Combination Treatment with MEK and AKT Inhibitors Is More Effective than Each Drug Alone in Human Non-Small Cell Lung Cancer In Vitro and In Vivo

AZD6244 and MK2206 are targeted small-molecule drugs that inhibit MEK and AKT respectively. The efficacy of this combination in lung cancer is unknown. Our previous work showed the importance of activated AKT in mediating resistance of non-small cell lung cancer (NSCLC) to AZD6244. Thus we hypothesized that dual inhibition of both downstream MEK and AKT pathways would induce synergistic antitumor activity. In this study, we evaluated the efficacy of AZD6244 and MK2206 individually on a large panel of lung cancer cell lines. Then, we treated 28 human lung cancer cell lines with a combination of AZD6244 and MK2206 at clinically applicable drug molar ratios. The AZD6244-MK2206 combination therapy resulted in a synergistic effect on inhibition of lung cancer cell growth compared to the results of single drug treatment alone. MK2206 enhanced AZD6244-induced Bim overexpression and apoptosis in A549 and H157 cells. When we tested the combination of AZD6244 and MK2206 at ratios of 8∶1, 4∶1, 2∶1, and 1∶8, we found that the synergistic effect of the combination therapy was ratio-dependent. At ratios of 8∶1, 4∶1, and 2∶1, the drug combination consistently demonstrated synergy, whereas decreasing the ratio to 1∶8 resulted in a loss of synergy and produced an additive or antagonistic effect in most cell lines. Furthermore, the AZD6244-MK2206 combination therapy showed synergy in the suppression of A549 and H157 xenograft tumor growth and increased mean animal survival time. The AZD6244-MK2206 combination therapy resulted in effective inhibition of both p-ERK and p-AKT expression in tumor tissue. In addition, a significant increase of apoptosis was detected in tumor tissue from mice treated with AZD6244-MK2206 compared with that from the single agent treated mice. Our study suggests that the combination of AZD6244 and MK2206 has a significant synergistic effect on tumor growth in vitro and in vivo and leads to increased survival rates in mice bearing highly aggressive human lung tumors

Elevated NSD3 histone methylation activity drives squamous cell lung cancer

Amplification of chromosomal region 8p11-12 is a common genetic alteration that has been implicated in the aetiology of lung squamous cell carcinoma (LUSC)(1-3). The FGFR1 gene is the main candidate driver of tumorigenesis within this region(4). However, clinical trials evaluating FGFR1 inhibition as a targeted therapy have been unsuccessful(5). Here we identify the histone H3 lysine 36 (H3K36) methyltransferase NSD3, the gene for which is located in the 8p11-12 amplicon, as a key regulator of LUSC tumorigenesis. In contrast to other 8p11-12 candidate LUSC drivers, increased expression of NSD3 correlated strongly with its gene amplification. Ablation of NSD3, but not of FGFR1, attenuated tumour growth and extended survival in a mouse model of LUSC. We identify an LUSC-associated variant NSD3(T1232A) that shows increased catalytic activity for dimethylation of H3K36 (H3K36me2) in vitro and in vivo. Structural dynamic analyses revealed that the T1232A substitution elicited localized mobility changes throughout the catalytic domain of NSD3 to relieve auto-inhibition and to increase accessibility of the H3 substrate. Expression of NSD3(T1232A) in vivo accelerated tumorigenesis and decreased overall survival in mouse models of LUSC. Pathological generation of H3K36me2 by NSD3(T1232A) reprograms the chromatin landscape to promote oncogenic gene expression signatures. Furthermore, NSD3, in a manner dependent on its catalytic activity, promoted transformation in human tracheobronchial cells and growth of xenografted human LUSC cell lines with amplification of 8p11-12. Depletion of NSD3 in patient-derived xenografts from primary LUSCs containing NSD3 amplification or the NSD3(T1232A)-encoding variant attenuated neoplastic growth in mice. Finally, NSD3-regulated LUSC-derived xenografts were hypersensitive to bromodomain inhibition. Thus, our work identifies NSD3 as a principal 8p11-12 amplicon-associated oncogenic driver in LUSC, and suggests that NSD3-dependency renders LUSC therapeutically vulnerable to bromodomain inhibition

The tumor suppressor gene TUSC2 (FUS1) sensitizes NSCLC to the AKT inhibitor MK2206 in LKB1-dependent manner.

