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Supplementary Figure 3 shows the activation of cell cycle-, and apoptosis-related proteins treated with lenvatinib or sorafenib in OCUT-1C and OCUT-2 cell subcutaneous tumors.

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PDF file, 115K, Supplementary Figure S2 E7050 inhibits tumor cell motility induced by HGF overexpression.

Supplementary Table 1, Figures 1-5 from E7080 Suppresses Hematogenous Multiple Organ Metastases of Lung Cancer Cells with Nonmutated Epidermal Growth Factor Receptor

While epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors improve the prognosis of patients with EGFR mutant lung cancer, the prognosis of patients with nonmutant EGFR lung cancer, especially those with metastases, is still extremely poor. We have assessed the therapeutic efficacy of E7080, an orally available inhibitor of multiple tyrosine kinases including VEGF receptor 2 (VEGFR-2) and VEGFR-3, in experimental multiple organ metastasis of lung cancer cell lines without EGFR mutations. E7080 markedly inhibited the in vitro proliferation of VEGF-stimulated microvascular endothelial cells. Intravenous inoculation into natural killer cell–depleted severe combined immunodeficient mice of the small cell lung cancer cell lines H1048 (producing low amounts of VEGF) and SBC-5 (producing intermediate amounts of VEGF) resulted in hematogenous metastases into multiple organs, including the liver, lungs, kidneys, and bones, whereas intravenous inoculation of PC14PE6, a non–small cell lung cancer cell line producing high amounts of VEGF, resulted in lung metastases followed by massive pleural effusion. Daily treatment with E7080 started after the establishment of micrometastases significantly reduced the number of large (>2 mm) metastatic nodules and the amount of pleural effusion, and prolonged mouse survival. Histologically, E7080 treatment reduced the numbers of endothelial and lymph endothelial cells and proliferating tumor cells and increased the number of apoptotic cells in metastatic nodules. These results suggest that E7080 has antiangiogenic and antilymphangiogenic activity and may be of potential therapeutic value in patients with nonmutant EGFR lung cancer and multiple organ metastases. Mol Cancer Ther; 10(7); 1218–28. ©2011 AACR.

Supplementary Table 2 shows the production of angiogenesis-related cytokines in the supernatant of ATC cells.

Anaplastic thyroid carcinoma (ATC) is a rare but aggressive undifferentiated tumor that frequently metastasizes to the brain. The multiple kinase inhibitor lenvatinib and sorafenib have been approved to treat unresectable differentiated thyroid cancer, and lenvatinib has been approved in Japan to treat ATC. This study compared the effects of lenvatinib and sorafenib in mouse models of central nervous system metastases of ATC. Immunodeficient mice were inoculated with ATC cells, and the effects of lenvatinib and sorafenib were evaluated in subcutaneous- and brain metastasis–mimicking models. Drug distribution was evaluated by imaging tandem mass spectrometry (ITMS). Neither lenvatinib nor sorafenib affected the viability of ATC cell lines, whereas both inhibited VEGF secretion by ATC cells. In the subcutaneous tumor model, both lenvatinib and sorafenib inhibited growth and were associated with reduced tumor microvessel density. In the brain metastasis–mimicking model, lenvatinib, but not sorafenib, inhibited the growth of ATC cells and reduced microvessel density in brain lesions. ITMS showed that lenvatinib was well-distributed in both subcutaneous and brain lesions, whereas the distribution of sorafenib was lower in brain than in subcutaneous lesions. These results demonstrate that lenvatinib is well-distributed in mouse models of ATC, and inhibited the growth of ATC brain lesions predominantly by inhibiting angiogenesis, suggesting that lenvatinib is highly potent against ATC brain metastases.

PDF file, 138K, Supplementary Figure S6 Treatment with WZ4002 and/or E7050 does not induce apoptosis in vivo.

PDF file, 181K, Supplementary Figure S3 Sustained inhibition of Akt and Erk phosphorylation by combining WZ4002 and E7050.

Supplementary Table 1: Patient characteristics; Supplementary Table 2: Primers for amplifying MET gene; Supplementary Figure 1: PC-9/LMC-GR did not express HGF or possess MET mutations; Supplementary Figure 2: Acquired gefitinib resistance of HCC827/luc cells in LMC model was associated with MET copy number gain; Supplementary Figure 3: Subcutaneous tumors with PC-9/LMC-GR cells are sensitive to gefitinib treatment.

PDF file, 423K, Supplementary Figure S4 Cytostatic effects of WZ4002 and E7050 treatment.

PDF file, 315K, Supplementary Figure S5 Relative body weight of mice during treatment with WZ4002 and/or E7050.

