Search

Fig. S1. Supplementary data for Fig. 1 Fig. S2. Supplementary drug sensitivity data to ALK inhibitors in KTOR1 and KTOR1-RE. Fig. S3. Supplementary results of the phosphoproteome analysis Fig. S4. Saracatinib sensitized KTOR1-RE cells to crizotinib Fig. S5. Combined knockdown of MET and SRC inhibited proliferation and induces apoptosis Fig. S6. Supplementary data for Fig. 6 Table S1. Primer sequences for direct sequence of ALK exons Table S2. Primer sequences for detection and sequence of EML4-ALK rearrangement Table S3. Primer sequences for qRT-PCR

Supplementary Figure S1-S6 and Table S1-S3

Alectinib is used as a first-line treatment for anaplastic lymphoma kinase (ALK)-rearranged non–small cell lung cancer (NSCLC). Whereas other ALK inhibitors have been reported to be involved in resistance to ATP-binding cassette (ABC) transporters, no data are available regarding the association between resistance to alectinib and ABC-transporters. To investigate whether ABC-transporters contribute to alectinib resistance, ABC-transporter expression in alectinib-resistant cell lines derived from a patient with ALK-rearranged NSCLC and from H2228 lung cancer cells was evaluated and compared with that in each parent cell type. ATP-binding cassette subfamily C member 11 (ABCC11) expression was significantly increased in alectinib-resistant cell lines compared with that in alectinib-sensitive lines. ABCC11 inhibition increased sensitivity to alectinib in vitro. ABCC11-overexpressing cells were established by transfection of an ABCC11 expression vector into H2228 cells, while control cells were established by transfecting H2228 cells with an empty vector. ABCC11-overexpressing cells exhibited decreased sensitivity to alectinib compared with that of control cells in vitro. Moreover, the tumor growth rate following alectinib treatment was higher in ABCC11-overexpressing cells than that in control cells in vivo. In addition, the intracellular alectinib concentration following exposure to 100 nmol/L alectinib was significantly lower in the ABCC11-overexpressing cell line compared with that in control cells. This is the first preclinical evidence showing that ABCC11 expression may be involved in acquired resistance to alectinib.

Figure S1. Schematic representation of the ABCC11 expression vector. Figure S2. Sequence analysis of the tyrosine kinase coding region of anaplastic lymphoma kinase (ALK). Figure S3. Cell viability assay of alectinib in H2228 and H2228-AR1S cells under low-attachment conditions. Figure S4. Gene expression of the ABC transporters in the five cell types. Figure S5. Intracellular concentration of alectinib. Figure S6. Tumor images on day 11 of treatment. The tumor volume at 10 days after alectinib administration. Figure S7. ALK phosphorylation in KTOR1 and KOR1-RE cell exposed to alectinib for 3 hours as determined using immunoblotting. Figure S8. IC50 values of H2228 and H2228-AR1S cell lines in the presence of crizotinib, ceritinib, lorlatinib, and brigatinib. Table S1. Immunoblotting antibodies. Table S2. Primers used for ABC transporters purchased from Thermo Fisher Scientific. Table S3. siRNA oligonucleotides purchased from Thermo Fisher Scientific. Table S4. Primer sequences for direct sequencing of ALK exons. Table S5. IC50 for alectinib in H2228-AR1S cells transfected with negative control or ABCC11 siRNA. Table S6. IC50 for alectinib in KTOR1-RE cells transfected with negative control or ABCC11 siRNA.

The mechanisms responsible for the development of resistance to alectinib, a second-generation anaplastic lymphoma kinase (ALK) inhibitor, are still unclear, and few cell lines are currently available for investigating ALK-rearranged lung cancer. To identify the mechanisms underlying acquired resistance to alectinib, two patient-derived cell lines were established from an alectinib-naïve ALK-rearranged lung cancer and then after development of alectinib resistance. The properties acquired during treatments were detected by comparisons of the two cell lines, and then functional analyses were performed. Coactivation of c-Src and MET was identified after the development of alectinib resistance. Combinatorial therapy against Src and MET significantly restored alectinib sensitivity in vitro (17.2-fold). Increased apoptosis, reduction of tumor volume, and inhibition of MAPK and PI3K/AKT signaling molecules for proliferation and survival were observed when the three kinases (Src, MET, and ALK) were inhibited. A patient-derived xenograft from the alectinib-resistant cells indicated that combination therapy with a saracatinib and crizotinib significantly decreased tumor size in vivo. To confirm the generality, a conventional alectinib-resistant cell line model (H2228-AR1S) was established from NCI-H2228 cells (EML4-ALK variant 3a/b). In H2228-AR1S, combination inhibition of Src and MET also restored alectinib sensitivity. These data reveal that dual salvage signaling from MET and Src is a potential therapeutic target in alectinib-resistant patients.Implications:This study demonstrates the feasibility to elucidate personalized drug-resistance mechanisms from individual patient samples.

Data sets of the phosphoproteome analysis in KTOR1 and KTOR1-RE cells

ABCC11 sequence inserted into the vector

Cancer cell resistance arises when tyrosine kinase inhibitor (TKI)-targeted therapies induce a drug-tolerant persister (DTP) state with growth via genetic aberrations, making DTP cells potential therapeutic targets. We screened an anti-cancer compound library and identified fibroblast growth factor receptor 1 (FGFR1) promoting alectinib-induced ALK fusion-positive DTP cell's survival. FGFR1 signaling promoted DTP cell survival generated from basal FGFR1- and FGF2-high expressing cells, following alectinib treatment, which is blocked by FGFR inhibition. The hazard ratio for progression-free survival of ALK-TKIs tended to increase in ALK fusion-positive non-small cell lung cancer patients with FGFR1- and FGF2-high expression. Combination of FGFR and targeted TKIs enhanced cell growth inhibition in FGFR1- and FGF2-high expressing cells with ALK fusion, HER2 amplification, and EGFR or BRAF mutations. Initial dual blockade of FGFR and various driver oncogenes based on FGFR1 and FGF2 expression levels before starting treatment would be a potent treatment strategy to prevent intrinsic resistance to targeted TKIs through DTP cells.

