Comment on Dasmahapatra et al, page 232, and Gao et al, page 241
Two small molecule combinations that induce leukemic cell apoptosis have disparate molecular mechanisms but converge in induction of reactive oxygen species and activation of c-Jun N-terminal kinase (JNK).
Despite the fundability of the catch phrase, to date few truly “targeted therapies” have been successfully developed for the treatment of leukemia. Effectively targeted therapy capitalizes on a significant biologic differential between malignant cells and their normal counterparts, providing a robust therapeutic index and diminishing toxicity. Unfortunately, our understanding of how leukemic stem cells differ from normal hematopoietic stem cells remains limited. Development of effective targeted therapies therefore requires some degree of serendipity in discovery of drugs or combinations that kill malignant cells with a high degree of specificity.
Steven Grant's laboratory has a long productive history of dissecting signaling pathways in leukemic cells and perturbing these pathways pharmacologically to achieve targeted apoptosis. In 2 articles in this issue, Grant and colleagues present 2 novel, apparently unrelated drug combinations, both of which demonstrate significant promise for the targeted killing of leukemic cells. Dissection of the pathways leading to apoptosis reveals convergence at activation of c-Jun N-terminal kinase (JNK).
In one article, Dasmahapatra and colleagues investigate interaction between a tyrosine kinase inhibitor adaphostin and proteasome inhibitors. Both classes of agents inactivate the Akt pathway, activating the JNK pathway through reactive oxygen species (ROS)-related mechanisms. Adaphostin and proteasome inhibitors induce apoptosis in leukemic cell lines synergistically. The combination, but not the individual drugs, induced increases in JNK phosphorylation, decreased MEK1, phospho-MEK, Raf-1, and phospho-ERK (but surprisingly little inactivation of Akt). The combination of adaphostin with either MG-132 or bortezomib led to increased generation of ROS. Apoptosis could be abrogated by addition of a free radical scavenger, which also prevented JNK activation. Expression of a dominant-negative c-Jun mutant or addition of a JNK inhibitor led to resistance to the drug combination, suggesting a central role of JNK activation for the activity of this combination (Figure). Finally, the combination effectively induced cell death in primary specimens of acute myeloid leukemia (AML) blasts associated with JNK activation, but was not toxic to normal CD34+ cells.
In the second study, Gao and colleagues combined a nonestrogen receptor-binding estrogen metabolite, 2-methoxyestradiol (ME2), with histone deacetylase inhibitors (HDACi's). ME2 generates ROS, activates JNK, and is apoptogenic. HDACi's have diverse molecular effects, which include generation of ROS. In U937 cells, ME2 plus the HDACi's sodium butyrate (NaB) or suberoylanilide hydroxamic acid (SAHA) induced apoptosis synergistically. The combination, but not individual molecules, reduced phosphorylated Akt and increased activated JNK. Akt inactivation and JNK activation preceded caspase activation and apoptosis; changes in the signaling pathways were caspase-independent. The drug combination potently induced oxidative damage; apoptosis was antagonized by the addition of a superoxide dismutase analog and by catalase. Constitutive expression of activated Akt abrogated the effects of the drug combination with the exception of oxidative injury; thus, ROS generation was presumed to be upstream of the signaling pathway changes. A pharmacologic JNK inhibitor and JNK1 siRNA diminished apoptosis in response to ME2 plus HDACi. As in the companion study, similar activity of the combinations was demonstrated in primary specimens of leukemic blasts, but not in normal CD34+ bone marrow cells.
These studies suggest that 2 different combinations of drugs with diverse molecular targets can synergistically induce apoptosis following induction of ROSs and dysregulation of signaling pathways, prominently including JNK activation. Significant selectivity appears when leukemic cell lines and primary leukemic blast specimens are compared with normal CD34+ cells. These studies do not specifically address the mechanism underlying the selectivity of these combinations. The authors reference 2 papers that suggest that neoplastic and normal cells differ in their ability to respond to ROS.1,2 Such innate differences could explain differential sensitivity to these combinations but need to be demonstrated in larger numbers of primary leukemic specimens in response to the specific treatments under investigation. Further studies with normal progenitors using these combinations should be performed to determine whether the signaling pathways are similarly perturbed in the normal cells: is the primary difference between malignant and normal cells their ability to protect themselves from ROS, or do they differ in extent of or response to JNK activation? Enthusiasm about these promising in vitro investigations must be tempered by the fact that the cells under investigation represent neither leukemic stem cells nor normal hematopoietic stem cells. True selectivity to stem cell populations should be tested in xenograft models using normal human progenitor cells and leukemic stem cells isolated from primary specimens. Despite these concerns, Grant and colleagues are to be commended for their ongoing efforts to systematically tease out molecular mechanisms leading to apoptosis in leukemic cells and their important leads in the identification of therapies that may ultimately be truly targeted. ▪