Targeting Anaplastic lymphoma kinase (ALK) is a promising therapeutic strategy for aberrant ALK-expressing malignancies including neuroblastoma, but resistance to ALK tyrosine kinase inhibitors (ALK TKI) is a distinct possibility necessitating drug combination therapeutic approaches. Using high-throughput, genome-wide CRISPR-Cas9 knockout screens, we identify miR-1304-5p loss as a desensitizer to ALK TKIs in aberrant ALK-expressing neuroblastoma; inhibition of miR-1304-5p decreases, while mimics of this miRNA increase the sensitivity of neuroblastoma cells to ALK TKIs. We show that miR-1304-5p targets NRAS, decreasing cell viability via induction of apoptosis. It follows that the farnesyltransferase inhibitor (FTI) lonafarnib in addition to ALK TKIs act synergistically in neuroblastoma, inducing apoptosis in vitro. In particular, on combined treatment of neuroblastoma patient derived xenografts with an FTI and an ALK TKI complete regression of tumour growth is observed although tumours rapidly regrow on cessation of therapy. Overall, our data suggests that combined use of ALK TKIs and FTIs, constitutes a therapeutic approach to treat high risk neuroblastoma although prolonged therapy is likely required to prevent relapse.

Center for Biomarker Research in Medicine Graz Austria

Chelsea and Westminster Hospital NHS Foundation Trust London SW10 9NH UK

Christian Doppler Laboratory for Applied Metabolomics Medical University of Vienna Vienna Austria

Department of Life Sciences Birmingham City University Birmingham UK

Department of Medicine University of Cambridge Addenbrookes Hospital Hills Road Cambridge CB2 0QQ UK

Department of Paediatric Haematology Oncology and Palliative Care Addenbrooke's Hospital Cambridge CB2 0QQ UK

Department of Pathology Division of Cellular and Molecular Pathology University of Cambridge Cambridge CB20QQ UK

Department of Pathology Division of Experimental and Translational Pathology Medical University of Vienna 1090 Vienna Austria

Department of Pathology Medical University of Vienna Vienna 1090 Austria

Department of Radiology University of Cambridge Cambridge Biomedical Campus Cambridge CB2 0QQ UK

European Research Initiative for ALK related malignancies Cambridge CB2 0QQ UK

Faculty of Medicine Masaryk University Brno Czech Republic

Functional Genomics GlaxoSmithKline Stevenage SG1 2NY UK

Laboratory of Cancer Biology and Genetics Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda MD 20814 USA

Merck and Co 2000 Galloping Hill Rd Kenilworth NJ 07033 USA

MRC Mitochondrial Biology Unit University of Cambridge The Keith Peters Building Cambridge Biomedical Campus Hills Road Cambridge CB2 0XY UK

Nottingham Trent University School of Science and Technology Clifton Lane Nottingham NG11 8NS UK

OncoSec San Diego CA 92121 USA

St Anna Children's Cancer Research Institute CCRI Zimmermannplatz 10 1090 Vienna Austria

Unit of Laboratory Animal Pathology University of Veterinary Medicine Vienna Vienna Austria

Zobrazit více v PubMed

Redaelli S, et al. Lorlatinib treatment elicits multiple on- and off-target mechanisms of resistance in ALK-driven cancer. Cancer Res. 2018;78:6866–6880. doi: 10.1158/0008-5472.CAN-18-1867. PubMed DOI

Mehlman C, et al. Ceritinib ALK T1151R resistance mutation in lung cancer with initial response to brigatinib. J. Thorac. Oncol. 2019;14:e95–e96. doi: 10.1016/j.jtho.2018.12.036. PubMed DOI

Friboulet L, et al. The ALK inhibitor ceritinib overcomes crizotinib resistance in non-small cell lung cancer. Cancer Discov. 2014;4:662–673. doi: 10.1158/2159-8290.CD-13-0846. PubMed DOI PMC

Sharma GG, et al. A compound L1196M/G1202R ALK mutation in a patient with ALK-positive lung cancer with acquired resistance to brigatinib also confers primary resistance to lorlatinib. J. Thorac. Oncol. 2019;14:e257–e259. doi: 10.1016/j.jtho.2019.06.028. PubMed DOI

