Early and late effects of pharmacological ALK inhibition on the neuroblastoma transcriptome

Background: Neuroblastoma is an aggressive childhood malignancy of the sympathetic nervous system. Despite multi-modal therapy, survival of high-risk patients remains disappointingly low, underscoring the need for novel treatment strategies. The discovery of ALK activating mutations opened the way to precision treatment in a subset of these patients. Previously, we investigated the transcriptional effects of pharmacological ALK inhibition on neuroblastoma cell lines, six hours after TAE684 administration, resulting in the 77-gene ALK signature, which was shown to gradually decrease from 120 minutes after TAE684 treatment, to gain deeper insight into the molecular effects of oncogenic ALK signaling. Aim: Here, we further dissected the transcriptional dynamic profiles of neuroblastoma cells upon TAE684 treatment in a detailed timeframe of ten minutes up to six hours after inhibition, in order to identify additional early targets for combination treatment. Results: We observed an unexpected initial upregulation of positively regulated MYCN target genes following subsequent downregulation of overall MYCN activity. In addition, we identified adrenomedullin (ADM), previously shown to be implicated in sunitinib resistance, as the earliest response gene upon ALK inhibition. Conclusions: We describe the early and late effects of ALK inhibitor TAE684 treatment on the neuroblastoma transcriptome. The observed unexpected upregulation of ADM warrants further investigation in relation to putative ALK resistance in neuroblastoma patients currently undergoing ALK inhibitor treatment

Green tea compound EGCG activates HBP1: an ALK downstream suppressor gene in neuroblastoma

The discovery of activating mutations in the ALK gene in primary neuroblastomas (NB) opened new opportunities to treat children with these tumors. Clinical trials for small molecule ALK inhibitors show that tumors acquire resistance to these inhibitors. This illustrates the need for additional compounds targeting downstream genes or alternative pathways. In order to identify new vulnerable nodes for targeted (and combinatorial) therapy, we aimed at deciphering downstream ALK signaling through expression profiling of a panel of 10 NB cell lines (ALK wild type, ALKF1174L, ALKR1275Q mutant or amplified) treated with ALK inhibitor NVP-TAE684. In depth data-mining lead to the identification of a 79-gene signature that recapitulates the transcriptional response upon ALK inhibition. Functional annotation analysis clearly showed that this signature is mainly composed of genes involved in MAPK/ERK, PI3K/AKT/mTOR, MYC/MYCN and neuronal differentiation signaling. Most interestingly iRegulon analysis (http://iregulon.aertslab.org) pinpointed transcription repressor HBP1 as a hub gene that acts downstream of ALK. In previous studies it is described that also MYC(N) regulates HBP1, and that it probably acts through repression by downstream miR-17-92. Indeed, we could confirm HBP1 repression by miR-17-92 in a neuroblastoma cell line with inducible miR-17-92 (SHEP-miR-17-92). Furthermore, in vitro studies in NB cell lines with stable HBP1 overexpression demonstrated reduction of cell growth and increase of cell death. Most interestingly, we identified a green tea component EGCG that can up-regulate HBP1 expression in cell lines and that lead to cell death. In conclusion, this study unraveled the transcriptional consequences of aberrant ALK signaling in human NB cell lines. Moreover, we identified HBP1 as a targetable component of ALK downstream signaling

Dual targeting of ALK and RET: establishing a novel basis for the treatment of neuroblastoma

