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Over the last decade Anaplastic Lymphoma Kinase (ALK), a receptor tyrosine kinase (RTK), has been identified as a fusion partner in a diverse variety of translocation events resulting in oncogenic signaling in many different cancer types. In tumors where full-length ALK RTK itself is mutated, such as neuroblastoma, the picture regarding the role of ALK as an oncogenic driver is less clear. Neuroblastoma is a complex and heterogeneous tumor that arises from the neural crest-derived peripheral nervous system. Although high-risk neuroblastoma is rare, it often relapses and becomes refractory to treatment. Thus, neuroblastoma accounts for 10-15% of all childhood cancer deaths. Since most cases are in children under the age of two, understanding the role and regulation of ALK during neural crest development is an important goal in addressing neuroblastoma tumorigenesis. An impressive array of tyrosine kinase inhibitors (TKIs) that act to inhibit ALK have been FDA approved for using in ALK-driven cancers. ALK TKIs bind differently within the ATP-binding pocket of the ALK kinase domain and have been associated with different resistance mutations within ALK itself that arise in response to therapeutic use, particularly in ALK fusion-positive NSCLC. This patient population has highlighted the importance of considering the relevant ALK TKI to be used for a given ALK mutant variant. In this poster we discuss ALK in neuroblastoma, as well as the use of ALK TKIs and other strategies to inhibit tumor growth. Current efforts combining novel approaches and increasing our understanding of the oncogenic role of ALK in neuroblastoma are aimed at improving efficacy of ALK TKIs as precision medicine options in the clinic. Citation Format: Jikui Guan, Diana Cervantes-Madrid, Joachim Siaw, Wasi Alam, Ganesh Umapathy, Marcus Borenäs, Dan Lind, Joanna Szydzik, Kathrin Pfeifer, Patricia Mendoza-Garcia, Ruth Palmer, Bengt Hallberg. Targeting anaplastic lymphoma kinase in neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A36.

The RAS-RAF-MEK-ERK pathway is a major player in initiation and progression of multiple cancers where it can be hyperactivated by upstream regulatory proteins, such as tyrosine kinases, phosphatases, and GTPases. Several inhibitors targeting the RAS-MAPK pathway exhibit anticancer activity and are approved as single agent in specific cancers. One such is the MEK inhibitor trametinib, which is included as a rational polytherapy strategy for treating EML4-ALK, EGFR, and K-RAS mutant lung cancer and neuroblastoma containing hyperactivating RAS-MAPK pathway mutations. In neuroblastoma, a heterogeneous disease, relapse cases display an increased rate of mutations of ALK, NRAS, and NF1 genes, leading to hyperactivation of RAS-MAPK signaling. Targeting the RAS-MAPK pathways offers attractive options for combinatorial treatment together with ALK inhibitors, since monotreatment has not yet produced strong clinical results in ALK-positive neuroblastoma patients. Here we investigate the response of ALK-positive neuroblastoma cell lines to MEK inhibition, employing trametinib. We show that pharmacologic inhibition of the MEK-ERK pathway in ALK-positive neuroblastoma cells results in increased levels of activation/phosphorylation of AKT and ERK5. This feedback response is regulated by the mTORC2 complex protein SIN1. Taken together, our results indicate that blocking MEK-ERK leads to increased activation of AKT signaling core in ALK-positive neuroblastoma, intensifying survival signals in these cells. Our results contraindicate use of MEK inhibitors as an effective single- and polytherapeutic strategy in ALK-positive neuroblastoma. Citation Format: Bengt Hallberg, Ganseh Umapathy, Jikui Guan, Dan Gustafsson, Niloufar Javanmardi, Diana Cervantes-Madrid, Anna Djoos, Tommy Martinsson, Ruth Palmer. Anaplastic lymphoma kinase addicted neuroblastoma cell lines are associated with growth upon treatment with MEK inhibitor trametinib [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B27.

Background: The protein kinase B (PKB/AKT) and RAF/MEK/ERK signaling pathways are activated in a wide range of human cancer types. In many cases, concomitant inhibition of both pathways is necessary to block proliferation and induce cell death and tumor shrinkage. Several feedback mechanisms have been described in which inhibition of one pathway leads to activation of a parallel signaling pathway, thereby decreasing the effectiveness of targeted monotherapies. Materials and Methods: In order to determine which signalling core should be blocked for combinatorial treatment, we treated a panel of EML4-ALK positive lung cancer cell lines using MEK-ERK inhibitors. Resazurin assay was performed to evaluate cell viability. Protein levels were determined using western blotting. Results: In this study, we describe a feedback mechanism in which MEK-ERK inhibition leads to increased activation of AKT signaling in EML4-ALK variant (E6a/b;A20) positive NSCLC cell lines. Interestingly, EML4-ALK variant (E13;A20) NSCLC cell lines responds to MEK-ERK inhibitors and shows synergism when combined with ALK tyrosine kinase inhibitors. We found that feedback response in EML4-ALK variant (E6a/b;A20) positive NSCLC cells was mediated by the mammalian target of rapamycin complex 2-associated protein SIN1, resulting in increased survival and proliferation that depended on AKT signaling. Conclusions: Taken together, these results elucidate an important feedback network and contraindicate the use of MEK inhibitors as effective therapeutic strategy in EML4-ALK variant (E6a/b;A20) positive NSCLC. Citation Format: Bengt Hallberg, Ruth H. Palmer, Ganesh Umapathy, Dan E. Gustafsson, Joachim T. Siaw, Wasi Alam, Jikui Guan, Robert Doebele, Anh T. Le, Andrea Doak. EML4-ALK variant E6a/b;A20 positive NSCLC cell lines are associated with growth upon blocking MEK-ERK pathway [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 5785.

