{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,13]],"date-time":"2026-07-13T20:28:28Z","timestamp":1783974508231,"version":"3.55.0"},"reference-count":43,"publisher":"Public Library of Science (PLoS)","issue":"1","license":[{"start":{"date-parts":[[2025,1,21]],"date-time":"2025-01-21T00:00:00Z","timestamp":1737417600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"El Consejo Nacional de Ciencia, Tecnolog\u00eda e Innovaci\u00f3n","award":["375-2019-FONDECYT"],"award-info":[{"award-number":["375-2019-FONDECYT"]}]},{"name":"El Consejo Nacional de Ciencia, Tecnolog\u00eda e Innovaci\u00f3n","award":["375-2019-FONDECYT"],"award-info":[{"award-number":["375-2019-FONDECYT"]}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>The anaplastic lymphoma kinase (ALK) oncoprotein plays a crucial role in non-small cell lung cancer (NSCLC) by activating signaling pathways involved in cell proliferation and survival through constitutive phosphorylation. While first-line crizotinib can regulate phosphorylation, mutations in the ALK gene can lead to resistance against ALK inhibitors (ALKi) such as ceritinib and alectinib. On the other hand, overexpression of BCL2, a protein involved in cell death regulation, has been observed in NSCLC and is considered a potential therapeutic target. In this study, we propose to inhibit BCL2 as a secondary therapeutic target in EML4-ALK cell models to overcome resistance caused by ALK mutations. Four Ba\/F3 EML4-ALK cell models (WT, C1156Y, L1196M, and G1202R) generated by site-directed mutagenesis exhibited varying levels of BCL2 expression. Both the WT and G1202R models showed overexpression of BCL2, while C1156Y and L1196M models approached baseline levels. We treated these cells with ABT-199, a selective BCL2 inhibitor, and found that models with high BCL2 expression exhibited resistance, while those with lower expression showed sensitivity to BCL2 inhibition. In addition, our analysis using bioinformatics indicated that ABT-199 not only targets BCL2 but also binds to the active site of all ALK mutants, it was contrasted by <jats:italic>in vitro<\/jats:italic> ALK kinase activity inhibition by ABT-199 (5.5 \u03bcM). This interaction was further supported by a significant decrease of ALK phosphorylation in single and combination treatment with 300nM ABT-199. Finally, when ABT-199 was combined with ALKi, we observed a wide range of synergistic effects in the WT and G1202R cell models, while the C1156Y and L1196M models showed limited synergy. In conclusion, our findings indicate that BCL2 targeting with ABT-199, in combination with ALKi, can significantly reduce tumor cell survival in Ba\/F3 EML4-ALK cell models.<\/jats:p>","DOI":"10.1371\/journal.pone.0308747","type":"journal-article","created":{"date-parts":[[2025,1,21]],"date-time":"2025-01-21T18:26:03Z","timestamp":1737483963000},"page":"e0308747","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":1,"title":["Inhibition of the anti-apoptotic protein BCL2 in EML4-ALK cell models as a second proposed therapeutic target for non-small cell lung cancer"],"prefix":"10.1371","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7634-1029","authenticated-orcid":true,"given":"Richard Junior","family":"Zapata Dongo","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1633-6310","authenticated-orcid":true,"given":"Diletta","family":"Fontana","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luca","family":"Mologni","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3408-7971","authenticated-orcid":true,"given":"Juan Enrique","family":"Faya Castillo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stefany Fiorella","family":"Infante Varillas","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2025,1,21]]},"reference":[{"key":"pone.0308747.ref001","first-page":"2024","article-title":"Key Statistics for Lung Cancer","volume":"11","author":"AC Society","year":"2024","journal-title":"Am Cancer Soc"},{"key":"pone.0308747.ref002","doi-asserted-by":"crossref","first-page":"337","DOI":"10.21037\/tlcr-22-631","article-title":"Early-stage anaplastic lymphoma kinase (ALK)-positive lung cancer: