Targeting ALK and WIP1 : neuroblastoma precision medicine under development

Neuroblastoma is the childhood solid tumor accountable for the largest number of deaths, calling for improved treatment. Some genetic alterations are considered crucial in driving initiation and progression of neuroblastoma. These are usually associated with poor prognosis and are viewed as potential therapeutic targets. Anaplastic lymphoma kinase (ALK) is one of the best-established disease drivers in neuroblastoma, harboring oncogenic mutations or amplifications in about 10-15 % of all cases. In 2013, results from the first phase I study of an ALK inhibitor in neuroblastoma, crizotinib, were announced showing disappointing clinical response. Improved later-generation ALK inhibitors have since become available while biological and clinical understanding of ALK mutations in neuroblastoma has increased. In Paper I, a patient with a metastatic high-risk neuroblastoma suffered extreme toxicity due to an underlying condition, Fanconi anemia, prohibiting further conventional treatment. Whole genome sequencing of tumor material revealed a novel ALK variant, ALK-I1171T, which was identified as a potent gain-of-function mutant resistant to crizotinib, but sensitive to later-generation ALK inhibitors including ceritinib. It was shown that ceritinib was equally effective as crizotinib in a panel of ALK-driven neuroblastoma cell lines. As a result, the child could be offered ceritinib in monotherapy, achieving complete metastatic remission and enabling resection of the primary tumor, and long-term survival. In Paper II, activation of ALK as well as tropomyosin-related kinase A (TRKA) was observed in the absence of corresponding genomic alterations in an infant with metastasized and treatment-refractory neuroblastoma. A novel germline mutation of the ALK ligand ALKAL-2 was revealed by whole genome sequencing, which in cell culture and drosophila experiments was shown to be functional. Since an inhibitor targeting both ALK and TRKA was available in entrectinib, this drug was tested in neuroblastoma cell lines. Subsequently entrectinib treatment could be offered to the patient, producing a prompt improvement of clinical status and a gradual decline of catecholamine markers and metastases over the course of several years. The majority of high-risk neuroblastomas contain a gain of chromosome 17q, a feature that correlates with aggressive disease and poor prognosis. The gene PPM1D is situated within the gained region and encodes the phosphatase WIP1, an inhibitor of p53 and a negative regulator of DNA damage response. WIP1 is often overexpressed in neuroblastoma, and overexpression in transgenic mice predisposes to tumor formation. In Paper III we showed that WIP1 expression correlates to 17q gain in neuroblastoma and medulloblastoma cell lines, which are also highly dependent on WIP1. Knockdown of WIP1 delayed tumor growth. Among different small molecule WIP1 inhibitors evaluated, SL-176 was demonstrated to be effective in all tested cell lines regardless of p53 mutational status. Pharmacologic inhibition of WIP1 with SL-176 in xenograft-bearing mice curbed tumor growth. To achieve an improved cytotoxic effect, a drug combination screening with the WIP1 inhibitor SL-176 was conducted in Paper IV, identifying combination with epigenetic modification by inhibition of the histone demethylase JMJD3 as the most synergistic strategy. Combination of SL-176 and the JMJD3 inhibitor GSK-J4 showed strong synergism in a panel of neuroblastoma cell lines with regard to cell viability, WIP1 downstream targets, cell cycle arrest and apoptosis. Pathway analysis of differentially expressed genes, as identified by RNA sequencing, revealed enrichment of genes involved in pathways associated with DNA damage response. While ALK inhibitors are already available to a subset of neuroblastoma patients, WIP1 is a promising therapeutic target where much work remains before patients may potentially benefit. It is our conviction that in the future, targeted therapy will be available to all high-risk neuro-blastoma patients. This thesis represents a few small steps on the road to accomplish that goal

The ENaC-overexpressing mouse as a model of cystic fibrosis lung disease

AbstractChronic lung disease remains the major cause of morbidity and mortality of cystic fibrosis (CF) patients. Cftr mutant mice developed severe intestinal obstruction, but did not exhibit the characteristic CF ion transport defects (i.e. deficient cAMP-dependent Cl− secretion and increased Na+ absorption) in the lower airways, and failed to develop CF-like lung disease. These observations led to the generation of transgenic mice with airway-specific overexpression of the epithelial Na+ channel (ENaC) as an alternative approach to mimic CF ion transport pathophysiology in the lung. Studies of the phenotype of βENaC-transgenic mice demonstrated that increased airway Na+ absorption causes airway surface liquid (ASL) depletion, reduced mucus transport and a spontaneous CF-like lung disease with airway mucus obstruction and chronic airway inflammation. Here, we summarize approaches that can be applied for studies of the complex in vivo pathogenesis and preclinical evaluation of novel therapeutic strategies in this model of CF lung disease

Multifocal Neuroblastoma and Central Hypoventilation in An Infant with Germline ALK F1174I Mutation

