Figure 4 from Passenger Gene Coamplifications Create Collateral Therapeutic Vulnerabilities in Cancer
Authors: Bei Yi , Bramé Luca , Kirchner Marieluise , Fritsche-Guenther Raphaela , Kunz Severine , Bhattacharya Animesh , Rusu Mara-Camelia , Gürgen Dennis , Dubios Frank P.B. , Köppke Julia K.C. , Proba Jutta , Wittstruck Nadine , Sidorova Olga Alexandra , Chamorro González Rocío , Dorado Garcia Heathcliff , Brückner Lotte , Xu Robin , Giurgiu Mădălina , Rodriguez-Fos Elias , Yu Qinghao , Spanjaard Bastiaan , Koche Richard P. , Schmitt Clemens A. , Schulte Johannes H. , Eggert Angelika , Haase Kerstin , Kirwan Jennifer , Hagemann Anja I.H. , Mertins Philipp , Dörr Jan R. , Henssen Anton G. 
Published: March 2024

<p>DDX1 interacts with α-KGDH complex members, and its interaction is required for mTORC1 pathway activation. <b>A,</b> A schematic of the DDX1 immunoprecipitation (IP) followed by LC-MS/MS. <b>B</b> and <b>C,</b> Volcano plot of proteins significantly enriched after immunoprecipitation of DDX1 in KELLY cells harboring <i>MYCN</i> amplifications without <i>DDX1</i> coamplifications (<b>B</b>) and in IMR5/75 cells harboring <i>DDX1-MYCN</i> coamplifications (<b>C</b>) measured using LC-MS/MS (significantly enriched proteins, blue; DDX1 marked in red; dotted line with blue filling marks α-KGDH complex members). <b>D,</b> 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 <i>DDX1</i> coamplifications compared with IMR5/75 cells harboring <i>DDX1-MYCN</i> coamplifications. <b>E,</b> 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). <b>F,</b> 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). <b>G,</b> Schematic illustration of protein domains in DDX1 as well as engineered DDX1 mutants (DDX1-ΔSPRY [core], Δ69-247aa; ΔRecA1, Δ13-472aa; ΔRecA2, Δ493-681aa). <b>H,</b> 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. <b>I,</b> 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. <b>J,</b> 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). <b>K,</b> Quantification of proximity ligation signal (magenta) using anti-DLST and anti-V5 antibodies in IMR5/75 cells expressing DDX1-V5 (<i>N</i> = 36) or DDX1-V5-Δ269-295aa (<i>N</i> = 34) as shown in <b>J</b> (Welch <i>t</i> test, <i>P</i> = 1.476e−07). <b>L,</b> 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 <i>t</i> test, <i>P</i> = 0.125, 0.006, 0.746, 0.064, 0.001, 0.399, respectively; data are shown as mean ± SE; <i>N</i> = 3 technical replicates).</p>