Preclinical Development of First-in-Class GSTO1 Degraders for Colorectal Cancer

谷胱甘肽S转移酶omega 1是一种非典型的谷胱甘肽转移酶亚型，在多种肿瘤中过度表达 并与抗药性有牵连。目前，还没有针对GSTO1的小分子药物处于临床阶段 发展。使用遗传工具，包括广泛的生物信息学分析与siRNA，shRNA， 蛋白质组学和CRISPR/Cas9技术；以及药理学小分子抑制剂和降解剂 已经证实GSTO1是肿瘤学中一个有效的可用药靶点。在此之前，我们确定了C1-27是一种有效的 GSTO1抑制剂，在体外和体内模型中都显示出对癌细胞的有效性。我们还合成了 并测试了第一个证实GSTO1在体外降解的GSTO1 PROTAC。通过转录 将bru-seq和rna-seq与蛋白质组学相结合，我们发现了新的药效学标记。 以及GSTO1调控肿瘤发生的关键细胞通路。综上所述，我们的发现验证了GSTO1 作为癌症治疗的重要药物靶点，以及作为有效和有效的原型抑制剂的C1-27。 在此之前，我们解决了C1-27(IC50=31 nM)和其他有效缓蚀剂的晶体结构 GSTO1。我们最新的铅优化活动使用了6种不同的共晶结构，结果是 迄今已知的有效GSTO1抑制剂(IC50=0.22±0.02 nM)。我们的CRISPR/Cas9 GSTO1基因敲除(KO)细胞 在体内不形成肿瘤或显示肿瘤生长延迟，在体外形成更小的3D球体。穿过 在GSTO1 KO细胞的多组学研究中，我们发现与细胞黏附分子和 干扰素反应途径，并与Myc转录信号呈显著负相关。重要的是 我们还确定了几种临床使用的化疗药物，这些药物显示出显著的合成致死性。 抑制GSTO1。我们发现组织因子(基因名，F3)转录和蛋白表达 对GSTO1 KO和C1-27处理的反应下调，进一步暗示GSTO1在先天性 免疫反应。综上所述，我们的结果表明GSTO1是癌症的治疗靶点，并提供了新的 对其在癌症进展中的重要作用的机械论见解。重要的是，我们的结果首次表明 抑制GSTO1可以激活免疫反应，下调F3。我们假设这种抑制作用 GSTO1将对肿瘤细胞产生双管齐下的作用：(A)通过减少Myc来损害癌细胞的存活 转录信号和F3下调，以及(B)通过干扰素增强免疫反应- 介导性的癌症先天免疫感应。我们进一步假设GSTO1抑制剂将致敏癌症 细胞选择化疗和免疫治疗。我们将通过以下三个方面来检验这些假设 明确的目标。目的1：阐明GSTO1通过激活GSTO1在增强免疫应答中的作用 I型干扰素通过F3和Myc的下调降低肿瘤细胞的存活率。目标2：优化GSTO1 基于共晶结构的降解剂选择临床候选药物。目的3：评估有希望的药物的体内疗效 新的GSTO1降解剂在PDX和结直肠癌同基因模型中作为单一药物和联合使用。

Glutathione S-transferase omega 1 (GSTO1) is an atypical GST isoform that is overexpressed in several cancers and has been implicated in drug resistance. Currently, no small-molecule drug targeting GSTO1 is under clinical development. Using genetic tools, including extensive bioinformatics analysis coupled with siRNA, shRNA, proteomics, and CRISPR/Cas9 technologies; and pharmacologic small-molecule inhibitors and degraders, we have validated GSTO1 as an impactful druggable target in oncology. Previously, we identified C1-27 as a potent GSTO1 inhibitor that shows efficacy against cancer cells in both in vitro and in vivo models. We also synthesized and tested the very first GSTO1 PROTAC confirming GSTO1 degradation in vitro. Through transcriptional profiling using Bru-seq and RNA-seq coupled with proteomics, we uncovered novel pharmacodynamic markers and cellular pathways critical for oncogenesis regulated by GSTO1. Taken together, our findings validate GSTO1 as an important drug target for cancer therapeutics and C1-27 as a potent and validated prototype inhibitor. Previously, we solved the crystal structure of C1-27 (IC50 = 31 nM) and other potent inhibitors in complex with GSTO1. Our most recent lead optimization campaign using 6 different co-crystal structures resulted in the most potent GSTO1 inhibitor (IC50 = 0.22 ± 0.02 nM) known to date. Our CRISPR/Cas9 GSTO1 knockout (KO) cell lines do not form tumors or display tumor growth delay in vivo and form smaller 3D spheroids in vitro. Through multi-omics studies in GSTO1 KO cells, we found a strong positive correlation with cell adhesion molecules and interferon response pathways, and a strong negative correlation with Myc transcriptional signature. Importantly, we also identified several clinically used chemotherapies showing significant synthetic lethality with loss or inhibition of GSTO1. We discovered that tissue factor (gene name, F3) transcription and protein expression are downregulated in response to GSTO1 KO and C1-27 treatment, further implicating a role for GSTO1 in the innate immune response. In summary, our results implicate GSTO1 as a therapeutic target in cancer and offer new mechanistic insights into its significant role in cancer progression. Importantly, our results show for the first time that inhibition of GSTO1 can activate immune responses and downregulate F3. We hypothesize that inhibiting GSTO1 will have a two-pronged effect on tumor cells: (A) impair cancer cell survival by reducing Myc transcriptional signature and F3 downregulation, and (B) enhance immune responses through interferon- mediated innate immune sensing of cancers. We further hypothesize that GSTO1 inhibitors will sensitize cancer cells to select chemotherapy and immunotherapy. We will test these hypotheses through the following three specific aims. Aim 1: Elucidate the functions of GSTO1 in enhancing immune response through activation of type-I interferon and reducing tumor cell viability through F3 and Myc downregulation. Aim 2: Optimize GSTO1 degraders based on co-crystal structures to select a clinical candidate. Aim 3: Assess in vivo efficacy of promising novel GSTO1 degraders in PDX and syngeneic models of CRC as single agents and in combination.