Tyrosine kinase inhibitors of anaplastic lymphoma kinase (ALK-TKIs) including alectinib have been the standard therapy against ALK fusion gene-positive non–small cell lung cancers (NSCLCs). Many ALK fusion variants have been identified in NSCLCs, and the predominant variants are echinoderm microtubule-associated protein-like 4-ALK (EML4-ALK) variant 1 (V1), V2 and V3a/b. However, there have been conflicting reports on the clinical responses of these variants to ALK-TKIs, and there are few reports on other less common ALK variants. To examine the influence of ALK variants on the efficacy of ALK-TKIs, we analyzed the sensitivity to alectinib of eight types of ALK variant: three major variants (V1, V2 and V3a) and five less common variants (V4; kinesin family member 5-ALK; kinesin light chain 1-ALK; striatin, calmodulin-binding protein-ALK; and tropomyosin-receptor kinase fused gene-ALK). Analysis was done by cell-free kinase assays using the recombinant proteins and by cell, growth assays using murine Ba/F3 cells expressing ALK variants. The kinase activity of each recombinant protein was significantly inhibited by alectinib. Intracellular ALK phosphorylation levels and its downstream signaling mediators, STAT3 and ERK, were suppressed by alectinib in each ALK variant-expressing Ba/F3 cell. Each cellular proliferation was markedly inhibited by alectinib treatment. There was no significant difference in the IC50 values between cells, with a

Rearranged during transfection (RET) fusion-positive non-small cell lung cancer (NSCLC) accounts for 1% of lung adenocarcinoma. Although small molecule agents with RET kinase inhibitory activity such as alectinib, vandetanib, and cabozantinib have been clinically evaluated in RET-fusion-positive NSCLC, an effective monotherapy regimen has not been established. We explored agents to use in combination with alectinib to enhance the antitumor effect of alectinib against RET-fusion cells. Cell proliferation under co-treatment with alectinib plus each of six chemotherapeutic agents or six molecularly targeted agents was evaluated in vitro. The combination effect was analyzed by IC50 isobologram and combination index using LC-2/ad and Ba/F3-KIF5B-RET cells. The in vivo combination effect was investigated in a Ba/F3-KIF5B-RET xenograft model. The phosphorylation levels of proteins regulating proliferation were measured by immunoblotting. Palbociclib, a CDK4/6 inhibitor, showed the greatest synergy against LC-2/ad cells in the isobologram analysis and combination index. This synergistic effect was also observed against Ba/F3-KIF5B-RET cells. Another CDK4/6 inhibitor, abemaciclib, also showed a synergistic effect. In vivo, the combination of alectinib plus palbociclib showed a more enhanced antitumor effect than each single agent in a mouse xenograft model with transplanted Ba/F3-KIF5B-RET cells. This combination suppressed the phosphorylation of S6 and Rb more intensely than did either single agent in both LC-2/ad and Ba/F3-KIF5B-RET cell lines, both in vitro and in vivo. Combination therapy with alectinib plus the CDK4/6 inhibitor enhanced the antitumor effect against RET-fusion-positive cells in vitro and in vivo.

Despite the promising clinical efficacy of the second-generation anaplastic lymphoma kinase (ALK) inhibitor alectinib in patients with ALK-rearranged lung cancer, some tumor cells survive and eventually relapse, which may be an obstacle to achieving a cure. Limited information is currently available on the mechanisms underlying the initial survival of tumor cells against alectinib. Using patient-derived cell line models, we herein demonstrate that cancer cells survive a treatment with alectinib by activating Yes-associated protein 1 (YAP1), which mediates the expression of the anti-apoptosis factors Mcl-1 and Bcl-xL, and combinatorial inhibition against both YAP1 and ALK provides a longer tumor remission in ALK-rearranged xenografts when compared with alectinib monotherapy. These results suggest that the inhibition of YAP1 is a candidate for combinatorial therapy with ALK inhibitors to achieve complete remission in patients with ALK-rearranged lung cancer., Relapse is a limitation for the efficacy of the anaplastic lymphoma kinase (ALK)-inhibitor alectinib in ALK-rearranged lung cancer. Here, the authors show that YAP1 activation upon alectinib treatment leads to therapy resistance and that inhibiting both YAP1 and ALK leads to longer tumor remission in mice.

Background Genetic alterations, including mutation of epidermal growth factor receptor or v-Ki-ras2 kirsten rat sarcoma viral oncogene homolog and fusion of anaplastic lymphoma kinase (ALK), RET proto-oncogene (RET), or v-ros UR2 sarcoma virus oncogene homolog 1 (ROS1), occur in non-small cell lung cancers, and these oncogenic drivers are important biomarkers for targeted therapies. A useful technique to screen for these fusions is the detection of native carboxy-terminal (C-terminal) protein by immunohistochemistry; however, the effects of other genetic alterations on C-terminal expression is not fully understood. In this study, we evaluated whether C-terminal expression is specifically elevated by fusion with or without typical genetic alterations of lung cancer. Methods In 37 human lung cancer cell lines and four tissue specimens, protein and mRNA levels were measured by capillary western blotting and reverse transcription–PCR, respectively. Results Compared with the median of all 37 cell lines, mRNA levels at the C-terminus of all five of the fusion-positive cell lines tested (three ALK, one RET, and one ROS1) were elevated at least 2000-, 300-, or 2000-fold, respectively, and high C-terminal protein expression was detected. In an ALK fusion–positive tissue specimen, the mRNA and protein levels of C-terminal ALK were also markedly elevated. Meanwhile, in one of 36 RET fusion–negative cell lines, RET mRNA levels at the C-terminus were elevated at least 500-fold compared with the median of all 37 cell lines, and high C-terminal protein expression was detected despite the absence of RET fusion. Conclusions This study of 37 cell lines and four tissue specimens shows the detection of C-terminal ALK or ROS1 proteins could be a comprehensive method to determine ALK or ROS1 fusion, whereas not only the detection of C-terminal RET protein but also other methods would be needed to determine RET fusion. Electronic supplementary material The online version of this article (10.1186/s12885-019-5527-2) contains supplementary material, which is available to authorized users.

Rearranged during transfection (RET) fusion–positive non–small cell lung cancer (NSCLC) accounts for 1% of lung adenocarcinoma. Although small molecule agents with RET kinase inhibitory activity such as alectinib, vandetanib, and cabozantinib have been clinically evaluated in RET-fusion–positive NSCLC, an effective monotherapy regimen has not been established. We explored agents to use in combination with alectinib to enhance the antitumor effect of alectinib against RET-fusion cells. Cell proliferation under co-treatment with alectinib plus each of six chemotherapeutic agents or six molecularly targeted agents was evaluated in vitro. The combination effect was analyzed by IC(50) isobologram and combination index using LC-2/ad and Ba/F3-KIF5B-RET cells. The in vivo combination effect was investigated in a Ba/F3-KIF5B-RET xenograft model. The phosphorylation levels of proteins regulating proliferation were measured by immunoblotting. Palbociclib, a CDK4/6 inhibitor, showed the greatest synergy against LC-2/ad cells in the isobologram analysis and combination index. This synergistic effect was also observed against Ba/F3-KIF5B-RET cells. Another CDK4/6 inhibitor, abemaciclib, also showed a synergistic effect. In vivo, the combination of alectinib plus palbociclib showed a more enhanced antitumor effect than each single agent in a mouse xenograft model with transplanted Ba/F3-KIF5B-RET cells. This combination suppressed the phosphorylation of S6 and Rb more intensely than did either single agent in both LC-2/ad and Ba/F3-KIF5B-RET cell lines, both in vitro and in vivo. Combination therapy with alectinib plus the CDK4/6 inhibitor enhanced the antitumor effect against RET-fusion–positive cells in vitro and in vivo.

