ALK F1174L Mutation Research Articles

Loss of ALK hotspot mutations in relapsed neuroblastoma.

ALK is the most commonly mutated oncogene in neuroblastoma with increased mutation frequency reported at relapse. Here we report the loss of an ALK mutation in two patients at relapse and a paired neuroblastoma cell line at relapse. ALK detection methods including Sanger sequencing, targeted next-generation sequencing and a new ALK Agena MassARRAY technique were used to detect common hotspot ALK variants in tumors at diagnosis and relapse from two high-risk neuroblastoma patients. Copy number analysis including single nucleotide polymorphism array and array comparative genomic hybridization confirmed adequate tumor cell content in DNA used for mutation testing. Case 1 presented with an ALK F1174L mutation at diagnosis with a variant allele frequency (VAF) ranging between 23.5% and 28.5%, but the mutation was undetectable at relapse. Case 2 presented with an ALK R1257Q mutation at diagnosis (VAF=39%-47.4%) which decreased to <0.01% at relapse. Segmental chromosomal aberrations were maintained between diagnosis and relapse confirming sufficient tumor cell content for mutation detection. The diagnostic SKNBE1n cell line harbors an ALK F1174S mutation, which was lost in the relapsed SKNBE2c cell line. To our knowledge, these are the first reported cases of loss of ALK mutations at relapse in neuroblastoma in the absence of ALK inhibitor therapy, reflecting intra-tumoral spatial and temporal heterogeneity. As ALK inhibitors are increasingly used in the treatment of refractory/relapsed neuroblastoma, our study highlights the importance of confirming whether an ALK mutation detected at diagnosis is still present in clones leading to relapse.

HGG-01. ENTRECTINIB IN RECURRENT OR REFRACTORY SOLID TUMORS INCLUDING PRIMARY CNS TUMORS: UPDATED DATA IN CHILDREN AND ADOLESCENTS

STARTRK-NG (phase 1/2) is evaluating entrectinib, a CNS-penetrant oral, TRK/ROS1/ALK tyrosine kinase inhibitor, in patients <21 years with recurrent/refractory solid tumors, including primary CNS tumors. After determining the recommended dose, 550mg/m2/day, in all-comers, expansion cohorts with gene-fusion-positive CNS/solid tumors (NTRK1/2/3, ROS1) are being enrolled. As of 5Nov2019 (data cut-off), 39 patients (4.9m–20y; median 7y) have been evaluated for response, classified as complete (CR) or partial response (PR), stable (SD) or progressive disease (PD) using RANO (CNS), RECIST (solid tumors), or Curie score (neuroblastoma). Responses in patients with fusion-positive tumors were Investigator-assessed (BICR assessments are ongoing) and occurred at doses ≥400mg/m2. Best responses in fusion-positive CNS tumors (n=14) were: 4 CR (GKAP1-NTRK2, ETV6-NTRK3 [n=2], EML1-NTRK2); 5 PR (KANK1-NTRK2, GOPC-ROS1, ETV6-NTRK3, TPR-NTRK1, EEF1G-ROS1); 3 SD (BCR-NTRK2, ARHGEF2-NTRK1, KIF21B-NTRK1); 2 PD (PARP6-NTRK3, EML4-ALK); and in fusion-positive solid tumors (n=8) were: 3 CR (ETV6-NTRK3 [n=2], DCTN1-ALK); 5 PR (EML4-NTRK3, TFG-ROS1 [n=3], KIF5B-ALK). Responses (Investigator-assessed) in non-fusion tumors (n=17) were: 1 CR (ALK F1174L mutation), 3 SD, 10 PD, 3 no data/unevaluable. The objective response rate (CR+PR/total) in patients with fusion-positive tumors was 77% (17/22) versus 6% (1/17) in those with non-fusion tumors. All 39 patients experienced ≥1 adverse event (AE); the most frequent AEs included weight gain and anemia (both 48.7%); increased ALT, increased AST, cough and pyrexia (all 46.2%); increased creatinine and vomiting (both 43.6%); and bone fractures (n=10, in 9 patients). Entrectinib has produced striking, rapid, and durable responses in solid tumors with target gene fusions, especially high-grade CNS neoplasms.

