Buyukcelebi Kadir, Chen Xintong, Abdula Fatih, Duval Alexander, Ozturk Harun, Seker-Polat Fidan, Jin Qiushi, Yin Ping, Feng Yue, Wei Jian-Jun, Bulun Serdar, Yue Feng, Adli Mazhar
Northwestern University.
Res Sq. 2023 Feb 9:rs.3.rs-2537075. doi: 10.21203/rs.3.rs-2537075/v1.
Uterine fibroid (UF) tumors originate from a mutated smooth muscle cell (SMC). Nearly 70% of these tumors are driven by hotspot recurrent somatic mutations in the gene; however, there are no tractable genetic models to study the biology of UF tumors because, under culture conditions, the non-mutant fibroblasts outgrow the mutant SMC cells, resulting in the conversion of the population to WT phenotype. The lack of faithful cellular models hampered our ability to delineate the molecular pathways downstream of MED12 mutations and identify therapeutics that may selectively target the mutant cells. To overcome this challenge, we employed CRISPR knock-in with a sensitive PCR-based screening strategy to precisely engineer cells with mutant MED12 Gly44, which constitutes 50% of MED12 exon two mutations. Critically, the engineered myometrial SMC cells recapitulate several UF-like cellular, transcriptional and metabolic alterations, including enhanced proliferation rates in 3D spheres and altered Tryptophan/kynurenine metabolism. Our transcriptomic analysis supported by DNA synthesis tracking reveals that MED12 mutant cells, like UF tumors, have heightened expression of DNA repair genes but reduced DNA synthesis rates. Consequently, these cells accumulate significantly higher rates of DNA damage and are selectively more sensitive to common DNA-damaging chemotherapy, indicating mutation-specific and therapeutically relevant vulnerabilities. Our high-resolution 3D chromatin interaction analysis demonstrates that the engineered MED12 mutations drive aberrant genomic activity due to a genome-wide chromatin compartmentalization switch. These findings indicate that the engineered cellular model faithfully models key features of UF tumors and provides a novel platform for the broader scientific community to characterize genomics of recurrent MED12 mutations and discover potential therapeutic targets.
子宫肌瘤（UF）肿瘤起源于突变的平滑肌细胞（SMC）。这些肿瘤中近70%是由该基因的热点复发性体细胞突变驱动的；然而，目前尚无易于处理的遗传模型来研究UF肿瘤的生物学特性，因为在培养条件下，非突变的成纤维细胞比突变的SMC细胞生长得更快，导致细胞群体转变为野生型表型。缺乏可靠的细胞模型阻碍了我们描绘MED12突变下游分子途径以及识别可能选择性靶向突变细胞的治疗方法的能力。为了克服这一挑战，我们采用了基于敏感PCR筛选策略的CRISPR敲入技术，精确构建携带突变MED12 Gly44的细胞，该突变占MED12外显子2突变的50%。至关重要的是，工程化的子宫肌层SMC细胞重现了几种类似UF的细胞、转录和代谢改变，包括三维球体中增殖率的提高以及色氨酸/犬尿氨酸代谢的改变。我们通过DNA合成追踪支持的转录组分析表明，与UF肿瘤一样，MED12突变细胞中DNA修复基因的表达升高，但DNA合成速率降低。因此，这些细胞积累了显著更高的DNA损伤率，并且对常见的DNA损伤化疗药物选择性更敏感，这表明了突变特异性和治疗相关的脆弱性。我们的高分辨率三维染色质相互作用分析表明，工程化的MED12突变由于全基因组染色质区室化转换而导致异常的基因组活性。这些发现表明，工程化细胞模型忠实地模拟了UF肿瘤的关键特征，并为更广泛的科学界提供了一个新平台，以表征复发性MED12突变的基因组学并发现潜在的治疗靶点。
