EML4-ALK Positive Non-small Cell Lung Cancer Research Articles

Abstract 3884: Targeting the ARID1A mutations overcomes primary resistance to ALK inhibitors in EML4-ALK positive NSCLC

Abstract Introduction: Anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitors (TKIs) have improved an initial clinical response of non-small-cell lung cancer (NSCLC) patients with ALK-rearrangements. However, a subset of patients shows a poor response to ALK-TKIs. Here, we aimed to identify novel mechanism of primary resistance in preclinical model from a patient who presented an impressive resistance to sequential treatment with ALK TKIs. Experimental design: The PDC YU-1076 cells were established from pleural effusion of the patient at the time he was receiving chemotherapy and radiotherapy after failure of ALK-TKI treatment. Sanger sequencing confirmed ML4-ALK variant 1 detected at the initial diagnosis. No resistance mutations in the ALK kinase domain were detected. To investigate co-occurring genetic alterations, we performed whole exome sequencing (WES) on YU-1076 cells and the matched blood sample. We further investigated the molecular profile of YU-1076 cells using RNA-sequencing analysis. Results: YU-1076 cells exhibited cross-resistance to clinically available ALK-TKIs including crizotinib, ceritinib, alectinib, and lorlatinib. However, immunoblot analysis of YU-1076 cells treated with incremental doses of TKIs unexpectedly revealed an effective reduction of ALK activity and downstream signals. We noticed that YU-1076 cells gradually changed from a small, round shape to a fibroblast-like shape following the treatment of ALK-TKIs. Transcriptome analysis confirmed enrichment for gene signatures related epithelial-to-mesenchymal transition (EMT) in ALK TKI-treated YU-1076 cells, suggesting that ALK-TKIs treatment promotes YU-1076 cells toward a more mesenchymal phenotype. WES analysis identified a major chromatin remodeling complex subunit, AT-rich interacting domain 1A (ARID1A), as well as MYC amplification, CDKN2A loss, and TP53 mutations. We focused on a synthetic lethal strategies using the SFK inhibitor dasatinib and the EZH2 inhibitor GSK126 in ARID1A mutant cancers. The combination of dasatinib with ALK-TKIs restored the sensitivity to the ALK TKIs in YU-1076 cells, accompanied by suppression of EMT marker genes VIM and CDH2 and increase of apoptotic markers BIM, PARP, and CAS3. Congruently, dasatinib significantly impaired tumor growth in YU-1076-xenografts. GSK126 also induced synergistic inhibition of cell growth with upregulation of apoptosis marker genes in YU-1076 cells, but did not affect the expression of EMT marker genes. Conclusion: Our data indicate that ARID1A could be potentially used as a predictive biomarker for unfavorable ALK-TKI response. In this context, a combination strategy of ALK TKI with dasatinib may be effective in overcoming primary resistance. Citation Format: Seung Yeon Oh, You Won Lee, Eun Ji Lee, Ju Young Kim, Sewon Park, Ju Yeon Park, Su-Jin Choi, Mi Ra Yu, Sowon Aum, Jiyun Lee, Chang Gon Kim, Jii Bum Lee, Sun Min Lim, Min Hee Hong, Mi Ran Yun, Byoung Chul Cho. Targeting the ARID1A mutations overcomes primary resistance to ALK inhibitors in EML4-ALK positive NSCLC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3884.