Abstract B56: Neuroblastoma patient-derived tumor-specific mRNA and DNA in platelets and extracellular vesicles

Abstract Background: Neuroblastoma is the most common extracranial pediatric solid tumor. Current diagnostic strategies involve imaging and histopathology of tumor tissue and bone marrow. PHOX2B is a specific mRNA marker for neuroblastoma that can be used for detection of circulating tumor cells (CTCs) in peripheral blood and bone marrow. Methylated RASFF1A and ALK F1174L mutation can be used as a tumor-specific cell-free DNA marker. In this study, we aim to detect tumor-specific mRNA in neuroblastoma patient-derived platelets and plasma. Furthermore, we investigate if there is a diagnostic potential for extracellular vesicles (EV) as a source of neuroblastoma-specific mRNA and DNA. Method: Blood was collected in EDTA tubes and was centrifuged to separate platelets, platelet-free plasma (PFP), and mononuclear cells (MNC, consisting of leukocytes and CTCs). Using size exclusion chromatography (SEC), extracellular vesicles were isolated from 500uL PFP. The presence of EV was confirmed with Western blot, NanoSight, and electron microscopy. RNA was isolated from PFP, platelets, MNC, and SEC fractions using TRIzolTM Reagent. DNA was isolated using the QIAamp DNA Blood Mini Kit®. To detect neuroblastoma-specific RNA, PHOX2B and GUSB as reference gene was measured by RT-qPCR. To detect cell-free tumor DNA, methylated RASSF1A was detected with RT-qPCR, and the ALK F1174L mutation by digital droplet PCR. As a reference gene for DNA, albumin was used. Results: We collected blood from one healthy control and two patients with neuroblastoma (one at relapse and one in remission). GUSB could be detected in PFP, platelets, and MNC from the control and both patients. PHOX2B was detected in PFP, platelets, and MNC from the patient with a relapse and not in the control or other patient sample. EV were isolated from plasma from one healthy control and one patient at diagnosis. GUSB was present in vesicle-enriched fractions from both samples. PHOX2B was only detected in the neuroblastoma patient vesicle-enriched fractions. In vesicle-enriched fractions of the patient, more total DNA (albumin) was present than in the control. Tumor-specific DNA (ALK F1174L mutation and methylated RASSF1A) was predominantly present in the vesicle-poor, protein-rich fractions. Conclusion: This study demonstrates that neuroblastoma-specific mRNA and DNA can be detected in platelets, PFP and EV. In particular, mRNA is enriched in EV. This finding suggests that cell-free mRNA from plasma is conserved in EV and implies a role for cell-free RNA from liquid biopsies for diagnostic approaches. In the future, we will investigate the presence and prognostic importance of cell-free RNA and DNA in neuroblastoma and other solid tumors, especially those with few circulating tumor cells. Citation Format: Nathalie Lak, Lieke van Zogchel, Lily Zappeij-Kannegieter, Janine Stutterheim, Ellen van der Schoot, Lieve Tytgat. Neuroblastoma patient-derived tumor-specific mRNA and DNA in platelets and extracellular vesicles [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B56.

Updated entrectinib data in children and adolescents with recurrent or refractory solid tumors, including primary CNS tumors.

107 Background: The phase 1/2 STARTRK-NG trial (NCT02650401) is evaluating entrectinib, a CNS-penetrant oral inhibitor of TRK, ROS1 and ALK tyrosine kinases, in children and adolescents &lt; 21 years old with recurrent/refractory solid tumors, including primary CNS tumors. Methods: After determining the recommended dose as 550mg/m2/day in all-comers, expansion cohorts with gene-fusion-positive CNS/solid tumors ( NTRK1/2/3 and ROS1) are being enrolled. Results: As of 1 July 2019 (data cut-off), 34 patients (4.9 months to 20 years old; median age 7 years) have been evaluated for response to treatment with entrectinib. Responses were classified as complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD) using RANO for CNS tumors, RECISTv1.1 for solid tumors, or Curie score for neuroblastomas. Responses in fusion-positive patients were assessed by blinded independent central review (BICR), and occurred at doses ≥400mg/m2. Best responses in patients with fusion-positive CNS tumors (n = 8) were four CR ( ETV6-NTRK3, EML1-NTRK2, GOPC-ROS1, and TPR-NTRK1), two PR ( KANK1-NTRK2 and EEF1G-ROS1), and two PD ( EML4-ALK and PARP6-NTRK3). In patients with fusion-positive solid tumors (n = 6) best responses were three CR ( DCTN1-ALK, ETV6-NTRK3, and ETV6-NTRK3), and three PR ( TFG-ROS1, EML4-NTRK3, and KIF5B-ALK). Responses (Investigator-assessed) in patients with non-fusion tumors (n = 20) were one CR ( ALK F1174L mutation), four SD, ten PD, and five patients were unevaluable or had no data. The objective response rate (defined as the total number of CR and PR) in fusion-positive patients was 86% (12/14) versus 5% (1/20) in non-fusion patients. Similarly, PFS was 17.5 months (95% CI 7.4–NE) in fusion-positive patients versus 1.9 months (1.8–5.7; p = 0.0002) in non-fusion patients. Most commonly reported treatment-related adverse events included weight gain (n = 14 [5 Grade 3/4]), elevated creatinine (n = 13), anemia (n = 13), nausea (n = 11), increased ALT (n = 10 [1 Grade 3/4]), increased AST (n = 10 [1 Grade 3/4]), decreased neutrophils (n = 9 [6 Grade 3/4]), and bone fractures (n = 7, of which 4 were treatment related). Conclusions: In children and adolescents &lt; 21 years old, entrectinib has produced striking, rapid, and durable responses in solid tumors with target gene fusions, especially in high-grade CNS neoplasms. Clinical trial information: NCT02650401.