Funding Information: This work has been supported by grants from the Swedish Cancer Society (TM 15-794; TM 20-1213; PK 19-566), the Swedish Childhood Cancer Foundation (TM 16-147; TM 17-166; TM 19-139; PK 17-122; SF 15-61, 18-99; DT 12-002, NC12-0026), the Swedish Research Council (TM 521-2014-3031), the Swedish state under the LUA/ALF agreement (TM ALFGBG-447171) and the Swedish Foundation for Strategic Research (TM/PK RB13-0204, www.nnbcr.se). SF was the recipient of a Research Assistant Fellowship (14-64), by the Swedish Childhood Cancer Foundation. Publisher Copyright: © 2022 by the authors.A preterm infant with central hypoventilation was diagnosed with multifocal neuroblastoma. Congenital anomalies of the autonomic nervous system in association with neuroblastoma are commonly associated with germline mutations in PHOX2B. Further, the ALK gene is frequently mutated in both familial and sporadic neuroblastoma. Sanger sequencing of ALK and PHOX2B, SNP microarray of three tumor samples and whole genome sequencing of tumor and blood were performed. Genetic testing revealed a germline ALK F1174I mutation that was present in all tumor samples as well as in normal tissue samples from the patient. Neither of the patient’s parents presented the ALK variant. Array profiling of the three tumor samples showed that two of them had only numerical aberrations, whereas one sample displayed segmental alterations, including a gain at chromosome 2p, resulting in two copies of the ALK-mutated allele. Whole genome sequencing confirmed the presence of the ALK variant and did not detect any aberrations in the coding or promotor region of PHOX2B. This study is to our knowledge the first to report a de novo ALK F1174I germline mutation. This may not only predispose to congenital multifocal neuroblastoma but may also contribute to the respiratory dysfunction seen in this patient.Peer reviewe

PPM1D Is a Therapeutic Target in Childhood Neural Tumors

Childhood medulloblastoma and high-risk neuroblastoma frequently present with segmental gain of chromosome 17q corresponding to aggressive tumors and poor patient prognosis. Located within the 17q-gained chromosomal segments is PPM1D at chromosome 17q23.2. PPM1D encodes a serine/threonine phosphatase, WIP1, that is a negative regulator of p53 activity as well as key proteins involved in cell cycle control, DNA repair and apoptosis. Here, we show that the level of PPM1D expression correlates with chromosome 17q gain in medulloblastoma and neuroblastoma cells, and both medulloblastoma and neuroblastoma cells are highly dependent on PPM1D expression for survival. Comparison of different inhibitors of WIP1 showed that SL-176 was the most potent compound inhibiting medulloblastoma and neuroblastoma growth and had similar or more potent effects on cell survival than the MDM2 inhibitor Nutlin-3 or the p53 activator RITA. SL-176 monotherapy significantly suppressed the growth of established medulloblastoma and neuroblastoma xenografts in nude mice. These results suggest that the development of clinically applicable compounds inhibiting the activity of WIP1 is of importance since PPM1D activating mutations, genetic gain or amplifications and/or overexpression of WIP1 are frequently detected in several different cancers

High Expression of PPM1D Induces Tumors Phenotypically Similar to TP53 Loss-of-Function Mutations in Mice

PPM1D is a negative regulator of p53 and genomic aberrations resulting in increased activity of PPM1D have been observed in cancers of different origins, indicating that PPM1D has oncogenic properties. We established a transgenic mouse model overexpressing PPM1D and showed that these mice developed a wide variety of cancers. PPM1D-expressing mice developed tumors phenotypically and genetically similar to tumors in mice with dysfunctional p53. T-cell lymphoblastic lymphoma was the most frequent cancer observed in these mice (55%) followed by adenocarcinomas (24%), leukemia (12%) and other solid tumors including neuroblastoma. Characterization of T-cell lymphomas in mice overexpressing PPM1D demonstrates Pten-deletion and p53-accumulation similar to mice with p53 loss-of-function. Also, Notch1 mutations which are recurrently observed in T-cell acute lymphoblastic lymphoma (T-ALL) were frequently detected in PPM1D-transgenic mice. Hence, PPM1D acts as an oncogenic driver in connection with cellular stress, suggesting that the PPM1D gene status and expression levels should be investigated in TP53 wild-type tumors

Development of Chronic Bronchitis and Emphysema in β-Epithelial Na+ Channel–Overexpressing Mice

Rationale: Chronic obstructive pulmonary disease is a leading cause of death worldwide, but its pathogenesis is not well understood. Previous studies have shown that airway surface dehydration in β-epithelial Na+ channel (βENaC)–overexpressing mice caused a chronic lung disease with high neonatal pulmonary mortality and chronic bronchitis in adult survivors

Clinical response of the novel activating ALK-I1171T mutation in neuroblastoma o 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