Ganesh Umapathy

The kinase ALK stimulates the kinase ERK5 to promote the expression of the oncogene MYCN in neuroblastoma

Targeting the kinase ERK5 may disrupt the activation of an oncogenic transcription factor in a subset of neuroblastoma patients. A New Target in Neuroblastoma Neuroblastoma is a common and aggressive pediatric cancer caused by various molecular abnormalites. Similar to other cancers, poor prognosis correlates with increased abundance or activation of the cell surface receptor tyrosine kinase ALK or increased abundance of the transcription factor MYCN. An ALK inhibitor used in the clinic is not wholly effective, and there are no therapies to directly target MYCN. Umapathy et al. found that ALK stimulated the expression of the gene encoding MYCN through a pathway involving several kinases in patient tumor cells. Targeting one of these kinases, ERK5, decreased the abundance of MYCN and suppressed proliferation in ALK-positive neuroblastoma cells in culture. Combined inhibition of ALK and ERK5 was more effective than the ALK inhibitor alone in limiting tumor growth in a mouse model. Thus, ERK5 represents a new target for treating ALK-driven cancers. Anaplastic lymphoma kinase (ALK) is an important molecular target in neuroblastoma. Although tyrosine kinase inhibitors abrogating ALK activity are currently in clinical use for the treatment of ALK-positive (ALK+) disease, monotherapy with ALK tyrosine kinase inhibitors may not be an adequate solution for ALK+ neuroblastoma patients. Increased expression of the gene encoding the transcription factor MYCN is common in neuroblastomas and correlates with poor prognosis. We found that the kinase ERK5 [also known as big mitogen-activated protein kinase (MAPK) 1 (BMK1)] is activated by ALK through a pathway mediated by phosphoinositide 3-kinase (PI3K), AKT, MAPK kinase kinase 3 (MEKK3), and MAPK kinase 5 (MEK5). ALK-induced transcription of MYCN and stimulation of cell proliferation required ERK5. Pharmacological or RNA interference–mediated inhibition of ERK5 suppressed the proliferation of neuroblastoma cells in culture and enhanced the antitumor efficacy of the ALK inhibitor crizotinib in both cells and xenograft models. Together, our results indicate that ERK5 mediates ALK-induced transcription of MYCN and proliferation of neuroblastoma, suggesting that targeting both ERK5 and ALK may be beneficial in neuroblastoma patients.

Phosphoproteomic analysis of anaplastic lymphoma kinase (ALK) downstream signaling pathways identifies signal transducer and activator of transcription 3 as a functional target of activated ALK in neuroblastoma cells

Activation of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase is a key oncogenic mechanism in a growing number of tumor types. In the majority of cases, ALK is activated by fusion with a dimerizing partner protein as a result of chromosomal translocation events, most studied in the case of the nucleophosmin–ALK and echinoderm microtubule‐associated protein‐like 4–ALK oncoproteins. It is now also appreciated that the full‐length ALK receptor can be activated by point mutations and by deletions within the extracellular domain, such as those observed in neuroblastoma. Several studies have employed phosphoproteomics approaches to find substrates of ALK fusion proteins. In this study, we used MS‐based phosphotyrosine profiling to characterize phosphotyrosine signaling events associated with the full‐length ALK receptor. A number of previously identified and novel targets were identified. One of these, signal transducer and activator of transcription 3 (STAT3), has previously been observed to be activated in response to oncogenic ALK signaling, but the significance of this in signaling from the full‐length ALK receptor has not been explored further. We show here that activated ALK robustly activates STAT3 on Tyr705 in a number of independent neuroblastoma cell lines. Furthermore, knockdown of STAT3 by RNA interference resulted in a reduction in myelocytomatosis neuroblastom (MYCN) protein levels downstream of ALK signaling. These observations, together with a decreased level of MYCN and inhibition of neuroblastoma cell growth in the presence of STAT3 inhibitors, suggest that activation of STAT3 is important for ALK signaling activity in neuroblastoma.

The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN.

ABSTRACT The first-in-class inhibitor of ALK, c-MET and ROS1, crizotinib (Xalkori), has shown remarkable clinical efficacy in treatment of ALK-positive non-small cell lung cancer. However, in neuroblastoma, activating mutations in the ALK kinase domain are typically refractory to crizotinib treatment, highlighting the need for more potent inhibitors. The next-generation ALK inhibitor PF-06463922 is predicted to exhibit increased affinity for ALK mutants prevalent in neuroblastoma. We examined PF-06463922 activity in ALK-driven neuroblastoma models in vitro and in vivo. In vitro kinase assays and cell-based experiments examining ALK mutations of increasing potency show that PF-06463922 is an effective inhibitor of ALK with greater activity towards ALK neuroblastoma mutants. In contrast to crizotinib, single agent administration of PF-06463922 caused dramatic tumor inhibition in both subcutaneous and orthotopic xenografts as well as a mouse model of high-risk neuroblastoma driven by Th-ALKF1174L/MYCN. Taken together, our results suggest PF-06463922 is a potent inhibitor of crizotinib-resistant ALK mutations, and highlights an important new treatment option for neuroblastoma patients. Summary: Our results suggest that PF-06463922 is a potent inhibitor of crizotinib-resistant ALK mutations, and highlights an important new treatment option for neuroblastoma patients.