Discovery of actionable genetic alterations with targeted panel sequencing in children with relapsed or refractory solid tumors.

Advances in genomic technologies and the development of targeted therapeutics are making the use of precision medicine increasingly possible. In this study, we explored whether precision medicine can be applied for the management of refractory/relapsed pediatric solid tumors by discovering actionable alterations using targeted panel sequencing. Samples of refractory/relapsed pediatric solid tumors were tested using a targeted sequencing panel covering the exonic DNA sequences of 381 cancer genes and introns across 22 genes to detect clinically significant genomic aberrations in tumors. The molecular targets were tiered from 1 to 5 based on the presence of actionable genetic alterations, strength of supporting evidence, and drug availability in the Republic of Korea. From January 2016 to October 2018, 55 patients were enrolled. The median time from tissue acquisition to drug selection was 29 d (range 14–39), and tumor profiling was successful in 53 (96.4%) patients. A total of 27 actionable alterations in tiers 1–4 were detected in 20 patients (36.4%), and the majority of actionable alterations were copy number variations. The tiers of molecular alterations were tier 1 (clinical evidence) in 4 variants, tier 2 (preclinical evidence) in 8 variants, tier 3 (consensus opinion) in 2 variants, and tier 4 (actionable variants with a drug that is available in other countries but not in the Republic of Korea) in 9 variants. In one patient with relapsed neuroblastoma with ALK F1174L mutation and ALK amplification, lorlatinib was used in a compassionate use program, and it showed some efficacy. In conclusion, using a targeted sequencing panel to discover actionable alterations in relapsed/refractory pediatric solid tumors was practical and feasible.

1219P - Maternal immunization against ALK as a weapon to fight neuroblastoma

BackgroundNeuroblastoma (NB) is the most common extracranial solid tumor in infancy. Some germline and somatic mutations associated with NB have been identified as activating mutations of the anaplastic lymphoma kinase (ALK) oncogene. Because of the nature of NB, which can occur in the early post-natal life or even during fetal life, we evaluated the efficacy of a DNA vaccine against ALK in a spontaneous preclinical model of NB, harboring ALKF1174L mutation in association with MYCN amplification (ALKF1174L/MYCN mice) by means of maternal immunization (MI). MethodsMYCN transgenic females were immunized against ALK by DNA vaccination by using a plasmid coding for the extracellular and trans-membrane domains of human ALK (ALK-ECTM) followed by electroporation, and then they were mated with an ALKF1174L-transgenic male. In ALKF1174L/MYCN offspring, the presence of abdominal, cervical and paraspinal tumors has been evaluated and quantified by Magnetic Resonance Imaging. The humoral immune response induced against ALK in the mothers and their offspring, as well as the presence of immune-complexes containing ALK, have been evaluated by ELISA. ALK expression in tumor tissue was assessed by Western blot. ResultsPre-birth immunization against ALK leads to an extended survival time and to a lower tumor growth kinetic in ALKF1174L/MYCN offspring born from ALK-ECTM-vaccinated mothers (ALK-ECTM offspring) as compared to controls born from control empty vector-vaccinated mothers. Maternally derived anti-ALK antibodies were successfully transferred from mothers to newborns. Moreover, anti-ALK IgM were found in the sera of five- and six-week old ALK-ECTM offspring, suggesting the induction of the pups’ own immune response against ALK. This effect could be due to the breast milk-mediated transfer of immune-complexes containing ALK, found in the milk of vaccinated mothers and in their offspring sera. Finally, MI against ALK induces a decrease in ALK expression in ALK-ECTM offspring’ tumor tissue. ConclusionsOverall, these results indicate that MI against ALK induces an active immunization against this oncoantigen in the offspring, impairing tumor development and enhancing survival time in a preclinical model of NB. Legal entity responsible for the studyDepartment of Molecular Biotechnology and Health Sciences. FundingAssociazione Italiana per la ricerca sul Cancro. DisclosureAll authors have declared no conflicts of interest.

ALK mutation dynamics and clonal evolution in a neuroblastoma model exhibiting two ALK mutations.