TUSC2-defective gene expression is detected in the majority of lung cancers and is associated with worse overall survival. We analyzed the effects of TUSC2 re-expression on tumor cell sensitivity to the AKT inhibitor, MK2206, and explored their mutual signaling connections, in vitro and in vivo. TUSC2 transient expression in three LKB1-defective non-small cell lung cancer (NSCLC) cell lines combined with MK2206 treatment resulted in increased repression of cell viability and colony formation, and increased apoptotic activity. In contrast, TUSC2 did not affect the response to MK2206 treatment for two LKB1-wild type NSCLC cell lines. In vivo, TUSC2 systemic delivery, by nanoparticle gene transfer, combined with MK2206 treatment markedly inhibited growth of tumors in a human LKB1-defective H322 lung cancer xenograft mouse model. Biochemical analysis showed that TUSC2 transient expression in LKB1-defective NSCLC cells significantly stimulated AMP-activated protein kinase (AMPK) phosphorylation and enzymatic activity. More importantly, AMPK gene knockdown abrogated TUSC2-MK2206 cooperation, as evidenced by reduced sensitivity to the combined treatment. Together, TUSC2 re-expression and MK2206 treatment was more effective in inhibiting the phosphorylation and kinase activities of AKT and mTOR proteins than either single agent alone. In conclusion, these findings support the hypothesis that TUSC2 expression status is a biological variable that potentiates MK2206 sensitivity in LKB1-defective NSCLC cells, and identifies the AMPK/AKT/mTOR signaling axis as an important regulator of this activity

Cisplatin enhances apoptosis induced by a tumor-selective adenovirus expressing tumor necrosis factor–related apoptosis-inducing ligand

Cancer cells frequently exhibit resistance to the cytotoxic effect of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL). Pretreatment of TRAIL-resistant cells with cisplatin sensitizes them to this ligand. Cisplatin also has been shown to enhance adenoviral transgene expression. This study aims to evaluate the ability of cisplatin to enhance the expression and the cytotoxic effect of the tumor-specific adenoviral vector Ad/gTRAIL, which expresses a green fluorescent protein–TRAIL fusion protein. Cultured cancer cells and normal human cells were infected with Ad/gTRAIL with or without cisplatin pretreatment. Adenoviral transgene expression was determined by using flow cytometry to measure green fluorescent protein fluorescence. Cytotoxicity was measured by using thiazolyl blue tetrazolium bromide assays and an apoptosis enzyme-linked immunosorbent assay kit. Green fluorescent protein–TRAIL fusion protein expression was significantly enhanced by cisplatin pretreatment in cancer cells. Cisplatin treatment before Ad/gTRAIL infection resulted in a 2- to 12-fold increase in green fluorescent protein fluorescence intensity across cancer lines. Although Ad/gTRAIL induced mild cytotoxicity in all cancer lines (inhibitory concentration of 50% values of >500 pfu/cell), pretreatment with cisplatin resulted in a dose-dependent enhancement of Ad/gTRAIL-mediated cytotoxicity, as indicated by the drastic reduction of inhibitory concentration of 50% values to 4 to 42 pfu/cell in all cell lines. There was no cytotoxicity noted in normal cells treated with both cisplatin and Ad/gTRAIL. Cisplatin pretreatment enhances Ad/gTRAIL cytotoxicity in malignant cells while not affecting normal cells. The mechanisms underlying this effect might include both enhancement of the susceptibility of cisplatin-treated cells to TRAIL and cisplatin-mediated enhancement of TRAIL expression in Ad/gTRAIL infected cells. These findings provide a rationale for development of Ad/gTRAIL-based therapy for thoracic malignancies

Inhibition of AKT and mTOR kinase activity by TUSC2 transfection and MK2206 combined treatment.

