Elucidating the molecular mechanism of Daple- FLT3 and Daple-PDGFRB gene fusion in blood cancers

PROJECT SUMMARY/ABSTRACT: Receptor tyrosine kinases (RTKs) are a major class of cell signaling receptors and they play major roles in cancer progression. In leukemia, and many cancers, aberrant activation of the RAS-RAF-MEK-ERK signal transduction pathway is often observed. Various RTK mutations, such as those found on PDGFRB and FLT3, are found in leukemia patients and often lead to hyperactivation of the kinase domain. This activation causes the subsequent increase in the RAS-RAF-MEK-ERK signaling pathway. Because the cancer is driven by kinase domain activation, many patients are responsive to tyrosine kinase inhibitor (TKI) treatment such as imatinib and sorafenib. However, in a subset of patients, they become unresponsive to TKIs over time due to a resistance in the cancer cells. One mechanism by which cancer cells can acquire resistance is through gaining a secondary mutation in the kinase domain that makes the kinase insensitive to the TKI. In response, patients are often given higher drug dosage or an approach to target multiple pathways simultaneously using combination therapy is taken. These approaches are often impractical due to cumulative side effects that may be life-threatening. Thus, further insights into the inner workings of cancer cells and mutations that lead to aggressive cancer phenotypes are essential to identifying new therapeutic targets, especially, those on which multiple cancer-fueling signaling pathways may converge. Daple/CCDC88C is scaffold protein that serves as a convergence point for three major signaling pathways in a cell: Wnts, G-proteins, and RTKs. Because protein scaffolds can serve as central hubs integrating cellular signaling, this raises the possibility of exploiting these proteins for therapeutic intervention. Several gene fusion between Daple and PDGFRB or FLT3 have been found in cases of myeloproliferative disorders and leukemias. The exact mechanism by which these gene fusions lead to constitutive activation of the kinase domain remains to be tested and whether regions on Daple that is involved in the gene fusion can be leveraged for therapy remains a mystery. Finally, because these mutations are rare, generating an in vivo model for therapeutic testing in mice is often seen as unpractical. This proposal seeks to address these questions and problems. Specifically, it seeks to understand how the Daple gene fusion affects hematopoietic cell expansion using cell-based models and zebrafish. Also planned in this proposal is to expose how the coiled-coil domain of the Daple-PDGFRB or Daple-FLT3 gene fusion affects protein subcellular localization and to determine if it is responsible for the dimerization and activation of the kinase domain in cells. The strategy that will be employed to target the dimerization point may also serve as a therapeutic strategy. Overall, these studies will give novel insights into how Daple associated mutations affect leukemia cell signaling and growth and it will also establish whether targeting this protein scaffold can be leveraged for cancer therapy.