The ALK gene is a major oncogene of neuroblastoma cases exhibiting ALK activating mutations. Here, we characterized two neuroblastoma cell lines established from a stage 4 patient at diagnosis either from the primary tumor (PT) or from the bone marrow (BM). Both cell lines exhibited similar genomic profiles. All cells in the BM-derived cell line exhibited an ALK F1174L mutation, whereas this mutation was present in only 5% of the cells in the earliest passages of the PT-derived cell line. The BM-derived cell line presented with a higher proliferation rate in vitro and injections in Nude mice resulted in tumor formation only for the BM-derived cell line. Next, we observed that the F1174L mutation frequency in the PT-derived cell line increased with successive passages. Further Whole Exome Sequencing revealed a second ALK mutation, L1196M, in this cell line. Digital droplet PCR documented that the allele fractions of both mutations changed upon passages, and that the F1174L mutation reached 50% in late passages, indicating clonal evolution. In vitro treatment of the PT-derived cell line exhibiting the F1174L and L1196M mutations with the alectinib inhibitor resulted in an enrichment of the L1196M mutation. Using xenografts, we documented a better efficacy of alectinib compared to crizotinib on tumor growth and an enrichment of the L1196M mutation at the end of both treatments. Finally, single-cell RNA-seq analysis was consistent with both mutations resulting in ALK activation. Altogether, this study provides novel insights into ALK mutation dynamics in a neuroblastoma model harbouring two ALK mutations.

Expression of the Neuroblastoma-Associated ALK-F1174L Activating Mutation During Embryogenesis Impairs the Differentiation of Neural Crest Progenitors in Sympathetic Ganglia.

Neuroblastoma (NB) is an embryonal malignancy derived from the abnormal differentiation of the sympathetic nervous system. The Anaplastic Lymphoma Kinase (ALK) gene is frequently altered in NB, through copy number alterations and activating mutations, and represents a predisposition in NB-genesis when mutated. Our previously published data suggested that ALK activating mutations may impair the differentiation potential of neural crest (NC) progenitor cells. Here, we demonstrated that the expression of the endogenous ALK gene starts at E10.5 in the developing sympathetic ganglia (SG). To decipher the impact of deregulated ALK signaling during embryogenesis on the formation and differentiation of sympathetic neuroblasts, Sox10-Cre;LSL-ALK-F1174L embryos were produced to restrict the expression of the human ALK-F1174L transgene to migrating NC cells (NCCs). First, ALK-F1174L mediated an embryonic lethality at mid-gestation and an enlargement of SG with a disorganized architecture in Sox10-Cre;LSL-ALK-F1174L embryos at E10.5 and E11.5. Second, early sympathetic differentiation was severely impaired in Sox10-Cre;LSL-ALK-F1174L embryos. Indeed, their SG displayed a marked increase in the proportion of NCCs and a decrease of sympathetic neuroblasts at both embryonic stages. Third, neuronal and noradrenergic differentiations were blocked in Sox10-Cre;LSL-ALK-F1174L SG, as a reduced proportion of Phox2b+ sympathoblasts expressed βIII-tubulin and almost none were Tyrosine Hydroxylase (TH) positive. Finally, at E10.5, ALK-F1174L mediated an important increase in the proliferation of Phox2b+ progenitors, affecting the transient cell cycle exit observed in normal SG at this embryonic stage. Altogether, we report for the first time that the expression of the human ALK-F1174L mutation in NCCs during embryonic development profoundly disturbs early sympathetic progenitor differentiation, in addition to increasing their proliferation, both mechanisms being potential crucial events in NB oncogenesis.

A kinome-wide RNAi screen identifies ALK as a target to sensitize neuroblastoma cells for HDAC8-inhibitor treatment

