Elimusertib has anti-tumor activity in preclinical patient-derived pediatric solid tumor models

The small molecule inhibitor of ataxia telangiectasia and Rad3-related protein (ATR), elimusertib, is currently being tested clinically in various cancer entities in adults and children. Its preclinical anti-tumor activity in pediatric malignancies, however, is largely unknown. We here assessed the preclinical activity of elimusertib in 38 cell lines and 32 patient-derived xenograft (PDX) models derived from common pediatric solid tumor entities. Detailed in vitro and in vivo molecular characterization of the treated models enabled the evaluation of response biomarkers. Pronounced objective response rates were observed for elimusertib monotherapy in PDX, when treated with a regimen currently used in clinical trials. Strikingly, elimusertib showed stronger anti-tumor effects than some standard of care chemotherapies, particularly in alveolar rhabdomysarcoma PDX. Thus, elimusertib has strong preclinical anti-tumor activity in pediatric solid tumor models, which may translate to clinically meaningful responses in patients

Parallel sequencing of extrachromosomal circular DNAs and transcriptomes in single cancer cells

Extrachromosomal DNAs (ecDNAs) are common in cancer, but many questions about their origin, structural dynamics and impact on intratumor heterogeneity are still unresolved. Here we describe single-cell extrachromosomal circular DNA and transcriptome sequencing (scEC&T-seq), a method for parallel sequencing of circular DNAs and full-length mRNA from single cells. By applying scEC&T-seq to cancer cells, we describe intercellular differences in ecDNA content while investigating their structural heterogeneity and transcriptional impact. Oncogene-containing ecDNAs were clonally present in cancer cells and drove intercellular oncogene expression differences. In contrast, other small circular DNAs were exclusive to individual cells, indicating differences in their selection and propagation. Intercellular differences in ecDNA structure pointed to circular recombination as a mechanism of ecDNA evolution. These results demonstrate scEC&T-seq as an approach to systematically characterize both small and large circular DNA in cancer cells, which will facilitate the analysis of these DNA elements in cancer and beyond

Sarcoma treatment in the era of molecular medicine

Sarcomas are heterogeneous and clinically challenging soft tissue and bone cancers. Although constituting only 1% of all human malignancies, sarcomas represent the second most common type of solid tumors in children and adolescents and comprise an important group of secondary malignancies. More than 100 histological subtypes have been characterized to date, and many more are being discovered due to molecular profiling. Owing to their mostly aggressive biological behavior, relative rarity, and occurrence at virtually every anatomical site, many sarcoma subtypes are in particular difficult-to-treat categories. Current multimodal treatment concepts combine surgery, polychemotherapy (with/without local hyperthermia), irradiation, immunotherapy, and/or targeted therapeutics. Recent scientific advancements have enabled a more precise molecular characterization of sarcoma subtypes and revealed novel therapeutic targets and prognostic/predictive biomarkers. This review aims at providing a comprehensive overview of the latest advances in the molecular biology of sarcomas and their effects on clinical oncology; it is meant for a broad readership ranging from novices to experts in the field of sarcoma.Peer reviewe

Mutations in ALK signaling pathways conferring resistance to ALK inhibitor treatment lead to collateral vulnerabilities in neuroblastoma cells

Background: Development of resistance to targeted therapies has tempered initial optimism that precision oncology would improve poor outcomes for cancer patients. Resistance mechanisms, however, can also confer new resistance-specific vulnerabilities, termed collateral sensitivities. Here we investigated anaplastic lymphoma kinase (ALK) inhibitor resistance in neuroblastoma, a childhood cancer frequently affected by activating ALK alterations. Methods: Genome-wide forward genetic CRISPR-Cas9 based screens were performed to identify genes associated with ALK inhibitor resistance in neuroblastoma cell lines. Furthermore, the neuroblastoma cell line NBLW-R was rendered resistant by continuous exposure to ALK inhibitors. Genes identified to be associated with ALK inhibitor resistance were further investigated by generating suitable cell line models. In addition, tumor and liquid biopsy samples of four patients with ALK-mutated neuroblastomas before ALK inhibitor treatment and during tumor progression under treatment were genomically profiled. Results: Both genome-wide CRISPR-Cas9-based screens and preclinical spontaneous ALKi resistance models identified NF1 loss and activating NRASQ61K mutations to confer resistance to chemically diverse ALKi. Moreover, human neuroblastomas recurrently developed de novo loss of NF1 and activating RAS mutations after ALKi treatment, leading to therapy resistance. Pathway-specific perturbations confirmed that NF1 loss and activating RAS mutations lead to RAS-MAPK signaling even in the presence of ALKi. Intriguingly, NF1 loss rendered neuroblastoma cells hypersensitive to MEK inhibition. Conclusions: Our results provide a clinically relevant mechanistic model of ALKi resistance in neuroblastoma and highlight new clinically actionable collateral sensitivities in resistant cells