The mechanisms responsible for the development of resistance to alectinib, a second-generation anaplastic lymphoma kinase (ALK) inhibitor, are still unclear, and few cell lines are currently available for investigating ALK-rearranged lung cancer. To identify the mechanisms underlying acquired resistance to alectinib, two patient-derived cell lines were established from an alectinib-naïve ALK-rearranged lung cancer and then after development of alectinib resistance. The properties acquired during treatments were detected by comparisons of the two cell lines, and then functional analyses were performed. Coactivation of c-Src and MET was identified after the development of alectinib resistance. Combinatorial therapy against Src and MET significantly restored alectinib sensitivity in vitro (17.2-fold). Increased apoptosis, reduction of tumor volume, and inhibition of MAPK and PI3K/AKT signaling molecules for proliferation and survival were observed when the three kinases (Src, MET, and ALK) were inhibited. A patient-derived xenograft from the alectinib-resistant cells indicated that combination therapy with a saracatinib and crizotinib significantly decreased tumor size in vivo. To confirm the generality, a conventional alectinib-resistant cell line model (H2228-AR1S) was established from NCI-H2228 cells (EML4-ALK variant 3a/b). In H2228-AR1S, combination inhibition of Src and MET also restored alectinib sensitivity. These data reveal that dual salvage signaling from MET and Src is a potential therapeutic target in alectinib-resistant patients. Implications: This study demonstrates the feasibility to elucidate personalized drug-resistance mechanisms from individual patient samples.

Erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), shows superior efficacy in patients with non-small cell lung cancer (NSCLC) harboring activating EGFR mutations (EGFR Mut+). However, almost all tumors eventually develop resistance to erlotinib. Recently, the Phase II JO25567 study reported significant prolongation of progression-free survival (PFS) by erlotinib plus bevacizumab combination compared with erlotinib in EGFR Mut+ NSCLC. Herein, we established a preclinical model which became refractory to erlotinib after long-term administration and elucidated the mode of action of this combination. In this model, tumor regrowth occurred after remarkable shrinkage by erlotinib; regrowth was successfully inhibited by erlotinib plus bevacizumab. Tumor vascular endothelial growth factor (VEGF) was greatly reduced by erlotinib in the erlotinib-sensitive phase but significantly increased in the erlotinib-refractory phase despite continued treatment with erlotinib. Although EGFR phosphorylation remained suppressed in the erlotinib-refractory phase, phosphorylated extracellular signal-regulated kinase (pERK), phosphorylated AKT, and phosphorylated signal transducer and activator of transcription 3 (pSTAT3) were markedly higher than in the erlotinib-sensitive phase; among these, pERK was suppressed by erlotinib plus bevacizumab. MVD was decreased significantly more with erlotinib plus bevacizumab than with each drug alone. In conclusion, the erlotinib plus bevacizumab combination demonstrated promising efficacy in the B901L xenograft model of EGFR Mut+ NSCLC. Re-induction of VEGF and subsequent direct or indirect VEGF-dependent tumor growth was suggested as a major mechanism of erlotinib resistance, and erlotinib plus bevacizumab achieved remarkably prolonged antitumor activity in this model.

Figure S8. RT-PCR analysis of EML4 in four tissue specimens. The relative EML4 mRNA expression at each exon in NAT specimens was calculated as the ratio of the normalized values with GAPDH mRNA to those in tumor tissues. Each bar represents the mean + SD (n = 3) (PPTX 45 kb)

Figure S5. RT-PCR analysis of ALK, RET, or ROS1 fusion in 4 tumor tissues. The mRNA expression of variant 1, 2, 3a, or 3b of EML4-ALK (a), KIF5B-RET, CCDC6-RET (b) or SLC34A2-ROS1 (c) was determined by RT-PCR using the Taqman probes shown in Additional file 1: Table S1. Red lines show fusion geneâ positive tumor tissue, and green lines show fusion geneâ negative tumor tissue (PPTX 122 kb)

Figure S1. RT-PCR analysis of ALK, RET, or ROS1 fusion in 37 cancer cell lines. The mRNA expression of variant 1, 2, 3a, or 3b of EML4-ALK (a), KIF5B-RET, CCDC6-RET (b) or SLC34A2-ROS1 (c) was determined by RT-PCR using the Taqman probes shown in Additional file 1: Table S1. Red lines show fusion geneâ positive cell lines and green lines show fusion geneâ negative cell lines (PPTX 129 kb)

Table S4. Exon count of mRNA (DOCX 26 kb)

Table S5. IC50s of ALK-TKIs and erlotinib (DOCX 28 kb)

Table S3. Target terminus of antibody (DOCX 28 kb)

Figure S4. Comparison of molecular weights between wild-type and fusion protein. After high exposure of N-terminal EML4 in the results shown in Fig. 2, two sizes of EML4 protein were detected in NCI-H2228 and SNU-2292 cell lines (left-hand figure), and we assumed that the larger protein was wild-type EML4 (arrow) and the smaller protein was ALK-fused EML4 (arrowhead), after referring to the protein weight of EML4-ALK detected by an ALK C-terminus antibody (right-hand figure) (a). In the same way, we assumed that the larger and smaller proteins were respectively wild-type EML4 (arrow) and ALK-fused EML4 (arrowhead) in the results for the ILS31007 tumor tissue specimen shown in Fig. 3b (b), and the larger and smaller proteins were respectively wild-type CCDC6 (arrow) and RET-fused CCDC6 (arrowhead) in the results for CCDC6 in the LC-2/ad cell line shown in Fig. 2 (c) (PPTX 600 kb)

Figure S2. Summary of ALK, RET, or ROS1 fusion in 37 cancer cell lines. Plot of normalized values calculated from the data in Additional file 5: Figure S1 (PPTX 50 kb)

Table S2. Taqman probes for exons of mRNA (DOCX 28 kb)

