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

a combination of both. Untreated cells received an equivalent dose of vehicle (DMSO). After 24 h
cells were washed with cold PBS and re-suspended in ice-cold radioimmunoprecipitation assay buffer
(50 mmol/L Tris- hydrochloric acid in pH 7.4, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium
dodecyl sulfate (SDS), 150 mmol/L sodium chloride, 1 mmol/L ethylenediaminetetraacetic acid, 1 mmol/L
sodium vanadate and 50 mmol/L sodium fluoride) containing protease inhibitor mixture (Roche Applied
Science, Penzberg, Germany). Following cell lysis by sonication and centrifugation at 18620 × g for 10 min
at 4 °C, the resulting supernatant was collected as the total cell lysate. Briefly, the lysates containing 45 μg
proteins were electrophoresed on 10% SDS-polyacrylamide gels (Life Technologies, Carlsbad, CA, USA)
and transferred to polyvinylidene difluoride membranes (Bio-Rad laboratories Inc., Hercules, CA, USA).
Membranes were blocked in Odyssey blocking buffer (Li-Cor Biosciences, Lincoln, NE, USA). All target
proteins were immunoblotted with appropriate primary and horseradish peroxidase (HRP)-conjugated
secondary antibodies. Chemiluminescent (HRP-conjugated) bands were detected in a ChemiDoc MP
Imaging System (Bio-Rad laboratories Inc.). β-actin was used as an internal control to confirm equal gel
loading.

Quantitative real time polymerase chain reaction
RNA was isolated from cell lines using phenol-chloroform-isoamyl alcohol, followed by precipitation with
isopropanol in the presence of glycogen and sodium acetate. RNA was re-suspended in water and treated
with DNAse I to avoid DNA contamination. Complementary DNA (cDNA) was synthesized using moloney
murine leukaemia virus reverse transcriptase enzyme. cDNA was added to Taqman Universal Master Mix
(Applied Biosystems, Carlsbad, CA, USA) in a 12.5 μL reaction with specific primers and probe for each
gene. The primer and probe sets were designed using Primer Express 3.0 Software (Applied Biosystems)
according to their Ref Seq (http://www.ncbi.nlm.nih.gov/LocusLink). Gene-specific primers are provided
in Chemicals and reagents. Quantification of gene expression was performed using the ABI Prism 7900HT
Sequence Detection System (Applied Biosystems) and was calculated according to the comparative Ct method.
Final results were determined as follows: 2^-(ΔCt sample-ΔCt calibrator), where ΔCt values of the calibrator
and sample are determined by subtracting the Ct value of the target gene from the value of the endogenous
gene (β-actin). Commercial RNA controls were used as calibrators (Liver and Lung; Stratagene, La Jolla, CA,
USA). In all quantitative experiments, a sample was considered not evaluable when the standard deviation
of the Ct values was > 0.30 in two independent analyses. As a result, the number of evaluable samples varied
among the genes examined.

RESULTS
Baseline mRNA expression of EGFR-mutation positive cells and sensitivity to PIM inhibition
[8]
We first evaluated the baseline mRNA expression of PIM, STAT3 and other relevant genes , in our panel of
five EGFR-mutation positive NSCLC cell lines. As can be seen in Figure 2A, both PC9 and H1975 cell lines
had higher PIM-1, STAT3 and JAK2 mRNA expression compared to the average gene expression of all five
cell lines. Hereafter, cell viability assays were performed and the IC of single AZD1208 treatment were
50s
[6]
determined. The IC of osimertinib have been previously calculated and reported . As expected, none of
50s
the cell lines was sensitive to single AZD1208 treatment, with IC ranging from 17.1 to 23.0 µmol/L [Figure
50s
[6]
2B, left panel], while all of them were sensitive to osimertinib [Figure 2B, right panel].
Combination of osimertinib plus a PIM inhibitor in EGFR-mutation positive cells
Taking into consideration our previous findings, that osimertinib alone induces the activation of resistant
[6]
signaling nodes , we then tried to evaluate if AZD1208 increases the effect of osimertinib on cell growth
inhibition. Dual treatment revealed moderate synergistic effects, with a CoI between 0.79 and 0.85 [Figure
3A, left panel]. The lowest CoIs and therefore stronger synergism, were seen in the PC9 [CoI of 0.79; 95%
confidence interval (CI): 0.66-0.92] and HCC827 (CoI of 0.79; 95%CI: 0.69-0.89) cell lines. A moderate
synergism was seen in HCC4006 cells, with a CoI of 0.83 (95%CI: 0.79-0.87). The combination was less