Sunday, November 24, 2024
11/24/2024
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.
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