PDF file, 40K, Supplementary Table S1 IC50 values of each compound in MTT assay.

Although the EGF receptor tyrosine kinase inhibitors (EGFR-TKI) erlotinib and gefitinib have shown dramatic effects against EGFR mutant lung cancer, patients become resistant by various mechanisms, including gatekeeper EGFR-T790M mutation, Met amplification, and HGF overexpression, thereafter relapsing. Thus, it is urgent to develop novel agents to overcome EGFR-TKI resistance. We have tested the effects of the mutant-selective EGFR-TKI WZ4002 and the mutant-selective Met-TKI E7050 on 3 EGFR mutant lung cancer cell lines resistant to erlotinib by different mechanisms: PC-9/HGF cells with an exon 19 deletion, H1975 with an L858R mutation, and HCC827ER with an exon 19 deletion, with acquired resistance to erlotinib because of HGF gene transfection, gatekeeper T790M mutation, and Met amplification, respectively. WZ4002 inhibited the growth of H1975 cells with a gatekeeper T790M mutation, but did not inhibit the growth of HCC827ER and PC-9/HGF cells. HGF triggered the resistance of H1975 cells to WZ4002, whereas E7050 sensitized HCC827ER, PC-9/HGF, and HGF-treated H1975 cells to WZ4002, inhibiting EGFR and Met phosphorylation and their downstream molecules. Combined treatment potently inhibited the growth of tumors induced in severe-combined immunodeficient mice by H1975, HCC827ER, and PC-9/HGF cells, without any marked adverse events. These therapeutic effects were associated with the inhibition of EGFR and Met phosphorylation in vivo. The combination of a mutant-selective EGFR-TKI and a Met-TKI was effective in suppressing the growth of erlotinib-resistant tumors caused by gatekeeper T790M mutation, Met amplification, and HGF overexpression. Further evaluations in clinical trials are warranted. Mol Cancer Ther; 11(10); 2149–57. ©2012 AACR.

Leptomeningeal carcinomatosis occurs frequently in EGFR-mutant lung cancer, and develops acquired resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs). This study aimed to clarify the mechanism of EGFR-TKI resistance in leptomeningeal carcinomatosis and seek for a novel therapeutic strategy. We examined EGFR mutations, including the T790M gatekeeper mutation, in 32 re-biopsy specimens from 12 leptomeningeal carcinomatosis and 20 extracranial lesions of EGFR-mutant lung cancer patients who became refractory to EGFR-TKI treatment. All the 32 specimens had the same baseline EGFR mutations, but the T790M mutation was less frequent in leptomeningeal carcinomatosis specimens than in extracranial specimens (8% vs. 55%, P < 0.01). To study molecular mechanisms of acquired EGFR-TKI resistance in leptomeningeal carcinomatosis, we utilized our previously developed mouse model of leptomeningeal carcinomatosis with the EGFR-mutant lung cancer cell line PC-9/ffluc cells, in which acquired resistance to gefitinib was induced by continuous oral treatment. Compared with subcutaneously inoculated gefitinib-resistant tumors, the T790M mutation was less frequent in leptomeningeal carcinomatosis that acquired resistance to gefitinib. PC-9/LMC-GR cells were established from the gefitinib-resistant leptomeningeal carcinomatosis model, and they were found to be intermediately resistant to gefitinib and osimertinib (third-generation EGFR-TKI). Although EGFR-T790M was negative, gefitinib resistance of PC-9/LMC-GR cells was related to MET copy number gain with MET activation. Moreover, combined use of EGFR-TKI and crizotinib, a MET inhibitor, dramatically regressed leptomeningeal carcinomatosis with acquired resistance to gefitinib or osimertinib. These findings suggest that combination therapy with MET inhibitors may be promising for controlling leptomeningeal carcinomatosis that acquires resistance to EGFR-TKIs. Mol Cancer Ther; 16(3); 506–15. ©2017 AACR.

FigureS1 show that epithelial-pehotype-and mesenchymal-phenotype-tumor cells were detected in the brain lesion of clinical samples from EML4-ALK lung cancer patients treated with crizotinib. FigureS2 show analysis of bypass signaling pathways in the crizotinib resistant clones. FigureS3 show that TGF-β1 treatmend did not induce crizotinib-resistance in A925LPE3 cells. FigureS4 show effect of ZEB1 knockdown on restoring sensitivity to crizotinib in #3 cells. FIgureS5 show that a nano-luc expression vector for the miR-200c/141 reporter assay was constructed. FIgureS6 show that quisinostat reduced the motility and induced epithelial morphology in #4 cels. FigureS7 show that pre-treatment of quisinostat restored sensitivity to the second generation ALK-TKIs. FigureS8 show that pretreatment of quisinostat reverted EMT and restored sensitivity to EGFR-TKI in the EGFR-mutated NSCLC cell lines. FigureS9 show that sequential therapy with quisinostat reverted EMT and desapeared pleural in vivo. TableS1 show compounds of a 200 kinase inhibitor library.

