Pub Date : 2018-09-01DOI: 10.1158/1557-3265.AACRIASLC18-IA18
A. Hata
Lung cancers harboring activating EGFR mutations are exquisitely sensitive to EGFR tyrosine kinase inhibitors; however, acquired drug resistance inevitably develops. Although genetic mechanisms of acquired resistance have been well characterized, less is known about how resistant clones evolve during treatment. Emerging data are beginning to illuminate the complex interplay of genetic clonal heterogeneity, adaptive signaling changes, epigenetic plasticity, and microenvironment factors that shapes the evolution of acquired resistance. Novel therapeutic strategies that specifically target these processes may prevent or delay the development of drug resistance. Citation Format: Aaron N. Hata. Evolution of acquired resistance in EGFR mutant NSCLC [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr IA18.
肺癌中含有活化的EGFR突变对EGFR酪氨酸激酶抑制剂非常敏感;然而，获得性耐药性不可避免地出现。虽然获得性耐药的遗传机制已被很好地描述，但对耐药克隆在治疗过程中如何进化知之甚少。新出现的数据开始阐明遗传克隆异质性、适应性信号变化、表观遗传可塑性和微环境因素之间复杂的相互作用，这些因素影响了获得性抗性的进化。专门针对这些过程的新治疗策略可能会预防或延缓耐药性的发展。引文格式:Aaron N. Hata。EGFR突变体NSCLC获得性耐药的进化[摘要]。第五届AACR-IASLC国际联合会议论文集:肺癌转化科学从实验室到临床;2018年1月8日至11日;费城(PA): AACR;临床肿瘤杂志，2018;24(17 -增刊):摘要1 - 18。
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Pub Date : 2018-09-01DOI: 10.1158/1557-3265.AACRIASLC18-PR08
J. Lee, Seongyeol Park, Joon Kim, T. Kim, Y. Ju
Purpose: Recently, we performed a whole-genome sequencing (WGS) study of serially acquired tumors and demonstrated that an early, complete inactivation of RB1 and TP53 is common among patients with EGFR-mutant lung adenocarcinoma (LADC) whose disease was transformed into small cell lung cancer (SCLC). To further understand this phenomenon, here we functionally characterized a patient-derived cell line of transformed SCLC and related in vitro models. We also deepened our genome analysis focusing on the complex rearrangements to probe their evolutionary paths in detail. Method: mRNA sequencing was performed for SNU-2962A cells and their transcriptome was analyzed with published mRNA sequencing datasets of primary LADCs (n = 87), and SCLCs (n = 81). A cell viability screening of SNU-2962A cells was performed to understand their therapeutic vulnerability using a kinome-targeting siRNA library. We also characterized isogenic RB1/TP53-knockout LADC cell lines with EGFR mutation (PC9, HCC-827 and HCC-4006). In addition, we reconstructed complex genomic rearrangements and analyzed them in relationship with whole-genome duplication to investigate their time points during clonal evolution. Results: The SNU-2962A cell line was established from the pleural effusion of the patient LC1. These cells exhibited adherent morphology in vitro and strongly expressed neuroendocrine markers. A hierarchical clustering of SNU-2962A cells with the published LADCs and SCLCs showed complete clustering with SCLCs. This cell line strongly expressed NEUROD1 and MYC, which was consistent with the variant-type SCLC. Kinome siRNA library screening showed a significantly reduced viability of SNU-2962A cells with knockdown of PI3K-MTOR pathway genes (PIK3CA or MTOR). Pharmacologic inhibition of this pathway using PI3K or AKT inhibitors showed moderate sensitivity. We tried to induce transformation into SCLCs using three different EGFR-mutant LADC cell lines by knockout of both RB1 and TP53 as well as knockin of PIK3CA E545K mutations using CRISPR, but we did not observe any meaningful evidence of neuroendocrine differentiation. We further analyzed our WGS data of 11 tumors from five patients, and found that many large-scaled genomic events such as whole-genome duplication, chromothripsis, or kataegis are frequently shared by LADCs and subsequent SCLCs, indicating their early occurrence. MYC amplification is frequently selected in transformed SCLCs. We also found a somatic deletion of BIM gene in LC3 patient, and this deletion was shared by the early LADC and the late SCLC. This may confer a dysfunctional apoptosis upon EGFR inhibition, which could predispose the early emergence of resistant tumor in this patient. Conclusion: Although a complete inactivation of both RB1 and TP53 is necessary for transformation from LADC into SCLC, it is not sufficient. Phylogenic reconstruction of large structural variations in our WGS dataset further clarified the mutational complexity of the commo