Background: Neuroblastoma (NB) is a pediatric cancer of the developing sympathetic nervous system for which survival rates for high-risk patients are still unsatisfactory. Moreover, current treatment is very harsh and toxic, causing severe short and longterm side effects. Therefore, the search for more effective and less toxic targeted drugs remains at the forefront of NB research. Activating mutations in the tyrosine kinase domain of the ALK transmembrane receptor are found in the majority of hereditary NB and occur as somatic defects in 7–10% of sporadic cases. Recently we showed that ALK mutations emerge or are selected for in relapsed NB. Small molecule inhibitors are available for targeted therapy in mutant ALK positive NB patients and open new possibilities for understanding the functional pathways through which ALK exerts its oncogenicity. With the aim of selecting novel nodes for therapy intervention in NB, we have generated a 77-gene signature list reminiscent of mutant ALK activation in NB cells. Cross species genomics analysis of MYCN and ALKF1174 driven tumors and targeted ALK inhibition studies in vitro indicated that ALKF1174L leads to upregulation of the oncogenic tyrosine kinase RET in mouse models of NB. Inhibition of mutant ALK in NB cells results in robust downregulation of RET, indicating a strong functional relationship between these two oncogenes. We have further determined the efficacy of dual versus single targeting of mutant ALK and RET in NB cell lines in vitro and in vivo. Material and Methods: Single and combinatorial targeting of mutant ALK and RET was performed on a panel of wild type, ALKF1174L and ALKR1275Q NB cell lines. Cell proliferation and apoptosis were assessed using Cell Titer Glow and Caspase Glo assays (Promega). Targeting compounds were selected based on their clinical applicability. Crizotinib (ALK-inhibitor) was combined with vandetanib, cabozantinib or sorafenib (RET-inhibitors). The combination index method by Chou and Talalay was used to determine an additive or synergistic effect between the drug combinations. In vivo experiments were performed using xenografted ALKR1275Q NB cells, carrying luciferase and GFP as reporter genes (CLBGA/Luc-GFP). Tumor response to drug treatment was determined by daily caliper measurements and bioluminescence imagining. Results: Our preliminary results indicate that dual RET and ALK inhibition by crizotinib and vandetanib in vitro has a very strong anti-proliferative effect on cells compared to either drug alone. We have established the growth parameters and dose response curve to crizotinib of the xenografted NB cell line CLBGA/Luc-GFP in immunocompromised mice. We show that targeting ALKR1275Q in vivo results in initial regression of the tumor, followed by regrowth upon discontinuation of the treatment. Further experiments are ongoing to determine the activation status of RET in the resistant cells and to establish the appropriate experimental parameters for combinatorial ALK and RET inhibition in vivo. Conclusions: Single compound treated tumors typically relapse following an initial response as is also the case for mutant ALK cells, either through acquisition of novel ALK mutations or mutations in downstream effectors of the ALK or other interfering signaling pathways. While higher affinity ALK inhibitors are underway, targeting the RET signaling pathway emerges as an important option for novel combination therapy which may render higher efficacy to future treatment in primary and relapsed NB patients

Synergistic combined molecular treatment targeting the HBP1 tumor suppressor gene in neuroblastoma

Neuroblastoma (NB) is a devastating childhood tumor of the peripheral nervous system. Poor survival rates warrant development of more efficient and less toxic treatment. In search for opportunities for combination molecular therapy in ALK-mutated NB, we established a 77-gene signature that recapitulates the transcriptional response upon ALK inhibition. Functional annotation analysis revealed predominantly MAPK/ERK, PI3K/AKT/mTOR, MYC/MYCN and neuronal differentiation signaling components. Amongst these, we identified HBP1, a transcriptional repressor and tumor suppressor gene. Based on a study describing HBP1 as a target of the PI3K/FOXO pathway and our observation of PI3K/AKT activation by ALK, we established an ALK-PI3K/AKT-FOXO3-HBP1 regulatory pathway in NB. In keeping with this, treatment with BEZ-235, an AKT/mTOR inhibitor, leads to loss of AKT and FOXO3 phosphorylation and subsequent HBP1 upregulation. Interestingly, we also found a functional connection between MYCN and HBP1. Given this interconnection, we hypothesized that combination treatment directly targeting both proteins could offer novel opportunities for chemotherapy-resistant tumors. To test this, we analysed effects on mouse xenografts with EGCG (epigallocatechin gallate) as tool compound for upregulating HBP1 levels in combination with the BRD4 inhibitor JQ1, effective for MYCN-amplified NB. Tumor growth was significantly delayed in the combination-treated group. Next, we reasoned that combining JQ1 with an histone deacetylase (HDAC) inhibitor could be effective, given the role of HDAC in the HBP1 repressive function. In a first proof-of-principle test, we observed strong synergistic effects with complete growth arrest in several cell lines. In conclusion, we identified HBP1 as a novel potent drugable target in NB

Integrated proximal proteomics reveals IRS2 as a determinant of cell survival in ALK-driven neuroblastoma

Oncogenic anaplastic lymphoma kinase (ALK) is one of the few druggable targets in neuroblastoma, and therapy resistance to ALK-targeting tyrosine kinase inhibitors (TKIs) comprises an inevitable clinical challenge. Therefore, a better understanding of the oncogenic signaling network rewiring driven by ALK is necessary to improve and guide future therapies. Here, we performed quantitative mass spectrometry-based proteomics on neuroblastoma cells treated with one of three clinically relevant ALK TKIs (crizotinib, LDK378, or lorlatinib) or an experimentally used ALK TKI (TAE684) to unravel aberrant ALK signaling pathways. Our integrated proximal proteomics (IPP) strategy included multiple signaling layers, such as the ALK interactome, phosphotyrosine interactome, phosphoproteome, and proteome. We identified the signaling adaptor protein IRS2 (insulin receptor substrate 2) as a major ALK target and an ALK TKI-sensitive signaling node in neuroblastoma cells driven by oncogenic ALK. TKI treatment decreased the recruitment of IRS2 to ALK and reduced the tyrosine phosphorylation of IRS2. Furthermore, siRNA-mediated depletion of ALK or IRS2 decreased the phosphorylation of the survival-promoting kinase Akt and of a downstream target, the transcription factor FoxO3, and reduced the viability of three ALK-driven neuroblastoma cell lines. Collectively, our IPP analysis provides insight into the proximal architecture of oncogenic ALK signaling by revealing IRS2 as an adaptor protein that links ALK to neuroblastoma cell survival through the Akt-FoxO3 signaling axis

