Anaplastic lymphoma kinase (ALK) inhibitor response in neuroblastoma is highly correlated with ALK mutation status, ALK mRNA and protein levels

Background In pediatric neuroblastoma (NBL), high anaplastic lymphoma kinase (ALK) levels appear to be correlated with an unfavorable prognosis, regardless of ALK mutation status. This suggests a therapeutic role for ALK inhibitors in NBL patients. We examined the correlation between levels of ALK, phosphorylated ALK (pALK) and downstream signaling proteins and response to ALK inhibition in a large panel of both ALK mutated and wild type (WT) NBL cell lines. Methods We measured protein levels by western blot and ALK inhibitor sensitivity (TAE684) by viability assays in 19 NBL cell lines of which 6 had a point mutation and 4 an amplification of the ALK gene. Results ALK 220 kDa (p=0.01) and ALK 140 kDa (p= 0.03) protein levels were higher in ALK mutant than WT cell lines. Response to ALK inhibition was significantly correlated with ALK protein levels (p<0.01). ALK mutant cell lines (n=4) were 14,9 fold (p<0,01) more sensitive to ALK inhibition than eight WT cell lines. Conclusion NBL cell lines often express ALK at high levels and are responsive to ALK inhibitors. Mutated cell lines express ALK at higher levels, which may define their superior response to ALK inhibition

Wild-type ALK and activating ALK-R1275Q and ALK-F1174L mutations upregulate Myc and initiate tumor formation in murine neural crest progenitor cells.

The anaplastic lymphoma kinase (ALK) gene is overexpressed, mutated or amplified in most neuroblastoma (NB), a pediatric neural crest-derived embryonal tumor. The two most frequent mutations, ALK-F1174L and ALK-R1275Q, contribute to NB tumorigenesis in mouse models, and cooperate with MYCN in the oncogenic process. However, the precise role of activating ALK mutations or ALK-wt overexpression in NB tumor initiation needs further clarification. Human ALK-wt, ALK-F1174L, or ALK-R1275Q were stably expressed in murine neural crest progenitor cells (NCPC), MONC-1 or JoMa1, immortalized with v-Myc or Tamoxifen-inducible Myc-ERT, respectively. While orthotopic implantations of MONC-1 parental cells in nude mice generated various tumor types, such as NB, osteo/chondrosarcoma, and undifferentiated tumors, due to v-Myc oncogenic activity, MONC-1-ALK-F1174L cells only produced undifferentiated tumors. Furthermore, our data represent the first demonstration of ALK-wt transforming capacity, as ALK-wt expression in JoMa1 cells, likewise ALK-F1174L, or ALK-R1275Q, in absence of exogenous Myc-ERT activity, was sufficient to induce the formation of aggressive and undifferentiated neural crest cell-derived tumors, but not to drive NB development. Interestingly, JoMa1-ALK tumors and their derived cell lines upregulated Myc endogenous expression, resulting from ALK activation, and both ALK and Myc activities were necessary to confer tumorigenic properties on tumor-derived JoMa1 cells in vitro

Mapping the Interactions between a RUN Domain from DENND5/Rab6IP1 and Sorting Nexin 1

Eukaryotic cells have developed a diverse repertoire of Rab GTPases to regulate vesicle trafficking pathways. Together with their effector proteins, Rabs mediate various aspects of vesicle formation, tethering, docking and fusion, but details of the biological roles elicited by effectors are largely unknown. Human Rab6 is involved in the trafficking of vesicles at the level of Golgi via interactions with numerous effector proteins. We have previously determined the crystal structure of Rab6 in complex with DENND5, alternatively called Rab6IP1, which comprises two RUN domains (RUN1 and RUN2) separated by a PLAT domain. The structure of Rab6/RUN1-PLAT (Rab6/R1P) revealed the molecular basis for Golgi recruitment of DENND5 via the RUN1 domain, but the functional role of the RUN2 domain has not been well characterized. Here we show that a soluble DENND5 construct encompassing the RUN2 domain binds to the N-terminal region of sorting nexin 1 by surface plasmon resonance analyses

How to minimise the effect of tumour cell content in detection of aberrant genetic markers in neuroblastoma