Enhancer hijacking determines extrachromosomal circular MYCN amplicon architecture in neuroblastoma

MYCN amplification drives one in six cases of neuroblastoma. The supernumerary gene copies are commonly found on highly rearranged, extrachromosomal circular DNA (ecDNA). The exact amplicon structure has not been described thus far and the functional relevance of its rearrangements is unknown. Here, we analyze the MYCN amplicon structure using short-read and Nanopore sequencing and its chromatin landscape using ChIP-seq, ATAC-seq and Hi-C. This reveals two distinct classes of amplicons which explain the regulatory requirements for MYCN overexpression. The first class always co-amplifies a proximal enhancer driven by the noradrenergic core regulatory circuit (CRC). The second class of MYCN amplicons is characterized by high structural complexity, lacks key local enhancers, and instead contains distal chromosomal fragments harboring CRC-driven enhancers. Thus, ectopic enhancer hijacking can compensate for the loss of local gene regulatory elements and explains a large component of the structural diversity observed in MYCN amplification. MYCN amplification is common in neuroblastomas. Here the authors analyse the MYCN amplicon structure and its epigenetic regulation by integrating short- and longread genomic and epigenomic data and find two classes of MYCN amplicons in neuroblastomas, one driven by local enhancers and the other by hijacking of distal regulatory elements

Mutations in ALK signaling pathways conferring resistance to ALK inhibitor treatment lead to collateral vulnerabilities in neuroblastoma cells.

BACKGROUND: Development of resistance to targeted therapies has tempered initial optimism that precision oncology would improve poor outcomes for cancer patients. Resistance mechanisms, however, can also confer new resistance-specific vulnerabilities, termed collateral sensitivities. Here we investigated anaplastic lymphoma kinase (ALK) inhibitor resistance in neuroblastoma, a childhood cancer frequently affected by activating ALK alterations. METHODS: Genome-wide forward genetic CRISPR-Cas9 based screens were performed to identify genes associated with ALK inhibitor resistance in neuroblastoma cell lines. Furthermore, the neuroblastoma cell line NBLW-R was rendered resistant by continuous exposure to ALK inhibitors. Genes identified to be associated with ALK inhibitor resistance were further investigated by generating suitable cell line models. In addition, tumor and liquid biopsy samples of four patients with ALK-mutated neuroblastomas before ALK inhibitor treatment and during tumor progression under treatment were genomically profiled. RESULTS: Both genome-wide CRISPR-Cas9-based screens and preclinical spontaneous ALKi resistance models identified NF1 loss and activating NRASQ61K mutations to confer resistance to chemically diverse ALKi. Moreover, human neuroblastomas recurrently developed de novo loss of NF1 and activating RAS mutations after ALKi treatment, leading to therapy resistance. Pathway-specific perturbations confirmed that NF1 loss and activating RAS mutations lead to RAS-MAPK signaling even in the presence of ALKi. Intriguingly, NF1 loss rendered neuroblastoma cells hypersensitive to MEK inhibition. CONCLUSIONS: Our results provide a clinically relevant mechanistic model of ALKi resistance in neuroblastoma and highlight new clinically actionable collateral sensitivities in resistant cells

Extrachromosomal circular DNA drives oncogenic genome remodeling in neuroblastoma

Extrachromosomal circularization of DNA is an important genomic feature in cancer. However, the structure, composition and genome-wide frequency of extrachromosomal circular DNA have not yet been profiled extensively. Here, we combine genomic and transcriptomic approaches to describe the landscape of extrachromosomal circular DNA in neuroblastoma, a tumor arising in childhood from primitive cells of the sympathetic nervous system. Our analysis identifies and characterizes a wide catalog of somatically acquired and undescribed extrachromosomal circular DNAs. Moreover, we find that extrachromosomal circular DNAs are an unanticipated major source of somatic rearrangements, contributing to oncogenic remodeling through chimeric circularization and reintegration of circular DNA into the linear genome. Cancer-causing lesions can emerge out of circle-derived rearrangements and are associated with adverse clinical outcome. It is highly probable that circle-derived rearrangements represent an ongoing mutagenic process. Thus, extrachromosomal circular DNAs represent a multihit mutagenic process, with important functional and clinical implications for the origins of genomic remodeling in cancer. Combined genomic and transcriptomic approaches identify the landscape of extrachromosomal circular DNA in neuroblastoma and reveal that extrachromosomal circular DNA is a major source of somatic rearrangements