Trigg RM, et al. The targetable kinase PIM1 drives ALK inhibitor resistance in high-risk neuroblastoma independent of MYCN status. Nat. Commun. 2019;10:5428. doi: 10.1038/s41467-019-13315-x. PubMed DOI PMC

Prokoph N, et al. IL10RA modulates crizotinib sensitivity in NPM1-ALK+ anaplastic large cell lymphoma. Blood. 2020;136:1657–1669. PubMed PMC

Arosio G, et al. Synergistic drug combinations prevent resistance in ALK+ anaplastic large cell lymphoma. Cancers (Basel) 2021;13:1–17. doi: 10.3390/cancers13174422. PubMed DOI PMC

Tucker ER, et al. Combination therapies targeting ALK-aberrant neuroblastoma in preclinical models. Clin. Cancer Res. 2023;29:1317–1331. doi: 10.1158/1078-0432.CCR-22-2274. PubMed DOI PMC

Westerhout EM, et al. Mesenchymal-type neuroblastoma cells escape ALK inhibitors. Cancer Res. 2022;82:484–496. doi: 10.1158/0008-5472.CAN-21-1621. PubMed DOI

Mossë YP, et al. Identification of ALK as the major familial neuroblastoma predisposition gene. Nature. 2008;455:930–935. doi: 10.1038/nature07261. PubMed DOI PMC

Zafar A, et al. Molecular targeting therapies for neuroblastoma: progress and challenges. Med. Res. Rev. 2021;41:961–1021. doi: 10.1002/med.21750. PubMed DOI PMC

Brady SW, et al. Pan-neuroblastoma analysis reveals age- and signature-associated driver alterations. Nat. Commun. 2020;11:1–13. doi: 10.1038/s41467-020-18987-4. PubMed DOI PMC

Almstedt E, et al. Integrative discovery of treatments for high-risk neuroblastoma. Nat. Commun. 2020;11:71. doi: 10.1038/s41467-019-13817-8. PubMed DOI PMC

Matthay, K. K. et al. Neuroblastoma. Nat. Rev. Dis. Prim. 2, 16078 (2016). PubMed

Schleiermacher G, Janoueix-Lerosey I, Delattre O. Recent insights into the biology of neuroblastoma. Int. J. Cancer. 2014;135:2249–2261. doi: 10.1002/ijc.29077. PubMed DOI

Brodeur GM. Spontaneous regression of neuroblastoma. Cell Tissue Res. 2018;372:277–286. doi: 10.1007/s00441-017-2761-2. PubMed DOI PMC

Pinto NR, et al. Advances in risk classification and treatment strategies for neuroblastoma. J. Clin. Oncol. 2015;33:3008–3017. doi: 10.1200/JCO.2014.59.4648. PubMed DOI PMC

Bresler SC, et al. ALK mutations confer differential oncogenic activation and sensitivity to ALK inhibition therapy in neuroblastoma. Cancer Cell. 2014;26:682–694. doi: 10.1016/j.ccell.2014.09.019. PubMed DOI PMC

Bellini A, et al. Frequency and prognostic impact of alk amplifications and mutations in the european neuroblastoma study group (siopen) high-risk neuroblastoma trial (hr-nbl1) J. Clin. Oncol. 2021;39:3377–3390. doi: 10.1200/JCO.21.00086. PubMed DOI PMC

Ackermann S, et al. A mechanistic classification of clinical phenotypes in neuroblastoma. Science. 2018;362:1165–1170. doi: 10.1126/science.aat6768. PubMed DOI PMC

Pearson ADJ, et al. Second paediatric strategy forum for anaplastic lymphoma kinase (ALK) inhibition in paediatric malignancies: ACCELERATE in collaboration with the European Medicines Agency with the participation of the Food and Drug Administration. Eur. J. Cancer. 2021;157:198–213. doi: 10.1016/j.ejca.2021.08.022. PubMed DOI

