A druggable dependency in low-MITF/high-AXL melanoma: preclinical efficacy and mechanism of action in a key treatment-resistant subclass.

低 MITF/高 AXL 黑色素瘤的药物依赖性：关键耐药亚类的临床前疗效和作用机制。

• 批准号: 10331800
• 负责人: DAVID E FISHER
• 金额: $ 36.61万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10331800
• 关键词: Ablation; BRAF gene; Behavior; Binding; Binding Sites; Biological Markers; Biopsy; C57BL/6 Mouse; Cell Death; Cell Line; Cell Nucleus; Cells; ChIP-seq; Complex; Computer Analysis; DNA; Databases; Dependence; Development; Elements; Engineering; Enzyme Tests; Enzymes; Exhibits; Failure; Genes; Genetic; Genetic Databases; Genetic Engineering; Genetic Transcription; Genomic approach; Histone H3; Histones; Human; Immunotherapy; In Vitro; KDM1A gene; Lysine; MEKs; Malignant Neoplasms; Mediating; Mediator of activation protein; Melanoma Cell; Messenger RNA; Modeling; Monitor; Mus; Mutation; NDRG1 gene; Oncogenic; PTEN gene; Pancreas; Patients; Pattern; Pharmacology; Phenocopy; Physiological; Population; Process; Protein Tyrosine Kinase; Publishing; Relapse; Reporting; Repression; Repressor Proteins; Resistance; Resistance development; Role; Small Interfering RNA; Systemic Therapy; Testing; The Cancer Genome Atlas; Therapeutic; Tumor Suppressor Genes; Undifferentiated; Work; Xenograft procedure; anti-PD-1; base; cancer cell; checkpoint inhibition; checkpoint therapy; clinical development; demethylation; drug candidate; experience; genetic corepressor; genome-wide; global run on sequencing; histone demethylase; in vitro testing; in vivo; inhibitor; insight; knock-down; loss of function; melanocyte; melanoma; mutant; overexpression; patient derived xenograft model; patient subsets; pharmacodynamic biomarker; pre-clinical; preclinical efficacy; response; single-cell RNA sequencing; small hairpin RNA; targeted treatment; therapeutic target; therapy resistant; transcriptome sequencing; triple-negative invasive breast carcinoma; tumor; tumorigenic

PROJECT SUMMARY Despite major advances in targeted and immune therapies for melanoma, most patients experience relapses or do not respond adequately. Acquired and intrinsic resistance to these therapies in both patient biopsies and cell lines have been associated with a unique undifferentiated cell state characterized by low levels of MITF, the master regulator of melanocyte development, and high levels of the AXL tyrosine kinase. This cell state is reported in a high fraction of melanomas exhibiting de novo resistance to BRAF inhibition and in 50-100% with acquired resistance. It is also a prominent feature of anti-PD1 resistant melanomas. Single-cell RNA-seq demonstrated that all melanomas, even high-MITF ones, contain low-MITF subpopulations that are positively selected by targeted therapy. To eradicate this population, AXL itself is not a therapeutic target, based on published tests of multiple inhibitors and shRNAs. We therefore interrogated genetic dependencies using Project Achilles—an unbiased genome-scale loss-of-function screen—and found that low-MITF/high-AXL melanomas exhibit a striking dependency, not on AXL, but on LSD1, a lysine-specific histone demethylase that has been implicated in oncogenic processes. We validated LSD1 dependence in multiple melanoma lines using both genetic and pharmacologic inhibition. Analysis of melanomas in the TCGA and CCLE databases for candidate LSD1 target genes mediating its dependency revealed three candidates that LSD1 selectively modulates in low-MITF melanomas. The first of these, NDRG1, was shown to be required for LSD1-targeted lethality. Concordance of these LSD1-target expression patterns is seen across both cell lines and tumors (TCGA). We also show that SP2509, an LSD1 inhibitor related to a compound in clinical development, is highly lethal to multiple low-MITF melanomas, an effect that is dependent upon (and modulated by) the low-MITF state. Deeper analysis of the Achilles database identified multiple subunits of CoREST (known LSD1 co- repressive complex) as strong dependency factors, phenocopying selective lethality of low-MITF/high-AXL melanomas and suggesting a mechanistic connection to LSD1-dependency. In Aim 1, we will test the hypothesis that therapeutic resistance of low-MITF melanomas can be mitigated by combining targeted or immune therapies with LSD inhibition in early passage melanoma cell lines and PDX. Our preliminary results demonstrate profound cooperative lethality by combining genetic or pharmacologic LSD1 inhibition with BRAF inhibitor in vitro and in xenografts. We will test the ability of LSD1 inhibition to antagonize the emergence of resistance in high-MITF melanoma, as well as to cooperate with agents that actively suppress MITF expression. In Aim 2, we will mechanistically examine functional roles of four candidate mediators of LSD1 dependency and use genomic approaches to systematically scrutinize the low-MITF state, identifying potential pharmacodynamic markers and mediators of LSD1 dependency. These findings provide a unique opportunity to reveal mechanistic insights and therapeutic opportunities for important treatment-resistant patient subsets.

