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

• 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
• 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

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 磷酸酶和激酶是肿瘤学中确定的药物靶标类别，这是由于存在结构化的 催化口袋或变构位点，其可以被小分子抑制剂靶向。来扩展我们的知识 磷酸酶和激酶以及AML中的依赖性，我们最近完成了以结构域为重点的CRISPR 筛选研究以寻找AML特异性依赖性。这些努力揭示了一个研究不足的磷酸酶 SCP 4是AML特有的依赖性，而正常的人类造血细胞可以耐受这种依赖性的丧失。 酵素我们的生物化学研究揭示了SCP 4与缺乏研究的 激酶旁系同源物STK 35和PDIK 1 L。我们已经证明，SCP 4与抑制性细胞因子相互作用，稳定并去除抑制性细胞因子。 STK 35和PDIK 1 L的活化环的磷酸化。虽然新奇和治疗潜力 我们的工作是明确的，机械的差距仍然存在于我们的知识，这种信号复合物。所以我们现在 寻求阐明AML中SCP 4-STK 35-PDIK 1 L功能的遗传、生化和结构机制 目的是建立可以揭示靶向这种疾病的治疗潜力的见解和测定， 通路本项目的两位主要研究者是Chris Vakoc博士（CSHL）和Yan杰西张博士 (UT奥斯汀），他们分别带来了表观基因组学和结构生物学方面的互补专业知识。第一 本项目的目的是评估SCP 4在原发性人类AML患者样本和正常人中的依赖性。 人造血干细胞和祖细胞（HSPC）。这些实验将依赖于新的CRISPR载体 由Junwei Shi博士（UPENN，共同研究者）开发，允许有效生成遗传 原代AML患者样品中的敲除。通过靶向不同AML患者样本和正常AML患者样本中的SCP 4， 人类造血细胞（体外和体内），我们试图了解哪种AML亚型最依赖于 在SCP 4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SCP 4， STK 35和PDIK 1 L，专注于X射线晶体学。一个主要的目标将是了解催化剂 机制和结构基础的基板识别，和大量的初步数据支持 这些努力的可行性。第三个目标将进行基因组规模上位筛选，寻找修饰剂 和SCP 4依赖性的决定因素。该战略将评估SCP 4作为一个更大的 信号网络，目的是揭示新的组成部分的途径。最后，第四个目标将寻求 将生物化学和表观基因组学方法应用于SCP 4、STK 35和PDIK 1 L以寻找下游效应物， 特别关注其功能直接或间接受该信号调节的转录因子 复杂.总之，我们的合作团队将应用严格和创新的基因，生化， 结构的方法，揭示了一个全新的信号通路的基本见解，并提供了一个 丰富的见解和分析资源，可推动AML新型靶向疗法的开发。