{"title":"Abstract PR08: Functional characterization and evolutionary reconstruction of small cell lung cancer transformation of EGFR-mutant lung adenocarcinomas","authors":"J. Lee, Seongyeol Park, Joon Kim, T. Kim, Y. Ju","doi":"10.1158/1557-3265.AACRIASLC18-PR08","DOIUrl":"https://doi.org/10.1158/1557-3265.AACRIASLC18-PR08","url":null,"abstract":"Purpose: Recently, we performed a whole-genome sequencing (WGS) study of serially acquired tumors and demonstrated that an early, complete inactivation of RB1 and TP53 is common among patients with EGFR-mutant lung adenocarcinoma (LADC) whose disease was transformed into small cell lung cancer (SCLC). To further understand this phenomenon, here we functionally characterized a patient-derived cell line of transformed SCLC and related in vitro models. We also deepened our genome analysis focusing on the complex rearrangements to probe their evolutionary paths in detail. Method: mRNA sequencing was performed for SNU-2962A cells and their transcriptome was analyzed with published mRNA sequencing datasets of primary LADCs (n = 87), and SCLCs (n = 81). A cell viability screening of SNU-2962A cells was performed to understand their therapeutic vulnerability using a kinome-targeting siRNA library. We also characterized isogenic RB1/TP53-knockout LADC cell lines with EGFR mutation (PC9, HCC-827 and HCC-4006). In addition, we reconstructed complex genomic rearrangements and analyzed them in relationship with whole-genome duplication to investigate their time points during clonal evolution. Results: The SNU-2962A cell line was established from the pleural effusion of the patient LC1. These cells exhibited adherent morphology in vitro and strongly expressed neuroendocrine markers. A hierarchical clustering of SNU-2962A cells with the published LADCs and SCLCs showed complete clustering with SCLCs. This cell line strongly expressed NEUROD1 and MYC, which was consistent with the variant-type SCLC. Kinome siRNA library screening showed a significantly reduced viability of SNU-2962A cells with knockdown of PI3K-MTOR pathway genes (PIK3CA or MTOR). Pharmacologic inhibition of this pathway using PI3K or AKT inhibitors showed moderate sensitivity. We tried to induce transformation into SCLCs using three different EGFR-mutant LADC cell lines by knockout of both RB1 and TP53 as well as knockin of PIK3CA E545K mutations using CRISPR, but we did not observe any meaningful evidence of neuroendocrine differentiation. We further analyzed our WGS data of 11 tumors from five patients, and found that many large-scaled genomic events such as whole-genome duplication, chromothripsis, or kataegis are frequently shared by LADCs and subsequent SCLCs, indicating their early occurrence. MYC amplification is frequently selected in transformed SCLCs. We also found a somatic deletion of BIM gene in LC3 patient, and this deletion was shared by the early LADC and the late SCLC. This may confer a dysfunctional apoptosis upon EGFR inhibition, which could predispose the early emergence of resistant tumor in this patient. Conclusion: Although a complete inactivation of both RB1 and TP53 is necessary for transformation from LADC into SCLC, it is not sufficient. Phylogenic reconstruction of large structural variations in our WGS dataset further clarified the mutational complexity of the commo","PeriodicalId":250896,"journal":{"name":"Heterogeneity and Evolution","volume":"18 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"128879380","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2018-09-01DOI: 10.1158/1557-3265.AACRIASLC18-B02
K. Fukuda, S. Takeuchi, S. Arai, Ryohei Katayama, S. Nanjo, Azusa Tanimoto, Akihiro Nisiyama, Takeshisa Suzuki, K. Takeuchi, M. Nishio, S. Yano
ALK rearrangement, most commonly EML4-ALK, is detected in approximately 3–5% of non-small cell lung cancer (NSCLC). While ALK tyrosine kinase inhibitor (TKI) shows dramatic clinical efficacy in ALK-rearranged NSCLC patients, almost all patients acquire resistance over time. ALK-secondary mutations, including ALK L1196M, are detected in ~40% of ALK-rearranged lung cancers resistant to ALK inhibitors. Epithelial–mesenchymal transition (EMT) was also reported to be associated with various targeted drugs; however, its involvement in ALK-inhibitor resistance is largely unknown. In this study, we continuously gave crizotinib treatment to SCID mice inoculated with EML4-ALK lung cancer cell line A925LPE into thoracic cavity and established crizotinib-resistant cells. We also obtained several single-cell clones with acquired EMT phenotypes (low E-cadherin, high vimentin and ZEB1). MicroRNA profile analysis revealed that EMT was induced by reducing the expression of miR-200 family members, including miR-200c and miR-141, which resulted in increasing ZEB1 and decreasing E-cadherin expression in the clone cells. A reporter assay on a 200-kinase inhibitor library indicated that the histone deacetylase (HDAC) inhibitor, quisinostat, had the highest potential to increase miR-200c-141 promoter activity. Interestingly, pretreatment of the cells with quisinostat reduced ZEB1 expression, increased E-cadherin expression, and thus restored sensitivity to crizotinib and alectinib, mediated by enhanced expression of miR-200c in vitro and in vivo. These results indicate that quisinostat induces