Goldsmith KC, et al. Lorlatinib with or without chemotherapy in ALK-driven refractory/relapsed neuroblastoma: phase 1 trial results. Nat. Med. 2023;29:1092–1102. doi: 10.1038/s41591-023-02297-5. PubMed DOI PMC

Facchinetti F, et al. Crizotinib-resistant ROS1 mutations reveal a predictive kinase inhibitor sensitivity model for ROS1- and ALK-rearranged lung cancers. Clin. Cancer Res. 2016;22:5983–5991. doi: 10.1158/1078-0432.CCR-16-0917. PubMed DOI

Gadgeel SM, et al. Safety and activity of alectinib against systemic disease and brain metastases in patients with crizotinib-resistant ALK-rearranged non-small-cell lung cancer (AF-002JG): results from the dose-finding portion of a phase 1/2 study. Lancet Oncol. 2014;15:1119–1128. doi: 10.1016/S1470-2045(14)70362-6. PubMed DOI

Passerini CG, et al. Crizotinib in advanced, chemoresistant anaplastic lymphoma kinase-positive lymphoma patients. J. Natl. Cancer Inst. 2014;106:2–5. PubMed

Shalem O, et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science= 2014;343:84–87. doi: 10.1126/science.1247005. PubMed DOI PMC

Sanjana NE, Shalem O, Zhang F. Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods. 2014;11:783–784. doi: 10.1038/nmeth.3047. PubMed DOI PMC

Subramanian A, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA. 2005;102:15545–15550. doi: 10.1073/pnas.0506580102. PubMed DOI PMC

Liberzon A, et al. Molecular signatures database (MSigDB) 3.0. Bioinformatics. 2011;27:1739–1740. doi: 10.1093/bioinformatics/btr260. PubMed DOI PMC

Liu T, et al. Exceptional response to the ALK and ROS1 inhibitor lorlatinib and subsequent mechanism of resistance in relapsed ALK F1174L-mutated neuroblastoma. Cold Spring Harb. Mol. Case Stud. 2021;7:1–15. doi: 10.1101/mcs.a006064. PubMed DOI PMC

Hrustanovic G, et al. RAS-MAPK dependence underlies a rational polytherapy strategy in EML4-ALK–positive lung cancer. Nat. Med. 2015;21:1038–1047. doi: 10.1038/nm.3930. PubMed DOI PMC

Valencia-Sama I, et al. NRAS status determines sensitivity to SHP2 inhibitor combination therapies targeting the RAS-MAPK pathway in neuroblastoma. Cancer Res. 2020;80:3413–3423. doi: 10.1158/0008-5472.CAN-19-3822. PubMed DOI

TARGET. TARGET, 2018, phs000218 (Study ID phs000467). https://ocg.cancer.gov/programs/target (2018).

Wang HQ, et al. Combined ALK and MDM2 inhibition increases antitumor activity and overcomes resistance in human ALK mutant neuroblastoma cell lines and xenograft models. Elife. 2017;6:1–19. doi: 10.7554/eLife.17137. PubMed DOI PMC

Yan J, et al. Analysis of NRAS gain in 657 patients with melanoma and evaluation of its sensitivity to a MEK inhibitor. Eur. J. Cancer. 2018;89:90–101. doi: 10.1016/j.ejca.2017.11.011. PubMed DOI

FDA. FDA Approves first treatment for hutchinson-gilford progeria syndrome and some progeroid laminopathies https://www.fda.gov/news-events/press-announcements/fda-approves-first-treatment-hutchinson-gilford-progeria-syndrome-and-some-progeroid-laminopathies (2020).