Erlotinib (ERL), an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, shows notable efficacy against non-small cell lung cancer (NSCLC) harboring EGFR mutations. Bevacizumab (BEV), a humanized monoclonal antibody to vascular endothelial cell growth factor (VEGF), in combination with ERL (BEV+ERL) significantly extended progression-free survival in patients with EGFR-mutated NSCLC compared with ERL alone. However, the efficacy of BEV+ERL against EGFR-mutated NSCLC harboring T790M mutation or MET amplification, is unclear. Here, we examined the antitumor activity of BEV+ERL in four xenograft models of EGFR-mutated NSCLC (three harboring ERL resistance mutations). In the HCC827 models (exon 19 deletion: DEL), ERL significantly inhibited tumor growth by blocking EGFR signal transduction. Although there was no difference between ERL and BEV+ERL in maximum tumor growth inhibition, BEV+ERL significantly suppressed tumor regrowth during a drug-cessation period. In the HCC827-EPR model (DEL+T790M) and HCC827-vTR model (DEL+MET amplification), ERL reduced EGFR signal transduction and showed less pronounced but still significant tumor growth inhibition than in the HCC827 model. In these models, tumor growth inhibition was significantly stronger with BEV+ERL than with each single agent. In the NCI-H1975 model (L858R+T790M), ERL did not inhibit growth or EGFR signal transduction, and BEV+ERL did not inhibit growth more than BEV. BEV alone significantly decreased microvessel density in each tumor. In conclusion, addition of BEV to ERL did not enhance antitumor activity in primarily ERL-resistant tumors with T790M mutation; however, BEV+ERL enhanced antitumor activity in T790M mutation- or MET amplification-positive tumors as long as their growth remained significantly suppressed by ERL.

Anaplastic lymphoma kinase (ALK) inhibitors, such as alectinib (ALC), have dramatic therapeutic effects on ALK-rearranged lung cancer, but cures are usually not achieved. We focused on tumor cells that survive ALK inhibitor administration and hypothesized that targeted therapy for these cells could provide complete remission. To explore survival factors, we established patient-derived cell lines and screened them using proteome analysis. Three ALK-rearranged ALC-sensitive cell lines (KTOR-1, KTOR-2, KTOR-3) were established from 3 patients; the 50% inhibitory concentrations (IC50)s for ALC were 24-65 nM. Comprehensive protein expression profiles of the 3 cells indicated that exposure to ALC significantly enriched proteins related to actin and extracellular matrix (ECM) adhesion. We focused on Yes-associated protein 1 (YAP1), which is activated by ECM adhesion and actin fiber accumulation. Nuclear localization of YAP1 (an activation marker of YAP1) was assessed using immunohistostaining. In KTOR1-3 and H2228 cells from an ALK-rearranged line purchased from ATCC, exposure to ALC in vitro promoted YAP1 accumulation in the nucleus. BALB/nu mice xenograft models of H2228 or KTOR1 were administered ALC (8 mg/kg/day, N=4) or a vehicle (N=4) for 7 days, and tumors were evaluated. In ALC-administered tumors, YAP1 was localized to the nucleus, which was rarely the case in vehicle-administered tumors. The expression of pro-apoptosis factors Mcl-1 and Bcl-xL also increased after exposure to ALC in vitro, but the increment was cancelled by YAP1 inhibition by siRNA or verteporfin (VER), a non-specific YAP1 inhibitor. Exposure to ALC with combinatorial YAP1 inhibition significantly increased Caspase 3/7 activity. To address the treatment effects of YAP1 inhibition, a YAP1-activated H2228 cell line (H2ARY) was established by exposing H2228 cells to 100-300 nM of ALC for 3 months and thorough subsequent cloning. The H2ARY had lower sensitivity to ALC in vitro than parental H2228 (IC50: 1.4 μM vs 315 nM, 96 h) and restored the sensitivity by YAP1 inhibition (208 nM with VER 1 μM, 312 nM with siYAP1). Twenty-four xenograft models (mean volume: 199 mm3) of H2ARY on BALB/nu mice were randomized (Day 0) into 4 treatment groups to receive ALC monotherapy (8 mg/kg daily, N=6), VER monotherapy (12.5 mg/kg twice a week, N=7), combination (N=7), or vehicle (N=5). On day 15, the tumor volume of the vehicle and VER monotherapy groups reached > 800 mm3, with no significant differences among the groups. On day 33, the tumors of the combination group were significantly smaller than those of the ALC monotherapy group (187 vs 761 mm3, P = 0.0125). Exposure to ALC-activated YAP1 may regulate anti-apoptotic activity by controlling the expression of Mcl-1 and Bcl-xL in ALK-rearranged lung cancer cells. This is the first evidence that combinatorial therapy against ALK and YAP1 could enhance ALK-rearranged tumor treatment. Citation Format: Takahiro Tsuji, Hiroaki Ozasa, Wataru Aoki, Shunsuke Aburaya, Tomoko Funazo, Koh Furugaki, Yasushi Yoshimura, Hitomi Ajimizu, Yuto Yasuda, Takashi Nomizo, Yuichi Sakamori, Hironori Yoshida, Mitsuyoshi Ueda, Young Hak Kim, Toyohiro Hirai. YAP1 mediates initial survival of alectinib therapy in ALK-rearranged lung cancer via pro-apoptotic protein regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 72.