a narrative review","volume":"12","author":"MF Chen","year":"2023","journal-title":"Transl Lung Cancer Res"},{"key":"pone.0308747.ref003","doi-asserted-by":"crossref","first-page":"423","DOI":"10.2147\/CLEP.S69718","article-title":"Prevalence and natural history of ALK positive non-small-cell lung cancer and the clinical impact of targeted therapy with ALK inhibitors","volume":"6","author":"PL Chia","year":"2014","journal-title":"Clin Epidemiol"},{"key":"pone.0308747.ref004","doi-asserted-by":"crossref","first-page":"1","DOI":"10.3390\/ph13100273","article-title":"Epidermal growth factor receptor (EGFR)-mutated non-small-cell lung cancer (NSCLC)","volume":"13","author":"C O\u2019Leary","year":"2020","journal-title":"Pharmaceuticals"},{"key":"pone.0308747.ref005","doi-asserted-by":"crossref","first-page":"1450","DOI":"10.1097\/JTO.0b013e3181c4dedb","article-title":"ALK gene rearrangements: A new therapeutic target in a molecularly defined subset of non-small cell lung cancer","volume":"4","author":"B Solomon","year":"2009","journal-title":"J Thorac Oncol"},{"key":"pone.0308747.ref006","unstructured":"ALK \u2010 My Cancer Genome n.d. https:\/\/www.mycancergenome.org\/content\/gene\/alk\/ (accessed May 17, 2024)."},{"key":"pone.0308747.ref007","doi-asserted-by":"crossref","first-page":"4403","DOI":"10.1158\/0008-5472.CAN-10-4041","article-title":"Anaplastic thyroid cancers harbor novel oncogenic mutations of the ALK gene","volume":"71","author":"AK Murugan","year":"2011","journal-title":"Cancer Res"},{"key":"pone.0308747.ref008","first-page":"15","article-title":"Analysis of alk, idh1, idh2 and mmp8 somatic mutations in differentiated thyroid cancers","author":"AK Murugan","year":"2021","journal-title":"Mol Clin Oncol"},{"key":"pone.0308747.ref009","doi-asserted-by":"crossref","first-page":"791","DOI":"10.1038\/modpathol.2017.181","article-title":"Genomic heterogeneity of ALK fusion breakpoints in non-small-cell lung cancer","volume":"31","author":"JN Rosenbaum","year":"2018","journal-title":"Mod Pathol"},{"key":"pone.0308747.ref010","first-page":"19","article-title":"Anaplastic lymphoma kinase (Alk) receptor tyrosine kinase: A catalytic receptor with many faces","author":"H. Huang","year":"2018","journal-title":"Int J Mol Sci"},{"key":"pone.0308747.ref011","doi-asserted-by":"crossref","first-page":"137","DOI":"10.1158\/2159-8290.CD-16-1123","article-title":"Targeting ALK: Precision medicine takes on drug resistance","volume":"7","author":"JJ Lin","year":"2017","journal-title":"Cancer Discov"},{"key":"pone.0308747.ref012","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1177\/1758834015617355","article-title":"ALK inhibitors in non-small cell lung cancer: The latest evidence and developments","volume":"8","author":"I Sullivan","year":"2016","journal-title":"Ther Adv Med Oncol"},{"key":"pone.0308747.ref013","first-page":"1","article-title":"A new ALK inhibitor overcomes resistance to first\u2010 and second\u2010generation inhibitors in NSCLC","volume":"14","author":"Y Lu","year":"2022","journal-title":"EMBO Mol Med"},{"key":"pone.0308747.ref014","first-page":"12","article-title":"Drug repositioning in non-small cell lung cancer (NSCLC) using gene co-expression and drug\u2013gene interaction networks analysis","author":"H MotieGhader","year":"2022","journal-title":"Sci Rep"},{"key":"pone.0308747.ref015","doi-asserted-by":"crossref","first-page":"e0295966","DOI":"10.1371\/journal.pone.0295966","article-title":"Mitoxantrone and abacavir: An ALK protein-targeted in silico proposal for the treatment of non-small cell lung cancer","volume":"19","author":"JE Faya Castillo","year":"2024","journal-title":"PLoS One"},{"key":"pone.0308747.ref016","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1158\/2159-8290.CD-21-1059","article-title":"Hallmarks of Cancer","volume":"12","author":"D. Hanahan","year":"2022","journal-title":"New Dimensions. Cancer Discov"},{"key":"pone.0308747.ref017","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1007\/s10495-022-01780-7","article-title":"BCL-2 protein family: attractive targets for cancer therapy","volume":"28","author":"D