The prognosis of advanced stage neuroblastoma patients remains poor and, despite intensive therapy, the 5-year survival rate remains less than 50%. We previously identified histone deacetylase (HDAC) 8 as an indicator of poor clinical outcome and a selective drug target for differentiation therapy in vitro and in vivo. Here, we performed kinome-wide RNAi screening to identify genes that are synthetically lethal with HDAC8 inhibitors. These experiments identified the neuroblastoma predisposition gene ALK as a candidate gene. Accordingly, the combination of the ALK/MET inhibitor crizotinib and selective HDAC8 inhibitors (3–6 µM PCI-34051 or 10 µM 20a) efficiently killed neuroblastoma cell lines carrying wildtype ALK (SK-N-BE(2)-C, IMR5/75), amplified ALK (NB-1), and those carrying the activating ALK F1174L mutation (Kelly), and, in cells carrying the activating R1275Q mutation (LAN-5), combination treatment decreased viable cell count. The effective dose of crizotinib in neuroblastoma cell lines ranged from 0.05 µM (ALK-amplified) to 0.8 µM (wildtype ALK). The combinatorial inhibition of ALK and HDAC8 also decreased tumor growth in an in vivo zebrafish xenograft model. Bioinformatic analyses revealed that the mRNA expression level of HDAC8 was significantly correlated with that of ALK in two independent patient cohorts, the Academic Medical Center cohort (n = 88) and the German Neuroblastoma Trial cohort (n = 649), and co-expression of both target genes identified patients with very poor outcome. Mechanistically, HDAC8 and ALK converge at the level of receptor tyrosine kinase (RTK) signaling and their downstream survival pathways, such as ERK signaling. Combination treatment of HDAC8 inhibitor with crizotinib efficiently blocked the activation of growth receptor survival signaling and shifted the cell cycle arrest and differentiation phenotype toward effective cell death of neuroblastoma cell lines, including sensitization of resistant models, but not of normal cells. These findings reveal combined targeting of ALK and HDAC8 as a novel strategy for the treatment of neuroblastoma.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract LB-C24: Resistance to ALK inhibition is associated with enhanced BRD4 dependence in ALK-mutated MYCN-amplified neuroblastoma

Abstract Despite substantial therapeutic advances, the long-term survival rate of patients with high-risk neuroblastoma (NB), a tumor of the peripheral sympathetic nervous system is still dismal. Activating mutations in the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase represent promising therapeutic targets in this disease and have prompted clinical trials of ALK inhibitors. But as with all molecularly targeted therapies, resistance is inevitable and will limit the ultimate utility of this therapeutic strategy. To elucidate the mechanisms of resistance to ALK inhibitors, we have generated isogenic NB cells, expressing the tumorigenic ALKF1174L mutation and amplified MYCN, with acquired resistance to the ALK inhibitor TAE684, through continuous exposure of increasing concentrations of the compound over time. We found that resistance was associated with downregulation of ALK activity, thus ruling out secondary mutations as a mechanism of resistance, and interestingly the other major oncogenic driver in neuroblastoma, MYCN, was downregulated as well. A comparative genome-wide expression analysis revealed global transcriptional downregulation in the resistant cells, as compared with their parental sensitive counterparts, in accordance with the aforementioned MYCN downregulation, as MYCN is known to act as a transcriptional amplifier in MYCN-amplified NB cells. Additionally, these resistant cells exhibited a 10-fold increased sensitivity to the BRD4 inhibitor JQ1, compared to sensitive cells, and MYCN as well as global gene expression were rescued after treatment. These results strongly suggest that resistance to ALK inhibition in ALKF1174L MYCN-amplified NB cells leads to a MYCN-independent enhanced BRD4 dependence, therefore providing a rationale for the use of BET inhibitors to overcome such resistance. Studies are currently underway to characterize the unique epigenetic landscape driving the resistant cells' survival and therefore identify novel targets that could be taken advantage of in future therapeutic strategies. Citation Format: David Debruyne, Bandana Sharma, Nathanael Gray, Rani George. Resistance to ALK inhibition is associated with enhanced BRD4 dependence in ALK-mutated MYCN-amplified neuroblastoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C24.

Abstract B74: Combined inhibition of ALK and CDKs involved in transcriptional regulation is synergistic in ALK-mutated neuroblastoma

Abstract Activating mutations in the anaplastic lymphoma receptor tyrosine kinase (ALK) represent important therapeutic targets in neuroblastoma (NB). One of the more common mutations, ALKF1174L, is sensitive to the FDA-approved ALK inhibitor, crizotinib, only at high doses and mediates acquired resistance to crizotinib in ALK-rearranged cancers. To identify compounds that would enhance the cytotoxic effect of crizotinib, we conducted a high throughput small molecule screen for compounds that synergize with crizotinib in NB cells expressing the ALKF1174L mutation. We identified two pan-selective cyclin dependent kinase (CDK) inhibitors, AT7519 and SNS-032, which have overlapping efficacy against the cell cycle-regulating CDK2 and transcriptional elongation-regulating CDK9. Both inhibitors demonstrated synergistic activity with crizotinib, leading to downregulation of pALK and downstream signaling and significantly increased apoptosis over that of either single agent alone. This effect was observed in NB cells expressing not only ALKF1174L, but also in the other commonly observed ALKR1275Q mutation. Synergy was also noted with ceritinib (LDK378), a structurally unrelated ALK inhibitor, in combination with both AT7519 and SNS-032. The combination of crizotinib and SNS-032 led to the inhibition of CDK9-mediated transcriptional elongation as indicated by downregulation of RNA polymerase II phosphorylation at serine 2. Additionally, the combination also induced proteolytic cleavage of elongation-regulating BRD4, suggesting alternative mechanisms contributing to transcriptional inhibition in addition to direct inhibition of CDK9 activity These findings were comparable with results obtained using compounds that were highly selective for CDK7, which has roles in transcriptional initiation as well as in CDK9 activation. Finally, in murine xenogaft models of ALK-mutated NB, the combination led to inhibition of tumor growth and prolongation of survival compared to single agents alone. Together, these data support further pre-clinical and clinical efforts to explore the therapeutic potential of combining ALK inhibitors with transcriptional CDK inhibitors in ALK-mutated NB. Citation Format: Nathan F. Moore, Edmond Chipumuro, Clark M. Hatheway, Tinghu Zhang, Nathanael S. Gray, Rani E. George. Combined inhibition of ALK and CDKs involved in transcriptional regulation is synergistic in ALK-mutated neuroblastoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B74.

