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.

项目概要 由于结构化的存在，磷酸酶和激酶是肿瘤学中已确定的药物靶标类别。 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 我们最近完成了针对 AML 的磷酸酶和激酶以及依赖性的 CRISPR 寻找 AML 特异性依赖性的筛选研究。这些努力揭示了一种研究不足的磷酸酶 SCP4 是 AML 特有的依赖性，而正常人类造血细胞可以耐受这种依赖性的丧失 酶。我们的生化调查揭示了 SCP4 与研究不足的物质之间存在密切联系 激酶旁系同源物 STK35 和 PDIK1L。我们已经证明 SCP4 与抑制相互作用、稳定并消除抑制 STK35 和 PDIK1L 激活环的磷酸化。虽然新颖性和治疗潜力 我们的工作很清楚，但我们对这种信号复合体的了解仍然存在机制差距。因此，我们现在 寻求阐明 SCP4-STK35-PDIK1L 在 AML 中的遗传、生化和结构机制 目的是建立见解和分析方法，以揭示针对这一点的治疗潜力 途径。该项目的两位首席研究员是 Chris Vakoc 博士（CSHL）和 Yan Jessie 张博士 （德克萨斯大学奥斯汀分校），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。第一个 该项目的目的是评估原发性人类 AML 患者样本和正常人中的 SCP4 依赖性 人类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，联合研究员）开发，可以有效生成遗传物质 原发性 AML 患者样本中的基因敲除。通过针对不同 AML 患者样本和正常人样本中的 SCP4 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 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 底物识别的机制和结构基础，以及大量的初步数据支持 这些努力的可行性。第三个目标是进行基因组规模的上位性筛选，寻找修饰剂 以及 SCP4 依赖性的决定因素。该策略将把 SCP4 评估为更大的项目的关键组成部分。 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 对 SCP4、STK35 和 PDIK1L 应用生化和表观基因组方法来寻找下游效应子， 特别关注其功能直接或间接受该信号传导调节的转录因子 复杂的。总之，我们的合作团队将应用严格和创新的遗传、生化和 结构方法揭示了对全新信号通路的基本见解并提供了 丰富的见解和分析资源可以推动 AML 新型靶向疗法的开发。