Figure 4 from Passenger Gene Coamplifications Create Collateral Therapeutic Vulnerabilities in Cancer

DDX1 interacts with α-KGDH complex members, and its interaction is required for mTORC1 pathway activation. A, A schematic of the DDX1 immunoprecipitation (IP) followed by LC-MS/MS. B and C, Volcano plot of proteins significantly enriched after immunoprecipitation of DDX1 in KELLY cells harboring MYCN amplifications without DDX1 coamplifications (B) and in IMR5/75 cells harboring DDX1-MYCN coamplifications (C) measured using LC-MS/MS (significantly enriched proteins, blue; DDX1 marked in red; dotted line with blue filling marks α-KGDH complex members). D, Schematic of the amplicon structure in KELLY and IMR5/75 cells (top). Venn diagram (bottom) of the proteins identified after immunoprecipitation of DDX1 in KELLY cells lacking DDX1 coamplifications compared with IMR5/75 cells harboring DDX1-MYCN coamplifications. E, Western immunoblot of DDX1, DLST, OGDH, and α-tubulin in IMR5/75 protein extracts before and after immunoprecipitation using antibodies directed against DDX1, DLST, OGDH, or nonspecific immunoglobulins (IgG). F, Representative confocal fluorescence imaging photomicrographs of KELLY cells expressing DDX1-mCherry (magenta), in which mitochondria were stained by MitoTracker DeepRed (yellow) and the nucleus is stained by Hoechst (blue; scale bar, 6 μm). G, Schematic illustration of protein domains in DDX1 as well as engineered DDX1 mutants (DDX1-ΔSPRY [core], Δ69-247aa; ΔRecA1, Δ13-472aa; ΔRecA2, Δ493-681aa). H, Western immunoblot of V5, DLST, and OGDH before and after immunoprecipitation using antibodies directed against V5, DLST, OGDH, or nonspecific immunoglobulins (IgG) in IMR5/75 cells expressing DDX1-V5 compared with DDX1-ΔSPRY (core), ΔRecA1, or ΔRecA2 truncation mutants, respectively. I, Western immunoblot of V5, DLST, and OGDH before and after immunoprecipitation using antibodies directed against V5, DLST, OGDH, or nonspecific immunoglobulins (IgG) in IMR5/75 cells expressing DDX1-V5 or DDX1-V5-Δ269-295aa. J, Representative confocal fluorescence imaging photomicrographs of proximity ligation assay signals (magenta dots) in IMR5/75 cells expressing DDX1-V5 or DDX1-V5-Δ269-295aa detected using anti-V5 and anti-DLST antibodies and counterstained with DAPI (blue) and phalloidin (yellow; scale bar, 7 μm). K, Quantification of proximity ligation signal (magenta) using anti-DLST and anti-V5 antibodies in IMR5/75 cells expressing DDX1-V5 (N = 36) or DDX1-V5-Δ269-295aa (N = 34) as shown in J (Welch t test, P = 1.476e−07). L, Relative gene expression of mTORC1 downstream pathway genes, as measured by quantitative PCR of KELLY cells inducibly expressing DDX1, DDX1-Δ269-295aa, or an empty vector in the presence and absence of doxycycline (1 μg/mL; Welch t test, P = 0.125, 0.006, 0.746, 0.064, 0.001, 0.399, respectively; data are shown as mean ± SE; N = 3 technical replicates).