PO-121 ALK dependant phosphoproteome and gene expression profiling of neuroblastoma cells

Introduction Receptor tyrosine kinases (RTKs) play important roles in cellular proliferation and differentiation. In addition, RTKs reveal oncogenic potential when their kinase activities are constitutively enhanced by point mutation, amplification, or rearrangement of the corresponding genes. We are investigating ALK (Anaplastic Lymphoma Kinase) tyrosine kinase in neuroblastoma (NB) - the most frequently occurring extracranial tumour in children, where ALK activating mutations are found. In this study we performed phosphoproteomic and a gene signature profiling of ALK activity from several different neuroblastoma cells comparing treatment with the first and third generations ALK inhibitors. Material and methods Crizotinib was one of the first ALK TKIs to be described; however the response in patients with ALK positive NB was poor. In contrast, robust and sustained clinical responses we seen in ALK rearranged IMT. Lorlatinib is a third generation ALK TKI with very positive responses in NSCLC. We treated two ALK addicted (CLB-BAR, CLB-GE) and one ALK non-addicted (SK-N-AS) NB cell lines with either crizotinib or lorlatinib and performed phosphoproteomic and RNAseq profiling to identify common targets for future combinational treatment for patients. Results and discussions Profiling of ALK addicted CLB-BAR and CLB-GE cell lines led to the identification of 3345 and 2252 phosphoproteins, respectively. Phosphorylation of the ALK receptor itself was decreased in both lines upon treatment with crizotinib or lorlatinib. Parallel RNAseq profiling was performed at 24 hours. Comparison with non-treated controls revealed 19 232 differently expressed genes. In ALK addicted cells phosphoproteomic analysis revealed more than 50 proteins that were dephosphorylated upon treatment with ALK TKIs. More than 400 genes were downregulated, and more than 600 were upregulated, upon treatment with either crizotinib or lorlatinib. As an outcome we obtained a list of predicted targets for further analysis. We focused on validation of downstream signalling molecules such as: FOXO3a/4, DUSP4 and ETV3/4. Conclusion In this study we established and compared phosphoproteomic and gene signature profiles of ALK activity in NB cells employing first and third generation ALK TKIs. This analysis has unveiled a number of leads for novel combinatorial treatment strategies for NB patients as well as an increased understanding ALK dependant signalling processes.

Clinical response of the novel activating ALK-I1171T mutation in neuroblastoma to the ALK inhibitor ceritinib

Tumors with anaplastic lymphoma kinase (ALK) fusion rearrangements, including non-small-cell lung cancer and anaplastic large cell lymphoma, are highly sensitive to ALK tyrosine kinase inhibitors (TKIs), underscoring the notion that such cancers are addicted to ALK activity. Although mutations in ALK are heavily implicated in childhood neuroblastoma, response to the ALK TKI crizotinib has been disappointing. Embryonal tumors in patients with DNA repair defects such as Fanconi anemia (FA) often have a poor prognosis, because of lack of therapeutic options. Here we report a child with underlying FA and ALK mutant high-risk neuroblastoma responding strongly to precision therapy with the ALK TKI ceritinib. Conventional chemotherapy treatment caused severe, life-threatening toxicity. Genomic analysis of the initial biopsy identified germline FANCA mutations as well as a novel ALK-I1171T variant. ALK-I1171T generates a potent gain-of-function mutant, as measured in PC12 cell neurite outgrowth and NIH3T3 transformation. Pharmacological inhibition profiling of ALK-I1171T in response to various ALK TKIs identified an 11-fold improved inhibition of ALK-I1171T with ceritinib when compared with crizotinib. Immunoaffinity-coupled LC-MS/MS phosphoproteomics analysis indicated a decrease in ALK signaling in response to ceritinib. Ceritinib was therefore selected for treatment in this child. Monotherapy with ceritinib was well tolerated and resulted in normalized catecholamine markers and tumor shrinkage. After 7.5 mo treatment, the residual primary tumor shrunk, was surgically removed, and exhibited hallmarks of differentiation together with reduced Ki67 levels. Clinical follow-up after 21 mo treatment revealed complete clinical remission including all metastatic sites. Therefore, ceritinib presents a viable therapeutic option for ALK-positive neuroblastoma.

Abstract B12: The ALK inhibitor PF-06463922 shows significant response as a single agent in ALK/MYCN driven models of neuroblastoma

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.