The acute and chronic myelogenous leukemias are common and often fatal malignancies derived from hematopoietic cells. Recent work has revealed new insights regarding the biological role of growth factors, lymphokines, and their receptors in myeloid lineage development and leukemogenesis. Capitalizing upon these insights, I am using genetic engineering to fuse the gene for the myeloid growth factor granulocyte- macrophage colony-stimulating factor (GMCSF) to the gene for Pseudomonas exotoxin A (ETA) to create a recombinant fusion toxin (GMCSF-ETA) that targets malignant myeloid cells. The recombinant GMCSF-ETA toxin will be used to study hematopoiesis and evaluated for clinical utility in purging marrow of myeloid leukemic cells for autologous transplantation. This work is an extension of previous studies in which it was first demonstrated that genetic fusions of diphtheria toxin (DT) and interleukin 2 could be efficiently expressed in yeast mutants resistant to DT and ETA (Perentesis et al., PNAS 85:8386-8390), and complements my cloning and structural studies of the target of these toxins, protein synthesis elongation factor 2 (Perentesis et al., J. Biol. Chem. 267:1190-1198). GMCSF has been selected for these prototypic studies because much is known regarding its biological activities, including its ability to recruit blast cells into the cell cycle and possible autocrine loops in some leukemias, as well as the developmental expression and internalization of its high affinity receptor. I thus plan to: (1) Optimize the expression and purification of the recombinant GMCSF-ETA toxin produced in bacteria and toxin-resistant yeast, (2) Evaluate the GMCSF-ETA toxin for binding, receptor signaling, and toxicity to targets and stem cells, (3) Test the ability of GMCSF-ETA toxin to alter myeloid proliferation and development in differentiation-inducible myeloid cell lines and examine the behavior of toxin-resistant mutants in these systems, (4) Use molecular modeling approaches to refine the GMCSF-toxin, (5) Test the activity of the GMCSF-ETA toxin ex vivo on fresh human leukemic cells from blood and bone marrow, (6) Test the activity of the GMCSF--ETA toxin in murine in vivo toxicity and efficacy studies. Because not all myeloid leukemias express functional receptors for a single growth factor such as GMCSF, toxin gene fusions with growth factors that act on earlier and later stages of hematopoietic development (e.g., IL-3, GCSF, and MCSF) will also be constructed and evaluated in the framework of the proposed studies.