Role of chemokine-mediated angiogenesis in resistance towards crizotinib and its reversal by anlotinib in EML4-ALK positive NSCLC

BackgroundThe identification of early plasma biomarkers for clinical outcomes and drug resistance has key importance for risk stratification in anaplastic lymphoma kinase (ALK)-positive advanced non-small cell lung cancer (NSCLC) patients. Moreover, it remains unclear whether the anti-angiogenic drug anlotinib can reverse the resistance of ALK-tyrosine kinase inhibitor (ALK-TKI) crizotinib, and no research has explored the effect of anlotinib combined with crizotinib on ALK-positive patients.MethodsIn this study, 76 baseline and longitudinal plasma samples from 61 ALK-positive NSCLC patients receiving crizotinib treatment were analyzed by Luminex liquid suspension chip for 40 chemokines. RNA sequence (RNA-seq) was used to identify differentially expressed genes (DEGs) between H3122 and H3122-crizotinib resistant (H3122CR) cells. Tube formation assay was performed to investigate the effect of chemokines on angiogenesis. And H3122CR-derived xenograft model was constructed to validate the efficacy and safety of anlotinib combined with crizotinib in vivo.ResultsBaseline and progression plasma samples detection suggested that CCL20 played a crucial role in monitoring and predicting the clinical response of crizotinib (hazard ratio for progression-free survival: 2.27 (1.13–4.58); for overall survival: 2.7 (1.23–5.8)). RNA-seq results for H3122 and H3122CR cells showed that high expression of chemokines and angiogenesis pathways were involved in crizotinib resistance. Subsequently, in vitro experiments indicated that CCL20 may induce crizotinib resistance by activation of angiogenesis via JAK2/STAT3-CCL20-VEGFA/IL6 axis. We further found that anti-angiogenic TKI anlotinib could reverse crizotinib resistance by inhibiting chemokines-induced angiogenesis, and anlotinib combined with crizotinib has a better antitumor effect than monotherapy in vitro & in vivo.ConclusionsOverall, CCL20-mediated angiogenesis is involved in crizotinib resistance and could be overcome by using anlotinib in EML4-ALK positive NSCLC. The combination of anlotinib and crizotinib is a promising strategy for patients resistant to ALK-TKIs.

MiR-100-5p confers resistance to ALK tyrosine kinase inhibitors Crizotinib and Lorlatinib in EML4-ALK positive NSCLC

Lung cancer causes the highest number of cancer-related deaths worldwide. Resistance to therapy is a major clinical issue contributing to the poor prognosis of lung cancer. In recent years, targeted therapy has become a concept where subgroups of non-small cell lung cancer (NSCLC) with genetically altered receptor tyrosine kinases are targeted by tyrosine kinase inhibitors (TKIs). One such subgroup harbors a gene fusion of echinoderm microtubule-associated protein-like 4 (EML4) with anaplastic lymphoma kinase (ALK). Although most NSCLC patients with EML4-ALK fusions initially respond to ALK TKI-therapy they eventually develop resistance. While ALK kinase domain mutations contribute to ALK TKI-refractoriness, they are only present in a fraction of all ALK TKI-resistant tumors. In this study we sought to explore a possible involvement of microRNAs (miRNAs) in conferring resistance to ALK TKIs in ALK TKI-refractory NSCLC cell lines. We subjected our ALK TKI-refractory cancer cells along with parental cancer cells to systematic miRNA expression arrays. Furthermore, ALK TKI-refractory cancer cells were exposed to a synthetic miRNA inhibitory Locked Nucleic Acid (LNA)-library in the presence of ALK TKIs Crizotinib or Lorlatinib. The outcome of the combined approaches uncovered miR-100-5p to confer resistance to Crizotinib and Lorlatinib in EML4-ALK NSCLC cells and to be a potential therapeutic target in drug resistance.

Abstract 2054: Cerivastatin overcomes the acquired resistance to crizotinib in EML4-ALK positive non-small cell lung cancer by controlling YAP activation

