A clinically annotated post-mortem approach to study multi-organ somatic mutational clonality in normal tissues.

Recent research on normal human tissues identified omnipresent clones of cells, driven by somatic mutations known to be responsible for carcinogenesis (e.g., in TP53 or NOTCH1). These new insights are fundamentally changing current tumor evolution models, with broad oncological implications. Most studies are based on surgical remnant tissues, which are not available for many organs and rarely in a pan-organ setting (multiple organs from the same individual). Here, we describe an approach based on clinically annotated post-mortem tissues, derived from whole-body donors that are routinely used for educational purposes at human anatomy units. We validated this post-mortem approach using UV-exposed and unexposed epidermal skin tissues and confirm the presence of positively selected NOTCH1/2-, TP53- and FAT1-driven clones. No selection signals were detected in a set of immune genes or housekeeping genes. Additionally, we provide the first evidence for smoking-induced clonal changes in oral epithelia, likely underlying the origin of head and neck carcinogenesis. In conclusion, the whole-body donor-based approach provides a nearly unlimited healthy tissue resource to study mutational clonality and gain fundamental mutagenic insights in the presumed earliest stages of tumor evolution.

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.

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, highlighting 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.

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