mesenchymal-epithelial transition (MET) by upregulating miR-200c expression that targets ZEB1 and thereby resensitizes to ALK-TKI. Furthermore, we analyzed tumor tissue obtained at autopsy from an ALK-rearranged NSCLC patient who acquired resistance to crizotinib. In specimens from the primary lung tumor, as well as from the brain and subcutaneous metastases, both ALK L1196M mutation and EMT were concomitantly detected in all crizotinib-resistant lesions. Therefore, we performed laser capture microdissection and measured the copy number of ALK L1196M in epithelial and mesenchymal type tumor lesions separately. Very interestingly, ALK L1196M mutation was predominantly detected in epithelial type tumor cell lesion; by sharp contrast, it was hardly detected in the mesenchymal type tumor cell lesion. These results clearly demonstrate that EMT is a clinically relevant independent mechanism for crizotinib resistance underlying ALK inhibitor-resistant cancers. Together, our study demonstrates the intratumor heterogeneity constituted by coexistence of resistance mutations and EMT in crizotinib-resistant tumors. HDAC inhibitor pretreatment, which reverts EMT, followed by a new-generation ALK inhibitor may be useful to circumvent resistance due to such intratumor heterogeneity. Citation Format: Koji Fukuda, Shinji Takeuchi, Sachiko Arai, Ryohei Katayama, Shigeki Nanjo, Azusa Tanimoto, Akih
{"title":"Abstract B02: Heterogeneity of epithelial-to-mesenchymal transition and resistance mutation in ALK inhibitor-resistant lung cancer and its circumvention","authors":"K. Fukuda, S. Takeuchi, S. Arai, Ryohei Katayama, S. Nanjo, Azusa Tanimoto, Akihiro Nisiyama, Takeshisa Suzuki, K. Takeuchi, M. Nishio, S. Yano","doi":"10.1158/1557-3265.AACRIASLC18-B02","DOIUrl":"https://doi.org/10.1158/1557-3265.AACRIASLC18-B02","url":null,"abstract":"ALK rearrangement, most commonly EML4-ALK, is detected in approximately 3–5% of non-small cell lung cancer (NSCLC). While ALK tyrosine kinase inhibitor (TKI) shows dramatic clinical efficacy in ALK-rearranged NSCLC patients, almost all patients acquire resistance over time. ALK-secondary mutations, including ALK L1196M, are detected in ~40% of ALK-rearranged lung cancers resistant to ALK inhibitors. Epithelial–mesenchymal transition (EMT) was also reported to be associated with various targeted drugs; however, its involvement in ALK-inhibitor resistance is largely unknown. In this study, we continuously gave crizotinib treatment to SCID mice inoculated with EML4-ALK lung cancer cell line A925LPE into thoracic cavity and established crizotinib-resistant cells. We also obtained several single-cell clones with acquired EMT phenotypes (low E-cadherin, high vimentin and ZEB1). MicroRNA profile analysis revealed that EMT was induced by reducing the expression of miR-200 family members, including miR-200c and miR-141, which resulted in increasing ZEB1 and decreasing E-cadherin expression in the clone cells. A reporter assay on a 200-kinase inhibitor library indicated that the histone deacetylase (HDAC) inhibitor, quisinostat, had the highest potential to increase miR-200c-141 promoter activity. Interestingly, pretreatment of the cells with quisinostat reduced ZEB1 expression, increased E-cadherin expression, and thus restored sensitivity to crizotinib and alectinib, mediated by enhanced expression of miR-200c in vitro and in vivo. These results indicate that quisinostat induces mesenchymal-epithelial transition (MET) by upregulating miR-200c expression that targets ZEB1 and thereby resensitizes to ALK-TKI. Furthermore, we analyzed tumor tissue obtained at autopsy from an ALK-rearranged NSCLC patient who acquired resistance to crizotinib. In specimens from the primary lung tumor, as well as from the brain and subcutaneous metastases, both ALK L1196M mutation and EMT were concomitantly detected in all crizotinib-resistant lesions. Therefore, we performed laser capture microdissection and measured the copy number of ALK L1196M in epithelial and mesenchymal type tumor lesions separately. Very interestingly, ALK L1196M mutation was predominantly detected in epithelial type tumor cell lesion; by sharp contrast, it was hardly detected in the mesenchymal type tumor cell lesion. These results clearly demonstrate that EMT is a clinically relevant independent mechanism for crizotinib resistance underlying ALK inhibitor-resistant cancers. Together, our study demonstrates the intratumor heterogeneity constituted by coexistence of resistance mutations and EMT in crizotinib-resistant tumors. HDAC inhibitor pretreatment, which reverts EMT, followed by a new-generation ALK inhibitor may be useful to circumvent resistance due to such intratumor heterogeneity. Citation Format: Koji Fukuda, Shinji Takeuchi, Sachiko Arai, Ryohei Katayama, Shigeki Nanjo, Azusa Tanimoto, Akih","PeriodicalId":250896,"journal":{"name":"Heterogeneity and Evolution","volume":"15 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"127741674","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2018-09-01DOI: 10.1158/1557-3265.AACRIASLC18-A24