KM12SM-ER cells were treated with siRNAs specific for NTRK1 (siNTRK), VEGFR-2 (siVEGFR-2), MET (siMET), or a scrambled control (siSCR). (A, C) The expression of TRK-A, VEGFR-2 and MET was determined by western blot. (B, D) Cell viability was determined using a CCK-8 kit. Data (mean {plus minus} SD) shown are representative of three independent experiments with similar results. *P < 0.05 as determined with the Student's t-test, compared with siSCR.

Purpose: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), such as gefitinib and erlotinib, show favorable response to EGFR mutant lung cancer. However, the responders acquire resistance almost without exception. We recently reported that hepatocyte growth factor (HGF) induces EGFR-TKI resistance by activating MET that restores downstream mitogen activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK)1/2 and phosphoinositide 3-kinase (PI3K)/Akt signaling. The purpose of this study was to determine whether inhibition of PI3K, a downstream molecule of both EGFR and MET, could overcome HGF-mediated EGFR-TKI resistance in EGFR mutant lung cancer cells PC-9 and HCC827.Experimental Design: We explored therapeutic effect of a class I PI3K inhibitor PI-103 on HGF-induced EGFR-TKI resistance in vitro and in vivo.Results: Unlike gefitinib or erlotinib, continuous exposure with PI-103 inhibited proliferation of PC-9 and HCC827 cells, even in the presence of HGF. On the other hand, in gefitinib-resistant xenograft model by using PC-9 cells mixed with HGF high producing fibroblasts, PI-103 monotherapy did not inhibit tumor growth. However, PI-103 combined with gefitinib successfully regressed gefitinib-resistant tumor. In vitro experiments by considering short half-life of PI-103 reveal that transient exposure of PI-103 combined with gefitinib caused sustained inhibition of Akt phosphorylation, but not ERK1/2 phosphorylation, resulting in induction of tumor cell apoptosis even in the presence of HGF.Conclusions: These results indicate that transient blockade of PI3K/Akt pathway by PI-103 and gefitinib could overcome HGF-mediated resistance to EGFR-TKIs by inducing apoptosis in EGFR mutant lung cancer. Clin Cancer Res; 17(8); 2260–9. ©2011 AACR.

Tumor cells were treated with 10nM of drugs for indicated time, and harvested lysates were assessed by western blotting.

Combination therapy of alectinib and ixazomib demonstrates feasibility enough to decrease tumor volume A, Percentage change in tumor volume after 28 days of treatment in A925L xenografts. B, Body weight of xenografted mice receiving indicated treatments. All data are expressed as the mean {plus minus} SE.

A, Comparison of ratio of BIM exon 3 to exon 4 transcripts in PC-9, PC-3, and PC-9BIMi2-/- cells. *, P < 0.05 PC-9 versus PC-3 or PC-3 versus PC-9BIMi2-/-. B, Alteration of transcripts containing BIM exon 2A, exon 3, and exon 4 in PC-9BIMi2-/- cells after treatment with gefitinib (1 µmol/L), osimertinib (1 µmol/L), and/or vorinostat (3 µmol/L) for 12 hours.

PDF file, 225KB, 1. EGFR ligands induce resistance to ALK inhibitors in a dose dependent manner in H2228 cells 2.H2228 cells were highly sensitive to siRNAs specific for ALK, whereas HGF and/or EGFR ligands (EGF, TGF-α, and HB-EGF) reduced the sensitivity of siRNAs specific for ALK 3.Induction of apoptosis in H2228 and H3122 cells by crizotinib 4.In MANA2 cells, HGF and EGFR ligands trigger ALK inhibitor resistance via Met and EGFR, respectively

All values are expressed as mean {plus minus} SE and assessed by one-way ANOVA. *, P < 0.05 osimertinib versus control in A, combination versus osimertinib in B.

Tumor tissues obtained in the liver metastasis model (Fig. 5) and brain metastasis-mimicking model (Fig. 6) were assessed for microvessel density. Sections of formalin-fixed and paraffin-embedded tissues were stained with anti-mouse CD31 antibody and evaluated by a standard IHC assay. (A) Representative pictures are shown. Bars indicate 100 µm. (B) The number of CD31-positive cells in the high-power field (200Ã-) was quantified. Data shown are mean {plus minus} SD of three independent areas. No significant difference was observed among the control, entrectinib-treated, and foretinib-treated groups.