项目摘要 尽管黑色素瘤的靶向和免疫疗法取得了重大进展，但大多数患者都会复发 或者没有做出适当的反应。在患者活检中对这些疗法的获得性和内在抗性， 细胞系与以低水平MITF为特征的独特的未分化细胞状态有关， 黑素细胞发育的主要调节因子，以及高水平的AXL酪氨酸激酶。这种细胞状态是 在对BRAF抑制表现出新发耐药性的黑色素瘤的高比例中报告， 获得性抵抗这也是抗PD 1耐药黑色素瘤的一个突出特征。单细胞RNA-seq 研究表明，所有的黑色素瘤，即使是高MITF的黑色素瘤，都含有低MITF亚群，这些亚群是阳性的。 通过靶向治疗选择。为了根除这一人群，AXL本身并不是一个治疗靶点， 发表了多种抑制剂和shRNA的测试。因此，我们使用 一个无偏见的基因组规模的功能丧失筛查项目，发现低MITF/高AXL 黑色素瘤表现出显著的依赖性，不是对AXL，而是对LSD 1，一种赖氨酸特异性组蛋白去甲基化酶， 与致癌过程有关我们在多种黑色素瘤细胞系中验证了LSD 1依赖性， 使用遗传和药理学抑制。TCGA和CCLE数据库中黑色素瘤的分析， 候选LSD 1靶基因介导其依赖性揭示了三个候选人，LSD 1选择性 调节低MITF黑色素瘤。其中第一个，NDRG 1，被证明是LSD 1靶向所必需的。 杀伤力在细胞系和肿瘤中观察到这些LSD 1靶表达模式的一致性 （TCGA）。我们还表明，SP2509是一种与临床开发中的化合物相关的LSD 1抑制剂， 对多发性低MITF黑色素瘤是致命的，这种效应依赖于低MITF（并受其调节） 状态对Achilles数据库的更深入分析确定了CoREST的多个亚基（已知的LSD 1 co- 抑制复合物）作为强依赖性因子，表型复制低MITF/高AXL的选择性致死性 黑色素瘤，并提出与LSD 1依赖性的机械联系。在目标1中，我们将测试 低MITF黑色素瘤的治疗耐药性可以通过靶向或 在早期传代黑色素瘤细胞系和PDX中使用LSD抑制的免疫疗法。我们的初步结果 通过将遗传或药理学LSD 1抑制与BRAF结合，证明了显著的协同致死性 抑制剂在体外和异种移植物中。我们将测试LSD 1抑制拮抗LSD 1的出现的能力。 高MITF黑色素瘤的耐药性，以及与积极抑制MITF的药物合作 表情在目标2中，我们将机械地检查LSD 1的四个候选介质的功能作用 依赖性，并使用基因组方法系统地检查低MITF状态，识别潜在的 LSD 1依赖性的药效学标志物和介质。这些发现提供了一个独特的机会 揭示机制的见解和重要的耐药患者亚群的治疗机会。

项目成果

Targeting TBK1 to overcome resistance to cancer immunotherapy.

• DOI: 10.1038/s41586-023-05704-6
• 发表时间: 2023-03
• 期刊: NATURE
• 影响因子: 64.8
• 作者: Sun, Yi;Revach, Or-yam;Anderson, Seth;Kessler, Emily A. A.;Wolfe, Clara H. H.;Jenney, Anne;Mills, Caitlin E. E.;Robitschek, Emily J. J.;Davis, Thomas G. R.;Kim, Sarah;Fu, Amina;Ma, Xiang;Gwee, Jia;Tiwari, Payal;Du, Peter P. P.;Sindurakar, Princy;Tian, Jun;Mehta, Arnav;Schneider, Alexis M. M.;Yizhak, Keren;Sade-Feldman, Moshe;LaSalle, Thomas;Sharova, Tatyana;Xie, Hongyan;Liu, Shuming;Michaud, William A. A.;Saad-Beretta, Rodrigo;Yates, Kathleen B. B.;Iracheta-Vellve, Arvin;Spetz, Johan K. E.;Qin, Xingping;Sarosiek, Kristopher A. A.;Zhang, Gao;Kim, Jong Wook;Su, Mack Y. Y.;Cicerchia, Angelina M. M.;Rasmussen, Martin Q. Q.;Klempner, Samuel J. J.;Juric, Dejan;Pai, Sara I. I.;Miller, David M. M.;Giobbie-Hurder, Anita;Chen, Jonathan H. H.;Pelka, Karin;Frederick, Dennie T. T.;Stinson, Susanna;Ivanova, Elena;Aref, Amir R. R.;Paweletz, Cloud P. P.;Barbie, David A. A.;Sen, Debattama R. R.;Fisher, David E. E.;Corcoran, Ryan B. B.;Hacohen, Nir;Sorger, Peter K. K.;Flaherty, Keith T. T.;Boland, Genevieve M. M.;Manguso, Robert T. T.;Jenkins, Russell W. W.
• 通讯作者: Jenkins, Russell W. W.