Elucidating the SCP4 pathway as a multi-catalytic signaling dependency in acute myeloid leukemia

阐明 SCP4 通路作为急性髓系白血病的多催化信号传导依赖性

基本信息

批准号：
10753227
负责人：
CHRISTOPHER VAKOC
金额：
$ 74.25万
依托单位：
COLD SPRING HARBOR LABORATORY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-01 至 2028-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10753227
关键词：
Active Sites Acute Acute Myelocytic Leukemia Allosteric Site Binding Biochemical Biological Assay Biological Markers Biological Models Bone Marrow Transplantation Bypass CD34 gene CRISPR screen Cell Nucleus Cells Clinical Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Companions Complex Coupled DNA-Binding Proteins Data Dependence Development Disease Drug Targeting Elements Enzymes Epistatic Gene Evaluation Gene Expression Generations Genetic Genetic Epistasis Genetic Screening Genetic Transcription Goals Hematopoietic Hematopoietic stem cells Human Immunodeficient Mouse Impairment In Vitro Investigation Knock-out Knowledge Literature Maintenance Malignant Neoplasms Maps Mediating Methods Mutagenesis Myeloid Progenitor Cells Nuclear Oncology Outcome Output Pathway interactions Patients Phenotype Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Principal Investigator Program Sustainability Proliferating Protein Dephosphorylation Protein phosphatase Proteins Publications Publishing Regulation Research Research Personnel Resources Sampling Signal Pathway Signal Transduction Signal Transduction Pathway Site Structure Substrate Specificity Surface Therapeutic Transplantation Work X-Ray Crystallography acute myeloid leukemia cell austin biomarker identification cancer genetics chemical genetics chemotherapy clinically relevant epigenomics experience experimental study gene complementation genetic approach genome editing genome-wide in vivo inhibitor innovation insight interdisciplinary approach metabolomics new therapeutic target novel novel therapeutics paralogous gene patient population pharmacologic phosphoproteomics rational design small molecule inhibitor structural biology tool tool development transcription factor transcriptomics vector

项目摘要

PROJECT SUMMARY Phosphatases and kinases are established classes of drug targets in oncology due to the presence of structured catalytic pockets or allosteric sites that can be targeted with small molecule inhibitors. To expand our knowledge of phosphatase and kinase and dependencies in AML, we recently completed domain-focused CRISPR screening studies in search of AML-specific dependencies. These efforts revealed a poorly studied phosphatase SCP4 as a dependency unique to AML, whereas normal human hematopoietic cells can tolerate loss of this enzyme. Our biochemical investigation revealed an intimate linkage between SCP4 and the poorly studied kinase paralogs STK35 and PDIK1L. We have shown that SCP4 interacts with, stabilizes, and removes inhibitory phosphorylation from the activation loops of STK35 and PDIK1L. While the novelty and the therapeutic potential of our work is clear, mechanistic gaps still remain in our knowledge of this signaling complex. Therefore, we now seek to elucidate the genetic, biochemical, and structural mechanisms of SCP4-STK35-PDIK1L function in AML with the intent of establishing insights and assays that can reveal the therapeutic potential of targeting this pathway. The two Principal Investigators of this Project are Dr. Chris Vakoc (CSHL) and Dr. Yan Jessie Zhang (UT Austin), who bring complementary expertise in epigenomics and structural biology, respectively. The first aim of this project will be to evaluate SCP4 dependency in primary human AML patient samples and in normal human hematopoietic stem and progenitor cells (HSPCs). These experiments will rely on new CRISPR vectors developed by Dr. Junwei Shi (UPENN, Co-Investigator) that allow for the efficient generation of genetic knockouts in primary AML patient samples. By targeting SCP4 in diverse AML patient samples and in normal human hematopoietic cells (in vitro and in vivo), we seek to understand which AML subtype is most dependent on SCP4 for disease maintenance. The second aim will seek to apply structural biology approaches to SCP4, STK35, and PDIK1L, with a focus on X-ray crystallography. A major objective will be to understand the catalytic mechanism and the structural basis of substrate recognition, and a substantial body of preliminary data supports the feasibility of these efforts. The third aim will perform genomescale epistasis screens, in search of modifiers and determinants of SCP4 dependency. This strategy will evaluate SCP4 as a critical component of a larger signaling network, with a goal of revealing novel components of the pathway. Finally, the fourth aim will seek to apply biochemical and epigenomic methods to SCP4, STK35, and PDIK1L in search of downstream effectors, with a particular focus on transcription factors whose function is directly or indirectly regulated by this signaling complex. In summary, our collaborative team will apply rigorous and innovative genetic, biochemical, and structural approaches to reveal fundamental insights into an entirely novel signaling pathway and to provide a rich resource of insights and assays that can drive the development novel targeted therapies for AML.

项目摘要由于存在结构化可以用小分子抑制剂靶向的催化口袋或变构位点。扩大我们的知识 AML中的磷酸酶和激酶以及依赖性的依赖性，我们最近完成了以域为中心的CRISPR 筛选研究以搜索AML特异性依赖性。这些努力表明了研究不足的磷酸酶 SCP4作为AML独有的依赖性，而正常的人造血细胞可以忍受这一损失酶。我们的生化研究揭示了SCP4与研究不佳之间的紧密联系激酶旁系同源物STK35和PDIK1L。我们已经证明SCP4与稳定和去除抑制作用相互作用， STK35和PDIK1L的激活环的磷酸化。而新颖性和治疗潜力我们的工作清晰，机械差距仍然存在于我们对这种信号传导复合物的了解中。因此，我们现在寻求阐明AML中SCP4-STK35-PDIK1L功能的遗传，生化和结构机制目的是建立可以揭示针对此目标的治疗潜力的见解和测定路径。该项目的两个主要研究人员是克里斯·瓦科克（Chris Vakoc）博士（CSHL）和Yan Jessie Zhang博士（UT Austin）分别带来了表观基因组学和结构生物学方面的补充专业知识。第一个该项目的目的是评估原代人AML患者样本中的SCP4依赖性人造血干和祖细胞（HSPC）。这些实验将依靠新的CRISPR媒介由Junwei Shi博士（UPenn，共同研究器）开发，可有效地产生遗传主要AML患者样品中的敲除。通过靶向不同的AML患者样品中的SCP4并在正常人造血细胞（体外和体内），我们寻求了解哪种AML亚型是最依赖的在SCP4上进行疾病维持。第二个目标将寻求将结构生物学方法应用于SCP4， STK35和PDIK1L，重点是X射线晶体学。一个主要目标是了解催化机制和底物识别的结构基础，以及大量的初步数据支持这些努力的可行性。第三个目标将执行基因组刻板屏幕，以寻找修饰符和SCP4依赖性的决定因素。该策略将评估SCP4作为较大的关键组成部分信号网络，目的是揭示该途径的新成分。最后，第四个目标将寻求将生化和表观基因组方法应用于SCP4，STK35和PDIK1L，以寻找下游效应子，特别关注转录因子的功能直接或间接地受到该信号的调节复杂的。总而言之，我们的协作团队将采用严格且创新的遗传，生化和结构方法揭示了对完全新颖的信号通路的基本见解，并提供丰富的见解和测定资源，可以推动开发新颖的针对性AML的目标疗法。