Abstract Crizotinib is highly effective in patients with non-small cell lung cancer (NSCLC) harboring the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion. However, its efficacy has been limited by the development of acquired resistance, and the mechanisms of such resistance remain largely unknown. Herein, we investigated a possible candidate for circumventing the acquired resistance to crizotinib. We established a model of acquired resistance to crizotinib (H3122-CR) by exposing EML4-ALK-positive H3122 lung cancer cells to increasing doses of crizotinib, and performed MTT screening using a library of FDA (Food and Drug Administration)-approved drugs composed of a collection of 640 clinically used compounds. Our MTT screening identified that cerivastatin, a drug targeting 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, significantly inhibited the cell growth in H3122-CR cells, which was completely restored by addition of geranylgeranyl pyrophosphate (GGPP), a key metabolite of mevalonate pathway. We also found that yes-associated protein (YAP), a major effector of the Hippo tumor suppressor pathway, was robustly accumulated in nucleus with a concomitant decrement of YAP phosphorylation in H3122-CR cells compared to parental cells. Importantly, inhibition of geranylgeranylation (GGylation) by GGTI-298 or cerivastatin markedly increased YAP phosphorylation, led to cytoplasmic translocation of YAP, and subsequently induced YAP inactivation. Moreover, the enrichment of EGFR signaling pathway and cell cycle signature including transcriptional targets of YAP was enhanced in H3122-CR cells, whose induction contributed YAP-mediated resistance to crizotinib. Inhibition of YAP function with siRNA or verteporfin as a YAP inhibitor greatly abrogated cell growth of H3122-CR cells by modulating cell cycle regulators and EGFR activation. Finally, we confirmed up-regulation of nuclear YAP expression in crizotinib-acquired resistant patient derived tumor xenograft (PDTX) models established from EML4-ALK positive NSCLC patients. Collectively, our findings define the GGylation-mediated YAP transcriptional activation as a new mechanism of resistance to crizotinib, providing a rationale for further exploring statins as a possible anticancer agent to overcome the acquired resistance to crizotinib in EML4-ALK positive NSCLC cells. Citation Format: Miran Yun, Hun Mi Choi, Kyoung Ho Pyo, Byoung Chul Cho. Cerivastatin overcomes the acquired resistance to crizotinib in EML4-ALK positive non-small cell lung cancer by controlling YAP activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2054. doi:10.1158/1538-7445.AM2017-2054

Abstract 3716: Overexpression of the adaptor protein CRKL as a mechanism of acquired resistance to Crizotinib in ALK-positive NSCLC

Abstract Background: The fusion of the anaplastic lymphoma kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) is a known oncogenic driver in a subset of non-small cell lung cancer (NSCLC). An ALK/ROS1/MET kinase inhibitor, Crizotinib, was approved in 2011 for the treatment of patients with metastatic ALK+ NSCLC. Despite the dramatic clinical effects of Crizotinib in EML4-ALK positive NSCLC, vast majority of patients subsequently acquire resistance and new therapeutic strategies are needed. Methods and Results: We performed a kinase (n=597) open reading frame screen to identify kinases and phosphoproteins capable of circumventing ALK inhibition in H3122 cell line sensitive to the ALK kinase inhibitor TAE684. The top hit of the screen imparting resistance to TAE684 was the adaptor phospho-protein CRK-like (CRKL), a known substrate of BCR-ABL. To validate our screen, we constructed a tagged form of CRKL and stably expressed it in H3122. H3122CRKL acquired a migratory phenotype, however, its growth rate was comparable to that of the parental cell line. The viability of H3122CRKL was assessed in response to drug dose escalation, confirming that overexpression of CRKL decreased sensitivity of H3122 to Crizotinib 47-fold compared to the control. Analysis of downstream signaling in response to Crizotinib treatment further revealed sustained activation of PI3K/AKT and MAPK/ERK1/2 pathways in CRKL overexpressing cells. Closer examination of signaling in H3122CRKL revealed additional signaling alterations. Most significantly, we found that overexpressing CRKL in H3122 resulted in activation of EGFR. We and others have previously demonstrated that EGFR signaling can mediate Crizotinib resistance. We further examined primary tumor cell lines (n=6), generated from ALK rearranged NSCLC patients who developed resistance to Crizotinib, and 4 demonstrated increased levels of both CRKL and phosphorylated EGFR. To determine if EGFR inhibition could be a therapeutic approach to overcome Crizotinib resistance in CRKL overexpressing cells, cell viability was assessed in response to dual treatment with Crizotinib and Gefitinib. The drug combination displayed robust synergistic effects on growth inhibition of ALK+ CRKL overexpressing cell lines but not of CRKLlow Crizotinib sensitive H3122, highlighting their co-dependence on EGFR. Finally, activation of FAK and SRC family kinases correlated with EGFR activation and CRKL overexpression in H3122CRKL, an observation further corroborated by a 9-fold increase in migratory capacity of H3122CRKL vs. control. Conclusion: Our findings indicate that CRKL overexpression can lead to acquired resistance to ALK inhibition in ALK rearranged NSCLC. CRKL overexpression results in EGFR activation suggesting that therapy directed at both EGFR and EML4-ALK may be effective in treating and/or preventing acquired resistance to Crizotinib. Citation Format: Magda Bahcall, Takaaki Sasaki, Atsuko Ogino, Marzia Capelletti, Mohit Butaney, Pasi A. Jänne. Overexpression of the adaptor protein CRKL as a mechanism of acquired resistance to Crizotinib in ALK-positive NSCLC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3716. doi:10.1158/1538-7445.AM2014-3716