Umapathy G, et al. MEK inhibitor trametinib does not prevent the growth of anaplastic lymphoma kinase (ALK)-addicted neuroblastomas. Sci. Signal. 2017;10:eaam7550. doi: 10.1126/scisignal.aam7550. PubMed DOI

Dhillon S. Lonafarnib: first approval. Drugs. 2021;81:283–289. doi: 10.1007/s40265-020-01464-z. PubMed DOI PMC

Ready NE, et al. Phase I study of the farnesyltransferase inhibitor lonafarnib with weekly paclitaxel in patients with solid tumors. Clin. Cancer Res. 2007;13:576–583. doi: 10.1158/1078-0432.CCR-06-1262. PubMed DOI

Katayama R, et al. Mechanisms of acquired crizotinib resistance in ALK- rearranged lung cancers. Bone. 2012;23:1–7. PubMed PMC

van der Velden DL, et al. The drug rediscovery protocol facilitates the expanded use of existing anticancer drugs. Nature. 2019;574:127–131. doi: 10.1038/s41586-019-1600-x. PubMed DOI

New Approaches to Neuroblastoma Therapy Consortium. NANT 2015-02: A Phase 1 Study of Lorlatinib (PF-06463922). https://clinicaltrials.gov/ct2/show/NCT03107988?term=ALK&cond=Neuroblastoma&draw=2&rank=4. (2024).

Pfizer. An investigational drug, crizotinib (PF-02341066), is being studied in tumors, except non-small cell lung cancer, that are positive for anaplastic lymphoma kinase (ALK). https://clinicaltrials.gov/ct2/show/NCT01121588?term=ALK&cond=Neuroblastoma&draw=2&rank=9. (2023).

National Cancer Institute. Ensartinib in treating patients with relapsed or refractory advanced solid tumors, non-Hodgkin lymphoma, or histiocytic disorders with ALK or ROS1 genomic alterations (a pediatric MATCH Treatment Trial). https://clinicaltrials.gov/ct2/show/NCT03213652?term=ALK&cond=Neuroblastoma&draw=2&rank=7. (2024).

BFox Chase Cancer Center. Brigatinib in relapsed or refractory ALK-positive anaplastic large cell lymphoma. https://clinicaltrials.gov/ct2/show/NCT03719898. (2024).

National Cancer Institute. Brentuximab vedotin or crizotinib and combination chemotherapy in treating patients with newly diagnosed stage II-IV anaplastic large cell lymphoma. https://classic.clinicaltrials.gov/ct2/show/NCT01979536 (2024).

Debruyne DN, et al. BORIS promotes chromatin regulatory interactions in treatment-resistant cancer cells. Nature. 2019;572:676–680. doi: 10.1038/s41586-019-1472-0. PubMed DOI PMC

Debruyne DN, et al. ALK inhibitor resistance in ALKF1174L-driven neuroblastoma is associated with AXL activation and induction of EMT. Oncogene. 2016;35:3681–3691. doi: 10.1038/onc.2015.434. PubMed DOI PMC

Berko ER, et al. Circulating tumor DNA reveals mechanisms of lorlatinib resistance in patients with relapsed/refractory ALK-driven neuroblastoma. Nat. Commun. 2023;14:2601. doi: 10.1038/s41467-023-38195-0. PubMed DOI PMC

Wang Z, Lei H, Sun Q. MicroRNA-141 and its associated gene FUS modulate proliferation, migration and cisplatin chemosensitivity in neuroblastoma cell lines. Oncol. Rep. 2016;35:2943–2951. doi: 10.3892/or.2016.4640. PubMed DOI PMC

Liu S, Liu B. Overexpression of nitrogen permease regulator like-2 (NPRL2) enhances sensitivity to irinotecan (CPT-11) in colon cancer cells by activating the DNA damage checkpoint pathway. Med. Sci. Monit. 2018;24:1424–1433. doi: 10.12659/MSM.909186. PubMed DOI PMC

Yu MJ, Zhao N, Shen H, Wang H. Long noncoding RNA MRPL39 inhibits gastric cancer proliferation and progression by directly targeting miR-130. Genet. Test Mol. Biomarkers. 2018;22:656–663. doi: 10.1089/gtmb.2018.0151. PubMed DOI

Kim BR, et al. The anti-tumor activator sMEK1 and paclitaxel additively decrease expression of HIF-1α and VEGF via mTORC1-S6K/4E-BP-dependent signaling pathways. Oncotarget. 2014;5:6540–6551. doi: 10.18632/oncotarget.2119. PubMed DOI PMC