Intrinsic susceptibility MRI identifies tumors with ALKF1174L mutation in genetically-engineered murine models of high-risk neuroblastoma.

The early identification of children presenting ALKF1174L-mutated neuroblastoma, which are associated with resistance to the promising ALK inhibitor crizotinib and a marked poorer prognosis, has become a clinical priority. In comparing the radiology of the novel Th-ALKF1174L/Th-MYCN and the well-established Th-MYCN genetically-engineered murine models of neuroblastoma using MRI, we have identified a marked ALKF1174L-driven vascular phenotype. We demonstrate that quantitation of the transverse relaxation rate R2* (s−1) using intrinsic susceptibility-MRI under baseline conditions and during hyperoxia, can robustly discriminate this differential vascular phenotype, and identify MYCN-driven tumors harboring the ALKF1174L mutation with high specificity and selectivity. Intrinsic susceptibility-MRI could thus potentially provide a non-invasive and clinically-exploitable method to help identifying children with MYCN-driven neuroblastoma harboring the ALKF1174L mutation at the time of diagnosis.

Abstract 5037: Intrinsic susceptibility magnetic resonance imaging identifies tumors with ALKF1174L mutation in transgenic murine models of high-risk neuroblastoma.

Abstract In neuroblastoma, mutations in the anaplastic lymphoma kinase (ALK) tyrosine kinase gene have been identified in 8-10 % of primary tumors. The most common and potent ALK mutation, ALKF1174L, leads to the constitutive activation of the ALK protein and is associated preferentially with MYCN amplification, a markedly poorer prognosis, and confers resistance to the promising ALK inhibitor crizotinib. The development of more efficacious ALK inhibitors will benefit from non-invasive imaging strategies for the rapid identification of children with high-risk ALK-expressing or mutated neuroblastoma. Intrinsic susceptibility magnetic resonance imaging (IS-MRI) data was acquired from tumors arising in TH-ALKF1174L/TH-MYCN and TH-MYCN mice, two genetically engineered mouse model of high-risk neuroblastoma. The native MRI transverse relaxation rate R2*, an imaging biomarker sensitive to the concentration of paramagnetic deoxyhemoglobin, and changes in R2* induced with 100% oxygen inhalation, were quantified. Tumors in the TH-ALKF1174L/TH-MYCN mice demonstrated significantly (p&amp;lt;0.0001) slower native R2* rates (61 ± 3s−1, n=23) than tumors in the TH-MYCN mice (101 ± 6s−1, n=21). With hyperoxic challenge, the TH-ALKF1174L/TH-MYCN mice demonstrated a significantly (p&amp;lt;0.0001) lower and negligible ΔR2*oxygen-air (-3 ± 1s−1, n=10) compared with tumors in TH-MYCN mice (-26 ± 3s−1, n=12). A sensitivity of 90% and a specificity of 81% for native R2*, and a sensitivity of 90% and a specificity of 94% for hyperoxia-induced ΔR2* was determined. Histological correlates revealed a significantly (p&amp;lt;0.05) higher uptake of the perfusion marker Hoechst 33342, and the presence of large hemorrhagic blood lakes filled with stagnant deoxygenated erythrocytes, in tumors within the TH-MYCN mice, but not the TH-ALKF1174L/TH-MYCN model. Together these corroborate the IS-MRI findings (relatively fast native R2* and significant ΔR2*, indicative of vascular instability, in tumors in the TH-MYCN mice). IS-MRI provides a robust method to discriminate and identify TH-MYCN transgenic mice harboring the ALKF1174L mutation based on a stark differential vascular phenotype, which may impact on impaired drug delivery. IS-MRI is suitable for the scanning of young children, and quantitation of native R2* easily incorporated into existing pediatric clinical imaging protocols. This approach could provide a robust and rapid indicator of ALK genotypic status of the tumor, enabling the early identification of children with ultra high-risk neuroblastoma at the time of diagnosis. Citation Format: Yann Jamin, Laura Glass, Albert Hallsworth, Rani E. George, Dow-Mu Koh, Andrew D.J. Pearson, Louis Chesler, Simon P. Robinson. Intrinsic susceptibility magnetic resonance imaging identifies tumors with ALKF1174L mutation in transgenic murine models of high-risk neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5037. doi:10.1158/1538-7445.AM2013-5037

Abstract 4454: The AXL tyrosine kinase receptor contributes to ALK-inhibitor resistance in neuroblastoma.