Kaloni","year":"2023","journal-title":"Apoptosis"},{"key":"pone.0308747.ref018","doi-asserted-by":"crossref","first-page":"1","DOI":"10.3390\/cancers13061292","article-title":"Targeting bcl-2 in cancer: Advances, challenges, and perspectives","volume":"13","author":"S Hafezi","year":"2021","journal-title":"Cancers (Basel)"},{"key":"pone.0308747.ref019","doi-asserted-by":"crossref","first-page":"1","DOI":"10.3389\/fonc.2020.562558","article-title":"Venetoclax-Based Combinations in Acute Myeloid Leukemia: Current Evidence and Future Directions","volume":"10","author":"B Samra","year":"2020","journal-title":"Front Oncol"},{"key":"pone.0308747.ref020","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1155\/2014\/943648","article-title":"Targeting BCL2-Proteins for the Treatment of Solid Tumours","volume":"2014","author":"M. Vogler","year":"2014","journal-title":"Adv Med"},{"key":"pone.0308747.ref021","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1002\/cncr.11461","article-title":"Expression of bcl-2 protein is associated with shorter survival in nonsmall cell lung carcinoma","volume":"98","author":"CI Huang","year":"2003","journal-title":"Cancer"},{"key":"pone.0308747.ref022","first-page":"901","article-title":"Upregulation of Bcl2 in NSCLC with acquired resistance to EGFR-TKI","volume":"15","author":"HT Cheong","year":"2018","journal-title":"Oncol Lett"},{"key":"pone.0308747.ref023","doi-asserted-by":"crossref","first-page":"3125","DOI":"10.1007\/s00204-020-02816-0","article-title":"Synergistic effects of Bcl-2 inhibitors with AZD9291 on overcoming the acquired resistance of AZD9291 in H1975 cells","volume":"94","author":"Z Liu","year":"2020","journal-title":"Arch Toxicol"},{"key":"pone.0308747.ref024","first-page":"8","article-title":"Therapeutic Targeting of Regulated Signaling Pathways of Non-Small Cell Lung Carcinoma","author":"GM Hasan","year":"2023","journal-title":"ACS Omega"},{"key":"pone.0308747.ref025","doi-asserted-by":"crossref","first-page":"953","DOI":"10.1002\/cam4.413","article-title":"Activity of second-generation ALK inhibitors against crizotinib-resistant mutants in an NPM-ALK model compared to EML4-ALK","volume":"4","author":"D Fontana","year":"2015","journal-title":"Cancer Med"},{"key":"pone.0308747.ref026","doi-asserted-by":"crossref","first-page":"114488","DOI":"10.1016\/j.ejmech.2022.114488","article-title":"Identification of non-ATP-competitive \u03b1-carboline inhibitors of the anaplastic lymphoma kinase","volume":"238","author":"L Mologni","year":"2022","journal-title":"Eur J Med Chem"},{"key":"pone.0308747.ref027","doi-asserted-by":"crossref","first-page":"2981","DOI":"10.1093\/bioinformatics\/btu426","article-title":"YASARA View \u2010 molecular graphics for all devices \u2010 from smartphones to workstations","volume":"30","author":"E Krieger","year":"2014","journal-title":"Bioinformatics"},{"key":"pone.0308747.ref028","doi-asserted-by":"crossref","first-page":"W530","DOI":"10.1093\/nar\/gkab294","article-title":"PLIP 2021: Expanding the scope of the protein-ligand interaction profiler to DNA and RNA","volume":"49","author":"MF Adasme","year":"2021","journal-title":"Nucleic Acids Res"},{"key":"pone.0308747.ref029","doi-asserted-by":"crossref","first-page":"11334","DOI":"10.3390\/ijms231911334","article-title":"In Planta, In Vitro and In Silico Studies of Chiral N6-Benzyladenine Derivatives: Discovery of Receptor-Specific S-Enantiomers with Cytokinin or Anticytokinin Activities","volume":"23","author":"EM Savelieva","year":"2022","journal-title":"Int J Mol Sci"},{"key":"pone.0308747.ref030","doi-asserted-by":"crossref","first-page":"268","DOI":"10.14573\/1803231","article-title":"Normalization of data for viability and relative cell function curves","volume":"35","author":"A Krebs","year":"2018","journal-title":"ALTEX"},{"key":"pone.0308747.ref031","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1007\/978-1-0716-2148-6_6","article-title":"Calculating Half Maximal Inhibitory Concentration (IC50) Values from Glycomics Microarray Data Using GraphPad Prism","volume":"2460","author":"M Le Berre","year":"2022","journal-title":"Methods Mol. Biol."