Background:Clinical heterogeneity reflects the complexity of genetic events associated with neuroblastoma (NB). To identify the status of all described genetic loci with possible prognostic interest, high-throughput approaches have been used, but only with tumour cell content >60%. In some tumours, necrotic, haemorrhagic and/or calcification areas influence the low amount of neuroblasts. We evaluated the effect of tumour cell content in the detection of relevant aberrant genetic markers (AGM) diagnosed by fluorescence in situ hybridisation (FISH) on tissue microarrays (TMA) in NB.Methods:Two hundred and thirty-three MYCN non-amplified primary NB included in 12 TMAs were analysed.Results:Presence of AGM reduced event-free survival (EFS) (P=0.004) as well as overall survival (OS) (P=0.004) of patients in the whole cohort. There were no differences in prognostic impact of presence of AGM according to tumour cell content.Conclusion:We propose the use of FISH to diagnose AGM of all NB samples having the above-mentioned areas to determine patient risk

Inhibition of N-linked glycosylation impairs ALK phosphorylation and disrupts pro-survival signaling in neuroblastoma cell lines

<p>Abstract</p> <p>Background</p> <p>The Anaplastic Lymphoma Kinase (ALK) is an orphan receptor tyrosine kinase, which undergoes post-translational N-linked glycosylation. The catalytic domain of ALK was originally identified in the t(2;5) translocation that produces the unglycosylated oncogenic protein NPM-ALK, which occurs in Anaplastic Large Cell Lymphoma (ALCL). Recently, both germline and somatic activating missense mutations of ALK have been identified in neuroblastoma (NB), a pediatric cancer arising from neural crest cells. Moreover, we previously reported that ALK expression is significantly upregulated in advanced/metastatic NB. We hypothesized that ALK function may depend on N-linked glycosylation and that disruption of this post-translational modification would impair ALK activation, regardless the presence of either gene mutations or overexpression.</p> <p>Methods</p> <p>We employed tunicamycin to inhibit N-linked glycosylation. The following ALK-positive NB cell lines were used: SH-SY5Y and KELLY (ALK mutation F1174L), UKF-NB3 (ALK mutation R1275Q) and NB1 (ALK amplification). As a control, we used the NB cell lines LA1-5S and NB5 (no ALK expression), and the ALCL cell line SU-DHL1 (NPM-ALK).</p> <p>Results</p> <p>Tunicamycin treatment of ALK-positive NB cells resulted in a hypoglycosylated ALK band and in decreased amounts of mature full size receptor. Concomitantly, we observed a marked reduction of mature ALK phosphorylation. On the contrary, tunicamycin had no effects on NPM-ALK phosphorylation in SU-DHL1 cells. Moreover, phosphorylation levels of ALK downstream effectors (AKT, ERK1/2, STAT3) were clearly impaired only in ALK mutated/amplified NB cell lines, whereas no significant reduction was observed in both ALK-negative and NPM-ALK-positive cell lines. Furthermore, inhibition of N-linked glycosylation considerably impaired cell viability only of ALK mutated/amplified NB cells. Finally, the cleavage of the Poly-ADP-ribose-polymerase (PARP) suggested that apoptotic pathways may be involved in cell death.</p> <p>Conclusions</p> <p>In this study we showed that inhibition of N-linked glycosylation affects ALK phosphorylation and disrupts downstream pro-survival signaling, indicating that inhibition of this post-translational modification may be a promising therapeutic approach. However, as tunicamycin is not a likely candidate for clinical use other approaches to alter N-linked glycosylation need to be explored. Future studies will assess whether the efficacy in inhibiting ALK activity might be enhanced by the combination of ALK specific small molecule and N-linked glycosylation inhibitors.</p

p21Waf1 expression is regulated by nuclear intermediate filament vimentin in neuroblastoma

