Long-Lasting Response to Lorlatinib in Patients with ALK-Driven Relapsed or Refractory Neuroblastoma Monitored with Circulating Tumor DNA Analysis.

Unlabelled: Patients with anaplastic lymphoma kinase (ALK)-driven neuroblastoma may respond to tyrosine kinase inhibitors, but resistance to treatment occurs and methods currently used for detection of residual disease have limited sensitivity. Here, we present a national unselected cohort of five patients with relapsed or refractory ALK-driven neuroblastoma treated with lorlatinib as monotherapy and test the potential of targeted circulating tumor DNA (ctDNA) analysis as a guide for treatment decisions in these patients. We developed a sequencing panel for ultrasensitive detection of ALK mutations associated with neuroblastoma or resistance to tyrosine kinase inhibitors and used it for ctDNA analysis in 83 plasma samples collected longitudinally from the four patients who harbored somatic ALK mutations.

Occurrence of cancer in Marfan syndrome: Report of two patients with neuroblastoma and review of the literature.

Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder caused by pathogenic variants in FBN1, with a hitherto unknown association with cancer. Here, we present two females with MFS who developed pediatric neuroblastoma. Patient 1 presented with neonatal MFS and developed an adrenal neuroblastoma with unfavorable tumor genetics at 10 months of age.

The Alk receptor tyrosine kinase regulates Sparkly, a novel activity regulating neuropeptide precursor in the central nervous system.

Numerous roles for the Alk receptor tyrosine kinase have been described in , including functions in the central nervous system (CNS), however the molecular details are poorly understood. To gain mechanistic insight, we employed Targeted DamID (TaDa) transcriptional profiling to identify targets of Alk signaling in the larval CNS. TaDa was employed in larval CNS tissues, while genetically manipulating Alk signaling output.

Anaplastic Lymphoma Kinase signaling stabilizes SLC3A2 expression via MARCH11 to promote neuroblastoma cell growth.

Solute Carrier Family 3, Member 2 (SLC3A2 or 4F2hc) is a multifunctional glycoprotein that mediates integrin-dependent signaling, acts as a trafficking chaperone for amino acid transporters, and is involved in polyamine transportation. We identified SLC3A2 as a potential Anaplastic Lymphoma Kinase (ALK) interacting partner in a BioID-proximity labeling screen in neuroblastoma (NB) cells. In this work we show that endogenous SLC3A2 and ALK interact in NB cells and that this SLC3A2:ALK interaction was abrogated upon treatment with the ALK inhibitor lorlatinib.

ALK F1174S mutation impairs ALK kinase activity in EML4-ALK variant 1 and sensitizes EML4-ALK variant 3 to crizotinib.

Objective: To assess the influence of F1174S mutation on kinase activity and drug sensitivity of the echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) fusion (EML4-ALK) variants 1 and 3.

Methods: We constructed mammalian expression plasmids of both wildtype and F1174 mutant EML4-ALK variants 1 and 3, and then characterized them with cell models by performing immunoblotting, neurite outgrowth assay, focus formation assay as well as protein stability assay. Drug sensitivity to ALK tyrosine kinase inhibitors was also compared between wildtype and F1174 mutant EML4-ALK fusions.

Preclinical exploration of the DNA damage response pathway using the interactive neuroblastoma cell line explorer CLEAN.

Neuroblastoma (NB) is the most common cancer in infancy with an urgent need for more efficient targeted therapies. The development of novel (combinatorial) treatment strategies relies on extensive explorations of signaling perturbations in neuroblastoma cell lines, using RNA-Seq or other high throughput technologies (e.g.

ALK signaling primes the DNA damage response sensitizing ALK-driven neuroblastoma to therapeutic ATR inhibition.

High-risk neuroblastoma (NB) is a significant clinical challenge. MYCN and Anaplastic Lymphoma Kinase (ALK), which are often involved in high-risk NB, lead to increased replication stress in cancer cells, suggesting therapeutic strategies. We previously identified an ATR (ataxia telangiectasia and Rad3-related)/ALK inhibitor (ATRi/ALKi) combination as such a strategy in two independent genetically modified mouse NB models.

IGF1R Contributes to Cell Proliferation in -Mutated Neuroblastoma with Preference for Activating the PI3K-AKT Signaling Pathway.