Purpose: Hepatocyte growth factor (HGF) induces resistance to reversible and irreversible epidermal growth factor receptor–tyrosine kinase inhibitors (EGFR-TKI) in EGFR mutant lung cancer cells by activating Met and the downstream phosphoinositide 3-kinase (PI3K)/Akt pathway. Moreover, continuous exposure to HGF accelerates the emergence of EGFR-TKI–resistant clones. We assayed whether a new Met kinase inhibitor, E7050, which is currently being evaluated in clinical trials, could overcome these three mechanisms of resistance to EGFR-TKIs.Experimental Design: The effects of E7050 on HGF-induced resistance to reversible (gefitinib), irreversible (BIBW2992), and mutant-selective (WZ4002) EGFR-TKIs were determined using the EGFR mutant human lung cancer cell lines PC-9 and HCC827 with an exon 19 deletion and H1975 with an T790M secondary mutation. PC-9 cells were mixed with HGF-producing fibroblasts, MRC-5 cells, and subcutaneously inoculated into severe combined immunodeficient mice, and the therapeutic effects of E7050 plus gefitinib were assayed.Results: E7050 circumvented resistance to all of the reversible, irreversible, and mutant-selective EGFR-TKIs induced by exogenous and/or endogenous HGF in EGFR mutant lung cancer cell lines, by blocking the Met/Gab1/PI3K/Akt pathway in vitro. E7050 also prevented the emergence of gefitinib-resistant HCC827 cells induced by continuous exposure to HGF. In the in vivo model, E7050 plus gefitinib resulted in marked regression of tumor growth associated with inhibition of Akt phosphorylation in cancer cells.Conclusions: A new Met kinase inhibitor, E7050, reverses the three HGF-induced mechanisms of gefitinib resistance, suggesting that E7050 may overcome HGF-induced resistance to gefitinib and next-generation EGFR-TKIs. Clin Cancer Res; 18(6); 1663–71. ©2012 AACR.

Western blotting (A) and BIM exon 4-containing isoform mRNA expression (B) in PC-9BIMi2-/- cells transfected with control (scramble) or three different HDAC3 specific siRNA. *, P < 0.05 versus siRNA control (scramble).

Supplementary Table 1. Predicted biological function of TP53 mutations detected in ALK-rearranged non-small cell lung cancer patients Supplementary Table 2. Baseline charcteristics of ALK-rearranged non-small cell lung cancer patients with and without TP53 mutations Supplementary Table 3. Predicted biological function of ATM mutations detected in ALK-rearranged non-small cell lung cancer patients

Purpose:In ALK-rearranged non–small cell lung cancer (NSCLC), impacts of concomitant genetic alterations on targeted therapies with ALK-tyrosine kinase inhibitors (ALK-TKI) are not yet well understood. Here, we investigated genetic alterations related to ALK-TKI resistance using clinico-genomic data and explored effective therapies to overcome the resistance in preclinical models through the identification of underlying molecular mechanisms.Experimental Design:We used integrated clinical and next-generation sequencing data generated in a nationwide lung cancer genome screening project (LC-SCRUM-Japan). ALK-rearranged NSCLC cell lines expressing wild-type or mutant TP53 were used to evaluate cellular apoptosis induced by ALK-TKIs.Results:In 90 patients with ALK-rearranged NSCLC who were treated with a selective ALK-TKI, alectinib, TP53 comutated patients showed significantly worse progression-free survival (PFS) than TP53 wild-type patients [median PFS, 11.7 months (95% confidence interval, CI, 6.3–not reached, NR) vs. NR (23.6–NR); P = 0.0008; HR, 0.33 (95% CI, 0.17–0.65)]. ALK-rearranged NSCLC cell lines that lost p53 function were resistant to alectinib-induced apoptosis, but a proteasome inhibitior, ixazomib, markedly induced apoptosis in the alectinib-treated cells by increasing the expression of a proapoptotic protein, Noxa, which bound to an antiapoptotic protein, Mcl-1. In subcutaneous tumor models, combination of ixazomib and alectinib prominently induced tumor regression and apoptosis even though the tumors were generated from ALK-rearranged NSCLC cells with nonfunctional p53.Conclusions:These clinical and preclinical results indicate concomitant TP53 mutations reduce the efficacy of alectinib for ALK-rearranged NSCLC and the combined use of a proteasome inhibitor with alectinib is a promising therapy for ALK-rearranged/TP53-mutated NSCLC.