Abstract 1901: Detection of EML4-ALK fusion gene expression in circulating tumor cells (CTCs) captured from non-small cell lung cancer (NSCLC) patients

Abstract Background & Aims: Detection of unique genomic profile such as EML4-ALK rearrangement in CTCs can be directly implicated to the course of treatment in patients with NSCLC. The aims of this study were to determine 1) if CTC could reliably be identified using microfabricated porous filter platform in advanced NSCLC; 2) feasibility of RT-PCR as a useful tool in detection of EML4-ALK rearrangement from CTCs. Method: Lung cancer cell lines with EML4-ALK rearrangement (NCI-H3122, NCI-H2228) were spiked into 5 ml of blood and were passed through the CTC platform. Twenty-five patients (19 FISH-proven ALK positive and 6 ALK negative in tissue) were prospectively enrolled in this study. Peripheral blood samples were drawn from the study patients and processed through CTC platform. Captured cells were characterized by IF staining; DAPI (nucleated cell marker), cancer cells by EpCAM and WBCs by CD45. RNA was extracted from the captured CTCs and RT-PCR was used to detect EML4-ALK fusion gene. Results: EpCAM expression was evaluated qualitatively and quantitatively in NSCLC cell lines by FACS analysis. Surprisingly, 100% of NSCLC cells expressed EpCAM, most at very high levels (165,000 ∼ 215,274 EpCAM antigens/cell). EpCAM was uniformly expressed throughout the population. The capture yield of ALK positive lung cancer cells were varied in spiking experiments (41-59%). There were 25 stage IV NSCLC patients whose clinical situations were variable in the aspect of previous cytotoxic chemotherapies and ALK-targeted tyrosine kinase inhibitors. CTCs were detected in 41% and EML4-ALK expressions were detected by RT-PCR in 58% of initially EML4-ALK positive NSCLC patients. Sequencing of PCR products identified the expression of EML4-ALK fusion gene. The sensitivity and the specificity of RT-PCR in detection of EML4-ALK fusion gene expression from the CTCs of NSCLCs were 58% and 100%, respectively. Conclusion We have proved the existence of EML4-ALK positive CTCs using RT-PCR after the enrichment of CTCs from the peripheral blood of NSCLC patients. Being able to reliably capture and molecularly characterize CTC may not only allow us to characterize a patient's cancer from a simple blood draw but investigation of genomic profile from CTCs may give us insights into the genetics and biology of tumor cell dissemination with further investigation. Citation Format: Joo Kyung Park, Ji Kon Ryu, Se-Hoon Lee, Eun-Hye Kim, Byung-Chul Kim. Detection of EML4-ALK fusion gene expression in circulating tumor cells (CTCs) captured from non-small cell lung cancer (NSCLC) patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1901. doi:10.1158/1538-7445.AM2014-1901