Figure 3 from Passenger Gene Coamplifications Create Collateral Therapeutic Vulnerabilities in Cancer

A proof-of-principle study identifies a selective mTOR pathway dependency in cells with DDX1-MYCN coamplification. A, Correlation between DDX1 copy-number and dependency scores (CERES) for RAPTOR in neuroblastoma cell lines (Pearson correlation analysis, R = −0.5996, P = 0.0152, N = 13). B, Western immunoblot of RAPTOR and DDX1 in the KELLY cells transduced with the doxycycline-inducible DDX1-mCherry vectors and with two pairs of sgRNAs targeting RAPTOR (sgRAPTOR) or a nontargeting sgRNA (sgNT) as well as Cas9 in the presence and absence of doxycycline (1 μg/mL). Tubulin serves as a loading control. C, Representative images of cell colonies formed by KELLY cells transduced with the doxycycline-inducible DDX1-mCherry vectors and with two pairs of sgRNA targeting RAPTOR (sgRAPTOR) or nontarget sgRNA (sgNT) as well as Cas9 in the presence and absence of doxycycline (1 μg/mL) and stained with crystal violet (left). Quantification of colony numbers (right, mean ± SE. N = 3 biological replicates; Welch t test, P = 0.564, 0.000117, and 0.00131 for sgNT, sgRAPTOR_1, and sgRAPTOR_2, respectively). D, Gene set enrichment analysis (GSEA) based on a set of genes regulated by mTORC1 measured in genes differentially expressed in tumors with high versus low DDX1 expression. E, GSEA based on a set of genes regulated by mTORC1 measured in genes differentially expressed in KELLY cells harboring a MYCN amplification with versus without ectopic DDX1 expression. F, Western blot of the relative protein expression of mTOR ser2448 phosphorylation and P70-S6K Thr389 phosphorylation in KELLY cell after inducible expression of DDX1 (1,000 ng/mL doxycycline treatment for 48 hours).</p

Supplementary Table S7 from Passenger Gene Coamplifications Create Collateral Therapeutic Vulnerabilities in Cancer

List of antibodies, materials, oligonucleotides, deposited data and software used in this study.</p

Figure 5 from Passenger Gene Coamplifications Create Collateral Therapeutic Vulnerabilities in Cancer

DDX1 hijacks the α-KGDH complex resulting in α-KG accumulation and OXPHOS reduction. A, Relative concentrations of α-KG, citrate, and isocitrate in cancer cell lines with DDX1-MYCN coamplifications (red) compared with cells only harboring MYCN amplifications (blue; Welch t test, P = 0.038764, 0.008224, and 0.025814 for α-KG, citrate, and isocitrate, respectively; N = 4 independent MYCN-amplified cancer cell lines versus N = 8 independent cancer cell lines with DDX1-MYCN coamplification). B, Relative α-KG concentrations measured by GC-MS in KELLY cells ectopically expressing DDX1 or the DDX1-Δ269-295aa for 48 hours. KELLY cells transduced with an empty vector and exposed to doxycycline were used as control (Wilcox test, P = 0.02778; data are shown as mean ± standard error). C, Western immunoblot of DDX1, P70-S6K, P70-S6K Thr389 phosphorylation, and α-tubulin in IMR5/75 cells treated with DM-αKG (2 mmol/L for 48 hours) and expressing shRNA targeting either DLST or GFP as control. D, Mitochondrial oxygen consumption rate (OCR) measured using live-cell metabolic analysis at basal respiration, maximal respiration, and ATP production in KELLY cells inducibly expressing DDX1 or DDX1-Δ269-295aa for 48 hours. KELLY cell transduced with a doxycycline-inducible empty vector served as negative control (Welch t test, P = 0.002, 0.010, and 0.002 for basal respiration, maximal respiration, and ATP production, respectively; data are shown as mean ± SE; N = 4 independent replicates). E, Exemplary photomicrographs taken on a transmission electron microscope of cells expressing DDX1 compared with cells expressing DDX1 Δ269-295aa. Cells transduced with an empty vector well as cells not treated with doxycycline served as negative controls. F, Quantification of mitochondrial length (longest axis in a cross-section) of cells shown in E (Wilcox test, P = 0.7954, 7.028e−10, and 0.1453, independently).</p