Figure 1 from SMYD3 Impedes Small Cell Lung Cancer Sensitivity to Alkylation Damage through RNF113A Methylation–Phosphorylation Cross-talk

Abstract

<p>SMYD3 is a candidate regulator of SCLC susceptibility to alkylating chemotherapy. <b>A,</b> Synthetic lethality screening using a library composed of 285 characterized inhibitors, testing H209 SCLC cell sensitivity to alkylation damage by preactivated form of CP (4H-CP). Data represent the relative growth of H209 cells treated with a combination of 4H-CP (2.5 μmol/L) and different inhibitors (1 μmol/L each) compared with 4H-CP only (see Supplementary Table S1 and detailed description in the Methods). <b>B,</b><i>SMYD3</i> expression in different histologic subtypes of human lung cancer (GSE30219). The box plots show the distribution of <i>SMYD3</i> expression in indicated lung cancer subtypes: lung squamous cell carcinomas (LUSC; <i>n</i> = 61), lung adenocarcinomas (LUAC; <i>n</i> = 85), large cell neuroendocrine tumors (LCNE; <i>n</i> = 56), SCLC (<i>n</i> = 20), and in adjacent normal lung tissue (<i>n</i> = 14). <i>P</i> values were calculated using the Kruskal−Wallis test. <b>C,</b> Representative IHC staining of SMYD3 in normal human lung (<i>n</i> = 8) and tumor biopsies obtained from patients with confirmed SCLC (<i>n</i> = 24). A magnification is provided. All 24 analyzed SCLC biopsies showed positive nuclear and cytoplasmic SMYD3 staining with H-score >180 in 20 samples and H-score >100 in 4 samples. Scale bars, 50 μm. <b>D,</b> Analysis of DMS-114 SCLC cell line growth response to increasing concentrations of 4H-CP with or without SMYD3i (EPZ031686) at the indicated concentrations. The percentage of viable cells was normalized to control vehicle-treated cells. <i>P</i> values were calculated by two-way ANOVA with the Tukey test for multiple comparisons. Data are represented as nonlinear regression with mean ± SEM. <b>E,</b> Quantification of 4H-CP and SMYD3i combination treatment synergy using the Loewe model. Loewe synergy score was calculated from DMS-114 cell survival assays (as in <b>D</b>, SynergyFinder 2.0). <b>F,</b> Schematic of xenografts and CP treatment schedule using SCLC H1092 cells modified to express a control nontargeting sgRNA (sgControl) or a Cas9/sgRNA targeting SMYD3 (sg<i>SMYD3</i>) complemented or not using either WT or F183 inactive mutant SMYD3, or treated with SMYD3i (EPZ031686). The cells were grafted subcutaneously to immunocompromised <i>NOD. SCID-IL2Rg</i><sup>−/−</sup> (NSG) mice. <b>G,</b> Quantification of H1092 xenograft tumor volume (<i>n</i> = 5 mice, for each treatment group) is shown. Animals in control groups received placebo (vehicle) treatment. values were calculated by two-way ANOVA with Tukey testing for multiple comparisons. Data are represented as mean ± SEM. <b>H,</b> Quantification of H1092 xenograft tumor volume (<i>n</i> = 5 mice, for each treatment group) is shown. <i>P</i> values were calculated by two-way ANOVA with Tukey testing for multiple comparisons. Data are represented as mean ± SEM. In all panels, representative of at least three independent experiments is shown unless stated otherwise.</p>

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