Relative mRNA expression of BIM exon 2A, exon 3, and exon 4, by RT-PCR and ratio of exon 3 to exon 4 transcripts in PC-9BIMi2-/- cells treated with droxinostat (3 µmol/L) (A) or RGFP966 (5 µmol/L) (B) for 12 hours. *, P < 0.05 versus control.

Purpose: Cancer cell microenvironments, including host cells, can critically affect cancer cell behaviors, including drug sensitivity. Although crizotinib, a dual tyrosine kinase inhibitor (TKI) of ALK and Met, shows dramatic effect against EML4-ALK lung cancer cells, these cells can acquire resistance to crizotinib by several mechanisms, including ALK amplification and gatekeeper mutation. We determined whether microenvironmental factors trigger ALK inhibitor resistance in EML4-ALK lung cancer cells.Experimental Design: We tested the effects of ligands produced by endothelial cells and fibroblasts, and the cells themselves, on the susceptibility of EML4-ALK lung cancer cell lines to crizotinib and TAE684, a selective ALK inhibitor active against cells with ALK amplification and gatekeeper mutations, both in vitro and in vivo.Results:EML4-ALK lung cancer cells were highly sensitive to ALK inhibitors. EGF receptor (EGFR) ligands, such as EGF, TGF-α, and HB-EGF, activated EGFR and triggered resistance to crizotinib and TAE684 by transducing bypass survival signaling through Erk1/2 and Akt. Hepatocyte growth factor (HGF) activated Met/Gab1 and triggered resistance to TAE684, but not crizotinib, which inhibits Met. Endothelial cells and fibroblasts, which produce the EGFR ligands and HGF, respectively, decreased the sensitivity of EML4-ALK lung cancer cells to crizotinib and TAE684, respectively. EGFR-TKIs resensitized these cells to crizotinib and Met-TKI to TAE684 even in the presence of EGFR ligands and HGF, respectively.Conclusions: Paracrine receptor activation by ligands from the microenvironment may trigger resistance to ALK inhibitors in EML4-ALK lung cancer cells, suggesting that receptor ligands from microenvironment may be additional targets during treatment with ALK inhibitors. Clin Cancer Res; 18(13); 3592–602. ©2012 AACR.

Tumor cells were treated with various concentrations of drugs for 4 h, and harvested lysates were assessed by western blotting.

KM12SM-ER cells were inoculated into the brain of SHO mice. The mice were treated daily with or without entrectinib (15 mg/kg or 30mg/kg) from day 8 to day 17. Mean {plus minus} SE of total flux are shown in the left panel. Representative pictures were shown in the right panel.

Purpose: The BIM deletion polymorphism is associated with apoptosis resistance to EGFR tyrosine kinase inhibitors (EGFR-TKI), such as gefitinib and erlotinib, in non–small cell lung cancer (NSCLC) harboring EGFR mutations. Here, we investigated whether the BIM deletion polymorphism contributes to resistance against osimertinib, a third-generation EGFR-TKI. In addition, we determined the efficacy of a histone deacetylase (HDAC) inhibitor, vorinostat, against this form of resistance and elucidated the underlying mechanism.Experimental Design: We used EGFR-mutated NSCLC cell lines, which were either heterozygous or homozygous for the BIM deletion polymorphism, to evaluate the effect of osimertinib in vitro and in vivo. Protein expression was examined by Western blotting. Alternative splicing of BIM mRNA was analyzed by RT-PCR.Results: EGFR-mutated NSCLC cell lines with the BIM deletion polymorphism exhibited apoptosis resistance to osimertinib in a polymorphism dosage–dependent manner, and this resistance was overcome by combined use with vorinostat. Experiments with homozygous BIM deletion–positive cells revealed that vorinostat affected the alternative splicing of BIM mRNA in the deletion allele, increased the expression of active BIM protein, and thereby induced apoptosis in osimertinib-treated cells. These effects were mediated predominantly by HDAC3 inhibition. In xenograft models, combined use of vorinostat with osimertinib could regress tumors in EGFR-mutated NSCLC cells homozygous for the BIM deletion polymorphism. Moreover, this combination could induce apoptosis even when tumor cells acquired EGFR-T790M mutations.Conclusions: These findings indicate the importance of developing HDAC3-selective inhibitors, and their combined use with osimertinib, for treating EGFR-mutated lung cancers carrying the BIM deletion polymorphism. Clin Cancer Res; 23(12); 3139–49. ©2016 AACR.

Supplementary Figures S1-S4.