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. phosphoproteomics). This typically requires dedicated bioinformatics support, which is not always available. Additionally, while data from published studies are highly valuable and raw data (e.g. fastq files) are nowadays released in public repositories, data processing is time-consuming and again difficult without bioinformatics support. To facilitate NB research, more user-friendly and immediately accessible platforms are needed to explore newly generated as well as existing high throughput data. To make this possible, we developed an interactive data centralization and visualization web application, called CLEAN (the Cell Line Explorer web Application of Neuroblastoma data; https://ccgg.ugent.be/shiny/clean/). By focusing on the regulation of the DNA damage response, a therapeutic target of major interest in neuroblastoma, we demonstrate how CLEAN can be used to gain novel mechanistic insights and identify putative drug targets in neuroblastoma.

Benchmark of tools for in silico prediction of MHC class I and class II genotypes from NGS data.

BACKGROUND: The Human Leukocyte Antigen (HLA) genes are a group of highly polymorphic genes that are located in the Major Histocompatibility Complex (MHC) region on chromosome 6. The HLA genotype affects the presentability of tumour antigens to the immune system. While knowledge of these genotypes is of utmost importance to study differences in immune responses between cancer patients, gold standard, PCR-derived genotypes are rarely available in large Next Generation Sequencing (NGS) datasets. Therefore, a variety of methods for in silico NGS-based HLA genotyping have been developed, bypassing the need to determine these genotypes with separate experiments. However, there is currently no consensus on the best performing tool. RESULTS: We evaluated 13 MHC class I and/or class II HLA callers that are currently available for free academic use and run on either Whole Exome Sequencing (WES) or RNA sequencing data. Computational resource requirements were highly variable between these tools. Three orthogonal approaches were used to evaluate the accuracy on several large publicly available datasets: a direct benchmark using PCR-derived gold standard HLA calls, a correlation analysis with population-based allele frequencies and an analysis of the concordance between the different tools. The highest MHC-I calling accuracies were found for Optitype (98.0%) and arcasHLA (99.4%) on WES and RNA sequencing data respectively, while for MHC-II HLA-HD was the most accurate tool for both data types (96.2% and 99.4% on WES and RNA data respectively). CONCLUSION: The optimal strategy for HLA genotyping from NGS data depends on the availability of either WES or RNA data, the size of the dataset and the available computational resources. If sufficient resources are available, we recommend Optitype and HLA-HD for MHC-I and MHC-II genotype calling respectively.

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.

Low immunogenicity of common cancer hot spot mutations resulting in false immunogenic selection signals.

Cancer is driven by somatic mutations that result in a cellular fitness advantage. This selective advantage is expected to be counterbalanced by the immune system when these driver mutations simultaneously lead to the generation of neoantigens, novel peptides that are presented at the cancer cell membrane via HLA molecules from the MHC complex. The presentability of these peptides is determined by a patient's MHC genotype and it has been suggested that this results in MHC genotype-specific restrictions of the oncogenic mutational landscape. Here, we generated a set of virtual patients, each with an identical and prototypical MHC genotype, and show that the earlier reported HLA affinity differences between observed and unobserved mutations are unrelated to MHC genotype variation. We demonstrate how these differences are secondary to high frequencies of 13 hot spot driver mutations in 6 different genes. Several oncogenic mechanisms were identified that lower the peptides' HLA affinity, including phospho-mimicking substitutions in BRAF, destabilizing tyrosine mutations in TP53 and glycine-rich mutational contexts in the GTP-binding KRAS domain. In line with our earlier findings, our results emphasize that HLA affinity predictions are easily misinterpreted when studying immunogenic selection processes.

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. 8-10% of primary NB patients are ALK-positive, a figure that increases in the relapsed population. ALK is activated by the ALKAL2 ligand located on chromosome 2p, along with ALK and MYCN, in the "2p-gain" region associated with NB. Dysregulation of ALK ligand in NB has not been addressed, although one of the first oncogenes described was v-sis that shares > 90% homology with PDGF. Therefore, we tested whether ALKAL2 ligand could potentiate NB progression in the absence of ALK mutation. We show that ALKAL2 overexpression in mice drives ALK TKI-sensitive NB in the absence of ALK mutation, suggesting that additional NB patients, such as those exhibiting 2p-gain, may benefit from ALK TKI-based therapeutic intervention.