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Page 4 of 10 Bracht et al. J Cancer Metastasis Treat 2019;5:22 I http://dx.doi.org/10.20517/2394-4722.2018.111

The ability of AZD1208 to induce cell cycle arrest and apoptosis by inhibition of all PIM kinase isoforms
indicates its potential as an anticancer treatment. Previous publications have shown that PIM inhibition
augments the efficacy of targeted therapies in diverse tumor types. Therefore, dual PIM-1 and EGFR
inhibition might modulate cell survival pathways and block progression and growth of cancer cells, by
preventing the EGFR TKI-induced STAT3 and RTKs activation in EGFR-mutation positive NSCLC cell
[6-8]
lines . This research aims to evaluate whether the pan-PIM inhibitor AZD1208 improves the efficacy of
osimertinib in EGFR-mutation positive cell lines.

METHODS
Chemicals and reagents
Human lung adenocarcinoma PC-9 cells, harboring EGFR exon 19 deletion and 11-18 cells, harboring EGFR
exon 21 L858R mutation were provided by F. Hoffmann-La Roche Ltd. (Basel, Switzerland), and by Dr.
Mayumi Ono, (Kyushu University, Fukuoka, Japan), respectively. EGFR exon 19 deletion positive HCC4006
and HCC827 cells were purchased from the American Type Culture Collection (ATCC). The H1975 cell line,
harboring both EGFR exon 21 L858R and resistant T790M mutation as well as the TNBC MBA-MB-231
cell line were purchased from ATCC. All cell lines were maintained in RPMI (Roswell Park Memorial
Institute medium) 1640 supplemented with 1% penicillin/streptomycin/glutamine (Gibco) and 10% fetal
bovine serum (FBS; Gibco) in a 5% CO 37 °C cell culture incubator and routinely evaluated for mycoplasma
2
contamination.

The pan-PIM inhibitor, AZD1208, and osimertinib were bought from Selleck Chemicals (Houston, TX,
USA). Drugs were prepared in dimethylsulfoxide (DMSO) at a concentration of 10-100 mmol/L stock
solutions and stored at -20 °C. Further dilutions were made in culture medium to final concentration before
use. All antibodies used in our study, including dilution and company catalog number can be found in
Supplementary Table 1. All primers used can be found in Supplementary Table 2.

Cell viability assay
3
3
3
Cells were seeded in 96-well plates at the following densities: 1.5 × 10 (PC9), 2.0 × 10 (H1975) 3.0 × 10
(HCC4006, HCC827 and 11-18), and incubated for 24 h, as previously described . For the combined treatment
[6]
3
3
cells were seeded in 96-well plates at the following densities: 1 × 10 (PC9 and H1975), 1.5 × 10 (HCC827,
HCC4006 and 11-18), and incubated for 24 h. Cell viability was assessed using the 3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide (MTT) assay (Sigma Aldrich, St Louis, MO, USA). Cells were treated with
serial dilutions of the drugs. For the half maximal inhibitory concentration (IC ) determination, AZD1208
50
doses ranged from 0-200 μmol/L and treatment lasted 72 h. For the MTT viability assays with a combination of
AZD1208 and osimertinib (IC were previously obtained) drug doses were as follows: PC9, H1975, HCC4006,
50s
HCC827 cells were treated with osimertinib ranging from 0-100 nmol/L, and AZD1208 ranging from
0-100 μmol/L, or with the combination of both. The 11-18 cells were treated with osimertinib ranging from 0-200
nmol/L, and AZD1208 ranging from 0-100 μmol/L, or with the combination of both. The combined treatment
lasted 96 h. After treatment incubation, 0.5 mg/mL of MTT reagent was added to the medium in the wells for 2
h at 37 °C and formazan crystals in viable cells were solubilized with 100 μL DMSO and spectrophotometrically
quantified using a microplate reader (Varioskan Flash; Thermo Fisher Scientific, Waltham, MA, USA) at 565
nm of absorbance. Fractional survival was then calculated as percentage to control cells. Data of combined drug
effects were subsequently analyzed by the Chou and Talalay method [32,33] . Combination index (CoI) values < 1, =
1 and > 1 indicated synergism, additive effect and antagonism, respectively.

Western blotting
For immunoblotting experiments, 1.5 million cells were seeded in T75 flasks (Starstedt, Newton, USA).
The next day, PC9 and H1975 cells were treated with 24 nmol/L osimertinib, 5 μmol/L AZD1208 and