Idelalisib

CLL cell lines were treated with UNC1999 (S7164; Selleckchem), GSK343 (S7165; Selleckchem), and the PI3Kδ inhibitor idelalisib (GS-1101; Selleckchem). DZNep (120964–45-6) and JQ-(1268524–70-4) (Sigma Aldrich) treatments were incubated for 3 days using a different range of concentrations as mentioned in the figures. The levels of active AKT was analysed using the Akt Kinase Activity Assay Kit (ab139436; Abcam) according to the manufacturer’s instructions.

The following drugs used in this study were purchased: ABT‐263 (AdooQ BioScience, Irwin, CA, USA), LY294002 (Cayman Chemical Company, Ann Arbor, MI, USA), Ibrutinib and Idelalisib (Selleck Chemicals, Houston, TX, USA). 293T and SU‐DHL4 (a kind gift from Dr Kunihiko Takeyama, Dana Farber Cancer Institute, Boston, MA, USA) cells were cultured in DMEM (Sigma Aldrich, St Louis, MO, USA) and RPMI (Sigma Aldrich), respectively. Both culture media were supplemented with 10% FCS, 2 mM l‐glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin and 1 mM sodium pyruvate.

HCT‐15 cells stably expressing PIK3CD (HCT‐15‐PIK3CD) and HCT‐15 cells stably expressing vector (HCT‐15‐Vector) (5 × 10⁶) were injected subcutaneously into the right flank of male Balb/C nude mice (6‐week old), respectively. All mice were housed under specific pathogen‐free conditions. Tumor size was measured every week using a slide caliper and tumor volume was calculated using the formula: tumor volume (mm³) = length × width² × .5. After 4 weeks, all mice were killed and the tumors were excised, weighted, fixed in formalin and embedded in paraffin. In addition, HCT‐116 cells (5 × 10⁶) were injected subcutaneously into the right flank of male Balb/C nude mice. One week after inoculation, the mice were orally administered vehicle DMSO (80 mg/mL, Vehicle group) or idelalisib (30 mg/kg/d) (Selleck Chemicals, Houston, TX, USA) for 10 days. Four weeks after inoculation, the tumors were removed, weighted, fixed and embedded. The 4‐μm‐thick sections from all tumors were prepared for immunohistochemical analysis and stained with hematoxylin. Animal experiments were approved by the Animal Experimentation Ethics Committee and performed in accordance with ethical guidelines for animal experimentation.

PI3K inhibitors (duvelisib, idelalisib, umbralisib, eganelisib [all Sellekchem, Munich, Germany]) were reconstituted at a concentration of 20 mM (duvelisib, idelalisib, umbralisib) or 40 mM (eganelisib) in dimethyl sulfoxide (DMSO, Genaxxon Bioscience, Ulm, Germany) and all used at a final concentration of 1 µM in complete medium according to literature.^{30 (link),31 (link)}

VL51 cells were cultured according to the recommended conditions, as previously described.^{12 (link)} To develop resistance cell lines were exposed to a 90% inhibitory concentration (IC₉₀) of idelalisib (Selleckchem, Houston, TX, USA) for several months until they acquired specific drug resistance (resistant cells). In parallel, cells were cultured under similar conditions in the absence of drug (parental cells). Proliferation of stable resistance was tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay after 2 weeks of drug-free culture. The multidrug resistance phenotype was assessed by real-time polymerase chain reaction for MDR1 and MDR2/3 genes using published primers.^{13 (link)} We developed biological replicates by splitting the resistant clones 1 month after the development of resistance, keeping them separate for 6 months before performing further experiments. Cell line identity was periodically authenticated by short tandem repeat DNA profiling, as previously described.^{14 (link)} Cells were periodically tested to confirm Mycoplasma negativity using the MycoAlert My-coplasma Detection Kit (Lonza, Visp, Switzerland).