Abstract
Ras mutations, which result in constitutive Ras activation, occur frequently in human malignancies, including leukemia. This finding has prompted the development of farnesyltransferase inhibitors (FTIs), which interfere with Ras farnesylation and membrane translocation necessary for Ras function. However, FTIs alone have not yet fulfilled their clinical potential, raising the possibility that their role may lie in combination with other agents. The present studies examined interactions between the farnesyltransferase inhibitor L744832 and the Chk1 inhibitor UCN-01 in human leukemia cells. Combined exposure of U937 cells to sub-toxic concentrations of UCN-01 (100nM) and the FTI L744352 (10μM) resulted in a dramatic and highly synergistic increase in mitochondrial dysfunction, apoptosis, and loss of clonogenicity. Similar interactions were noted in other leukemia cells (HL-60, Raji, Jurkat) and primary AML blasts, and with other farnesyltransferase inhibitors (e.g., FTI-277). These events were accompanied by cleavage of the anti-apoptotic proteins Bcl-2, XIAP, and Mcl-1. Co-administration of L744832 blocked UCN-01-mediated phosphorylation of MEK/ERK, leading to down-regulation of phospho-CREB and -p90RSK, and activation of p34cdc2 and SEK/JNK. Combined treatment also resulted in pronounced reductions in levels of phospho-Akt, -GSK, -p70S6K, -mTOR, -FKHR, -caspase-9, and -Bad. Ectopic expression of Bcl-2 or Bcl-xL but not dominant-negative caspase-8 blocked UCN-01/L744832-mediated mitochondrial dysfunction and apoptosis, but did not prevent activation of p34cdc2 and JNK, or inactivation of MEK/ERK and Akt. Enforced expression of myristolated Akt but not constitutively-active MEK significantly attenuated UCN-01/L744832-induced apoptosis. However, dual transfection with constitutively active Akt and MEK resulted in further protection from UCN-01/L744832-mediated lethality. Finally, down-regulation of JNK1 by siRNA significantly reduced the lethality of the UCN-01/L744832 regimen. Together, these findings suggest that farnesyltransferase inhibitors interrupt the cytoprotective Akt and MAPK pathways while reciprocally activating SAPK/JNK in leukemia cells exposed to UCN-01, and in so doing, dramatically increase mitochondria-dependent apoptosis. They also raise the possibility that combined treatment with FTIs and UCN-01 may represent a novel therapeutic strategy in leukemia.
Polymorphism of thymidylate synthase gene and chemosensitivity of 5-fluorouracil regimen in metastatic gastrointestinal cancer
