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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 7 of 10

Figure 3. The effects of AZD1208 and osimertinib on RTKs and downstream components in EGFR-mutation positive NSCLC cells.
A: in all cell lines, cell viability assays were performed to explore the effect of combined osimertinib and AZD1208 treatment. The
obtained CoI was calculated based on the Chou and Talalay method, and values < 1, = 1 and > 1 indicate synergism, additive effect and
antagonism, respectively. Experiments were performed in biological triplicates, and the average and standard deviations plus 95%CIs are
shown; B: two EGFR-mutation positive NSCLC cell lines (H1975 and PC9) were treated with single osimertinib (0.024 μmol/L), single
AZD1208 (5 μmol/L) or with the combination. Untreated cells received an equivalent dose of vehicle (DMSO). Cell lysates were used for
immunoblotting and changes in RTKs and non-RTKs upon the different treatments were investigated in the two cell lines. Experiments
were performed in biological triplicates with similar results, and a representative blot is shown. RTK: receptor tyrosine kinase; EGFR:
epidermal growth factor receptor; NSCLC: non-small cell lung cancer; CoI: combination index; 95%CI: 95% confidence interval; DMSO:
dimethylsulfoxide; CDCP1: CUB domain -containing protein 1; YAP1: yes-associated protein 1; STAT3: signal transducer and activator of
transcription 3

combination reversed the osimertinib-induced STAT3 phosphorylation in both cell lines, and diminished
paxillin and Src phosphorylation in PC9 and H1975 cells, respectively [Figure 3B, right panel]. Overall these
data reconfirm our previous findings that, even in oncogene addicted EGFR-mutation positive cancer cells,
single EGFR TKIs are insufficient [6-8,34] . Concomitant PIM-1 inhibition may reverse some of the deleterious
effects of osimertinib on the activation of oncogenic signaling pathways.

DISCUSSION
Herein, we have reviewed the available literature on PIM and we have found that this kinase is involved in
[26]
multiple tumor types, including lung cancer . The survival of lung cancer patients has been improved in
the last decade due to the availability of new and more effective targeted therapies. EGFR-mutation positive
patients are treated with EGFR TKIs, and the last years we have seen the development and approval of three
[35]
generations of these agents . However, ultimately resistance mechanisms take over and treatment options
are not indefinite. Furthermore, about 20% of EGFR-mutation positive patients have intrinsic resistance to
EGFR TKIs [8,34] , indicating that molecularly targeted therapy is not a one-size-fits-all approach. Therefore,
defining biomarkers of resistance and designing a truly personalized therapy is of great relevance. We
have previously seen, and we confirm in the present work, that EGFR TKIs, including osimertinib, induce
[6-8]
STAT3 and Src-YAP1 activation . Rational double or triple combinations prevented this phenomenon and
increased the efficacy of EGFR TKI monotherapy in EGFR-mutation positive models . Since PIM was
[6-8]
shown to be a key player in IL-6/STAT3 signaling [14,18,21,28,29] , we hypothesized that by combining the PIM
[6-8]
inhibitor, AZD1208 with osimertinib we may obtain similar results .