<p>Abstract</p> <p>Background</p> <p>Human neuroblastoma (NB) cell lines may present with either one of the so-called S-and N-subtypes. We have previously reported a strong correlation between protein expression levels of vimentin, an S-subtype marker, and the p21^Waf1cyclin-dependent kinase inhibitor. We here investigated whether this correlation extend to the mRNA level in NB cell lines as well as in patients' tumors. We also further explored the relationship between expression of vimentin and p21, by asking whether vimentin could regulate p21 expression.</p> <p>Methods</p> <p>Vimentin and p21 mRNA levels in NB cell lines as well as in patients' tumors (<it>n </it>= 77) were quantified using Q-PCR. Q-PCR data obtained from tumors of high risk NB patients (<it>n </it>= 40) were analyzed in relation with the overall survival using the Log-rank Kaplan-Meier estimation. siRNA-mediated depletion or overexpression of vimentin in highly or low expressing vimentin cell lines, respectively, followed by protein expression and promoter activation assays were used to assess the role of vimentin in modulating p21 expression.</p> <p>Results</p> <p>We extend the significant correlation between vimentin and p21 expression to the mRNA level in NB cell lines as well as in patients' tumors. Overall survival analysis from Q-PCR data obtained from tumors of high risk patients suggests that lower levels of p21 expression could be associated with a poorer outcome. Our data additionally indicate that the correlation observed between p21 and vimentin expression levels results from p21 transcriptional activity being regulated by vimentin. Indeed, downregulating vimentin resulted in a significant decrease in p21 mRNA and protein expression as well as in p21 promoter activity. Conversely, overexpressing vimentin triggered an increase in p21 promoter activity in cells with a nuclear expression of vimentin.</p> <p>Conclusion</p> <p>Our results suggest that p21 mRNA tumor expression level could represent a refined prognostic factor for high risk NB patients. Our data also show that vimentin regulates p21 transcription; this is the first demonstration of a gene regulating function for this type III-intermediate filament.</p

Combination Therapies Targeting Alk-Aberrant Neuroblastoma in Preclinical Models.

BACKGROUND: ALK activating mutations are identified in approximately 10% of newly diagnosed neuroblastomas and ALK amplifications in a further 1-2% of cases. Lorlatinib, a third generation ALK inhibitor, will soon be given alongside induction chemotherapy for children with ALK-aberrant neuroblastoma. However, resistance to single agent treatment has been reported and therapies that improve the response duration are urgently required. We studied the preclinical combination of lorlatinib with chemotherapy, or with the MDM2 inhibitor, idasanutlin, as recent data has suggested that ALK inhibitor resistance can be overcome through activation of the p53-MDM2 pathway. AIMS: To study the preclinical activity of ALK inhibitors alone and combined with chemotherapy or idasanutlin. METHODS: We compared different ALK inhibitors in preclinical models prior to evaluating lorlatinib in combination with chemotherapy or idasanutlin. We developed a triple chemotherapy (CAV: cyclophosphamide, doxorubicin and vincristine) in vivo dosing schedule and applied this to both neuroblastoma genetically engineered mouse models (GEMM) and patient derived xenografts (PDX). RESULTS: Lorlatinib in combination with chemotherapy was synergistic in immunocompetent neuroblastoma GEMM. Significant growth inhibition in response to lorlatinib was only observed in the ALK-amplified PDX model with high ALK expression. In this PDX lorlatinib combined with idasanutlin resulted in complete tumor regression and significantly delayed tumor regrowth. CONCLUSION: In our preclinical neuroblastoma models, high ALK expression was associated with lorlatinib response alone or in combination with either chemotherapy or idasanutlin. The synergy between MDM2 and ALK inhibition warrants further evaluation of this combination as a potential clinical approach for children with neuroblastoma

Hyperactivation of Alk induces neonatal lethality in knock-in AlkF1178L mice

The ALK (Anaplastic Lymphoma Kinase) gene encodes a tyrosine kinase receptor preferentially expressed in the central and peripheral nervous systems. A syndromic presentation associating congenital neuroblastoma with severe encephalopathy and an abnormal shape of the brainstem has been described in patients harbouring de novo germline F1174V and F1245V ALK mutations. Here, we investigated the phenotype of knock-in (KI) mice bearing the AlkF1178L mutation (F1174L in human). Although heterozygous KI mice did not reproduce the severe breathing and feeding difficulties observed in human patients, behavioral tests documented a reduced activity during dark phases and an increased anxiety of mutated mice. Matings of heterozygotes yielded the expected proportions of wild-type, heterozygotes and homozygotes at birth but a high neonatal lethality was noticed for homozygotes. We documented Alk expression in several motor nuclei of the brainstem involved in the control of sucking and swallowing. Evaluation of basic physiological functions 12 hours after birth revealed slightly more apneas but a dramatic reduced milk intake for homozygotes compared to control littermates. Overall, our data demonstrate that Alk activation above a critical threshold is not compatible with survival in mice, in agreement with the extremely severe phenotype of patients carrying aggressive de novo ALK germline mutations

