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. DLG2 is expressed in the murine "bridge signature'' that represents the transcriptional transition state when neural crest cells or Schwann cell precursors differentiate to chromaffin cells of the adrenal gland. We show that the restoration of DLG2 expression spontaneously drives neuroblastoma cell differentiation, high-lighting the importance of DLG2 in this process. These findings are supported by genetic analyses of high-risk 11q deletion neuroblastomas, which identified genetic lesions in the DLG2 gene. Our data also suggest that further exploration of other bridge genes may help elucidate the mechanisms underlying the differentiation of NC-derived progenitors and their contribution to neuroblastomas

Anaplastic lymphoma kinase activity, a therapeutic target, suppresses neuroblastoma cell differentiation

Neuroblastoma (NB) is the most common extracranial pediatric solid malignancy caused by the failed differentiation of precursor cells of the developing sympathetic nervous system. NB accounts for about 15% of childhood cancer-related deaths. Treatment failure and relapse are common in NB patients despite intensive chemotherapy and immunotherapy interventions, suggesting the need for new and effective treatment options. Common genetic aberrations associated with NB include MYCN amplification, chromosome 11q deletion, 1p deletion, 17q gain, 2p gain, and recurrent mutations in Anaplastic Lymphoma Kinase (ALK). While treatment of some categories of ALK-positive pediatric cancer patients such as non-Hodgkin lymphoma and inflammatory myofibroblastic tumour (IMT) with the first-generation ALK tyrosine kinase inhibitor (TKI), crizotinib, produced promising results, the outcome for ALK-positive NB patients was less encouraging, hence the need for more potent ALK TKIs for treatment of NB patients. This thesis aimed to further our understanding of ALK signalling and its role in NB differentiation and explore novel ALK TKIs in a neuroblastoma setting. In the first study, we investigated the therapeutic efficacy of the second-generation ALK TKI, brigatinib, in an NB preclinical setting. Brigatinib was reported to be effective against ALK fusion-positive non-small cell lung tumours. We found that brigatinib potently inhibited both the activity of ALK full-length and growth of ALK-addicted NB cells in-vitro, in xenograft and Drosophila models. Compared to crizotinib, brigatinib inhibited the activities of different ALK-mutant alleles more effectively and potently inhibited crizotinib resistant ALK mutants in vitro. In the second study, we characterized a novel ALK-I1171T mutant allele which we identified in a tumour from a 16 month old NB patient. We showed that ALK-I1171T is a gain-of-function mutation, which is resistant to crizotinib, but can be effectively inhibited by second- and third-generation ALK TKIs such as brigatinib, ceritinib and lorlatinib. Based on these results and the severe toxic side effect of the initially administered chemotherapy, ceritinib monotherapy was chosen for this child. After 7.5 months of ceritinib treatment, the primary tumour shrunk in size and was removed surgically. The patient showed complete metastatic remission and remains in remission at 58 months post-treatment. In the third and last study, we investigated Disk large homologue 2 (DLG2), a gene reported to be uniquely upregulated in transient intermediary cells during Schwann cell precursor (SCP) differentiation to adrenal chromaffin cells. We found that DLG2, a gene located on the frequently deleted chromosome 11q in NB, is an NB tumour suppressor gene whose expression is lost in NB cell lines. Restoration of DLG2 expression inhibited NB cell growth and promoted NB cell differentiation. High expression of DLG2 in NB tumours is associated with good prognosis. Mechanistically we showed that oncogenic ALK maintains an undifferentiated NB cell phenotype by repressing DLG2 expression via the ERK1/2-SP1 signalling cascade. In summary, these findings highlight the role of ALK in differentiation and therapeutic potential of targeting ALK in ALK-positive NB tumours

ATR inhibition enables complete tumour regression in ALK-driven NB mouse models

High-risk neuroblastoma (NB) often involves MYCN amplification as well as mutations in ALK. Currently, high-risk NB presents significant clinical challenges, and additional therapeutic options are needed. Oncogenes like MYCN and ALK result in increased replication stress in cancer cells, offering therapeutically exploitable options. We have pursued phosphoproteomic analyses highlighting ATR activity in ALK-driven NB cells, identifying the BAY1895344 ATR inhibitor as a potent inhibitor of NB cell growth and proliferation. Using RNA-Seq, proteomics and phosphoproteomics we characterize NB cell and tumour responses to ATR inhibition, identifying key components of the DNA damage response as ATR targets in NB cells. ATR inhibition also produces robust responses in mouse models. Remarkably, a 2-week combined ATR/ALK inhibition protocol leads to complete tumor regression in two independent genetically modified mouse NB models. These results suggest that NB patients, particularly in high-risk groups with oncogene-induced replication stress, may benefit from ATR inhibition as therapeutic intervention. Effective therapeutic options are still needed in neuroblastoma treatment. Here, the authors, through a comprehensive proteomics analysis, identify ATR as a potential therapeutic target of neuroblastoma and demonstrate the efficacy of the ATR inhibitor BAY1895344 in combination with the ALK tyrosine kinase inhibitor lorlatinib

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