Non-small cell lung cancer is known to have a poor prognosis. One reason for this is resistance to anticancer drugs. Various mechanisms for resistance to anticancer drugs have been reported. Herein we focus on ABCC11, an adenosine triphosphate (ATP)-binding cassette transporter. ABCC11 is ubiquitously expressed in various adult human tissues, including liver, lung, and kidney, and confers drug resistance to some cytotoxic agents such as 5-fluorouracil (5-FU), pemetrexed, and methotrexate. However, the association between ABCC11 and resistance to molecularly-targeted therapeutic drugs is still unknown. We hypothesized that alectinib, a molecularly-targeted therapeutic agent for anaplastic lymphoma kinase (ALK)-rearranged lung cancer, was a substrate for ABCC11. To evaluate the expression of ABCC11 in alectinib-resistant cells, an alectinib-resistant cell line model (AR1S) was established by exposing NCI-H2228, an ALK-rearranged cell line, to alectinib for 3 months. Patient-derived cell lines that were sensitive or resistant to alectinib were also established from a treatment-naïve patient (KTOR-1), and after disease progression (KTOR-1 RE). The protein expression of ABCC11 was increased in both alectinib-resistant cell lines (AR1S and KTOR-1 RE), compared to naïve cell lines (H2228 and KTOR-1). To investigate the role of ABCC11 in alectinib resistance, ABCC11 overexpression cell lines (OE-A and OE-B) were established by introducing an ABCC11 expression construct into H2228. A negative control cell line (mock) was established by introducing the control empty vector into H2228. The gene expression of ABCC11 in OE-A and OE-B was higher than that in mock (133-fold increase, P < 0.0001 and 109-fold increase, P < 0.0001 respectively), and the protein expression of ABCC11 was also higher in OE-A and OE-B. The IC50 for alectinib was higher in OE-A (8.0 times) and OE-B (10.8 times) compared to mock. ABCC11 was knocked down using siRNA in AR1S to evaluate alectinib susceptibility. Knockdown of ABCC11 improved the IC50 for alectinib, compared with a negative control (0.299-fold decrease). Next, the tumor responses to alectinib in OE-A and OE-B were evaluated in vivo. Xenograft models of OE-A, OE-B, and mock on BALB/nu mice were administered daily alectinib (8 mg/kg/day) or vehicle for 10 days. In mice administered alectinib (N = 6-7), the tumor shrinkage rate of OE-A (−23.6%) and OE-B (−34.3%) was significantly lower than that of mock (−76.8%). There results have provided the first of preclinical evidence that ABCC11 is involved in resistance to alectinib. Citation Format: Tomoko Yamamoto Funazo, Hiroaki Ozasa, Takahiro Tsuji, Koh Furugaki, Yasushi Yoshimura, Hitomi Ajimizu, Yuto Yasuda, Takashi Nomizo, Yuichi Sakamori, Hironori Yoshida, Young Hak Kim, Toyohiro Hirai. ABCC11 is involved in resistance to alectinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1271.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) show notable effects against non-small cell lung cancers (NSCLCs) harboring EGFR-activating mutations. However, almost all patients eventually acquire resistance to EGFR-TKIs. In this study, we established novel erlotinib resistant NSCLC cells and examined their resistant mechanisms. Resistant cells were established in 14, 3, and 0 wells exposed to 0.1, 1, and 10μM erlotinib, respectively. The IC50 values of these cells were 47- to 1209-fold higher than that of the parent cells. No secondary T790M mutation was detected in any resistant cells. However, in 13/17 resistant cells, EGFR copy number was reduced less than approximately one-eighth of parent cells, and in one resistant cell (B10), >99.99% of the population was EGFR-unamplified cells. Most (97.5%) parent cells showed EGFR amplification, but 2.5% of the population comprised EGFR-unamplified cells. An EGFR-unamplified clone (4D8) isolated from parent cells in erlotinib-free normal medium also showed erlotinib resistance comparable to the resistant B10 cells. Loss of an EGFR-amplified chromosome 7 (EGFR-ampch7) was observed in 4D8 and B10 cells. EGFR-unamplified cells were constantly maintained as a minor population of the parent cells under normal cell culture conditions. In conclusion, loss of an EGFR-ampch7 causes acquired resistance in EGFR-mutated HCC827 cells exposed to a relatively low concentration of erlotinib, but a high concentration prevents the emergence of resistance.

Purpose: We investigated the antitumor activity of the combination of two different humanized monoclonal human epidermal growth factor receptor (HER) 2 antibodies, pertuzumab and trastuzumab, for gastric cancer. Experimental Design: Tumor mouse xenograft models were used to examine antitumor activity. Cell proliferation was examined using crystal violet staining. HER family proteins' expression was analyzed by ELISA and immunohistochemistry. Phosphorylated proteins and heterodimers were detected by Western blotting and in situ proximity ligation assay (PLA), respectively. Apoptosis activity was examined by caspase 3/7 activity. Antibody-dependent cellular cytotoxicity (ADCC) activity was detected by xCELLigence. Microvessel density was examined by CD31 staining. Results: Pertuzumab in combination with trastuzumab showed significant antitumor activity compared with each monotherapy in NCI-N87, an HER2-positive human gastric cancer xenograft model. The efficacy was stronger than that of the maximum effective dose with each monotherapy. Similar antitumor activity was shown in 4-1ST, another HER2-positive gastric cancer model, but not in MKN-28, an HER2-negative model. Combining pertuzumab with trastuzumab enhanced cell growth inhibition and apoptosis activity by inhibiting EGFR-HER2 heterodimerization and the phosphorylation of these receptors and their downstream factors. This effect was also seen in HER2-HER3 signaling. Furthermore, pertuzumab in combination with trastuzumab potentiated the ADCC activity of those antibodies and reduced tumor microvessel density. Conclusions: We showed the significantly enhanced efficacy of pertuzumab combining with trastuzumab for HER2 overexpressing gastric cancer through the potentiation of cell growth inhibition, apoptosis activity, cell killing activity by ADCC, and antiangiogenic activity. This study suggests the clinical benefit of combination therapy with pertuzumab and trastuzumab for patients with HER2-positive gastric cancers. Clin Cancer Res; 17(15); 5060–70. ©2011 AACR.

It has been reported that bevacizumab in combination with paclitaxel significantly prolongs progression-free survival compared with paclitaxel alone in the initial treatment for metastatic breast cancer. To understand how bevacizumab enhances the efficacy of paclitaxel, we investigated the mechanism in a MX-1 human breast cancer xenograft model. The antitumor activity of bevacizumab at 5 mg/kg in combination with paclitaxel at 20 or 30 mg/kg was significantly higher than that of either agent alone. First, we measured the paclitaxel concentration in tumor to see whether bevacizumab enhances the activity by increasing the tumor concentration of paclitaxel. When given in combination with bevacizumab, the levels of paclitaxel in the tumor increased. Paclitaxel at 30 mg/kg with bevacizumab showed a similar tumor concentration as paclitaxel alone at either 60 or 100 mg/kg, with a similar degree of tumor growth inhibition. In contrast, no remarkable differences in paclitaxel concentration in the plasma or liver were observed between the paclitaxel monotherapy group and the paclitaxel plus bevacizumab group. An increase in paclitaxel concentration by bevacizumab was also found in another model, A549. In the same MX-1 model, vascular permeability in the tumor was significantly decreased by treatment with bevacizumab. There was no difference in microvessel density between the bevacizumab alone group and the combination group. Results suggest that the synergistic antitumor activity of paclitaxel and bevacizumab in combination may be a result of the increase in paclitaxel concentration in tumor resulting from the downregulation of vascular permeability when co-administered with bevacizumab.