},{"key":"pone.0308747.ref032","doi-asserted-by":"crossref","first-page":"2866","DOI":"10.1093\/bioinformatics\/btw230","article-title":"Combenefit: an interactive platform for the analysis and visualization of drug combinations","volume":"32","author":"GY Di Veroli","year":"2016","journal-title":"Bioinformatics"},{"key":"pone.0308747.ref033","first-page":"11","article-title":"The Resistance Mechanisms and Treatment Strategies for ALK-Rearranged Non-Small Cell Lung Cancer","author":"Y Pan","year":"2021","journal-title":"Front Oncol"},{"key":"pone.0308747.ref034","doi-asserted-by":"crossref","first-page":"1472","DOI":"10.1158\/1078-0432.CCR-11-2906","article-title":"Mechanisms of resistance to crizotinib in patients with ALK gene rearranged non-small cell lung cancer","volume":"18","author":"RC Doebele","year":"2012","journal-title":"Clin Cancer Res"},{"key":"pone.0308747.ref035","doi-asserted-by":"crossref","first-page":"300","DOI":"10.1016\/j.ctrv.2013.07.002","article-title":"ALK inhibitors in the treatment of advanced NSCLC","volume":"40","author":"C Gridelli","year":"2014","journal-title":"Cancer Treat Rev"},{"key":"pone.0308747.ref036","doi-asserted-by":"crossref","first-page":"1209","DOI":"10.1007\/s00018-015-2117-6","article-title":"Molecular mechanisms that underpin EML4-ALK driven cancers and their response to targeted drugs","volume":"73","author":"R Bayliss","year":"2016","journal-title":"Cell Mol Life Sci"},{"key":"pone.0308747.ref037","first-page":"1230","article-title":"BCL2 Protein in non-small-cell lung carcinoma","volume":"29","author":"F Pezzella","year":"1993","journal-title":"N Engl J Med"},{"key":"pone.0308747.ref038","doi-asserted-by":"crossref","first-page":"1850","DOI":"10.1038\/s41416-023-02219-9","article-title":"Dual targeting of BCL-2 and MCL-1 in the presence of BAX breaks venetoclax resistance in human small cell lung cancer","volume":"128","author":"Z Valko","year":"2023","journal-title":"Br J Cancer"},{"key":"pone.0308747.ref039","doi-asserted-by":"crossref","first-page":"360","DOI":"10.1158\/1078-0432.CCR-17-1606","article-title":"Venetoclax is effective in small-cell lung cancers with high BCL-2 expression","volume":"24","author":"TL Lochmann","year":"2018","journal-title":"Clin Cancer Res"},{"key":"pone.0308747.ref040","doi-asserted-by":"crossref","first-page":"1170","DOI":"10.1021\/jm401805h","article-title":"Design of Potent and Selective Inhibitors to Overcome Clinical Anaplastic Lymphoma Kinase Mutations Resistant to Crizotinib","volume":"57","author":"Q Huang","year":"2014","journal-title":"J Med Chem"},{"key":"pone.0308747.ref041","doi-asserted-by":"crossref","first-page":"679","DOI":"10.1016\/j.ccr.2011.04.004","article-title":"CH5424802, a Selective ALK Inhibitor Capable of Blocking the Resistant Gatekeeper Mutant","volume":"19","author":"H Sakamoto","year":"2011","journal-title":"Cancer Cell"},{"key":"pone.0308747.ref042","doi-asserted-by":"crossref","first-page":"1","DOI":"10.3390\/molecules28020783","article-title":"In Silico Mining of Natural Products Atlas (NPAtlas) Database for Identifying Effective Bcl-2 Inhibitors: Molecular Docking, Molecular Dynamics, and Pharmacokinetics Characteristics","volume":"28","author":"NM Almansour","year":"2023","journal-title":"Molecules"},{"key":"pone.0308747.ref043","doi-asserted-by":"crossref","first-page":"1238093","DOI":"10.3389\/fmolb.2023.1238093","article-title":"Progress of non-small-cell lung cancer with ROS1 rearrangement","volume":"10","author":"X Yang","year":"2023","journal-title":"Front Mol Biosci"}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0308747","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,1,21]],"date-time":"2025-01-21T18:26:28Z","timestamp":1737483988000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0308747"}},"subtitle":[],"editor":[{"given":"Avaniyapuram Kannan","family":"Murugan","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2025,1,21]]},"references-count":43,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2025,1,21]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0308747","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,1,21]]}}}