<p>LKB1-defective HCC366, H322, and A549 were transfected with TUSC2 for 24 hours, starved for 24 hours and either treated with 1 µM MK2206 for 24 hours or left untreated. Cell lysates were collected for western blot analysis for the levels of A) p-AKT(S473) and p-AKT(Th308); and B) p-mTOR(S2448). AKT and mTOR was precipitated from 200 ug cell lysis using A) AKT or B) mTOR antibodies. The kinase activity of AKT and mTOR were measured with KLISA AKT and mTOR assay kit, respectively, using GSK-3α and S6K GST fusion proteins as substrates, respectively. Kinase activities were determined by ELISA, as substrate absorbance was measured at 450 nm, and reference wavelengths were measured at 540/595 nm using a Synergy 2 Multi-detection microplate reader. Columns, mean of three different experiments, each with duplicate samples; bars, SD. *, <i>P</i><0.05, compared with EV control; **, <i>P</i><0.05, compared with EV+MK2206.</p

IC₅₀ values and gene mutation status of five lung cancer cell lines.

<p>IC₅₀ values and gene mutation status of five lung cancer cell lines.</p

Endogenous and overexpressed levels of TUSC2 in LKB1-defective and wild type NSCLC cells.

<p>Cells were transfected with DC-TUSC2 for 24 hours, and TUSC2 protein levels were detected by western blot with a rabbit anti-TUSC2 polyclonal antibody.</p

Stimulation of AMPK phosphorylation and kinase activity by TUSC2 in LKB1-defective cells.

<p>A) HCC366, H322, and A549 were transfected with TUSC2 then either treated with 1 µM MK2206 for 24 hours or left untreated. AMPK kinase activity in the immunocomplexes was measured by phosphorylation of SAMS peptide as described in Materials and Methods. Columns, mean of three different experiments, each with duplicate samples; bars, SD. *, <i>P</i><0.05, compared with EV control; **, <i>P</i><0.05, compared with EV+MK2206. LKB1- defective cells HCC366 and H322 cells were co-transfected with 2 µg TUSC2 plasmid and 50 nM AMPK siRNA with Lipofectamine™ 2000. Twenty-four hours after transfection, cells were starved for 24 hours and treated with 1 µM MK2206 for an additional 48 hours. B) Cell lysis were collected for western blot analysis to assess levels of AMPK and p-AMPK proteins; or C) Cells were assayed for apoptosis as described in Materials and Methods. Columns, mean of three different experiments, each with duplicate samples; bars, SD. ^#, <i>P</i><0.05, compared with TUSC2; ^##, <i>P</i><0.05, compared with TUSC2+MK2206.</p

Effective <i>in vivo</i> inhibition of tumor growth by TUSC2 systematic restoration and MK2206 combined treatment.

<p>A subcutaneous mouse model of human NSCLC H322 was used to evaluate the combined effect of systemic delivery of the DC–based TUSC2 nanoparticles and MK2206 treatment on tumor growth inhibition. A) Tumor volume was calculated, taking length to be the longest diameter across the tumor and width to be the corresponding perpendicular diameter, using the following formula: length × width²×0.52. Tumor growth inhibition rate was calculated as 100%× (tumor size_treated/tumor size_control) on each measurement day. Bars, SD; B) Tumors were resected, fixed with 4% paraformaldehyde, paraffin-embedded for immunohistochemistry staining with the indicated antibodies, and examined under a Nikon TC200 fluorescence microscope equipped with a digital camera.</p

Inhibition of tumor cell viability and colony formation by TUSC2 transfection and MK2206 combined treatment in LKB1-defective and wild type NSCLC cells.

<p>A) Forty-eight hours post-treatment, cells were assayed for viability as described in Materials and Methods. Cell viability was plotted against concentration of MK2206. B) Cells were transfected with DC-TUSC2. Twenty four hours post-transfection, cells were split, replated in triplicate, and grown in medium containing 400 µg/ml of the antibiotic G418 before treatment with 1 µM MK2206. Colonies were fixed with glutaraldehyde (6.0% v/v), stained with crystal violet (0.5% w/v), and counted using a stereomicroscope. Columns, mean of three different experiments, each with duplicate samples; bars, SD. *, <i>P</i><0.05, compared with EV control; **, <i>P</i><0.05, compared with EV+MK2206</p