Fig 2: TRF1 regulation by PI3K and AKT inhibitors. a Structurally diverse PI3K and PI3K/mTOR inhibitors used in the study. b PI3K and mTOR data generated internally and reported in literature. c Representative western blot images of phosphorylated AKT-Ser473 and total AKT in CHA-9.3 mouse lung tumor cell line at 24 h after treatment with PI3K, AKT, and mTOR inhibitors as indicated. d Percent inhibition of TRF1 foci by immunofluorescence and of AKT phosphorylation at S473 (pAKT) in CHA-9.3 mouse lung tumor cell line at 24 h after treatment with the indicated inhibitors relative to TRF1 levels and to pAKT levels in control cells treated with DMSO. The inhibitors were tested at 10 μM except GSK-2126458 that was used at 1.0 μM (in c and d). Error bars represent standard deviation. The Student’s t test was used for statistical analysis; P values are shown. n number of independent experiments

Fig 3: PI3K/AKT inhibitor post-translationally downregulate TRF1 levels. a Quantification of total nuclear TRF1 levels in lung cancer-derived cells (CHA 9-3) treated either with DMSO, AKT inhibitor (AKTi) (10 μM), ETP47037 (10 μM), or ETP47228 (10 μM). A representative western blot image is shown below. b Quantification of total nuclear TRF1 levels in in immortalized p53 −/− MEFs treated either with DMSO, AKT inhibitor (AKTi) (10 μM), or ETP47037 (10 μM). A representative western blot image is shown below. c Quantification of total nuclear TRF1 levels in P110α lox/lox MEFs cells transduced with pBabe-GFP or with pBabe-Cre recombinase. A representative WB images is shown below. d Quantification by immunofluorescence of mean TRF1 foci intensity in CHA 9-3 cells treated either with DMSO, DMSO plus bortezomib (50 nM), ETP47037 (10 μM) plus bortezomib (50 nM), ETP47228 (10 μM) plus bortezomib (50 nM), and AKTi (10 μM) plus bortezomib (50 nM). Student’s t test was used for statistical analysis; P values are shown. Error bars represent standard error. n number of independent experiments

Fig 4: (a) Chemical structure of eight PI3Kα-selective inhibiting chemical probes. (b) Modelling of compound 7 bound covalently to Cys862 of p110α (PDB ID: 7R9V was used as a starting point). H-bonds are depicted as dashed black lines. (c) Time-dependent IC50 shift derived from time-resolved fluorescence resonance energy transfer (TR-FRET) ratios (Fig. S2† for all compounds), comparing compound 7 and its reversible analogue 7r. (d) IC50 values for PKB phosphorylation (pPKB, Ser473) measured in SKOV3 cells by in-cell western (ICW) plotted against log D values (measured by Bienta Enamine Biology Services). Data shown are mean ± SD from at least n = 3 independent experiments. Error bars are not shown when smaller than symbols. (e) LC-SRM quantification of covalent Cys862-modification by 7 and no covalent modification by 7r. (f) Bioluminescence resonance energy transfer (BRET) target occupancy assay in live HEK293 cells. The cells were transiently transfected with constructs encoding a Nanoluciferase (Nanoluc) fused to different PI3K isoforms (PI3Kα, PI3Kβ and PI3Kδ) or a Cys862 to Ser mutated PI3Kα. 24 h after transfection, cells were incubated with 3 μM of 7 or 7r for 2 h. The probes were washed out (twice for 10 min with Opti-MEM) and a cell-permeable Py-BODIPY moiety bearing a fluorescent energy transfer probe (BRET tracer) was added to the cells (0.2 μM final concentration). Recovery of the BRET signal, resulting from displacement of the inhibitor from the ATP-binding pocket, was monitored for 1.5 h. Prolonged target occupancy after probe washout indicates covalent bond formation. Data shown are mean ± SEM (n = 3).

Fig 5: (a) Schematic diagram of PI3K pathway stimulation in cancer cell lines to deconvolute PI3Kα isoform contribution downstream of membrane receptors. Serum starved cells (24 h) were incubated with 1 μM 9 or DMSO for 2 h followed by washout (labelled 9-wo or DMSO-wo). Receptor ligands (L) were then added to the medium in the absence or presence of the PI3Kβ-selective inhibitor TGX221 (1 μM) for the indicated times (5, 30, 180 min) prior to lysis. Total PKB and pPKB (Ser473) levels were measured by western blot (Fig. S9†). All treatments and media changes were carried out using serum-free medium. Depicted in the schematic are the active and inactive (red line-crossed) PI3K isoforms resulting from inhibitor treatments. (b) PI3K signaling in MCF7 cells treated with inhibitors and stimulated with 10 ng mL−1 EGF (left), 10 μg mL−1 insulin (center) or 50 ng mL−1 CXCL12 (right). (c) Quantification of PI3K isoform contribution (area under curve) to PI3K signaling in response to EGF, insulin and CXCL12 in MCF7 cells. (d) PI3K signaling in SKOV3 cells treated with inhibitors and stimulated with 10 ng mL−1 EGF (left), 10 μg mL−1 insulin (center) or 50 ng mL−1 CXCL12 (right). (e) Quantification of PI3K isoform contribution (area under curve) to PI3K signaling in response to EGF, insulin and CXCL12 in SKOV3 cells.