Abstract 5530: OTX015, a novel pan BET-BRD inhibitor is active in non-small-cell lung cancer (NSCLC) cell lines bearing the fusion protein EML4-ALK

Abstract Background: Various inhibitors targeting the activity of BET proteins have been recently developed and have shown potent anti-proliferative effects in several tumors, including NSCLC. The fusion of the echinoderm microtubule-associated protein-like 4 (EML4) and the anaplastic lymphoma kinase (ALK) genes results in the chimeric oncogene EML4-ALK, identified as a distinct entity of NSCLC patients that enables effective ALK-targeted therapy. However, most of these patients invariably acquire resistance within few months. Herein, we report preclinical findings obtained with a novel oral pan-BET-BRD inhibitor, OTX015, in a panel of NSCLC cell lines, some of which bear the fusion protein EML4-ALK. Materials and Methods: Five established NSCLC cell lines (i.e. H2228, H3122, A549, HOP62 and HOP92) were exposed to increasing concentrations of OTX015 (OncoEthix SA, Switzerland). In each case, the effect on cell viability was determined by the MTT assay after 72 h of exposure. GI50 values, representing 50% growth inhibition concentration, were calculated using GraphPad Prism 5.0 software. Protein levels were determined by Western blot using commercial antibodies. RNA was extracted with the Qiagen RNAEasy kit and reverse-transcribed using the Superscript First-Strand Synthesis System for RT-PCR kit following manufacturer's instructions. RT-PCR was performed using Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System. Results: OTX015 displayed anti-proliferative activity in EML4-ALK-positive H2228 and H3122 cells after a 72-h treatment, with GI50 values of 629 and 627nM, respectively. The corresponding GI50 values for the benchmark compound, JQ-1, were 2161 and 1265 nM. Interestingly, OTX015 was also active in the EML4-ALK-negative A549 cell line, with a GI50 of 432 nM. BRD4/3/2, c-MYC, BCL-2, p21 and CyclinD1 were detected at protein and mRNA levels in all cell lines. Both OTX015-sensitive and -resistant cells exhibited similar basal expression levels for the aforementioned proteins. EML4-ALK variants 1 and 3 were identified in H3122 and H2228 cells, respectively. Assessment of the signaling pathways involved in the anti-proliferative activity of OTX015 in these cells showed a transient up-regulation of STAT3, followed by a down-regulation after 24 h and up to 72 h of exposure. These results indicate that the key downstream effector of OTX015 is STAT3, frequently found to be overexpressed in crizotinib-resistant cell lines. Interestingly, c-MYC protein and mRNA levels were not altered by OTX015. EML4-ALK-positive H3122 cells showed down-regulation of n-MYC mRNA levels after OTX015 treatment. Conclusion: Our results indicate that NSCLC cell lines with genomic ALK alterations are sensitive to BET-BRD inhibition by OTX015, suggesting its potential clinical use as anticancer agent in EML4-ALK positive NSCLC patients. Citation Format: Ramiro Vázquez, Lucile Astorgues-Xerri, Mohamed Bekradda, Esteban Cvitkovic, Patrice Herait, Michela Boi, Giorgio Inghirami, Maurizio D'Incalci, María E. Riveiro, Eric Raymond. OTX015, a novel pan BET-BRD inhibitor is active in non-small-cell lung cancer (NSCLC) cell lines bearing the fusion protein EML4-ALK. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5530. doi:10.1158/1538-7445.AM2014-5530

Abstract C256: Expanded clinical opportunities for crizotinib from an analysis of over 5,000 cancer patient exomes.

