Abstract
<p>A proof-of-principle study identifies a selective mTOR pathway dependency in cells with <i>DDX1-MYCN</i> coamplification. <b>A,</b> Correlation between <i>DDX1</i> copy-number and dependency scores (CERES) for <i>RAPTOR</i> in neuroblastoma cell lines (Pearson correlation analysis, <i>R</i> = −0.5996, <i>P</i> = 0.0152, <i>N</i> = 13). <b>B,</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 <i>RAPTOR (</i>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. <b>C,</b> Representative images of cell colonies formed by KELLY cells transduced with the doxycycline-inducible DDX1-mCherry vectors and with two pairs of sgRNA targeting <i>RAPTOR</i> (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. <i>N</i> = 3 biological replicates; Welch <i>t</i> test, <i>P</i> = 0.564, 0.000117, and 0.00131 for sgNT, sgRAPTOR_1, and sgRAPTOR_2, respectively). <b>D,</b> 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. <b>E,</b> GSEA based on a set of genes regulated by mTORC1 measured in genes differentially expressed in KELLY cells harboring a <i>MYCN</i> amplification with versus without ectopic DDX1 expression. <b>F,</b> 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>
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