The RAS-RAF-MEK-ERK pathway is a major player in initiation and progression of multiple cancers where it can be hyper-activated by upstream regulatory proteins, such as tyrosine kinases, phosphatases, and GTPases. Several inhibitors targeting the RAS-MAPK pathway exhibit anticancer activity and are approved as single agent in specific cancers. One such is the MEK inhibitor trametinib, which is included as a rational poly therapy strategy for treating EML4-ALK, EGFR, and K-RAS mutant lung cancer and neuroblastoma containing hyper-activating RAS-MAPK pathway mutations. In neuroblastoma, a heterogeneous disease, relapse cases display an increased rate of mutations of ALK, NRAS, NF1 genes, leading to hyper-activation of RAS-MAPK signaling. Targeting the RAS-MAPK pathways offers attractive options for combinatorial treatment together with ALK inhibitors, since monotreatment has not yet produced strong clinical results in ALK-positive neuroblastoma patients. Here we investigate the response of ALK-positive neuroblastoma cell lines to MEK inhibition, employing trametinib. We show that pharmacologic inhibition of the MEK-ERK pathway in ALK-positive neuroblastoma cells results in increased levels of activation/phosphorylation of AKT and ERK5. This feedback response is regulated by the mTORC2 complex protein SIN1. Taken together, our results indicate that blocking MEK-ERK leads to "increased activation" of AKT signalling core in ALK-positive neuroblastoma, intensifying survival signals in these cells. Our results contraindicate use of MEK inhibitors as effective single and poly-therapeutic strategy in ALK-positive neuroblastoma. Citation Format: Bengt Hallberg, Ganesh Umapathy, Diana Cervantes-Madrid, Jikui Guan, Dan E. Gustafsson, Ruth H. Palmer. Anaplastic lymphoma kinase addicted neuroblastoma cell lines are associated with growth upon treatment with MEK inhibitor trametinib [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B038.

Mutations in the kinase domain of Anaplastic Lymphoma Kinase (ALK) have been suggested to be important players in the genetics and progression of the childhood tumor neuroblastoma. Here we report the appearance of a novel ALK mutation in neuroblastoma together with other chromosomal aberrations that mediate neuroblastoma initiation and progression. Analyses of genomic tumor DNA from biopsy samples initially showed an 11q minus, 17q gain with a mutation of the ALK gene at protein position 1171, which mediate an amino acid change from isoleucine to threonine. We show that mutation of I1171 to threonine generates a potent gain-of-function mutant, as observed in two independent systems. Firstly, in PC12 cell lines expressing ALKI1171T display ligand independent activation of ALK, neurite outgrowth and further downstream signaling activation. Secondly, ALKI1171T meditate foci formation in a NIH3T3 transformation analysis. Finally, pharmacological inhibition of ALKI1171T employing ceritinib, an FDA approved ALK inhibitor show 14-fold better ability to abrogate ALKI1171T compared with crizotinib. Citation Format: Joachim T. Siaw, Jikui Guan, Tommy Martinsson, Magnus Hansson, Per Kogner, Ruth Palmer. A novel activating I1171T ALK mutation in neuroblastoma responds better to ceritinib compare to the first generation inhibitor crizotinib [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B40.

ALK inhibitors such as the ALK/MET/ROS1 inhibitor crizotinib (Xalkori) have shown clinical efficacy in a number of tumour types. However, in ALK positive neuroblastoma treatment with the ALK inhibitor crizotinib has proved more difficult, highlighting the exploration of new drugs as a clinical priority. A recent report of an increased percentage of ALK positive cases in the relapsed neuroblastoma patient population, together with the increased repertoire of ALK inhibitors now available, led to the investigation of alternative ALK inhibitors with potential for use in treatment of neuroblastoma. Here we report an investigation of the activity of a next generation ALK inhibitor in a range of in vitro and pre-clinical ALK driven neuroblastoma models. Initially PF-06463922 was tested in various neuroblastoma cell lines and a range of gain-of-function ALK neuroblastoma mutations were subsequently analyzed in more detail in engineered Ba/F3 and PC12 cell models and by in vitro kinase assays, comparing the effect of PF-06463922 in abrogating cell growth and induced pharmacodynamics markers of response with the ALK inhibitor crizotinib. These results clearly show PF-06463922 to be a superior inhibitor of ALK kinase activity inhibiting all neuroblastoma mutant ALK forms assayed. Finally, single agent oral administration of PF-06463922 lead to induction of apoptosis and a dramatic reduction in tumour volume in a genetically engineered mouse model of treatment-resistant high-risk neuroblastoma driven by aberrant expression of MYCN and activated ALK. Taken together, our results suggest that PF-06463922 represents an important potential step forward in the treatment of relapsed neuroblastoma with mutated ALK. Statement of significance: Our results together with PK/PD analysis of PF-06463922 suggest future clinical trial investigation of ALK positive neuroblastoma Citation Format: J. Guan, L. Danielson, D. Chand, Y. Jamin, K. Ruuth, E. Tucker, G. Umapathy, A. El Wakil, B. Witek, T. W. Johnson, T. Smeal, L. Chesler, R. H. Palmer, B. Hallberg. The ALK inhibitor PF-06463922 shows significant response as a single agent in ALK/MYCN driven models of neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B12.