MET blockade offers a new targeted therapy particularly in those cancers with MET amplification. (ALK) rearrangement achieved rapid response to crizotinib (10), a small-molecule inhibitor of MET and ALK. Clinical improvement and radiographic regression have also been reported in patients with and gene amplification (14), and the acquisition of a mutation in the MET activation loop (Y1230H) (15). Multiple mechanisms could arise simultaneously in a single patient to allow for MET resistance (15). Pim kinases are serine/threonine kinases that are constitutively active in cells (16,17) and the activity of Pim kinases is largely regulated at the transcriptional and translational levels (18). Recently, we have shown that Pim-1 is an important regulator of MET expression and signaling through the regulation of protein translation in part mediated by the ability of Pim to control the phosphorylation of eIF4B (19). The Pim family of serine/threonine kinases are known to modulate cell survival pathways, regulate the progression and growth of human cancers, and induce resistance to chemotherapy (18,20). Increased Pim levels have been shown to phosphorylate BH3 protein BAD and sequester its activity Thbd blocking apoptosis (16,21,22). Small-molecule AKT inhibitors induce dramatic upregulation of Pim-1 expression, and Pim-1 then functions to increase Laquinimod expression of a subset of RTKs that play an important part in the resistance to these drugs (23). Here, we examine the role of Pim kinases in the mechanisms underlying acquired resistance to small-molecule MET inhibitors in cells and tumors with amplification and, thus, addiction to the MET signaling pathway. Based on this evidence, we explore the activity of combining MET and Pim inhibitors to overcome cancer resistance to MET inhibitor therapy. Materials and Methods Antibodies and Reagents The following antibodies were purchased from Cell Signaling Technology: Anti-Pim-1 (Cat#3247), anti-Pim2 (Cat#4730), anti-Pim3 (Cat#4165) anti-MET (Cat#8198), anti-phospho-MET (Cat#3077), anti-BAD (Cat#9239), anti-phospho-BAD (Cat#5284), anti-eIF4B (Cat#3592), anti-phospho-eIF4B (S406, Cat#8151), anti-eIF4G (Cat#2498), anti-eIF4E(Cat#2067), anti-Myc-Tag(Cat#2272), anti-AKT (pan, Cat#4691), anti-phospho-AKT (S473, Cat#4058), anti-phospho-4E-BP1 (Cat#2855), anti-4E-BP1 (Cat#9452), anti-phospho-S6 (Cat#2215), anti-ERK (Cat#9102), anti-phospho-ERK (Cat#9101), anti-Bcl-2(Cat#4223), anti-Bim(Cat#2933), and anti-cleaved PARP(Cat#5625). Anti–actin (Cat#A3854), anti–tubulin (Cat#T4026), anti-FLAG (Cat#F1804) antibodies were purchased from Sigma. Anti-Mcl-1 antibody (Cat#sc-12756) was from Santa Cruz Biotechnology. A neutralizing antibody against MET was from Abcam (Cat#ab10728). HRP-linked enhanced chemiluminescence (ECL) mouse (Cat#NA931V) and rabbit IgG (Cat#NAV934V) were purchased from GE Healthcare Life Sciences. The small molecule inhibitors PP242, BEZ235, ABT199, and ABT737 were obtained from Selleck Biochemicals. PHA665752 was from Cayman Chemical. LGB321 was provided by Norvatis. AZD6094 and AZD1208 were provided by AstraZeneca. LY2801653 was from Eli Lilly. Cell culture MKN45, SNU5, and H1993 cells were from American Type Culture Collection. EBC-1 cells were from the Japanese Collection of Research Bioresources (JCRB) Cell Bank. All cell lines were authenticated by providers utilizing Short Tandem Repeat (STR) profiling. Cells were used over a course of no more than 3 months after resuscitation of frozen aliquots. Cells were grown in RPMI supplemented with 2 mM Glutamax (Life Technologies) and 10% fetal bovine serum (BioAbChem) at 37C under 5% CO2. Establishment of MET inhibitor-resistant cells Laquinimod MKN45 and EBC-1 cells were exposed Laquinimod to increasing concentrations of PHA665752 or AZD6094 every 3 weeks starting from 50 nM until a concentration of 5 M was reached at the end of a 6-month period. MET inhibitor-resistant cells were Laquinimod successfully expanded in 10% FBS culture medium containing 1 M of either MET inhibitors. Established resistant sublines were designated PHAR and AZDR. Plasmids and siRNAs The Pim-1 expressing construct pTripZ-Pim-1 was described previously (24). The bicistronic luciferase construct phpRL-BCL2-FL-pA (25) was a gift from Richard Lloyd (Addgene plasmid #42595). Bicistronic luciferase plasmids containing HCV IRES has been previously described (23). The source of the siRNAs were as follows: On-TARGEThuman Pim-1, human MET, and human BCL2, Dharmacon; human Pim-3, Life Technologies Silencer Select product with the following sequence: 5GCACGUGGUGAAGGAGCGG3; human BAD, SignalSilence? Bad siRNA II, Cell Signaling. All transfections were done with with lipofectamine 3000 reagent with both plasmids and siRNAs according to the manufacturers instructions. Real-time PCR analyses SYBR Green reactions were done using a BioRad iQ5 real-time quantitative PCR system. For data analysis, raw counts were normalized to the housekeeping.