Studies on GM-CSF Induced Signaling and Cell Growth in JMML: An Experimental Model To Identify Mechanism Directed Targeted Therapeutics.

Juvenile myelomonocytic leukemia (JMML) is considered to be one of the most difficult to treat and deadliest diseases of early childhood. The pathogenesis of JMML involves deregulated cytokine signal transduction, especially a selective hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). Current treatment modalities do not provide significant long-term disease control. We describe an experimental model system in which the effect of targeted agents can be studied in the context of GM-CSF mediated growth and survival of JMML cells.

Bone marrow and peripheral mononuclear cells from JMML patients were enriched by immunoaffinity chromatography and grown in the presence of GM-CSF. Purity of these cells was confirmed by FACS and their absolute dependence on GM-CSF for growth and survival was assessed. A direct role for GM-CSF was confirmed by the addition of neutralizing antibodies. Molecular techniques showed the lack of excess GM-CSF secretion by these cells that proved receptor hypersensitivity as the principal driving force behind the excessive proliferation. To understand the signaling pathways involved in this process, we constructed a capture antibody array with antibodies to critical signaling molecules. GM-CSF treated and control lysates were incubated with the arrays and the expression and phosphorylation of the signaling molecules were detected by anti p-tyr, p-Ser and p-Thr antibodies. Blots were then quantified by an automated image analysis program. Results showed consistent activation of a group of signaling molecules mediated by the addition of GM-CSF. These include IRS-1, FAK, IkappaB and LIFR. Using this assay system we then looked at the effects of a number of novel therapeutic agents to inhibit the growth and specifically interfere with the GM-CSF mediated signaling cascade. These novel therapeutic agents and targeted kinase inhibitors such as 17-AAG, DMAG, arsenic trioxide, rebeccamycin, AMN-107, and Prima-1. (IC50: 0.1, 0.01. 0.5, 0.1, 0 and 10, uM respectively). These agents were studied in combination to identify agents that exhibit synergistic and additive properties. Significant additive effects were seen with certain combinations, notably in combination with Hsp90 inhibitors.

We describe an effective experimental model to identify the players and pathways involved in the abnormal growth properties of JMML. We discuss in detail the implications of these findings in the development of potential anti JMML therapies in the future.