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 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 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

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

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 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

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