N. Marjanovic, S. R. Ng, A. Regev, T. Jacks
Small cell lung cancer (SCLC) is one of the most aggressive cancer types, and patients in clinic usually (>60% cases) present with metastasis. Current therapies for SCLC have not changed from 1980, and they include a combination therapy of cisplatin and etoposide. Most patients relapse after initial response, and the 5-year survival for extensive stage SCLC is 2%. One possible explanation for the failure of conventional and targeted therapies in cancers is the cellular heterogeneity that exists within tumors. Thus, understanding phenotypic heterogeneity at the single-cell level can be leveraged to predict mechanisms of resistance, which enables the design of effective combination therapies. In this project, we used as a model system an autochthonous mouse model of human SCLC, in which we deleted p53, RB, and p130. We collected multiple primary tumors, circulating tumor cells (CTC), and lymph node (proximal site) and liver (distant site) metastases from the same mouse, and across different mice. These samples were collected at the stage that should correspond to the limited/extensive stage of human SCLC, which is most commonly seen in clinic. Then we used single-cell RNA-seq (sc-RNAseq) methods to define and understand transcriptional heterogeneity in these samples. Furthermore, we aimed to understand the evolutionary relationships between primary tumors, CTCs, lymph node and liver metastases using different computational approaches such as diffusion maps. Further defining and functionally annotating transcriptional heterogeneity will help us better understand the disease, and find the new Achilles heel for targeting. Note: This abstract was not presented at the conference. Citation Format: Nemanja Despot Marjanovic, Sheng Rong Ng, Aviv Regev, Tyler Jacks. Using single-cell RNA-seq approaches to decipher heterogeneity in autochthonous mouse models of small cell lung cancer [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A24.
{"title":"Abstract A24: Using single-cell RNA-seq approaches to decipher heterogeneity in autochthonous mouse models of small cell lung cancer","authors":"N. Marjanovic, S. R. Ng, A. Regev, T. Jacks","doi":"10.1158/1557-3265.AACRIASLC18-A24","DOIUrl":"https://doi.org/10.1158/1557-3265.AACRIASLC18-A24","url":null,"abstract":"Small cell lung cancer (SCLC) is one of the most aggressive cancer types, and patients in clinic usually (>60% cases) present with metastasis. Current therapies for SCLC have not changed from 1980, and they include a combination therapy of cisplatin and etoposide. Most patients relapse after initial response, and the 5-year survival for extensive stage SCLC is 2%. One possible explanation for the failure of conventional and targeted therapies in cancers is the cellular heterogeneity that exists within tumors. Thus, understanding phenotypic heterogeneity at the single-cell level can be leveraged to predict mechanisms of resistance, which enables the design of effective combination therapies. In this project, we used as a model system an autochthonous mouse model of human SCLC, in which we deleted p53, RB, and p130. We collected multiple primary tumors, circulating tumor cells (CTC), and lymph node (proximal site) and liver (distant site) metastases from the same mouse, and across different mice. These samples were collected at the stage that should correspond to the limited/extensive stage of human SCLC, which is most commonly seen in clinic. Then we used single-cell RNA-seq (sc-RNAseq) methods to define and understand transcriptional heterogeneity in these samples. Furthermore, we aimed to understand the evolutionary relationships between primary tumors, CTCs, lymph node and liver metastases using different computational approaches such as diffusion maps. Further defining and functionally annotating transcriptional heterogeneity will help us better understand the disease, and find the new Achilles heel for targeting. Note: This abstract was not presented at the conference. Citation Format: Nemanja Despot Marjanovic, Sheng Rong Ng, Aviv Regev, Tyler Jacks. Using single-cell RNA-seq approaches to decipher heterogeneity in autochthonous mouse models of small cell lung cancer [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A24.","PeriodicalId":250896,"journal":{"name":"Heterogeneity and Evolution","volume":"66 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2018-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"116177262","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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