Role of FH loss in development of HLRCC heriditary kidney cancer

在2018年，(1)我们论证了在NF2缺陷性乳头状肾癌中靶向缺失Hippo信号通路的可行性。乳头状肾细胞癌(PRCC)是一组在组织和遗传上不同的肿瘤，占所有肾脏肿瘤的15-20%，可能需要不同的治疗方法。编码Hippo信号通路关键调节因子的NF2抑癌基因在22.5%的PRCC中被观察到改变。河马信号通路通过调节YAP1转录活性来控制细胞增殖。NF2的缺失会导致YAP1的异常激活。Src家族成员YES也调节YAP1的转录活性。当我们在三个NF2缺失的PRCC细胞系中研究YAP和Yes活性的重要性时，我们发现NF2缺乏与YAP1转录靶标的表达增加相关，基于siRNA的YAP1和Yes1的敲除下调了这一途径，并显著降低了细胞活力。Dasatinib和Saracatinib对Yes有很强的抑制作用，两者均可导致YAP1转录靶标下调，细胞活力降低，G0-G1细胞周期停滞。NF2缺陷型PRCC的异种移植模型也显示，达沙替尼对肿瘤生长有抑制作用。因此，抑制YAP1的转录活性和抑制YAP1的转录活性在体内和体外都具有显著的抗肿瘤细胞作用，并可能为NF2缺陷的PRCC患者提供一种可行的治疗方法。(2)为了更好地靶向糖酵解癌细胞的糖酵解，我们参与开发了一种新型的、有效的、基于细胞活性的吡唑类乳酸脱氢酶(LDH)抑制剂。利用定量的高通量筛选范例促进了HIT鉴定，而基于结构的设计和多参数优化使得能够开发出具有有效的生化和基于细胞的LDH酶活性抑制的化合物。先导化合物表现出对LDHA和LDHB的低NM抑制，对腹膜乳酸生成的亚微摩尔抑制，以及对胰腺癌和A673尤文肉瘤细胞糖酵解的抑制。此外，通过细胞热位移分析(CETSA)证明了先导化合物对LDHA的强靶向结合，并通过SPR测定了药物-靶向停留时间。对这些数据的分析表明，药物靶向滞留时间(脱落率)可能是获得有效的基于细胞的、持久的体内抑制这种癌症代谢靶点的一个重要因素。事实上，我们的主要乳酸脱氢酶抑制剂在体内显示出强大的靶向活性。

In 2018, (1) we demonstrated the feasibility of targeting loss of the Hippo signaling pathway in NF2-deficient papillary kidney cancers. Papillary renal cell carcinomas (PRCC) are a histologically and genetically heterogeneous group of tumors that represent 15-20% of all kidney neoplasms and may require diverse therapeutic approaches. Alteration of the NF2 tumor suppressor gene, encoding a key regulator of the Hippo signaling pathway, is observed in 22.5% of PRCC. The Hippo signaling pathway controls cell proliferation by regulating the transcriptional activity of Yes-Associated Protein, YAP1. Loss of NF2 results in aberrant YAP1 activation. The Src family kinase member Yes also regulates YAP1 transcriptional activity. When we investigated the importance of YAP and Yes activity in three NF2-deficient PRCC cell lines, we found that NF2-deficiency correlated with increased expression of YAP1 transcriptional targets, and siRNA-based knockdown of YAP1 and Yes1 downregulated this pathway and dramatically reduced cell viability. Dasatinib and saracatinib have potent inhibitory effects on Yes and treatment with either resulted in downregulation of YAP1 transcription targets, reduced cell viability, and G0-G1 cell cycle arrest. Xenograft models for NF2-deficient PRCC also demonstrated reduced tumor growth in response to dasatinib. Thus, inhibiting Yes and the subsequent transcriptional activity of YAP1 had a substantial anti-tumor cell effect both in vitro and in vivo and may provide a viable therapeutic approach for patients with NF2-deficient PRCC. (2) In order to better target glycolysis in glycolytic cancer cells, we participated in developing a novel, potent, cell-active pyrazole-based inhibitor of lactate dehydrogenase (LDH). Utilization of a quantitative high-throughput screening paradigm facilitated hit identification, while structure-based design and multiparameter optimization enabled development of compounds with potent biochemical and cell-based inhibition of LDH enzymatic activity. Lead compounds exhibit low nM inhibition of both LDHA and LDHB, submicromolar inhibition of celular lactate production, and inhibition of glycolysis in MiaPaCa2 pancreatic cancer and A673 Ewing's sarcoma cells. Moreover, robust target engagement of LDHA by lead compounds was demonstrated using the cellular thermal shift assay (CETSA), and drug-target residence time was determined via SPR. Analysis of these data suggests that drug-target residence time (off-rate) may be an important attribute to consider for obtaining potent cell-based and durable in vivo inhibition of this cancer metabolism target. Indeed, our lead LDH inhibitor demonstrates potent in vivo on-target activity.