ETV5 functionally connects ALK and CXCR4 signaling in neuroblastoma

Activating ALK mutations occur in 10% of neuroblastomas (NB) and represent an important druggable target for more effective treatment of high-risk patients. In order to design effective novel targeted therapeutic approaches, gaining detailed insights into downstream ALK signaling is crucial. We and others identified PI3K/AKT and RAS/MAPK as major downstream signaling axes. Also, we connected FOXO3a controlled RET expression to the PI3K/AKT axis. Here, using multiple ALK activating and inhibiting cell models, we firmly establish ETV5 as a major RAS/MAPK downstream target upregulated through mutant ALK. ETV5 is known to act as regulator of epithelial-mesenchymal transition (EMT) and controls stem cell properties and neuronal cell fate decisions. Knockdown of ETV5 reduced the clonogenic potential and growth of NB cells in vitro and in vivo. RNAseq transcriptome profiling following ETV5 knock down provided an ETV5 signature score which identifies patients with poor overall survival and was enriched in gene sets controlling EMT in keeping with observed reduced invasive properties in ETV5 depleted NB cell lines. Finally, the chemokine receptor CXCR4 emerged as a crucial ETV5 target gene thus opening unexpected novel opportunities for drugging, as CXCR4 inhibitors are available. Our data highlight ETV5 as an intrinsic component of ALK downstream and RAS/MAPK signaling in NB. The presence of RAS/MAPK and acquired ALK mutations in relapsed NB tumors highlights the significance of the ETV5 signaling pathway in NB pathogenesis. Moreover, ETV5 provides a functional link between the ALK and chemotaxis pathways involved in cancer metastasis and identifies CXCR4 as novel drug target

The HBP1 tumor suppressor is a druggable ALK downregulated gene controlling MYCN activity

ALK is an important druggable target in ALK mutant neuroblastoma (NB). In this study, we sought novel vulnerable nodes downstream of ALK for combinatorial therapy. First, we identified a 79-gene signature that recapitulates the transcriptional response upon ALK inhibition based on transcriptome profiling of ALK wild type, ALKF1174L or ALKR1275Q mutant and ALK amplified NB cell lines following ALK inhibition using NVP-TAE684, LDK-378, X-396 and Crizotinib. This signature was validated in primary tumor samples and in the SK-N-AS cell line with regulated expression of ALK wild type, ALKF1174L and ALKR1275Q constructs. The majority of the mutant ALK dependent downstream signaling components were implicated in MAPK/ERK, PI3K/AKT/mTOR, MYC/MYCN and neuronal differentiation signaling. Bioinformatic analysis using the iRegulon Cytoscape plugin (http://iregulon.aertslab.org) resulted in the identification of the transcriptional repressor HBP1 as a hub gene, acting downstream of ALK. HPB1 is a known negative regulator of MYC/MYCN activity. Using MYCN and miR-17-92 inducible SHEP model systems, we established a MYCN/miR-17-92/HBP1 positive feedback regulatory loop in which MYCN represses the negative control of HBP1 through upregulation of miR-17-92 which targets the HBP1 3'UTR. Next, we showed that stable HBP1 overexpression inhibits growth and induces apoptosis of NB cells. Finally, we explored the effects of the green tea compound epigallocatechin gallate (EGCG) on HBP1 upregulation in NB cells. EGCG treatment induced HBP1 expression and had a strong negative effect on cell growth and survival. EGCG/ALK inhibitor combination treatment in a panel of cell lines showed clear additive effects. In conclusion, we identified HBP1 as a novel important ALK downregulated gene controlling MYCN activity, further stressing the important interconnection between oncogenic MYCN and ALK signaling. EGCG upregulates HBP1 levels and is a strong candidate for further in vivo testing for additive or synergistic effects with ALK inhibitors or MYCN targeting compounds such as JQ1