Current and future treatment strategies for rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children, and can be subcategorized histologically and/or based on PAX-FOXO1 fusion gene status. Over the last four decades, there have been no significant improvements in clinical outcomes for advanced and metastatic RMS patients, underscoring a need for new treatment options for these groups. Despite significant advancements in our understanding of the genomic landscape and underlying biological mechanisms governing RMS that have informed the identification of novel therapeutic targets, development of these therapies in clinical trials has lagged far behind. In this review, we summarize the current frontline multi-modality therapy for RMS according to pediatric protocols, highlight emerging targeted therapies and immunotherapies identified by preclinical studies, and discuss early clinical trial data and the implications they hold for future clinical development

Forward genetic screen of human transposase genomic rearrangements

BACKGROUND: Numerous human genes encode potentially active DNA transposases or recombinases, but our understanding of their functions remains limited due to shortage of methods to profile their activities on endogenous genomic substrates. RESULTS: To enable functional analysis of human transposase-derived genes, we combined forward chemical genetic hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) screening with massively parallel paired-end DNA sequencing and structural variant genome assembly and analysis. Here, we report the HPRT1 mutational spectrum induced by the human transposase PGBD5, including PGBD5-specific signal sequences (PSS) that serve as potential genomic rearrangement substrates. CONCLUSIONS: The discovered PSS motifs and high-throughput forward chemical genomic screening approach should prove useful for the elucidation of endogenous genome remodeling activities of PGBD5 and other domesticated human DNA transposases and recombinases

Genomic DNA transposition induced by human PGBD5

Transposons are mobile genetic elements that are found in nearly all organisms, including humans. Mobilization of DNA transposons by transposase enzymes can cause genomic rearrangements, but our knowledge of human genes derived from transposases is limited. In this study, we find that the protein encoded by human PGBD5, the most evolutionarily conserved transposable element-derived gene in vertebrates, can induce stereotypical cut-and-paste DNA transposition in human cells. Genomic integration activity of PGBD5 requires distinct aspartic acid residues in its transposase domain, and specific DNA sequences containing inverted terminal repeats with similarity to piggyBac transposons. DNA transposition catalyzed by PGBD5 in human cells occurs genome-wide, with precise transposon excision and preference for insertion at TTAA sites. The apparent conservation of DNA transposition activity by PGBD5 suggests that genomic remodeling contributes to its biological function

Detection and validation of circular DNA fragments using nanopore sequencing

Occurrence of extra-chromosomal circular DNA is a phenomenon frequently observed in tumor cells, and the presence of such DNA has been recognized as a marker of adverse outcome across cancer types. We here describe a computational workflow for identification of DNA circles from long-read sequencing data. The workflow is implemented based on the Snakemake workflow management system. Its key step uses a graph-theoretic approach to identify putative circular fragments validated on simulated reads. We then demonstrate robustness of our approach using nanopore sequencing of selectively enriched circular DNA by highly sensitive and specific recovery of plasmids and the mitochondrial genome, which is the only circular DNA in normal human cells. Finally, we show that the workflow facilitates detection of larger circular DNA fragments containing extrachromosomal copies of the MYCN oncogene and the respective breakpoints, which is a potentially useful application in disease monitoring of several cancer types

Partner independent fusion gene detection by multiplexed CRISPR-Cas9 enrichment and long read nanopore sequencing

Fusion genes are hallmarks of various cancer types and important determinants for diagnosis, prognosis and treatment. Fusion gene partner choice and breakpoint-position promiscuity restricts diagnostic detection, even for known and recurrent configurations. Here, we develop FUDGE (FUsion Detection from Gene Enrichment) to accurately and impartially identify fusions. FUDGE couples target-selected and strand-specific CRISPR-Cas9 activity for fusion gene driver enrichment - without prior knowledge of fusion partner or breakpoint-location - to long read nanopore sequencing with the bioinformatics pipeline NanoFG. FUDGE has flexible target-loci choices and enables multiplexed enrichment for simultaneous analysis of several genes in multiple samples in one sequencing run. We observe on-average 665 fold breakpoint-site enrichment and identify nucleotide resolution fusion breakpoints within 2 days. The assay identifies cancer cell line and tumor sample fusions irrespective of partner gene or breakpoint-position. FUDGE is a rapid and versatile fusion detection assay for diagnostic pan-cancer fusion detection

Small-molecule dual PLK1 and BRD4 inhibitors are active against preclinical models of pediatric solid tumors

