GDF15 gene suppression by BET inhibition and gene signature based personalized chemotherapy to counteract cachexia [Quant-seq]. GDF15 gene suppression by BET inhibition and gene signature based personalized chemotherapy to counteract cachexia [Quant-seq]

Elevation of growth differentiation factor 15 (GDF15) has recently emerged as a key driver of cancer cachexia, a syndrome of extreme weight loss that affects patients with various types of cancer. Chemotherapy, as well as the tumour itself have been reported to contribute to increased GDF15 secretion in both cancer and host tissues. Bromodomain and extraterminal (BET) domain proteins have been shown to regulate GDF15 expression in pancreatic cancer cells. With currently neither preventive strategies nor specific therapeutic options for cachexia available, antibodies targeting GDF15 or its receptor are being developed. However, the mechanism of chemotherapy-induced GDF15 upregulation in tumour and host cells is still unknown, leaving uncertainty about safety and efficacy of therapies targeting retrospective reduction of GDF15 in cancer cachexia. We tested various cardiotoxic drugs for their effect on GDF15 regulation in cardiomyocyte cell culture and analysed gene expression, mutational status, and pathway activities of the NCI-60 cancer cell line panels to get further insights into the regulation of GDF15 expression in both cancer and host cells upon treatment with anticancer drugs. To investigate the regulatory effect of BET inhibition on GDF expression more extensively, we screened BET inhibitors in doxorubicin treated human cardiomyocyte cell culture as well as in 21 cancer cell lines and performed differential analysis of gene expression, metabolites, and pathway activities. Our results reveal that DNA interacting compounds induce GDF15 expression in human cardiomyocytes up to 20-fold, whereas cardiotoxic drugs with other modes of action led, if at all, to less than 3-fold induction. We discovered that BET inhibitors suppress doxorubicin-induced GDF15 overexpression in human cardiomyocytes up to 7.4-fold (p=0.0051). In cancer cell lines, we identified compound-specific gene signatures that correlate with the extent of GDF15 induction upon treatment in the espective cell line. BET inhibition also reduces GDF15 expression in a defined subset of cancer cell lines that differentiate from the other tested cell lines in a less active PI3K/Akt axis and significantly higher extracellular pantothenate concentration (p=0.00095). We identified compound- and cell-line-specific gene signatures that predict GDF15 upregulation upon treatment, providing indication that the risk for drug-induced GDF15 overexpression and concomitant cachexia can be reduced by a biomarker-driven selection of patient-specific anticancer agents. Our results suggest that BET inhibitors could counteract cachexia at the transcriptional level in tumour and host tissues by reducing GDF15 expression. We identified characteristic gene and metabolite expressions of responsive cancer cell lines that can serve as biomarkers for patient selection. Overall design: Colon cancer cell lines were grown in media and treated with either DMSO (control) or compound. BI 894999 (WO2014076237) is the clinical compound synthesized by Boehringer Ingelheim. The experiment was performed in biological duplicates per condition/cell lines. Information about sampling time points, compounds, compound concentrations, and replicates are detailed out in the respective sample descriptions. All samples were subjected to RNA isolation, followed by library preparation (QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina, Lexogen), and messenger RNA sequencing on an Illumina NextSeq 500 system.

生长分化因子15（GDF15）水平升高近来被认定为癌症恶病质的关键驱动因素，癌症恶病质是一种可导致多种癌症患者出现极端体重下降的综合征。化疗与肿瘤本身均可促进癌症患者及宿主组织中GDF15的分泌增加。溴结构域与额外结构域（BET）蛋白已被证实可调控胰腺癌细胞内GDF15的表达。目前针对癌症恶病质既无有效的预防策略，也无特异性治疗手段，靶向GDF15或其受体的抗体正处于研发阶段。然而，化疗诱导肿瘤与宿主细胞中GDF15上调的具体机制仍未明确，这使得靶向降低癌症恶病质患者体内GDF15水平的治疗方案的安全性与有效性存在诸多不确定性。

我们在心肌细胞培养体系中测试了多种心脏毒性药物对GDF15调控的影响，并分析了NCI-60癌细胞系面板的基因表达、突变状态及通路活性，以进一步探究抗癌药物处理后，癌细胞与宿主细胞内GDF15表达的调控机制。为更全面地研究BET抑制对GDF家族表达的调控作用，我们在阿霉素处理的人心肌细胞培养体系以及21株癌细胞系中筛选了BET抑制剂，并开展了基因表达、代谢物及通路活性的差异分析。

本研究结果显示，可与DNA相互作用的化合物可使人心肌细胞内GDF15的表达上调最高达20倍；而其他作用机制的心脏毒性药物即便能诱导GDF15表达，其上调幅度也不足3倍。我们发现，BET抑制剂可将阿霉素诱导的人心肌细胞GDF15过表达抑制最高达7.4倍（p=0.0051）。在癌细胞系中，我们鉴定出了与对应细胞系经处理后GDF15诱导幅度相关的化合物特异性基因特征。BET抑制还可在特定的癌细胞系亚群中降低GDF15的表达，该亚群癌细胞与其他受试细胞系的区别在于PI3K/Akt通路活性更低，且细胞外泛酸浓度显著更高（p=0.00095）。我们鉴定出了可预测处理后GDF15上调的化合物与细胞系特异性基因特征，这表明通过生物标志物驱动的患者特异性抗癌药物选择，可降低药物诱导GDF15过表达及伴随恶病质的风险。本研究结果提示，BET抑制剂可通过降低GDF15表达，在肿瘤与宿主组织的转录层面对抗癌症恶病质。我们还鉴定出了响应癌细胞系的特征性基因与代谢物表达特征，可作为患者筛选的生物标志物。

总体实验设计：结肠癌细胞系在培养基中培养后，分别用二甲基亚砜（DMSO，对照组）或受试化合物处理。BI 894999（WO2014076237）是勃林格殷格翰（Boehringer Ingelheim）合成的临床化合物。每个条件/细胞系均设置生物学重复两次。采样时间点、化合物信息、化合物浓度及重复设置的详细信息见对应样本描述。所有样本均进行RNA提取，随后进行文库制备（采用Lexogen公司的QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina），并在Illumina NextSeq 500系统上完成信使RNA测序。