Abstract The crizotinib-resistant ALKF1174L mutation occurs de novo in neuroblastoma (NB) and as an acquired mechanism of resistance in ALK translocation-driven cancers, lending impetus to the development of novel ALK inhibitors with different modes of action. One such molecule is TAE684, a structurally unrelated ATP-competitive diaminopyrimidine derivative that has shown activity against ALKF1174L in both in vitro and in vivo models of ALK-driven cancers, such as lung cancer and NB. To identify mechanisms of acquired resistance to TAE684, we established in vitro models of resistance by serially exposing TAE684-sensitive SH-SY5Y NB cells (expressing ALKF1174L) to increasing concentrations of the compound over a prolonged period (&amp;gt; 1 year). The resulting TAE684-resistant SY5Y-TR cells exhibited &amp;gt;10-fold reduced sensitivity to TAE684 compared with parental cells. Three TAE684-resistant clonal lines (TR1-TR3) were analyzed, all of which showed downregulation of pALK. In addition, TR1 cells showed downregulation of pAKT but increased expression of pERK1/2 compared to parental cells, suggesting activation of compensatory signaling pathways. To identify candidate receptor tyrosine kinases (RTKs) that may account for this observation, we analyzed TR1 cells using phospho-RTK arrays. Upregulation of the AXL RTK was seen in the resistant cells as compared to the parental cells, and this was confirmed by immunoblotting in all the resistant clones. Concurrent with the role of AXL in tumor cell invasion, TR1 cells exhibited increased invasiveness in matrigel assays. Depletion of AXL expression by shRNA knockdown in TR1 cells led to their growth inhibition. TR1 cells were more sensitive to SKI-606, a Src/Abl inhibitor with activity against AXL, than parental cells. However, exposure to an Hsp90 inhibitor led to significant cytotoxicity in all the clones (IC50s&amp;lt;30nM), as compared to parental cells (IC50=304nM). Inhibiting Hsp90 in the TR1 cells led to a decrease in pAXL and pERK1/2 levels with a concomitant decrease in the binding of AXL to Hsp90. Analysis of AXL in TR1 cells ruled out mutation, genomic amplification or promoter demethylation as a basis for its increased expression; however, the AXL ligand, GAS6, was increased in the resistant clones. These studies demonstrate that AXL activation contributes to TAE684 resistance in ALKF1174L-expressing cells, and suggests that the same would be true for derivatives of TAE684 in clinical development. The finding that AXL is also involved in EGFR-targeted therapy in lung cancer suggests that AXL activation could be a general mechanism of resistance to tyrosine kinase inhibitor therapy. Citation Format: Namrata Bhatnagar, William Luther, Bandana Sharma, Qingsong Liu, Nathanael Gray, Rani E. George. The AXL tyrosine kinase receptor contributes to ALK-inhibitor resistance in neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4454. doi:10.1158/1538-7445.AM2013-4454

Abstract 1683: ALK F1174L kinase activity as driver of cell proliferation in neuroblastoma cell line models and neuroblastoma tumors