Aberrant activation of anaplastic lymphoma kinase (ALK) by activating point mutation or amplification drives 5-12% of neuroblastoma (NB). Previous work has identified the involvement of the insulin-like growth factor 1 receptor (IGF1R) receptor tyrosine kinase (RTK) in a wide range of cancers. We show here that many NB cell lines exhibit IGF1R activity, and that IGF1R inhibition led to decreased cell proliferation to varying degrees in ALK-driven NB cells.

ALK fusion NSCLC oncogenes promote survival and inhibit NK cell responses via expression.

Anaplastic lymphoma kinase (ALK) fusion variants in Non-Small Cell Lung Cancer (NSCLC) consist of numerous dimerizing fusion partners. Retrospective investigations suggest that treatment benefit in response to ALK tyrosine kinase inhibitors (TKIs) differs dependent on the fusion variant present in the patient tumor. Therefore, understanding the oncogenic signaling networks driven by different ALK fusion variants is important.

Patient-associated mutations in Drosophila Alk perturb neuronal differentiation and promote survival.

Activating anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (RTK) mutations occur in pediatric neuroblastoma and are associated with poor prognosis. To study ALK-activating mutations in a genetically controllable system, we employed CRIPSR/Cas9, incorporating orthologs of the human oncogenic mutations ALKF1174L and ALKY1278S in the Drosophila Alk locus. AlkF1251L and AlkY1355S mutant Drosophila exhibited enhanced Alk signaling phenotypes, but unexpectedly depended on the Jelly belly (Jeb) ligand for activation.

Characterization of Endogenous Protein Interactomes in Larval Brain, Using a CRISPR/Cas9-based Strategy and BioID-based Proximity Labeling.

Understanding protein-protein interactions (PPIs) and interactome networks is essential to reveal molecular mechanisms mediating various cellular processes. The most common method to study PPIs is affinity purification combined with mass spectrometry (AP-MS). Although AP-MS is a powerful method, loss of weak and transient interactions is still a major limitation.

The autocrine loop of ALK receptor and ALKAL2 ligand is an actionable target in consensus molecular subtype 1 colon cancer.

Background: In the last years, several efforts have been made to classify colorectal cancer (CRC) into well-defined molecular subgroups, representing the intrinsic inter-patient heterogeneity, known as Consensus Molecular Subtypes (CMSs).

Methods: In this work, we performed a meta-analysis of CRC patients stratified into four CMSs. We identified a negative correlation between a high level of anaplastic lymphoma kinase (ALK) expression and relapse-free survival, exclusively in CMS1 subtype.

DamID transcriptional profiling identifies the Snail/Scratch transcription factor Kahuli as an Alk target in the Drosophila visceral mesoderm.

Development of the Drosophila visceral muscle depends on Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) signaling, which specifies founder cells (FCs) in the circular visceral mesoderm (VM). Although Alk activation by its ligand Jelly Belly (Jeb) is well characterized, few target molecules have been identified. Here, we used targeted DamID (TaDa) to identify Alk targets in embryos overexpressing Jeb versus embryos with abrogated Alk activity, revealing differentially expressed genes, including the Snail/Scratch family transcription factor Kahuli (Kah).

Chromosome Imbalances in Neuroblastoma-Recent Molecular Insight into Chromosome 1p-deletion, 2p-gain, and 11q-deletion Identifies New Friends and Foes for the Future.

Neuroblastoma is the most common extracranial solid pediatric tumor, with around 15% childhood cancer-related mortality. High-risk neuroblastomas exhibit a range of genetic, morphological, and clinical heterogeneities, which add complexity to diagnosis and treatment with existing modalities. Identification of novel therapies is a high priority in high-risk neuroblastoma, and the combination of genetic analysis with increased mechanistic understanding-including identification of key signaling and developmental events-provides optimism for the future.

In vivo Profiling of the Alk Proximitome in the Developing Drosophila Brain.

Anaplastic lymphoma kinase (Alk) is an evolutionary conserved receptor tyrosine kinase belonging to the insulin receptor superfamily. In addition to its well-studied role in cancer, numerous studies have revealed that Alk signaling is associated with a variety of complex traits such as: regulation of growth and metabolism, hibernation, regulation of neurotransmitters, synaptic coupling, axon targeting, decision making, memory formation and learning, alcohol use disorder, as well as steroid hormone metabolism. In this study, we used BioID-based in vivo proximity labeling to identify molecules that interact with Alk in the Drosophila central nervous system (CNS).

