Cell proliferation was measured by Cell Titer 96 AQueous One Solution Cell Proliferation Assay. The protein expression and phosphorylation status were analyzed by immunoblotting. The ATP production rate was analyzed by Seahorse XF ATP Real-Time rate assay. Flow cytometry was used for cell cycle, apoptosis, and mitochondrial analysis.
Results: The cytotoxicity of PIM447 was detected in Kasumi-1 and SKNO-1 with IC50 values of 1591.54 nM and 202.28 nM, respectively, indicating SKNO-1 cells responded well to PIM447. The IC50 of 202.28 nM was much lower than plasma level of 3.1 μM, indicating the feasibility of PIM447 for subtype-specific clinical applications. Subsequent examination showed that PIM447 could induce cell cycle arrest in G0/G1 phase while decreasing cyclin E protein content but increasing p27 protein content in SKNO-1 cells. The doubling time of SKNO-1 cells increased from 39 hours to 132 hours after treated with PIM447, indicating that PIM447 could inhibit cell proliferation. As expected, the phosphorylation of PIM1 downstream molecules mTOR, P70S6K and 4E-BP1 were decreased after PIM447 treatment. Given that the most significantly different pathway was “metabolic pathway”, we performed Seahorse bioenergy analysis and found a significant reduction of total ATP production rate either through glycolysis or oxidative phosphorylation, indicating that PIM447 can inhibit glycolysis and oxidative phosphorylation of SKNO-1 cells. Furthermore, the mitochondrial membrane potential and mitochondrial mass were significantly decreased following PIM447 treatment, while increased mitochondrial reactive oxygen species substance.
Summary/Conclusion
We concluded that PIM447 not only inhibited PIM1 activity, but also modulate glycolysis, oxidative phosphorylation, and mitochondria function in SKNO-1 cells. These results indicate further in vivo study and clinical trials for refractory myeloid leukemia are warranted.
Keywords: Pim-1, relapsed/refractory, AML1-ETO, Kit