PIM kinases alter mitochondrial dynamics and chemosensitivity in lung cancer.

Plasmids: pCIP and hPIM1 constructs were created by subcloning into the expression vector pCIG3 (pCMV-IRES-GFP, a gift from Dr. Felicia Goodrum, Addgene, plasmid #78264), modified to replace the GFP cassette with puromycin resistance gene. pSuper-Retro-puro-human shDrp1 was previously described ( 9 ). Reagents and antibodies: AZD1208 was acquired from AdooQ Biosciences. Docetaxel and Mdivi-1 were obtained from Selleck Chemicals. JC-1, mitoSOX-red and H 2 -DCFDA were purchased from Invitrogen. Primary antibodies for immunoblot and immunofluorescence analyses were purchased from BD Biosciences [Actin, 612656], Santa Cruz [Drp1, sc-271583, PIM1, sc-13513 and Bcl-2, sc-7382], and Cell Signaling Technology [TOM20, 4240S; pIRS (S1101), 2385S; PIM1, 3247S; pDRP1 (S616), 3455S; pDRP1 (S637), 4867S; and cleaved caspase-3, 9661S. All other materials and chemicals were of reagent grade. Cell transfection and immunoblotting: Wild type (WT) mouse embryonic fibroblasts (MEFs), triple-knockout (TKO; Pim1 −/− , Pim2 −/− , and Pim3 −/− ) MEFs ( 21 ), TKO MEFs stably expressing PIM1 (TKO-PIM1) ( 22 ), H1299, H1299Keap1 −/− , A549, and H460 cells were maintained in DMEM containing 10% FBS and 1% penicillin/streptomycin. All cell lines were maintained at 37°C in 5% CO 2 and were authenticated by short tandem repeat DNA profiling performed by the University of Arizona Genetics Core Facility. The cell lines were used for fewer than 50 passages and routinely tested for mycoplasma contamination. Stable retroviral transfections were carried out to generate PIM1 overexpressing (hPIM1) and Drp1 knockout (shDrp1) cells. Immunoblotting was performed as described previously ( 23 ). Mitochondrial superoxide assay: MitoSOX red staining was performed to measure mitochondrial superoxide production. Cells were plated in 6-well plates containing microscope coverslips and treated as indicated. Post-treatment, cells were incubated with 5 μM MitoSOX red dissolved in Hank’s Balanced Salt Solution (HBSS) for 30 minutes. Then, cells were washed with 1X DPBS and associated staining was determined either on mounted slides or in live cells using fluorescent microscopy. ROS assay: Electron paramagnetic resonance (EPR) was used to measure ROS in H1299, A549, and H460 cells treated with PIM447 for 48 h. Post-treatment, cells were incubated in Krebs-HEPES buffer, pH 7.4 containing 200 μM CMH (1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine) probe for 30 minutes at 37°C. Cells treated with 500 μM hydrogen peroxide for the same incubation period served as a positive control. Fifty microliters of the Incubation solution were added to a glass EPR capillary tube (Noxygen Science Transfer & Diagnostics, Elzach, Germany) that was placed inside the cavity of the E-scan spectrometer for data acquisition. The parameter settings for acquisition were as follows: center field 1.99 g, microwave power 1 mW, modulation amplitude 9 G, sweep time 10 seconds, number of scans 10, and field sweep 60 G. Sample temperature was stabilized and kept at 37°C by the Temperature & Gas Controller “Bio III” unit, interfaced to the spectrometer. Spectra were recorded and analyzed by using Win EPR software (2.11 version). ROS was also measured by DCF staining, as described previously ( 20 ). Immunofluorescence: Cells were plated in 6-well plates containing microscope coverslips and treated as indicated. Post-treatment, cells were fixed with 10% buffered formalin for 20 min and kept in blocking solution (5% NGS and 0.3% TritonX-100 in PBS) for 60 min. Then, cells were incubated with anti-TOM20 (rabbit mAb, 1:500 dilution), or anti-PIM1 (mouse mAb, 1: 100 dilution) or anti-DRP1 (mouse mAb, 1:200 dilution) antibodies for 60 min. Following primary antibody incubation, cells were washed with 1× PBS and incubated in secondary antibodies (Alexa Fluor 568 goat anti-mouse and Alexa Fluor 488 goat anti-rabbit, 1:500 dilution) for 60 min. Finally, cells were mounted on glass slides with mounting media (Cell Signaling Technology 8961S, Prolong® Gold) containing DAPI. Images were taken at 60× magnification using a fluorescent microscope. Transmission electron microscopy: Cells or tumor tissue samples were fixed with 2.5% glutaraldehyde in 0.1 M PIPES buffer, pH 7.4 overnight at 4°C. The samples were then washed with 0.1M PIPES, pH 7.4 three times for 10 minutes each. The samples were then post-fixed with 1% osmium tetroxide in PIPES, pH7.4 for 1 h, washed with deionized water two times for 10 min, followed by 20 min in aqueous 2% uranyl acetate, and washed again with deionized water for 10 min. The samples were then dehydrated with a graded series of increasing concentrations