Abstract Patients with chromosomal rearrangements resulting in fusion proteins are amongst the most responsive to targeted therapy. For example, targeting of the BCR-ABL fusion in chronic myelogenous leukemia (CML) with imatinib and the EML4-ALK fusion in non-small cell lung cancer (NSCLC) with crizotinib has led to dramatic patient responses in these diseases. While crizotinib is approved for use in EML4-ALK positive NSCLC through its inhibition of ALK, the drug also inhibits ROS1, MST1R (RON), MET, and more recently has been shown to inhibit the ALK homolog, LTK. To gain a more comprehensive understanding of the full therapeutic potential of crizotinib, we undertook a genomic survey of ALK, LTK, ROS1, MET and MST1R across thousands of patients subjected to full exome sequencing including patients from The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC), as well as tens thousands of patients from Oncomine® databases. We confirmed the presence of EML4-ALK fusions in both lung and colorectal cancer (CRC), identified a PRKAR1A-ALK fusion in CRC, and found evidence of novel recurrent ALK fusions in kidney papillary renal cell carcinoma and thyroid gland carcinoma. ALK hotspot mutations and focal amplifications were confined to neuroblastoma, as previously described. We also report the first instance of an LTK fusion, identified in thyroid gland carcinoma. LTK amplifications were also observed in 1.4% of gastric cancers and rarely in medulloblastoma and breast cancer. LTK was prominently over-expressed in leukemia, and in an analysis of over 4,000 PML-RARA fusion positive leukemia patients, LTK was amongst the most significantly over-expressed genes. In addition to confirming previously published ROS1 fusions, our survey of ROS1 identified rare novel fusions in NSCLC and glioblastoma. High-level MET amplifications were observed in 1-5% of papillary renal cell carcinoma, the intestinal subtype of gastric adenocarcinoma, oligodendroglioma, glioblastoma and lung adenocarcinoma. Hotspot mutations in MET were frequently observed in head and neck squamous cell carcinoma (HNSCC) (11%), and observed in a third of metastatic HNSCC samples. Additional hotspot mutations were also observed in lung adenocarcinoma (2%) and small cell lung cancer (2%). Aberrations in MST1R were rare. These results leverage all available genomic profiling data to provide a broadened scope of therapeutic opportunity for inhibitors like crizotinib. With the growing availability of next-generation sequencing data and analyses, such surveys can support hypothesis-driven development of targeted therapies and help expand opportunities for clinical stage therapies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C256. Citation Format: Sean Eddy, Mark Tomilo, Mary Ellen Urick, Nickolay A. Khazanov, Paul Williams, Armand Bankhead, Dinesh Cyanam, Supra Gajjala, Peter Wyngaard, Emma Bowden, Dan R. Rhodes. Expanded clinical opportunities for crizotinib from an analysis of over 5,000 cancer patient exomes. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C256.

Abstract 918: ASP3026, a selective ALK inhibitor, shows anti-tumor activity in a mouse model xenografted with NCI-H2228 intracranially.

