Fig. 4: AAMDC knockdowns downregulate the PI3K-AKT-mTOR axis through translational suppression of MYC and ATF4 leading to transcriptional downregulation of AAMDC-dependent targets.

a Regulation of the PI3K-AKT-mTOR pathway in SUM52PE cells transduced with AAMDC shRNAs assessed by immunoblotting. WT wild-type (untransduced cells), EV empty vector. Source data are provided as a Source Data file. b Modulation of the PI3K-AKT-mTOR pathway by pharmacological inhibition. SUM52PE cells were treated for 24 h with the indicated PI3K-AKT-mTOR inhibitors. Source data are provided as a Source Data file. c Transcriptional regulation of selected AAMDC-dependent transcripts involved in cell cycle and epigenetic regulation (left) and metabolic control (right). The results are normalized to vehicle-treated cells. Statistical significance is determined by multiple t-test using the Holm-Sidak method with alpha = 0.001 (left) and alpha = 0.05 (right), and presented as mean values ± SD, *p < 0.0001. n = 3 biologically independent experiments. d, Decreased promoter occupancy of ATF4 and MYC transcription factors at predicted promoter sites in two AAMDC targets: ASNS and MTHFD1L determined by promoter-specific chromatin immunoprecipitation (ChIP). SUM52PE cells are transduced with either empty vector (EV) or with AAMDC shRNA #2 (sh2), or treated with either vehicle control or 100 nM dactolisib (24 h). The position of ATF4 and MYC and the primers used for quantification are shown (top). Enrichment is determined by qRT-PCR and normalized to control cells and presented as mean values ± SEM. p-values are determined by two-tailed unpaired t-test. For ASNS promoter: *p = 0.0102 and *p = 0.0447 for dactolisib and sh2, respectively; **p = 0.0019, ***p = 0.0003. For MTHFD1L promoter: *p = 0.0201. n = 3 biologically independent experiments. TSS transcription start site, F forward, R reverse.