of ethanol (50%, 70%, 90%, and 100%) in Pelco Biowave Pro microwave, set at 250W, 20°C, and vacuum for 40 seconds. The samples were then infiltrated (microwave, 1:1 Spurr’s resin ethanol, 250W, 20°C, vacuum 3 minutes and Spurr’s resin, 25W, 20°C vacuum twice three minutes each) and embedded in Spurr’s resin overnight at 60 0 °C. Ultrathin (60nm) sections were cut onto uncoated copper mesh grids and stained with 2% lead acetate for 2 min. The samples were examined using FEI CM12 transmission electron microscope operated at 80kV. Digital images were obtained in 8-bit TIFF format using a 4×4 digital camera. Cell viability assay and drug interaction assessment: The cell viability was measured by crystal violet staining. Briefly, cells were plated in 96-well plates, treated with inhibitor/drugs for 72 h, fixed in 4% formaldehyde, and stained with 0.1% crystal violet. The cells were lysed in a 1% sodium dodecyl sulfate solution, and absorbance was measured using microplate reader at a wavelength of 595 nm. Synergy between PIM inhibitor and chemotherapies was assessed by calculating the combination index (CI) value using CompuSyn software. CI < 1 indicated synergy (the smaller the value, the greater the degree of synergy), CI = 1 indicated an additive effect, and CI > 1 indicated antagonism. JC-1 assay: Mitochondrial membrane potential (MMP) was determined by JC-1 assay. A549 and H460 cells were plated in 96-well plates and allowed to adhere overnight. Then, the cells were washed once with 1× DPBS, stained, and treated as indicated. At the end of the treatment period, absorbance was recorded using a fluorescence microplate reader with an excitation of 485 nm and emissions of 540 nm and 590 nm. Hydrogen peroxide was used as a positive control. MMP was determined using the ratio of the fluorescence of J-aggregates (590 nm) to monomers (540 nm) and normalized to the respective DMSO control. For JC-1 imaging, cells were seeded on 6-well plate, treated as indicated and processed as described above. Finally, images were taken for green and red channels at 20× magnification using a fluorescent microscope. In vivo studies: The sample size justification of 4 mice (8 tumors) per group is based on comparing the combination to each individual agent alone. We will have 80% statistical power to detect a standardized decrease of 1.325 between groups (difference divided by standard deviation) assuming a two-sided alpha of 0.05. Five million A549 cells in PBS were injected subcutaneously into each flank of SCID mice in PBS. Once average tumor size reached approximately 100 mm 3 , mice were randomized for treatment with vehicle (Cremophore EL/Ethanol/PBS-24/6/70 ratio, p.o. daily; 5% DMSO + 30% PEG + 5% Tween80 + ddH 2 O, i.p. every 3 rd day), AZD1208 (30mg/kg by p.o. daily), docetaxel (5 mg/kg by i.p. every 3 rd day), or AZD1208 + docetaxel. Tumor volume was monitored by caliper measurements. Thirty-six days after injection, animals were sacrificed, and tumors were harvested. Tumors were fixed, embedded in paraffin, and sectioned for staining with hematoxylin and eosin (H&E) or antibodies specific for Ki67, Cleaved caspase-3 (CC3), PIM1, and DRP1. Percent positive staining for the above mentioned proteins was calculated using ImageJ analysis software. Investigators were blinded to the sample information prior to software-based analysis. For Transmission electron microscopy (TEM) analysis, tumors from vehicle and AZD1208-treated mice were fixed in 2.5% glutaraldehyde. All animal studies were approved by the Institutional Animal Care and Use Committee at the University of Arizona. Statistical analysis: Tumor growth was analyzed by fitting a mixed linear model of tumor volume vs. time for each mouse. The resulting slopes (growth rate) was compared using a factorial model with vehicle, AZD alone, Docetaxel alone, and the AZD + Docetaxel dual treatment. Differences in proliferation and apoptosis among treatment groups was analyzed using linear mixed models adjusted for the correlation among measurements within the same mouse. All immunofluorescence staining and western blots are representative of at least three independent experiments. Differences across groups were determined by unpaired 2-tailed Student’s t-test. One-way analysis of variance (ANOVA) was used to analyze differences between more than two groups across one timepoint. P values were adjusted using Bonferroni’s multiple comparison test. The data is presented as the mean SD or mean SEM as indicated, and a p-value < 0.05 was considered statistically significant.

Supplementary Material 1625053_Supp_Fig_Caption 1625053_Supp_Fig6 1625053_Supp_Fig4 1625053_Supp_Fig5 1625053_Supp_Fig7 1625053_Supp_Fig3 1625053_Supp_Fig1 1625053_Supp_Fig2