Genome-Wide Analysis of Neuroblastomas using High-Density Single Nucleotide Polymorphism Arrays

BACKGROUND: Neuroblastomas are characterized by chromosomal alterations with biological and clinical significance. We analyzed paired blood and primary tumor samples from 22 children with high-risk neuroblastoma for loss of heterozygosity (LOH) and DNA copy number change using the Affymetrix 10K single nucleotide polymorphism (SNP) array. FINDINGS: Multiple areas of LOH and copy number gain were seen. The most commonly observed area of LOH was on chromosome arm 11q (15/22 samples; 68%). Chromosome 11q LOH was highly associated with occurrence of chromosome 3p LOH: 9 of the 15 samples with 11q LOH had concomitant 3p LOH (P = 0.016). Chromosome 1p LOH was seen in one-third of cases. LOH events on chromosomes 11q and 1p were generally accompanied by copy number loss, indicating hemizygous deletion within these regions. The one exception was on chromosome 11p, where LOH in all four cases was accompanied by normal copy number or diploidy, implying uniparental disomy. Gain of copy number was most frequently observed on chromosome arm 17q (21/22 samples; 95%) and was associated with allelic imbalance in six samples. Amplification of MYCN was also noted, and also amplification of a second gene, ALK, in a single case. CONCLUSIONS: This analysis demonstrates the power of SNP arrays for high-resolution determination of LOH and DNA copy number change in neuroblastoma, a tumor in which specific allelic changes drive clinical outcome and selection of therapy

Differential expression of genes mapping to recurrently abnormal chromosomal regions characterize neuroblastic tumours with distinct ploidy status

<p>Abstract</p> <p>Background</p> <p>Neuroblastic tumours (NBTs) represent a heterogeneous spectrum of neoplastic diseases associated with multiple genetic alterations. Structural and numerical chromosomal changes are frequent and are predictive parameters of NBTs outcome. We performed a comparative analysis of the biological entities constituted by NBTs with different ploidy status.</p> <p>Methods</p> <p>Gene expression profiling of 49 diagnostic primary NBTs with ploidy data was performed using oligonucleotide microarray. Further analyses using Quantitative Real-Time Polymerase Chain Reaction (Q-PCR); array-Comparative Genomic Hybridization (aCGH); and Fluorescent <it>in situ </it>Hybridization (FISH) were performed to investigate the correlation between aneuploidy, chromosomal changes and gene expression profiles.</p> <p>Results</p> <p>Gene expression profiling of 49 primary near-triploid and near-diploid/tetraploid NBTs revealed distinct expression profiles associated with each NBT subgroup. A statistically significant portion of genes mapped to 1p36 (<it>P </it>= 0.01) and 17p13-q21 (<it>P </it>< 0.0001), described as recurrently altered in NBTs. Over 90% of these genes showed higher expression in near-triploid NBTs and the majority are involved in cell differentiation pathways. Specific chromosomal abnormalities observed in NBTs, 1p loss, 17q and whole chromosome 17 gains, were reflected in the gene expression profiles. Comparison between gene copy number and expression levels suggests that differential expression might be only partly dependent on gene copy number. Intratumoural clonal heterogeneity was observed in all NBTs, with marked interclonal variability in near-diploid/tetraploid tumours.</p> <p>Conclusion</p> <p>NBTs with different cellular DNA content display distinct transcriptional profiles with a significant portion of differentially expressed genes mapping to specific chromosomal regions known to be associated with outcome. Furthermore, our results demonstrate that these specific genetic abnormalities are highly heterogeneous in all NBTs, and suggest that NBTs with different ploidy status may result from different mechanisms of aneuploidy driving tumourigenesis.</p