Simultaneous inhibition of multiple molecular targets is an established strategy to improve the continuance of clinical response to therapy. Here, we screened 49 molecules with dual nanomolar inhibitory activity against BRD4 and PLK1, best classified as dual kinase-bromodomain inhibitors, in pediatric tumor cell lines for their antitumor activity. We identified two candidate dual kinase-bromodomain inhibitors with strong and tumor-specific activity against neuroblastoma, medulloblastoma, and rhabdomyosarcoma tumor cells. Dual PLK1 and BRD4 inhibitor treatment suppressed proliferation and induced apoptosis in pediatric tumor cell lines at low nanomolar concentrations. This was associated with reduced MYCN-driven gene expression as assessed by RNA sequencing. Treatment of patient-derived xenografts with dual inhibitor UMB103 led to significant tumor regression. We demonstrate that concurrent inhibition of two central regulators of MYC protein family of protooncogenes, BRD4, and PLK1, with single small molecules has strong and specific antitumor effects in preclinical pediatric cancer models

Reflection of neuroblastoma intratumor heterogeneity in the new OHC-NB1 disease model

Accurate modeling of intratumor heterogeneity presents a bottleneck against drug testing. Flexibility in a preclinical platform is also desirable to support assessment of different endpoints. We established the model system, OHC-NB1, from a bone marrow metastasis from a patient diagnosed with MYCN-amplified neuroblastoma and performed whole-exome sequencing on the source metastasis and the different models and passages during model development (monolayer cell line, 3D spheroid culture and subcutaneous xenograft tumors propagated in mice). OHC-NB1 harbors a MYCN amplification in double minutes, 1p deletion, 17q gain and diploid karyotype, which persisted in all models. A total of 80-540 single-nucleotide variants (SNVs) was detected in each sample, and comparisons between the source metastasis and models identified 34 of 80 somatic SNVs to be propagated in the models. Clonal reconstruction using the combined copy number and SNV data revealed marked clonal heterogeneity in the originating metastasis, with 4 clones being reflected in the model systems. The set of OHC-NB1 models represents 43% of somatic SNVs and 23% of the cellularity in the originating metastasis with varying clonal compositions, indicating that heterogeneity is partially preserved in our model system

Long-read sequencing of diagnosis and post-therapy medulloblastoma reveals complex rearrangement patterns and epigenetic signatures

Cancer genomes harbor a broad spectrum of structural variants (SVs) driving tumorigenesis, a relevant subset of which escape discovery using short-read sequencing. We employed Oxford Nanopore Technologies (ONT) long-read sequencing in a paired diagnostic and post-therapy medulloblastoma to unravel the haplotype-resolved somatic genetic and epigenetic landscape. We assembled complex rearrangements, including a 1.55-Mbp chromothripsis event, and we uncover a complex SV pattern termed templated insertion (TI) thread, characterized by short (mostly <1 kb) insertions showing prevalent self-concatenation into highly amplified structures of up to 50 kbp in size. TI threads occur in 3% of cancers, with a prevalence up to 74% in liposarcoma, and frequent colocalization with chromothripsis. We also perform long-read-based methylome profiling and discover allele-specific methylation (ASM) effects, complex rearrangements exhibiting differential methylation, and differential promoter methylation in cancer-driver genes. Our study shows the advantage of long-read sequencing in the discovery and characterization of complex somatic rearrangements

Target actionability review to evaluate CDK4/6 as a therapeutic target in paediatric solid and brain tumours

BACKGROUND: Childhood cancer is still a leading cause of death around the world. To improve outcomes, there is an urgent need for tailored treatment. The systematic evaluation of existing preclinical data can provide an overview of what is known and identify gaps in the current knowledge. Here, we applied the target actionability review (TAR) methodology to assess the strength and weaknesses of available scientific literature on CDK4/6 as a therapeutic target in paediatric solid and brain tumours by structured critical appraisal. METHODS: Using relevant search terms in PubMed, a list of original publications investigating CDK4/6 in paediatric solid tumour types was identified based on relevancy criteria. Each publication was annotated for the tumour type and categorised into separate proof-of-concept (PoC) data modules. Based on rubrics, quality and experimental outcomes were scored independently by two reviewers. A third reviewer evaluated and adjudicated score discrepancies. Scores for each PoC module were averaged for each tumour type and visualised in a heatmap matrix in the publicly available R2 data portal. RESULTS AND CONCLUSIONS: This CDK4/6 TAR, generated by analysis of 151 data entries from 71 publications, showed frequent genomic aberrations of CDK4/6 in rhabdomyosarcoma, osteosarcoma, high-grade glioma, medulloblastoma, and neuroblastoma. However, a clear correlation between CDK4/6 aberrations and compound efficacy is not coming forth from the literature. Our analysis indicates that several paediatric indications would need (further) preclinical evaluation to allow for better recommendations, especially regarding the dependence of tumours on CDK4/6, predictive biomarkers, resistance mechanisms, and combination strategies. Nevertheless, our TAR heatmap provides support for the relevance of CDK4/6 inhibition in Ewing sarcoma, medulloblastoma, malignant peripheral nerve sheath tumour and to a lesser extent neuroblastoma, rhabdomyosarcoma, rhabdoid tumour and high-grade glioma. The interactive heatmap is accessible through R2 [r2platform.com/TAR/CDK4_6]