Conventional drug-resistant cancer cell line models have contributed to the elucidation of drug-resistant mechanisms. However, whether or not these models reflect patients in clinical settings is often controversial. We herein report a patient-derived drug resistant model system named the “clinical paired resistant model”. This model consists of 2 cell lines derived from a treatment-naïve patient (drug sensitive cell line model) and again after disease progression (drug resistant cell line model). The clinical paired resistant model of alectinib, the second generation ALK inhibitor, revealed the properties that cancer cells acquired during treatment using phosphoproteome and immunoblotting analyses; the proto-oncogene protein tyrosine kinase Src and hepatocyte growth factor receptor MET were activated after the development of alectinib resistance. No secondary mutations were detected in the coding region of ALK tyrosine kinase in the alectinib resistant model. In this alectinib-resistant model, the inhibition of Src and MET using saracatinib and PHA-665752 significantly restored alectinib sensitivity in vitro (17.2-fold change in IC50). Downstream signaling molecules for proliferation and survival, phosphorylation of Akt and ERK1/2, were inhibited and caspase 3/7 activity was significantly increased when the cells were treated with all three inhibitors (saracatinib, PHA-665752, and alectinib). Combined knockdown of SRC and MET restored alectinib sensitivity and inhibited downstream signaling in combination with ALK inhibition using alectinib, suggesting that the dual salvage signaling of MET and Src conferred alectinib resistance. A xenograft generated from our paired resistant model (N=5-6, in each group) indicated that combination therapy with a saracatinib and crizotinib, the first generation ALK inhibitor which also inhibits MET, significantly decreased tumor size in vivo as compared with saracatinib or crizotinib monotherapy. We also established a conventional alectinib resistant cell line model in vitro by exposing NCI-H2228 cells (EML4-ALK variant 3a/b) to 300 nM of alectinib for 3 months and found MET and Src were also activated in the model. Our clinical paired resistant model permits the detection of drug-resistant mechanisms without exploring the common characteristics of numerous drug-resistant patients. Our results demonstrate that MET and Src are potential therapeutic targets in patients with alectinib resistance and that the clinical paired resistant model may be a new strategy to elucidate drug-resistant mechanisms in relatively rare cancers. Citation Format: Takahiro Tsuji, Hiroaki Ozasa, Takashi Nomizo, Tomoko Funazo, Yuto Yasuda, Yuichi Sakamori, Hironori Yoshida, Kiyoshi Uemasu, Hitomi Ajimizu, Ryoko Okutani, Shunsuke Aburaya, Wataru Aoki, Mitsuyoshi Ueda, Koh Furugaki, Yasushi Yoshimura, Toyohiro Hirai, Young Hak Kim. A clinical paired resistant model elucidated novel dual salvage signaling that confers alectinib resistance in ALK-rearranged lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1830.

Erlotinib treatment in combination with gemcitabine is a standard therapy for patients with locally advanced pancreatic cancer in many countries, including the US and the EU. Since mutations of the K-ras oncogene (KRAS) occur in approximately 90% of pancreatic cancers, we examined the antitumor activity of erlotinib in combination with gemcitabine in KRAS-mutated pancreatic cancer cell lines, HPAC and Capan-1, which have the KRAS mutation G12D and G12V, respectively. We analyzed the mode of inhibition of in vitro tumor cell proliferation by means of a combination index and found that a combination treatment of erlotinib plus gemcitabine had an additive effect in the two cell lines. We then examined the effect of erlotinib and gemcitabine on the phosphorylation of epidermal growth factor receptor (EGFR). Erlotinib strongly suppressed, while gemcitabine augmented the phosphorylation of EGFR, which was completely blocked by erlotinib in the two cell lines. An in vivo tumor growth inhibition test was then performed using the HPAC tumor xenograft model. The combination therapy of erlotinib and gemcitabine resulted in a significant inhibition of tumor growth compared with erlotinib or gemcitabine monotherapy. To the best of our knowledge, this is the first study to show the combination effect of erlotinib and gemcitabine in vivo using a xenograft model of a KRAS-mutated pancreatic cancer cell line.

256 Background: Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate composed of trastuzumab and DM1 (an anti-microtubule agent derived from maytansine) that is approved for the treatment of HER2-positive metastatic breast cancer. HER2-overexpression rate in biliary tract cancer (BTC) is reported as approximately 10%, and HER2-targeted therapy may enhance therapeutic efficacy in HER2-positive BTC. In this study, we investigated the antitumor activity of T-DM1 in several BTC cell lines and xenograft models. Methods: HER2 expression was detected by flow cytometry and Western blotting in 17 BTC cell lines. The cell growth inhibition activity of T-DM1 and DM1 was examined by quantifying the DNA with Hoechst 33258 nucleic acid stain. In vivo antitumor activity of T-DM1 was evaluated in three xenograft mouse models using cell lines with different levels of HER2 expression. HER2 expression in xenografts was assessed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), according to gastric cancer criteria. Cell cycle was assessed by flow cytometry after staining with propidium iodide. Apoptosis was assessed by caspase 3/7 activity measurement. Results: In the 17 BTC cell lines, HER2 expression was high in KMCH-1, Mz-ChA-1, and TGBC-18-TKB. Although cell growth inhibition of DM1 was similar in all cell lines (50% inhibitory concentration (IC50): 0.79–7.2 nM), that of T-DM1 was HER2-expression-dependent, with IC50 values in KMCH-1, Mz-ChA-1, and KKU-100 (which has low HER2 expression) of 0.031, 1.3, and 4.3 μg/mL, respectively. In KMCH-1 and Mz-ChA-1 xenograft models, T-DM1 treatment once every 3 weeks for 6 weeks showed significant dose-dependent antitumor activity, and tumor growth inhibition 21 days after first dose at 20 mg/kg T-DM1 was 108% (KMCH-1) and 75% (Mz-ChA-1), whereas there was no significant efficacy in KKU-100 xenograft model. Values for HER2 status (IHC score/FISH ratio of HER2 to CEP17) in KMCH-1, Mz-ChA-1, and KKU-100 tumor tissues were 3+/3.5, 2+/4.7, and 0/1.1, respectively. In KMCH-1, T-DM1 induced M phase arrest and apoptosis, as is reported in HER2-positive breast cancer cells. Conclusions: T-DM1 could be an effective therapy for HER2-overexpressed BTC.

Glypican 3 (GPC3), a GPI-anchored heparan sulfate proteoglycan, is expressed in the majority of hepatocellular carcinoma (HCC) tissues. Using MRL/lpr mice, we successfully generated a series of anti-GPC3 monoclonal antibodies (mAbs). GPC3 was partially cleaved between Arg358 and Ser359, generating a C-terminal 30-kDa fragment and an N-terminal 40-kDa fragment. All mAbs that induced antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) against cells expressing GPC3 recognized the 30-kDa fragment, indicating that the C-terminal region of GPC3 serves as an epitope for mAb with ADCC and/or CDC inducing activities. Chimeric mAbs with Fc replaced by human IgG1 were created from GC33, one of the mAbs that reacted with the C-terminal 30-kDa fragment. Chimeric GC33 induced not only ADCC against GPC3-positive human HCC cells but also was efficacious against the Huh-7 human HCC xenograft. Thus, mAbs against the C-terminal 30-kDa fragment such as GC33 are useful in therapy targeting HCC.