Liu, Q., Zhou, Q. & Zhong, P. circ_0067934 increases bladder cancer cell proliferation, migration and invasion through suppressing miR‑1304 expression and increasing Myc expression levels. Exp. Ther. Med. 19, 3751–3759 (2020). PubMed PMC

Du W, et al. Circ-PRMT5 promotes gastric cancer progression by sponging miR-145 and miR-1304 to upregulate MYC. Artif. Cells Nanomed. Biotechnol. 2019;47:4120–4130. doi: 10.1080/21691401.2019.1671857. PubMed DOI

Schirripa M, et al. Early modifications of circulating microRNAs levels in metastatic colorectal cancer patients treated with regorafenib. Pharmacogenomics J. 2019;19:455–464. doi: 10.1038/s41397-019-0075-3. PubMed DOI

Di Paolo D, et al. Combined replenishment of miR-34a and let-7b by targeted nanoparticles inhibits tumor growth in neuroblastoma preclinical models. Small. 2020;16:1–15. doi: 10.1002/smll.201906426. PubMed DOI

Marengo B, et al. Etoposide-resistance in a neuroblastoma model cell line is associated with 13q14.3 mono-allelic deletion and miRNA-15a/16-1 down-regulation. Sci. Rep. 2018;8:1–15. doi: 10.1038/s41598-018-32195-7. PubMed DOI PMC

Pucci P. Combination therapy and noncoding RNAs: a new era of cancer personalized medicine. Epigenomics. 2022;14:117–120. doi: 10.2217/epi-2021-0405. PubMed DOI

Naveed A, et al. NEAT1 polyA-modulating antisense oligonucleotides reveal opposing functions for both long non-coding RNA isoforms in neuroblastoma. Cell. Mol. Life Sci. 2021;78:2213–2230. doi: 10.1007/s00018-020-03632-6. PubMed DOI PMC

Li CG, et al. MicroRNA-1304 suppresses human non-small cell lung cancer cell growth in vitro by targeting heme oxygenase-1. Acta Pharmacol. Sin. 2017;38:110–119. doi: 10.1038/aps.2016.92. PubMed DOI PMC

Kumar V, et al. Pharmacokinetics and biodistribution of polymeric micelles containing miRNA and small-molecule drug in orthotopic pancreatic tumor-bearing mice. Theranostics. 2018;8:4033–4049. doi: 10.7150/thno.24945. PubMed DOI PMC

Daige CL, et al. Systemic delivery of a miR34a mimic as a potential therapeutic for liver cancer. Mol. Cancer Ther. 2014;13:2352–2360. doi: 10.1158/1535-7163.MCT-14-0209. PubMed DOI

van Zandwijk N, et al. Safety and activity of microRNA-loaded minicells in patients with recurrent malignant pleural mesothelioma: a first-in-man, phase 1, open-label, dose-escalation study. Lancet Oncol. 2017;18:1386–1396. doi: 10.1016/S1470-2045(17)30621-6. PubMed DOI

Hong DS, et al. Phase 1 study of MRX34, a liposomal miR-34a mimic, in patients with advanced solid tumours. Br. J. Cancer. 2020;122:1630–1637. doi: 10.1038/s41416-020-0802-1. PubMed DOI PMC

Khan AA, et al. Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs. Nat. Biotechnol. 2009;27:549–555. doi: 10.1038/nbt.1543. PubMed DOI PMC

Grimm D, et al. Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature. 2006;441:537–541. doi: 10.1038/nature04791. PubMed DOI

Zhang S, Cheng Z, Wang Y, Han T. The risks of mirna therapeutics: In a drug target perspective. Drug Des. Devel. Ther. 2021;15:721–733. doi: 10.2147/DDDT.S288859. PubMed DOI PMC

Zhou B, Cox AD. Up-front polytherapy for ALK-positive lung cancer. Nat. Med. 2015;21:974–975. doi: 10.1038/nm.3942. PubMed DOI

Kwong LN, et al. Oncogenic NRAS signaling differentially regulates survival and proliferation in melanoma. Nat. Med. 2012;18:1503–1510. doi: 10.1038/nm.2941. PubMed DOI PMC

