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 S3 from Passenger Gene Coamplifications Create Collateral Therapeutic Vulnerabilities in Cancer

Gene sets enriched in in cell lines after ectopic DDX1 expression.</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

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

Supplementary Table S5 from Passenger Gene Coamplifications Create Collateral Therapeutic Vulnerabilities in Cancer

Metabolite levels in cells with DDX1-MYCN co-amplification compared to cell lines without such co-amplification.</p

Figure 1 from Passenger Gene Coamplifications Create Collateral Therapeutic Vulnerabilities in Cancer

Overview of the systematic approach to identify collateral lethal dependencies associated with passenger gene coamplifications in cancers.</p

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 S4 from Passenger Gene Coamplifications Create Collateral Therapeutic Vulnerabilities in Cancer

Peptides significantly enriched after DDX1 immunoprecipitation as measured using LC-MS/MS.</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