Co-Targeting PIM Kinase and PI3K/mTOR in NSCLC.

Gillian Moore,Lauren Brady,Kenneth J O’Byrne,Stephen P Finn,Samira Elbai,Michael O’Neill,Sinead Cuffe,Siobhan Nicholson,Ronan Ryan,Katie E O’Sullivan,Kathy Gately,Carmen Blanco-Aparicio,Clara Lightner,Susan Heavey

doi:10.3390/cancers13092139

Abstract

Simple SummaryPIM kinases interact with major oncogenic players, including the PI3K/Akt pathway, and provide an escape mechanism leading to drug resistance. This study examined PIM kinase expression in NSCLC and the potential of PIM1 as a prognostic marker. The effect on cell signaling of novel preclinical PI3K/mTOR/PIM kinase inhibitor IBL-301 was compared to PI3K/mTOR inhibition in vitro and ex vivo. PI3K-mTOR inhibitor sensitive (H1975P) and resistant (H1975GR) cells were compared for altered IL6/STAT3 pathway expression and sensitivity to IBL-301. All three PIM kinases are expressed in NSCLC and PIM1 is a marker of poor prognosis. IBL-301 inhibited c-Myc, the PI3K-Akt and JAK/STAT pathways in vitro and in NSCLC tumor tissue explants. IBL-301 also inhibited secreted pro-inflammatory cytokine MCP-1. PIM kinases were activated in H1975GR cells which were more sensitive to IBL-301 than H1975P cells. A miRNA signature of PI3K-mTOR resistance was validated. Co-targeting PIM kinase and PI3K-mTOR warrants further clinical investigation.PIM kinases are constitutively active proto-oncogenic serine/threonine kinases that play a role in cell cycle progression, metabolism, inflammation and drug resistance. PIM kinases interact with and stabilize p53, c-Myc and parallel signaling pathway PI3K/Akt. This study evaluated PIM kinase expression in NSCLC and in response to PI3K/mTOR inhibition. It investigated a novel preclinical PI3K/mTOR/PIM inhibitor (IBL-301) in vitro and in patient-derived NSCLC tumor tissues. Western blot analysis confirmed PIM1, PIM2 and PIM3 are expressed in NSCLC cell lines and PIM1 is a marker of poor prognosis in patients with NSCLC. IBL-301 decreased PIM1, c-Myc, pBAD and p4EBP1 (Thr37/46) and peIF4B (S406) protein levels in-vitro and MAP kinase, PI3K-Akt and JAK/STAT pathways in tumor tissue explants. IBL-301 significantly decreased secreted pro-inflammatory cytokine MCP-1. Altered mRNA expression, including activated PIM kinase and c-Myc, was identified in Apitolisib resistant cells (H1975GR) by an IL-6/STAT3 pathway array and validated by Western blot. H1975GR cells were more sensitive to IBL-301 than parent cells. A miRNA array identified a dysregulated miRNA signature of PI3K/mTOR drug resistance consisting of regulators of PIM kinase and c-Myc (miR17-5p, miR19b-3p, miR20a-5p, miR15b-5p, miR203a, miR-206). Our data provides a rationale for co-targeting PIM kinase and PI3K-mTOR to improve therapeutic response in NSCLC.

Highlights

The pro-viral insertion site in Moloney murine leukemia virus (PIM) proteins are evolutionarily conserved serine/threonine kinases in eukaryotes consisting of PIM1, PIM2 and PIM3
PIM1, PIM2 and PIM3 proteins were expressed in HCC827, H460 and H1975 cell lines as determined by Western blot analysis
The H460 cell line had high expression of both the 34 kDa and 40 kDa PIM2 isoforms while HCC827 cells had weak expression of both PIM2 isoforms and H1975 cells had very weak expression of the 34 kDa isoform and high expression of the 40 kDa isoform

Summary

Introduction

The pro-viral insertion site in Moloney murine leukemia virus (PIM) proteins are evolutionarily conserved serine/threonine kinases in eukaryotes consisting of PIM1, PIM2 and PIM3. PIM kinases regulate cell survival pathways, are implicated in the progression of lung cancer [1,2] and play a role in enhanced resistance to chemotherapy [3,4] and molecular targeted therapies of EGFR, MET and PI3K/Akt/mTOR signaling [5,6,7]. The tumorigenic potential of the PIM family is mainly mediated by its interactions with other pathways commonly upregulated in cancer such as the PI3K (Phosphoinositide 3-kinase) pathway. Both PIM and PI3K phosphorylate overlapping substrates to activate common pathways that control various physiological processes that dictate the balance between cell survival and apoptosis. We have shown that a co-targeted approach is more effective compared to single PIM kinase or PI3K-mTOR inhibition, as previously reported in prostate cancer [10]

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