Chordoma preclinical models are insensitive to BAF complex inhibitor FHD-286, alone or in combination with gemcitabine

To evaluate BRG1(SMARCA4)/BRM(SMARCA2) as a potential therapeutic target, chordoma cell lines were treated with FHD-286. A panel of 4 chordoma cell lines were seeded in a white-walled 96-well plate so that at the start of treatment, 36 hours after plating, the confluence was 15%. The assay endpoint for each cell line was the amount of time for untreated cells to reach 80-90% confluence. Cell line-specific seeding densities and assay durations can be found through the link at the bottom of this page. CH22, UM-Chor1, U-CHCF365, and U-CH2 cell lines were plated in 100uL of (4:1) IMDM:RPMI 1640 + 10% FBS + 1% NEAA + 1% GlutaMAX. Cells were treated in triplicate with a 3-fold serial dilution of each drug with a top concentration of 5 uM to the lowest concentration of 0.762 nM. At the assay endpoint, cell viability was measured using CellTiter-Glo 2.0 Cell Viability Assay. Representative dose-response curves of the mean +/- the standard deviation are shown. The data was normalized to the DMSO-only control. Experiments for treatment with FHD-286 were repeated in all lines to ensure reproducibility. The absolute EC50 of all lines toward the single agent FHD-286 was >1 uM.To evaluate if exacerbating replication stress induces sensitivity to BAF complex inhibition, cells were plated as noted above except for the addition of either 2.5 nM gemcitabine to CH22 and U-CHCF365 and 25 nM gemcitabine to U-CH2 and UM-Chor1 to the FHD-286 titration. The FHD-286 titration + gemcitabine addition was normalized to the gemcitabine only treatment instead of to the DMSO only control. Gemcitabine did not significantly increase sensitivity to FHD-286 in CH22 and UM-Chor1. The potency was increased in U-CH2; however, in U-CHCF365, the sensitivity toward FHD-286 appears to decrease with increasing FHD-286 concentration.

为评估BRG1(SMARCA4)/BRM(SMARCA2)作为潜在治疗靶点的可行性，本研究采用FHD-286处理脊索瘤细胞系。我们选取4株脊索瘤细胞系，接种于白壁96孔板中，确保给药开始时（即接种后36小时）细胞汇合度为15%。各细胞系的试验终点为：未处理细胞达到80%~90%汇合度所需的时长。细胞系特异性接种密度与试验周期可通过本页面底部链接获取。
CH22、UM-Chor1、U-CHCF365及U-CH2细胞系均以100 μL体系接种于体积比为4:1的IMDM:RPMI 1640完全培养基中，该培养基添加有10%胎牛血清(FBS)、1%非必需氨基酸(NEAA)及1% GlutaMAX。
每株细胞均设置3个复孔，采用3倍系列梯度稀释法配制FHD-286药液，最高作用浓度为5 μM，最低浓度为0.762 nM。
于试验终点时，采用CellTiter-Glo 2.0细胞活力检测试剂盒测定细胞活力。结果以平均值±标准差的代表性剂量-反应曲线展示，所有数据均以仅含二甲基亚砜(DMSO)的对照组进行归一化处理。
所有细胞系均重复开展FHD-286单药处理实验以确保结果可重复性。所有细胞系对单药FHD-286的绝对半数有效浓度(EC50)均大于1 μM。
为探究加剧复制应激是否可诱导细胞对BAF复合体(BAF complex)抑制产生敏感性，我们按照前述方法接种细胞，仅在FHD-286梯度稀释给药时额外添加吉西他滨(gemcitabine)：向CH22与U-CHCF365细胞系添加2.5 nM吉西他滨，向U-CH2与UM-Chor1细胞系添加25 nM吉西他滨。
FHD-286联合吉西他滨给药组的数据归一化处理以仅添加吉西他滨的对照组为参照，而非仅含DMSO的对照组。
实验结果显示，在CH22与UM-Chor1细胞系中，吉西他滨未显著增强细胞对FHD-286的敏感性；U-CH2细胞系的药物效价有所提升；但在U-CHCF365细胞系中，随着FHD-286浓度升高，细胞对其的敏感性反而呈下降趋势。