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NSCLC cell lines investigated. Among the EGFR-mutation positive cell lines examined, the H1975 and PC9 cell lines
had the highest PIM1 and STAT3 mRNA expression. The combination decreased the osimertinib-induced STAT3
phosphorylation.

Conclusion: This study provides a short review on PIM kinases, and shows our results of combined PIM and EGFR
inhibition in EGFR-mutation positive NSCLC cell lines. The combination was moderately synergistic but decreased
STAT3 phosphorylation, an important signaling node in therapy resistance.

Keywords: Non-small cell lung cancer, EGFR, PIM-1, AZD1208, tyrosine kinase inhibitors

INTRODUCTION
Activation of the epidermal growth factor receptor (EGFR) leads to downstream induction of signaling
pathways, including phosphatidylinositol 3-kinase/AKT, Ras/mitogen-activated protein kinase and Janus
kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) [Figure 1]. Consequently,
[1,2]
these pathways stimulate cell proliferation, cell motility, cell cycle progression and cell survival . EGFR
[1]
mutations were discovered in 2004 . This finding has radically changed the therapy of EGFR-mutation
positive non-small cell lung cancer (NSCLC) patients, replacing standard chemotherapy with EGFR
[1,3]
tyrosine kinase inhibitors (TKIs) . Treatment with first-, second-, and third-generation EGFR TKIs has
[4]
significantly improved the progression free survival of NSCLC patients, harboring EGFR mutations . The
[5]
third-generation EGFR TKI, osimertinib is now the standard first-line therapy of these patients . However,
although initially effective, monotherapy with EGFR TKIs, including osimertinib, triggers intrinsic or
[6-8]
acquired resistance pathways , and the patients eventually succumb to their disease.
Signaling nodes, including signal transducer and activator of transcription 3 (STAT3) and Src/Yes-associated
protein 1 (YAP1), are not abrogated or even activated upon EGFR blockade. Moreover, besides EGFR, other
receptor tyrosine kinases (RTKs) are present and induce downstream signaling, independent of EGFR
inhibition [9-13] . We have demonstrated that simultaneous inhibition of EGFR, STAT3 and Src-YAP1 was
highly synergistic in vitro and in vivo. Moreover, multiple RTKs and non-RTKs were up regulated at baseline
[6-8]
or after treatment with EGFR TKIs, limiting their efficacy . Proviral integration site for Moloney murine
leukemia virus-1 (PIM-1) is a serine/threonine kinase, involved in cell cycle progression, cell growth, cell
survival and therapy resistance. PIM-1 is activated in various types of cancer, amongst which prostate [14-16] ,
colorectal [15,17-19] , triple negative breast cancer (TNBC) [20,21] , hematologic malignancies [22-24] , neuroblastoma [25]
[26]
and lung cancer . For this reason, PIM kinases could provide a common target for the treatment of diverse
malignancies.

PIM activation is induced by changes in the tumor microenvironment, such as hypoxia, and causes resistance
[15]
to antiangiogenic drugs . The combination of PIM inhibitors with antiangiogenic compounds, down
regulates the expression of hypoxia inducible factor 1 (HIF1) and decreases tumor cell proliferation, the
formation of tumor vasculature and the ability of the cells to metastasize . A second study has also shown
[15]
the involvement of PIM kinases in therapy resistance through hypoxia. Although the authors in this study
propose a HIF1-independent, nuclear factor-erythroid 2 p45-related factor 2 induced hypoxia, PIM kinase
[27]
inhibitors are again the solution to prevent therapy resistance and induce cell death in different tumor types .

PIM-1 is involved in RTKs, STAT3 and interleukin-6 (IL-6) signaling [Figure 1]. Specifically, STAT
proteins bind to the promoter of the PIM-1 gene, and increase PIM-1 expression. A feedback loop, in turn,
regulates the expression of STAT proteins [14,18,21,28,29] . Considering the fact that there are no approved STAT3
inhibitors, the combination of EGFR TKIs with a PIM-1 inhibitor could be of interest to block STAT3