Kinase experiment:

IC50 values for CAL-130 inhibition of PI3K isoforms are determined in ex vivo PI3 kinase assays using recombinant PI3K. A ten-point kinase inhibitory profile is determined with ATP at a concentration consistent with the KM for each enzyme[1].

Cell experiment:

Cell proliferation of CCRF-CEM cells or shRNA-transfected CCRF-CEM cells, in presence or absence of CAL-130 (1, 2.5 and 5 μM), is followed by cell counting of samples in triplicate using a hemocytometer and trypan blue. For apoptosis determinations of untransfected or shRNA-transfected CCRF-CEMs, cells are stained with APC-conjugated Annexin-V in Annexin Binding Buffer and analyzed by flow cytometry. For primary T-ALL samples, cell viability is assessed using the BD Cell Viability kit coupled with the use of fluorescentcounting beads. For this, cells are plated with MS5-DL1 stroma cells, and after 72 hr following CAL-130 treatment, cells are harvested and stained with an APC-conjugated antihuman CD45 followed by a staining with the aforementioned kit[1].

Animal experiment:

Mice[1]For subcutaneous xenograft experiments, luminescent CCRF-CEM (CEMluc) cells are generated by lentiviral infection with FUW-luc and selection with Neomycin. Luciferase expression is verified with the Dual-Luciferase Reporter Assay kit. 2.5×106 CEM-luc cells embedded in Matrigel are injected in the flank of NOD.Cg-Prkdcscid Il2rgtm1Wjl/Sz mice. After 1 week, mice are treated by oral gavage with vehicle (0.5% methyl cellulose, 0.1% Tween 80), or CAL-130 (10 mg/kg) every 8 hr daily for 4 days, and then tumors are imaged as follows: mice anesthetized by isoflurane inhalation are injected intraperitoneally with D-luciferin (50 mg/kg). Photonic emission is imaged with the in vivo imaging system. Tumor bioluminescence is quantified by integrating the photonic flux (photons per second) through a region encircling each tumor using the Living Image software package. Administration of D-luciferin and detection of tumor bioluminescence in Lck/Ptenfl/fl/Gt(ROSA)26Sortm1(Luc)Kael/J mice are performed in a similar manner.

Class I phosphoinositide 3-kinase (PI3K) activate fundamental pathways controlling cell survival, metabolism, proliferation and and therefore play crucial role in cancer development. It is reported that p110γ is mainly expressed in leukocytes, whose role in cancer development is recently starting to be studied. CAL-130 is a specific dual inhibitor of p110γ and p110δ.
In vitro: To demonstrate that CAL-130 can block the activities of both PI3Kγ and PI3Kδ in thymocytes, previous autors evaluated its ability of preventing phosphorylation of Akt (Ser473) and calcium flux in response to T cell receptor (TCR). Consistently, CAL-130 treated thymocytes from 6-week-old WT animals prevented TCR-induced Akt phosphorylation and attenuated calcium flux to levels observed for their Pik3cγ-/-; Pik3cδ-/- counterparts [1].
In vivo: To assess the in vivo efficacy, previous study determined its effects on 6-week-old WT mice thymi. Mice orally received 10 mg/kg of the inhibitor, which was sufficient to maintain plasma concentrations of 0.33 μM at the end of 8 hrs. Moreover, such dose did not affect either plasma glucose or insulin levels. In contrast, CAL-130 treatment (10 mg/kg every 8 hrs) for a period of 7 days greatly affected the size, cellularity, and overall architecture of the thymus. Notely there was a 18-fold reduction in total thymocyte number when comparred to controls, which was mainly due to the DP population loss [1].
Clinical trial: CAL-130 is currently in the preclinical developlent stage and no clinical data are available.
Reference:
[1] Subramaniam PS, Whye DW, Efimenko E, Chen J, Tosello V, De Keersmaecker K, Kashishian A, Thompson MA, Castillo M, Cordon-Cardo C, Davé UP, Ferrando A, Lannutti BJ, Diacovo TG. Targeting nonclassical oncogenes for therapy in T-ALL. Cancer Cell. 2012;21(4):459-72.