TSR3 knockout activates a p53-dependent response and resensitizes resistant NPM1c+ AML cells to Ven. (A) Volcano plot revealing differentially expressed genes in OCI-AML3 cells transduced with a TSR3 gRNA (gTSR3) vs a nontargeting control gRNA (gControl). (B) Representative histogram plot of Annexin V staining in gTSR3 vs gControl-transduced OCI-AML3 cells. (C) Annexin V staining, (D) TUNEL staining, and (E) cell cycle distribution in gTSR3 vs gControl-transduced OCI-AML3 cells. (F) Schematic representation of the OCI-AML3 presensitization experiment. OCI-AML3 cells were transduced with gTSR3 or gControl and their response to venetoclax (Ven) was assessed using the MTS assay. (G) Proliferation of gTSR3 vs gControl-transduced OCI-AML3 cells after 4 days of incubation with Ven at the indicated doses (MTS assay). (H) IC50 values of gTSR3 vs gControl-transduced OCI-AML3 cells after 4 days of incubation with Ven (MTS assay). (I) Schematic representation of resensitization experiment using OCI-AML3 cells. Cells were transduced with BFP-expressing gTSR3 or gControl and were mixed with BFP-negative untransduced cells. Next, they were exposed to Ven and the BFP+ percentage was monitored for a course of 5 days by flow cytometry. (J) Percentage of BFP+gTSR3 or gControl-transduced cells on treatment with Ven at the indicated doses. BFP percentage was normalized to the reading of day 1. (A) Unpaired t test with multiple-hypothesis–testing correction (FDR, Benjamini-Hochberg method). (C-E,G-H,J) t test was used to calculate P values between groups. ∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001; ∗∗∗∗P ≤ .0001. Data represent 3 to 6 independent experiments (mean ± SD). (G-H) Absorbance values were normalized to DMSO-treated cells. Data represent 3 independent experiments (mean ± SD). MFI, mean fluorescence intensity. Panels F and I were created with BioRender.com. Vassiliou, G. (2025) https://BioRender.com/9tt8kww.
