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

Supplementary Table S1 from Passenger Gene Coamplifications Create Collateral Therapeutic Vulnerabilities in Cancer

List of gene dependencies associated with DDX1 co-amplification.</p

Supplementary Table S2 from Passenger Gene Coamplifications Create Collateral Therapeutic Vulnerabilities in Cancer

Gene sets enriched in in primary neuroblastomas with DDX1 co-amplification.</p

Supplementary Table S6 from Passenger Gene Coamplifications Create Collateral Therapeutic Vulnerabilities in Cancer

QC sample reporting for Gas chromatography–mass spectrometry (GS-MS)</p

Supplementary Figures S1-S9 from Passenger Gene Coamplifications Create Collateral Therapeutic Vulnerabilities in Cancer

Legends for supplementarytables and supplementary figures. Supplementary Figure S1. Passenger genes are frequently co-amplified with oncogenes in cancers. Supplementary Figure S2. DDX1 is highly expressed when co-amplified with MYCN. Supplementary Figure S3. Neuroblastoma cell lines with DDX1-MYCN co-amplification depend on mTORC1. Supplementary Figure S4. Ectopic DDX1 expression does not alter MYCN-driven tumorigenesis in zebrafish. Supplementary Figure S5.DDX1 expression does not affect tumorigenic properties of cancer cell lines but induces changes in cell size. Supplementary Figure S6. Aberrant DDX1 overexpression results in mTOCR1 pathway activation. Supplementary Figure S7. DDX1 interacts with alpha-KGDH complex members and disruption of the DDX1:DLST interaction reduces mTORC1 pathway activation. Supplementary Figure S8. High DDX1 expression is associated with -KG accumulation and OXPHOS reduction. Supplementary Figure S9. Aberrant DDX1 expression is associated with increased sensitivity to KG and pharmacological mTORC1 inhibition.</p

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

Figure 6 from Passenger Gene Coamplifications Create Collateral Therapeutic Vulnerabilities in Cancer

High DDX1 expression is sufficient to increase sensitivity to pharmacologic mTORC1 inhibition in vitro and in vivo. A, Relative cell viability of different neuroblastoma cell lines with DDX1-MYCN coamplification (red, N = 3) or with MYCN amplifications (blue, N = 2) treated with rapamycin (2.5 μmol/L for 72 hours) compared with cell viability after DMSO (vehicle control) treatment (Welch t test, P = 2.291e−05 DDX1-MYCN vs. MYCN; each data point represents a technical replicate). B, Relative cell viability of KELLY cells inducibly expressing DDX1, DDX1-Δ269-295aa, or an empty vector and treated with rapamycin (2.5 μmol/L for 72 hours) compared with cell viability after DMSO (vehicle control) treatment (Welch t test, P = 3.943e−05; data are shown as mean ± SE; N = 3 technical replicates). C, Relative cell viability of IMR5/75 cells expressing shRNAs directed against DDX1 (blue) or GFP (red) and treated with rapamycin (2.5 μmol/L for 72 hours) compared with cell viability after DMSO (vehicle control) treatment (Pairwise t test adjusted by Benjamini–Hochberg correction, P = 1.3e−05, 4.1e−05, and 2.2e−05 for each independent shRNA directed against DDX1 vs. shGFP, respectively; data are shown as mean ± SE; N = 3 technical replicates). D, Correlation between the DDX1 copy number and the IC50 value of rapamycin in different neuroblastoma cell lines derived from the GDSC2 database (Pearson correlation, R = −0.05043, P = 0.0394, N = 13 independent cancer cell lines). E, Relative cell viability of neuroblastic tumor cells derived from transgenic zebrafish expressing MYCN or MYCN and DDX1 and treated with rapamycin (2.5 μmol/L for 72 hours) compared with cell viability after DMSO (vehicle control) treatment (Welch t test, P = 0.02707; data are shown as mean ± SE; N = 3 independent replicates from cells derived from different zebrafish). F, Nanopore sequencing read coverage over the MYCN amplicon region in MYCN-amplified neuroblastoma PDX with or without DDX1 coamplification (log-scaled). G, Relative change in tumor volume of MYCN-amplified NB PDX with or without DDX1 coamplification treated with rapamycin compared with mice treated with vehicle controls (N  =  4 mice per group; *, P H, Tumor volumes after treatment with rapamycin compared with vehicle treatment in MYCN-amplified PDX (N  =  4 independent mice per treatment group; P  =  0.7918, 0.01286, respectively). I, Representative photomicrographs of PDX after IHC staining for cleaved caspase-3 or Ki-67 (scale bar, 50 μm). J, Quantification of cleaved caspase-3 or Ki-67-positive cells in PDX shown in I (N  =  10 sections of 200 μm × 200 μm; P  = 0.7454, 1.717e−08, 0.886, and 1.218e−11 independently).</p