Abstract Background. Whereas ALK translocations play a role in tumor growth in selected NSCLC cases, mutated forms of ALK have been shown to drive development of neuroblastoma, the most common malignant extracranial tumor of childhood. The mutation F1147L in the oncogene ALK increases the kinase activity, causing deregulated proliferation of sympatho-adrenal progenitor cells. We explored the use of kinase activity measurements to identify ALK activity in cell lysates and patient derived tissue lysates with the aim of identifying biomarkers to trace the activity of ALK in tumors and the effect of ALK inhibitors on these marker signals. Methods. Protein kinase activities were measured on PamChip® peptide microarrays comprising 144 Tyr containing peptide sequences from known human phosphorylation sites. The activity profiling experiments involved recombinant ALK kinase, lysates of cell lines overexpressing ALK and neuroblastoma tumors with elevated ALK F1174L presence. Per microarray analysis 5 ug of total protein was used in the presence of 100 uM ATP. Experiments were performed in the presence and absence of ALK kinase inhibitors crizotinib and NVP-TAE684. Real time kinetics of 144 peptide phosphorylations were measured with a fluorescently labeled antibody. Results. Phosphorylation profiles for neuroblastoma tumors, ALK overexpressing cell lines and recALK were generated. For recALK 48 novel peptide substrates were identified that showed signals depending on both ATP and kinase concentration and inhibition by the ALK inhibitors. In ALK overexpressing cell lines, but not in control cell lysates, signals on many ALK substrate peptides were significantly elevated, and returned to baseline level in the presence of ALK inhibitors. Whereas neuroblastomas with low ALK expression gave similar kinase activity profiles, the tumors harboring the ALK F1174L mutation were different, suggesting a more complex regulation of phosphorylation activity in these tumors. All tumors showed on-chip responses with ALK inhibitor drugs, but to different extent, which could be a basis for response prediction biomarker discovery. Conclusion. ALK activity of recombinant kinases, cell lysates and tumor lysates can be detected on PamChip® peptide microarrays. Based on results with model systems, this method shows promise to identify tumors that express elevated ALK activity and to study response of this class to ALK inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1683. doi:1538-7445.AM2012-1683

Internalization and Down-Regulation of the ALK Receptor in Neuroblastoma Cell Lines upon Monoclonal Antibodies Treatment

Recently, activating mutations of the full length ALK receptor, with two hot spots at positions F1174 and R1275, have been characterized in sporadic cases of neuroblastoma. Here, we report similar basal patterns of ALK phosphorylation between the neuroblastoma IMR-32 cell line, which expresses only the wild-type receptor (ALKWT), and the SH-SY5Y cell line, which exhibits a heterozygous ALK F1174L mutation and expresses both ALKWT and ALKF1174L receptors. We demonstrate that this lack of detectable increased phosphorylation in SH-SY5Y cells is a result of intracellular retention and proteasomal degradation of the mutated receptor. As a consequence, in SH-SY5Y cells, plasma membrane appears strongly enriched for ALKWT whereas both ALKWT and ALKF1174L were present in intracellular compartments. We further explored ALK receptor trafficking by investigating the effect of agonist and antagonist mAb (monoclonal antibodies) on ALK internalization and down-regulation, either in SH-SY5Y cells or in cells expressing only ALKWT. We observe that treatment with agonist mAbs resulted in ALK internalization and lysosomal targeting for receptor degradation. In contrast, antagonist mAb induced ALK internalization and recycling to the plasma membrane. Importantly, we correlate this differential trafficking of ALK in response to mAb with the recruitment of the ubiquitin ligase Cbl and ALK ubiquitylation only after agonist stimulation. This study provides novel insights into the mechanisms regulating ALK trafficking and degradation, showing that various ALK receptor pools are regulated by proteasome or lysosome pathways according to their intracellular localization.

Abstract 4757: The ALK-F1174L mutation accelerates MYCN-driven tumorigenesis in a murine transgenic neuroblastoma model

Abstract Activating mutations in the anaplastic lymphoma receptor tyrosine kinase gene (ALK) have been identified in neuroblastoma and represent a novel therapeutic target in this childhood tumor of the sympathetic nervous system. The ALK-F1174L mutation, in particular, occurs at a higher frequency in tumors with MYCN gene amplification, is associated with a worse prognosis and with resistance to pharmacologic ALK inhibitors in adults with ALK-translocated cancers. To investigate the role of mutated ALK in MYCN-driven neuroblastoma, we generated transgenic mice that overexpress ALK-F1174L in neural crest-derived cells under the control of the tyrosine hydroxylase (TH) promoter, and performed genetic crosses against TH-MYCN transgenic mice. Doubly transgenic TH-ALK-F1174L/TH-MYCN animals developed aggressive tumors of the adrenal glands and abdominal and thoracic sympathetic ganglia, with 100% penetrance and a very short latency (31 days). In contrast, TH-MYCN littermates failed to develop tumors by this time. Furthermore, TH-ALK-F1174L hemizygote or homozygote animals failed to initiate tumors in the absence of MYCN expression. On histologic examination, ALK-F1174L/MYCN tumors were stroma poor and displayed undifferentiated small round blue cells with numerous apoptotic figures and scattered mitoses, and exhibited strong expression of both MYCN and aberrant ALK-F1174L transcripts and protein. Taken together, our studies demonstrate that the ALK-F1174L mutation plays a potent and significant role in the genesis of neuroblastoma, cooperating with MYCN to drive tumor formation. To our knowledge, this is the first mouse model available for investigations into the role of mutant ALK in neuroblastoma, especially its interaction with MYCN, and should provide a valuable new tool for development of ALK-targeted therapeutics in neuroblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4757. doi:10.1158/1538-7445.AM2011-4757