Flex E, et al. Activating mutations in RRAS underlie a phenotype within the RASopathy spectrum and contribute to leukaemogenesis. Hum. Mol. Genet. 2014;23:4315–4327. doi: 10.1093/hmg/ddu148. PubMed DOI PMC

Jameson KatherineL, et al. IQGAP1 scaffold-kinase interaction blockade selectively targets RAS-MAP kinase–driven tumors. Nat. Med. 2013;19:626–630. doi: 10.1038/nm.3165. PubMed DOI PMC

Roy M, Li Z, Sacks DB. IQGAP1 is a scaffold for mitogen-activated protein kinase signaling. Mol. Cell. Biol. 2005;25:7940–7952. doi: 10.1128/MCB.25.18.7940-7952.2005. PubMed DOI PMC

Dietrich P, et al. Neuroblastoma RAS viral oncogene homolog (NRAS) is a novel prognostic marker and contributes to sorafenib resistance in hepatocellular carcinoma. Neoplasia (United States) 2019;21:257–268. doi: 10.1016/j.neo.2018.11.011. PubMed DOI PMC

Linardou H, et al. Assessment of somatic k-RAS mutations as a mechanism associated with resistance to EGFR-targeted agents: a systematic review and meta-analysis of studies in advanced non-small-cell lung cancer and metastatic colorectal cancer. Lancet Oncol. 2008;9:962–972. doi: 10.1016/S1470-2045(08)70206-7. PubMed DOI

Ninomiya K, et al. MET or NRAS amplification is an acquired resistance mechanism to the third-generation EGFR inhibitor naquotinib. Sci. Rep. 2018;8:1–11. doi: 10.1038/s41598-018-20326-z. PubMed DOI PMC

Berlak M, et al. Mutations in ALK signaling pathways conferring resistance to ALK inhibitor treatment lead to collateral vulnerabilities in neuroblastoma cells. Mol. Cancer. 2022;21:1–19. doi: 10.1186/s12943-022-01583-z. PubMed DOI PMC

Asati V, Mahapatra DK, Bharti SK. PI3K/Akt/mTOR and Ras/Raf/MEK/ERK signaling pathways inhibitors as anticancer agents: Structural and pharmacological perspectives. Eur. J. Med. Chem. 2016;109:314–341. doi: 10.1016/j.ejmech.2016.01.012. PubMed DOI

Yaari S, et al. Disruption of cooperation between Ras and MycN in human neuroblastoma cells promotes growth arrest. Clin. Cancer Res. 2005;11:4321–4330. doi: 10.1158/1078-0432.CCR-04-2071. PubMed DOI

Bachireddy P, Bendapudi PK, Felsher D. Getting at MYC through RAS. Clin. Cancer Res. 2005;11:4278–4281. doi: 10.1158/1078-0432.CCR-05-0534. PubMed DOI

Healy JR, et al. Limited anti-tumor activity of combined BET and MEK inhibition in neuroblastoma. Pediatr. Blood Cancer. 2021;67:1–15. PubMed PMC

Zhang Xiaoling, et al. Critical role for GAB2 in neuroblastoma pathogenesis through the promotion of SHP2/MYCN cooperation. Cell Rep. 2017;18:2932–2942. doi: 10.1016/j.celrep.2017.02.065. PubMed DOI PMC

Hart LS, et al. Preclinical therapeutic synergy of MEK1/2 and CDK4/6 inhibition in neuroblastoma. Clin. Cancer Res. 2017;23:1785–1796. doi: 10.1158/1078-0432.CCR-16-1131. PubMed DOI

Li W, et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 2014;15:554. doi: 10.1186/s13059-014-0554-4. PubMed DOI PMC

LOEWE, S. The problem of synergism and antagonism of combined drugs. Arzneimittelforschung3, 285–290 (1953). PubMed

Zheng S, et al. SynergyFinder Plus: toward better interpretation and annotation of drug combination screening datasets. Genomics, Proteom. Bioinform. 2022;20:587–596. doi: 10.1016/j.gpb.2022.01.004. PubMed DOI PMC