Erlotinib (ERL), an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, shows notable efficacy against non-small cell lung cancer (NSCLC) harboring EGFR mutations. Bevacizumab (BEV), a humanized monoclonal antibody to vascular endothelial cell growth factor (VEGF), in combination with ERL (BEV+ERL) significantly extended progression-free survival in patients with EGFR-mutated NSCLC compared with ERL alone. However, the efficacy of BEV+ERL against EGFR-mutated NSCLC harboring T790M mutation or MET amplification, is unclear. Here, we examined the antitumor activity of BEV+ERL in four xenograft models of EGFR-mutated NSCLC (three harboring ERL resistance mutations). In the HCC827 models (exon 19 deletion: DEL), ERL significantly inhibited tumor growth by blocking EGFR signal transduction. Although there was no difference between ERL and BEV+ERL in maximum tumor growth inhibition, BEV+ERL significantly suppressed tumor regrowth during a drug-cessation period. In the HCC827-EPR model (DEL+T790M) and HCC827-vTR model (DEL+MET amplification), ERL reduced EGFR signal transduction and showed less pronounced but still significant tumor growth inhibition than in the HCC827 model. In these models, tumor growth inhibition was significantly stronger with BEV+ERL than with each single agent. In the NCI-H1975 model (L858R+T790M), ERL did not inhibit growth or EGFR signal transduction, and BEV+ERL did not inhibit growth more than BEV. BEV alone significantly decreased microvessel density in each tumor. In conclusion, addition of BEV to ERL did not enhance antitumor activity in primarily ERL-resistant tumors with T790M mutation; however, BEV+ERL enhanced antitumor activity in T790M mutation- or MET amplification-positive tumors as long as their growth remained significantly suppressed by ERL.

Epidermal growth factor receptor (EGFR) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations are common in non-small cell lung cancer (NSCLC) and colorectal cancer (CRC). The aim of the present study was to develop a simple and versatile tool to determine EGFR and KRAS mutations for pre-clinical research in the laboratory. We developed a melting curve analysis to detect exon 19 deletion, L858R mutation, and T790M mutation of EGFR, and codon 12/13 and codon 61 mutations of KRAS using LightCycler480 with mutation-specific sensor and anchor probes. The analytical method was applicable to determine the approximate rate of heterogeneity of mutation in the genomic DNA of cancer cell lines. In conclusion, our melting curve analysis is a rapid and semi-quantitative method to screen for exon 19 deletion, L858R or T790M mutations of EGFR and codon 12/13/61 mutations of KRAS in cancer cell lines.

Erlotinib is used as a standard treatment for recurrent advanced non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) mutations in NSCLC have been shown to be a predictive factor of erlotinib, although the relationship between K-ras oncogene (KRAS) mutations and erlotinib resistance is controversial. Recently, in vitro sequence-dependent interactions of erlotinib and docetaxel have been studied on as a novel therapeutic approach against NSCLC. The purpose of the present study was to determine the optimum novel regimen of erlotinib and docetaxel against NSCLC cells which have EGFR mutation (HCC827 cells), KRAS mutation (A549 cells) or both wild-type (NCI-H292 cells). First, we analyzed the effects of in vitro combination for cell proliferation-inhibition using a combination index. In all cell lines, docetaxel followed by erlotinib treatment showed nearly additive effects. On the other hand, erlotinib followed by docetaxel treatment showed remarkable antagonistic interactions. Second, we examined the effect of combinations on the in vitro apoptosis induction. Erlotinib followed by docetaxel treatment reduced apoptosis induction compared with docetaxel alone; in contrast, docetaxel followed by erlotinib treatment had no inhibitory effects on docetaxel-induced apoptosis in any of the cell lines. Finally, an in vivo tumor growth inhibition test was performed using xenograft models. Docetaxel followed by erlotinib administration resulted in significant tumor growth inhibition compared with erlotinib or docetaxel monotherapy in all models. In conclusion, we demonstrated that docetaxel followed by erlotinib therapy was a potentially optimum regimen against NSCLC regardless of the mutation status of EGFR and KRAS.

We have been investigating the functional display of multipass membrane protein such as transporter or G-protein coupled receptor on the budded baculovirus (BV). We tested the use of a viral envelope protein gp64 transgenic mouse for the direct immunization of these membrane proteins displayed on BVs. The gp64 transgenic mice showed only a weak response to virus compared to wild type BALB/c mice. Immunizing gp64 transgenic mice with the BV expressing peptide transporter PepT1, we obtained 47 monoclonal antibodies (mAbs). These mAbs were specific to the PepT1 on the pancreatic cancer cells AsPC-1 by fluorocytometric analysis and exhibited antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity to AsPC-1. We also generated 7 mAbs by immunizing gp64 transgenic mice on a CCR2-deficient background with the BV expressing chemokine receptor CCR2 together with partially purified CCR2. These mAbs possessed specific binding to CCR2 in CHO cells on fluorocytometric analysis, and exhibited neutralizing activities for ligand-dependent inhibition of cyclic AMP production. This method provides a powerful tool for the generation of therapeutic/diagnostic mAbs against membrane proteins.

A major goal of current human genome-wide studies is to identify the genetic basis of complex disorders. However, the availability of an unbiased, reliable, cost efficient and comprehensive methodology to analyze the entire genome for complex disease association is still largely lacking or problematic. Therefore, we have developed a practical and efficient strategy for whole genome association studies of complex diseases by charting the human genome at 100 kb intervals using a collection of 27 039 microsatellites and the DNA pooling method in three successive genomic screens of independent case-control populations. The final step in our methodology consists of fine mapping of the candidate susceptible DNA regions by single nucleotide polymorphisms (SNPs) analysis. This approach was validated upon application to rheumatoid arthritis, a destructive joint disease affecting up to 1% of the population. A total of 47 candidate regions were identified. The top seven loci, withstanding the most stringent statistical tests, were dissected down to individual genes and/or SNPs on four chromosomes, including the previously known 6p21.3-encoded Major Histocompatibility Complex gene, HLA-DRB1. Hence, microsatellite-based genome-wide association analysis complemented by end stage SNP typing provides a new tool for genetic dissection of multifactorial pathologies including common diseases.

Background The epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) show notable effects for non-small cell lung cancers (NSCLC) harboring EGFR activating mutations. However, almost all patients eventually acquire resistance to EGFR-TKIs within several years. The major mechanisms of acquired resistance including 2nd mutation of T790M and amplification of MET have been identified. However, the mechanisms of other resistance remain unclear. In this study, we established novel erlotinib-resistant NSCLC cells and examined their resistant mechanisms. Methods NSCLC HCC827 cells have an EGFR Exon 19 deletion and gene amplification and are highly sensitive to erlotinib. The resistant cells were established by continuously exposing HCC827 cells to 0.1, 1 or 10 µM of erlotinib in 96 well plates for three months. The resistant mechanisms were determined by direct sequence analysis and EGFR FISH analysis. Results The fourteen and three resistant cells were established by 0.1 µM and 1 µM of erlotinib exposure, respectively. No resistant cells appeared in the wells of 10 µM of erlotinib. The IC50 values of these resistant cells were more than 25-fold higher than that of parental cells. No 2nd mutation of T790M was detected in any of the resistant cells and MET gene amplification was detected in only one resistant cells. Instead, we found that 13/17 resistant cells were dominated by EGFR not amplified cells and one of resistant cells B10 consisted of more than 99.9% of EGFR not amplified cells, and that the parental cells consisted of only 2.5% of EGFR not amplified cells and 97.5% of EGFR amplified cells. EGFR not amplified clone 4D8 isolated from parental cells and showed resistance to erlotinib comparable to the resistant cells B10. Furthermore, we found that EGFR not amplified cells were constantly emerged from EGFR amplified clone isolated from parental cells under normal cell culture condition. Conclusion Loss of amplified EGFR gene with mutation causes acquired resistance in HCC827 cells when exposed to relatively low concentration of erlotinib while high concentration of erlotinib deprives HCC827 cells of the chance of emergence of resistant cells. Disclosure All authors have declared no conflicts of interest.