Abstract EML4-ALK translocation has been validated as a therapeutic target in a subset of non-small cell lung cancer (NSCLC) patients. Crizotinib, an FDA-approved ALK inhibitor, is effective against several types of human cancers with ALK abnormalities including EML4-ALK, RANBP2-ALK and ALK mutations. However, long-term treatment is often limited by the development of resistant tumors and distant metastases. Multiple brain metastases are a critical issue because of their poor prognosis despite the standard radiotherapy. The reason for metastases and development of tumor in the brain could be attributable to the poor penetration of crizotinib into central nervous system, as previously reported. ASP3026 is a selective ALK inhibitor which shows antitumor activities in several crizotinib-refractory models including gate keeper mutants. Here, we established an intracranial xenograft model by implanting NCI-H2228 cells directly in the brain of immunocompromised mice. Xenografted cells develop intracranial tumors which grow in murine brain and eventually become lethal, resembling the clinical tumors that metastasized in the brain. To determine the anti-tumor activity against tumors in the brain, mice were treated with ASP3026 or crizotinib for 2 weeks and held to observe survival duration. Ten, 30 and 100 mg/kg daily oral administration of ASP3026 dose dependently inhibited tumor growth in brain, and tumor regression was achieved in 30 and 100 mg/kg groups confirmed by MR imaging. These results were supported by the observation that Ktrans and IAUC (90s) were significantly decreased in 30 and 100 mg/kg groups using dynamic contrast enhanced MR imaging (DCE-MRI). Furthermore, ASP3026 significantly prolonged the survival of tumor bearing mice compared to vehicle treatment group. On the other hand, 30mg/kg daily oral administration of crizotinib inhibited the tumor growth during the treatment period but did not produce significant survival benefit. Taken together, these results indicate that ASP3026 shows better efficacy than crizotinib and improves overall survival in an intracranial mouse xenograft model, suggesting that ASP3026 may have potential to benefit EML4-ALK positive NSCLC patients with brain metastases. Citation Format: Satoshi Konagai, Hiroshi Fushiki, Hideki Sakagami, Yoko Ueno, Masamichi Mori, Itsuro Shimada, Yutaka Kondoh, Sousuke Miyoshi, Shintaro Nishimura, Sadao Kuromitsu. ASP3026, a selective ALK inhibitor, shows anti-tumor activity in a mouse model xenografted with NCI-H2228 intracranially. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 918. doi:10.1158/1538-7445.AM2013-918

Abstract 866: ASP3026, a selective ALK inhibitor, induces tumor regression in a crizotinib-refractory model and prolongs survival in an intrapleurally xenograft model

Abstract EML4-ALK translocation has been validated as a therapeutic target in a subset of non-small cell lung cancer (NSCLC) patients. In clinical settings, crizotinib has shown promising response rates in patients with EML4-ALK positive NSCLC, while disease relapse has been observed in several sites, including the lungs. Our previous studies found that ASP3026, a selective ALK inhibitor, had higher kinase selectivity than crizotinib and induced regression of tumors expressing EML4-ALK with a gate-keeper mutation that confers resistance to crizotinib. Here, we evaluated the antitumor activity of ASP3026 and crizotinib against orthotopic lung and intrapleural tumors in mice using luciferase-expressing NCI-H2228, a human NSCLC tumor cell line endogenously expressing EML4-ALK. In an orthotopic lung model, implanted cells were monitored using bioluminescent imaging (BLI) of the chest area. ASP3026 and crizotinib were administered orally at once daily doses ranging from 3 to 30 mg/kg (starting after confirmation of tumor growth). ASP3026 at 30 mg/kg induced tumor regression by 27 days after the start of administration; in contrast, crizotinib at 30 mg/kg induced regression during the first 7 days of administration, although tumors subsequently regrew despite continuous drug administration. After tumor regrowth had been established by the first cycle treatment of crizotinib, substantial regression was achieved by subsequent administration of ASP3026 at 30 mg/kg against the crizotinib-refractory tumors. In an intrapleural xenograft model, disease-related mortality was observed in tested animals, with a median survival time (MST) of 39 days in vehicle-treated control mice and 71 days in crizotinib-treated animals at 30 mg/kg. In contrast, no mice receiving ASP3026 treatment at 30 mg/kg died during the experimental period (90 days). These effects were accompanied by change in the bioluminescent emissions, with crizotinib-treated mice showing increased emissions following the initial reduction, while ASP3026-induced reduction continuing throughout the experimental period. The mean bioluminescent emission in the group treated with ASP3026 was significantly lower than in the crizotinib-treated group, indicating that ASP3026-treated animals exhibited lower tumor burden than crizotinib-treated ones.Taken together, these results suggest that ASP3026 is effective in producing shrinkage of tumors refractory to crizotinib and shows superior efficacy to crizotinib against intrapleural metastatic tumors with respect to prolonging survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 866. doi:1538-7445.AM2012-866