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

项目总结 由于结构性的存在，磷酸酶和激酶是肿瘤学中确定的一类药物靶点。 可以被小分子抑制剂靶向的催化口袋或变构位置。扩大我们的知识面 我们最近完成了针对AML的结构域的CRISPR 筛选研究以寻找AML特异性相关性。这些努力揭示了一种研究很少的磷酸酶。 SCP4是AML独有的依赖性，而正常的人类造血细胞可以容忍这种缺失 酵素。我们的生化研究揭示了SCP4与研究较少的 蛋白激酶与STK35和PDIK1L同源。我们已经证明了SCP4与抑制物相互作用、稳定和去除抑制物 STK35和PDIK1L激活环的磷酸化。虽然新颖性和治疗潜力 我们的工作是显而易见的，我们对这个信号复合体的认识中仍然存在机械上的差距。因此，我们现在 目的阐明SCP4-STK35-PDIK1L在AML中作用的遗传、生化和结构机制 目的是建立洞察力和分析方法，以揭示靶向治疗的潜力 路径。该项目的两名首席研究员是克里斯·瓦科克博士(CSHL)和严杰西·张博士 (UT Austin)，他们分别带来了表观基因组学和结构生物学的互补专业知识。第一 该项目的目的将是评估原发人类AML患者样本和正常人群中SCP4的依赖性 人造血干祖细胞(HSPC)。这些实验将依赖于新的CRISPR载体 由施俊伟博士(宾夕法尼亚大学，共同研究员)开发，允许高效地产生基因 原发AML患者样本中的基因敲除。通过在不同的AML患者样本和正常对照中靶向SCP4 人类造血细胞(体外和体内)，我们试图了解哪个AML亚型最依赖 用于疾病维护的SCP4。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射线结晶学。一个主要的目标将是理解催化作用 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。第三个目标将进行基因组规模的上位性筛选，以寻找修饰物 和SCP4依赖的决定因素。这一战略将评估SCP4作为一个更大的 信号网络，目的是揭示该途径的新组成部分。最后，第四个目标将寻求 将生化和表观基因组学方法应用于SCP4、STK35和PDIK1L，以寻找下游效应子， 特别关注转录因子，这些转录因子的功能直接或间接受该信号调节 很复杂。总而言之，我们的协作团队将应用严格和创新的遗传、生化和 结构性方法，揭示对全新信号通路的基本见解，并提供 丰富的洞察力和分析资源，可以推动AML新的靶向治疗的开发。

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.