Regulation of Oxidative Energy Metabolism by G0S2 in FLT3+ AML
Introduction
Acute myeloid leukemia (AML) patients with mutations in the FMS-like tyrosine kinase 3 (FLT3) gene represent 20-30% of all AML cases. These patients can be treated with tyrosine kinase inhibitors (TKIs) targeting FLT3; however, up to 60% of patients develop resistance or experience adverse side effects, which makes identifying mechanisms of drug resistance a priority. Our previous work demonstrated a tumor suppressor role for G0/G1 switch gene 2 (G0S2) in chronic myeloid leukemia (CML), which is profoundly downregulated in TKI resistance (Gonzalez et al. Clin Transl Med, 2022). G0S2 regulates multiple cellular functions, including lipolysis, de novo lipogenesis, and oxidative phosphorylation. Using publically available data, we found that G0S2 mRNA expression does not correlate with overall survival (OS) in AML, but is significantly downregulated in AML versus normal mononuclear cells (MNCs, p<0.001), and further reduced in AML patients with mutated FLT3 (p=0.006). Therefore, we hypothesized that reduced G0S2 expression in FLT3+ AML results in altered oxidative energy metabolism, promoting therapy resistance.
Methods
To address this hypothesis, we first used lentiviral vectors to transduce primary FLT3+ AML mononuclear cells (MNCs) with an empty vector (EV) and an ectopic G0S2 expression vector (pLVX-G0S2). We also transduced the FLT3+ AML cell line, MOLM-13, with a non-targeting control vector (shNT), a G0S2 knockdown vector (shG0S2), or pLVX-G0S2. We assessed colony formation ability by plating cells in 0.9% Methocult plus cytokines for primary cells and measured apoptosis using AnnexinV and 7-aminoactinomycin D (7AAD). Cell lines were treated with doxycycline (100 ng/ml) for vector induction. For in vivo experiments, NOD-scid IL2Rgammanull (NSG) mice received intravenous injections of 3 million cells/mouse of the MOLM-13 cell lines with the shNT and shG0S2 vectors, and all mice were immediately placed in doxycycline hyclate chow to induce vector expression. To assess the role of G0S2 in FTL3+ AML cells, we used the correlation feature available at UALCAN to calculate the genes co-expressed with G0S2 in The Cancer Genome Atlas (TCGA) AML data. We performed immunoblotting using SDS page, PVDF membranes, and a human antibody cocktail for the 5-electron transport chain (ETC) complexes. In addition, we measured oxygen consumption rates (OCR) using the Cell Mito Stress Test kit for the Agilent Seahorse XFp Bioanalyzer. Finally, we performed mass-spectrometry based metabolomics and lipidomics on the MOLM-13 cell line.
Results
In primary FLT3+ AML MNCs, ectopic G0S2 expression resulted in a significant reduction of colony formation, which correlated with increased induction of apoptosis when compared to the empty vector control. Importantly, mice receiving shG0S2-expressing FLT3+ cell lines resulted in a significant reduction in overall survival when compared with NSG mice receiving shNT-expressing control cells (p=0.0246). Furthermore, pathway enrichment analysis using data from TCGA revealed that the genes co-expressed with G0S2 in AML implicated a role in energy metabolism. After confirming G0S2 ectopic expression in the MOLM-13 cell line, immunoblotting showed reduced ETC expression of complexes III and V, which correlated with reductions in oxygen consumption rates using the Agilent Seahorse Bioanalyzer. Not surprisingly, our metabolomics and lipidomics analysis from MOLM-13 shNT versus shG0S2 cells also revealed alterations in amino acid and glycerophospholipid metabolism.
Conclusion
Altogether, our data demonstrate that loss of G0S2 expression in FLT3+ AML dysregulates oxygen metabolism by altering the expression of ETC components. Ultimately, we showed that G0S2 is a tumor suppressor that is downregulated in FLT3+ AML, which might promote therapy resistance by altering oxidative energy metabolism.