CAL-101 inhibits PI3K signaling and cellular viability. (A) CAL-101 screening of primary cells from patients with ALL, CLL, acute myeloid leukemia (AML), or myeloproliferative neoplasm (MPN) or from normal healthy volunteers (NHV). Cell viability was determined using an 3-carboxymethoxyphenyl)-2-(4-sulphenyl)-2H-tetrazolium assay, and the sensitivity of each sample relative to untreated cells was calculated for estimation of the EC₅₀. The heat map readout shown was generated using GenePattern Version 3.2 software (Broad Institute) and indicates the percentage EC₅₀ for each sample relative to the maximum drug concentration tested (10μM). (B) CAL-101 inhibition of p110δ blocks PI3K signaling in malignant B-cell lines and primary patient tumor cells. Serum-starved cells were incubated with 1μM CAL-101, and total cell lysates were subjected to Western blot analysis using anti–phospho-Akt^S473, anti-Akt, anti-phospho S6^S235/236, and anti-S6 antibodies (supplemental data). Starved cells were incubated with vehicle or serial dilution of CAL-101 for 1 hour, and pAkt^T308, pS6^S235/236, total Akt, and total S6 were detected by PathScan sandwich ELISA (supplemental data). (C-D) CLL (n = 5) and MCL (n = 5) patient whole blood samples were subjected to Ficoll-Hypaque separation. Isolated cells were incubated in RPMI with vehicle or serial dilutions of CAL-101 before fixation and staining with anti–phospho-Akt^T308 Alexa Fluor 488 or isotype-matched Alexa Fluor 488 antibody. Cells pretreated with CAL-101 or vehicle for 1 hour and then stimulated with 10 μg/mL anti-IgM (BCR activation), or 50 ng/mL sCD40L (CD40 stimulation) for 10 minutes before fixation and staining with anti–phospho-Akt^S473 Alexa Fluor 488 or isotype-matched Alexa Fluor 488 antibody. FITC-CD5⁺ cells were gated and analyzed by 2-color flow cytometry to quantify intracellular p-Akt^T308 levels using the Beckman Coulter Cytomics FC 500MPL using CXP Version 2.2 software. Bar graphs represent the percentage difference in mean fluorescence intensity values between isotype-matched control Ig and phospho-Akt^T308. (E) CAL-101 induces apoptosis in diffuse large B-cell lymphoma, follicular lymphoma, and B-ALL cell lines. Cells were treated with vehicle or 0.5μM or 1.0μM CAL-101 for 24 hours. The percentage of apoptotic cells was determined by annexin V–FITC/7-AAD staining followed by 2-color flow cytometric analysis. Percentages represent both annexin V–FITC/7-AAD negative and annexin V–FITC/7-AAD double-positive. (F) Cells were cultured in RPMI/10% fetal bovine serum with CAL-101 or vehicle alone for 24 hours, and cells were lysed and analyzed by PathScan Sandwich 96-well ELISA for the detection of cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase as indicated (supplemental data). Results are expressed as mean ± SD. Statistically significant differences between means were determined using a one-way analysis of variance. Data are expressed as the fold change and are representative of 3 separate experiments.