Aim: Erlotinib (ERL), a specific inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, benefits patients with non-small cell lung cancer (NSCLC), especially those with EGFR active mutations. However, progressive disease occurs when resistance is acquired by T790M mutation or MET amplification. Bevacizumab (BEV), a humanized anti-vascular endothelial cell growth factor monoclonal antibody, has been demonstrated to be effective in combination with standard chemotherapies for advanced NSCLC patients. We previously reported the promising efficacy of combining ERL and BEV in mouse models of NSCLC harboring EGFR exon 19 deletion. In the present study, we examined the antitumor activity of BEV combined with ERL in NSCLC models harboring T790M resistance mutation. Methods: BALB-nu/nu mice were subcutaneously inoculated with NSCLC cell lines, NCI-H1975 (L858R + T790M mutation) or HCC827-EPR (exon19 deletion + T790M mutation). After randomization on Day 1, BEV (5 mg/kg) was intraperitoneally administered once a week, and ERL (75 mg/kg) was orally given daily. Antitumor activity was evaluated by tumor growth inhibition (TGI) with measuring tumor volume. In tumor tissues, phosphorylations of EGFR signaling molecules were evaluated by western blot analysis, and microvessel density was evaluated by CD31 immunohistochemistry. Results: In the NCI-H1975 model, the TGI on Day 11 of ERL, BEV and combination was 8%, 45% and 35%, respectively. NCI-H1975 tumors were resistant to ERL, and the combination of ERL and BEV did not enhance the antitumor activity of BEV monotherapy. In the HCC827-EPR model, the TGI on Day 50 of ERL, BEV and combination was 96%, 63% and 111%, respectively. HCC827-EPR tumors exhibited significant sensitivity to ERL (p ≤ 0.05) in vivo, and combining the two agents showed significantly higher antitumor activity than each monotherapy (p ≤ 0.05). The phosphorylation levels of EGFR, AKT or ERK in HCC827-EPR tumors were suppressed by ERL. The microvessel density was significantly decreased by BEV in both models. Conclusions: The combination treatment of ERL and BEV has the potential to be effective for ERL-responding NSCLC models with T790M resistance mutation. Disclosure: T. Mitsudomi: Dr.Mitsudomi has received research funds from Astra Zeneca, Chugai Pharma, Boehringer Ingelheim, Roche, Pfizer. All other authors have declared no conflicts of interest.

e18091 Background: The epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) show notable effects for non-small cell lung cancers (NSCLC) harboring EGFR activating mutations. However, almost all patients eventually acquire resistance to EGFR-TKIs within several years. The major mechanisms of acquired resistance are acquisition of a secondary EGFR mutation T790M and amplification of MET. However, the mechanisms of other resistance remain unclear. In this study, we established novel erlotinib-resistant NSCLC cells and examined their resistant mechanism. Methods: Resistant cells were established by continuously exposing EGFR mutated NSCLC cells HCC827 to 0.1, 1 or 10 μM of erlotinib in 96 well plates for three months. The resistant mechanisms were determined by direct sequence analysis and EGFR FISH analysis. Results: Resistant cells were emerged from 14/96 and 3/96 wells by 0.1 μM and 1 μM of erlotinib exposure, respectively. No resistant cells appeared in the wells of 10 μM of erlotinib. The IC50 values of these resistant cells were more than 100-fold higher than that of parental cells. No secondary mutation of T790M was detected in any of the resistant cells. Instead, we found that 13/17 resistant cells were dominated by EGFR not amplified cells and one of resistant cells B10 consisted of more than 99.9% of them, while the parental cells consisted of 2.5% of EGFR not amplified cells. Then, we isolated EGFR not amplified clone 4D8 from parental cells and found that this clone also had a resistance to erlotinib comparable to the resistant cells B10. Metaphase FISH analysis showed that EGFR amplified cells in parental cells had a chromosome 7 containing multiple copies of EGFR, while EGFR not amplified cells in B10 did not have it. Furthermore, we found that EGFR not amplified cells were constantly emerged from EGFR amplified clone isolated from parental cells under normal cell culture condition. Conclusions: Loss of a chromosome 7 containing multiple copies of EGFR causes acquired resistance in HCC827 cells when exposed to relatively low concentration of erlotinib, while high concentration of erlotinib deprives HCC827 cells of the chance of emergence of resistant cells.

Previous preclinical and clinical findings have suggested a potential role of epidermal growth factor receptor (EGFR) in osteoclast differentiation and the pathogenesis of bone metastasis in cancer. In this study, we investigated the effect of erlotinib, an orally active EGFR tyrosine kinase inhibitor (TKI), on the bone invasion of human non-small-cell lung cancer (NSCLC) cell line NCI-H292. First, we established a novel osteolytic bone invasion model of NCI-H292 cells which was made by inoculating cancer cells into the tibia of scid mice. In this model, NCI-H292 cells markedly activated osteoclasts in tibia, which resulted in osteolytic bone destruction. Erlotinib treatment suppressed osteoclast activation to the basal level through suppressing receptor activator of NF-κB ligand (RANKL) expression in osteoblast/stromal cell at the bone metastatic sites, which leads to inhibition of osteolytic bone destruction caused by NCI-H292 cells. Erlotinib inhibited the proliferation of NCI-H292 cells in in vitro. Erlotinib suppressed the production of osteolytic factors, such as parathyroid hormone-related protein (PTHrP), IL-8, IL-11 and vascular endothelial growth factor (VEGF) in NCI-H292 cells. Furthermore, erlotinib also inhibited osteoblast/stromal cell proliferation in vitro and the development of osteoclasts induced by RANKL in vitro. In conclusion, erlotinib inhibits tumor-induced osteolytic invasion in bone metastasis by suppressing osteoclast activation through inhibiting tumor growth at the bone metastatic sites, osteolytic factor production in tumor cells, osteoblast/stromal cell proliferation and osteoclast differentiation from mouse bone marrow cells.