Mapping the Phospho-dependent ALK Interactome to Identify Novel Components in ALK Signaling.

Protein-protein interactions (PPIs) play essential roles in Anaplastic Lymphoma Kinase (ALK) signaling. Systematic characterization of ALK interactors helps elucidate novel ALK signaling mechanisms and may aid in the identification of novel therapeutics targeting related diseases. In this study, we used the Mammalian Membrane Two-Hybrid (MaMTH) system to map the phospho-dependent ALK interactome.

Neuroblastoma xenograft models demonstrate the therapeutic potential of Lu-octreotate.

Background: Neuroblastoma (NB) is one of the most frequently diagnosed tumors in infants. NB is a neuroendocrine tumor type with various characteristics and features, and with diverse outcome. The most malignant NBs have a 5-year survival rate of only 40-50%, indicating the need for novel and improved treatment options.

BioID-Screening Identifies PEAK1 and SHP2 as Components of the ALK Proximitome in Neuroblastoma Cells.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) that is mutated in approximately 10% of pediatric neuroblastoma (NB). To shed light on ALK-driven signaling processes, we employed BioID-based in vivo proximity labeling to identify molecules that interact intracellularly with ALK. NB-derived SK-N-AS and SK-N-BE(2) cells expressing inducible ALK-BirA* fusion proteins were generated and stimulated with ALKAL ligands in the presence and absence of the ALK tyrosine kinase inhibitor (TKI) lorlatinib.

Extracellular domain shedding of the ALK receptor mediates neuroblastoma cell migration.

Although activating mutations of the anaplastic lymphoma kinase (ALK) membrane receptor occur in ∼10% of neuroblastoma (NB) tumors, the role of the wild-type (WT) receptor, which is aberrantly expressed in most non-mutated cases, is unclear. Both WT and mutant proteins undergo extracellular domain (ECD) cleavage. Here, we map the cleavage site to Asn654-Leu655 and demonstrate that cleavage inhibition of WT ALK significantly impedes NB cell migration with subsequent prolongation of survival in mouse models.

Loss of RET Promotes Mesenchymal Identity in Neuroblastoma Cells.

Aberrant activation of anaplastic lymphoma kinase (ALK) drives neuroblastoma (NB). Previous work identified the RET receptor tyrosine kinase (RTK) as a downstream target of ALK activity in NB models. We show here that ALK activation in response to ALKAL2 ligand results in the rapid phosphorylation of RET in NB cells, providing additional insight into the contribution of RET to the ALK-driven gene signature in NB.

ALK ligand ALKAL2 potentiates MYCN-driven neuroblastoma in the absence of ALK mutation.

High-risk neuroblastoma (NB) is responsible for a disproportionate number of childhood deaths due to cancer. One indicator of high-risk NB is amplification of the neural MYC (MYCN) oncogene, which is currently therapeutically intractable. Identification of anaplastic lymphoma kinase (ALK) as an NB oncogene raised the possibility of using ALK tyrosine kinase inhibitors (TKIs) in treatment of patients with activating ALK mutations.

11q Deletion or ALK Activity Curbs DLG2 Expression to Maintain an Undifferentiated State in Neuroblastoma.

High-risk neuroblastomas typically display an undifferentiated or poorly differentiated morphology. It is therefore vital to understand molecular mechanisms that block the differentiation process. We identify an important role for oncogenic ALK-ERK1/2-SP1 signaling in the maintenance of undifferentiated neural crest-derived progenitors through the repression of DLG2, a candidate tumor suppressor gene in neuroblastoma.

Identification of the Wallenda JNKKK as an Alk suppressor reveals increased competitiveness of Alk-expressing cells.

Anaplastic lymphoma kinase (Alk) is a receptor tyrosine kinase of the insulin receptor super-family that functions as oncogenic driver in a range of human cancers such as neuroblastoma. In order to investigate mechanisms underlying Alk oncogenic signaling, we conducted a genetic suppressor screen in Drosophila melanogaster. Our screen identified multiple loci important for Alk signaling, including members of Ras/Raf/ERK-, Pi3K-, and STAT-pathways as well as tailless (tll) and foxo whose orthologues NR2E1/TLX and FOXO3 are transcription factors implicated in human neuroblastoma.