Targeting Pim Kinase Activity with the Novel Small Molecule Inhibitor SGI-1776 Significantly Augments the Efficacy of Cytarabine In Models of Acute Myeloid Leukemia

Abstract 3253

Drug resistance is a major cause of treatment failure for patients with acute myeloid leukemia (AML) and novel strategies that circumvent resistance mechanisms are urgently needed. The Pim kinases (PIM1, PIM2, PIM3) are a small family of proto-oncogenes within the CAMK superfamily with essential roles in the regulation of signal transduction cascades that promote cell survival, proliferation, and drug resistance. Accordingly, overexpression of Pim kinases has been reported in a wide range of malignancies including AML. However, the specific role of Pim kinases as regulators of sensitivity to standard agents utilized in AML therapy remains to be elucidated. We hypothesized that inhibiting Pim kinase activity would significantly increase the efficacy of the standard of care drug cytarabine by disrupting signaling events that enable cells to resist cytotoxic stress.

In order to investigate a potential role for Pim kinases as mediators of cytarabine resistance, we first evaluated their expression levels in paired HL-60 cell lines that are sensitive and resistant to cytarabine. Our results showed that the levels of Pim-1 are significantly higher in cytarabine-resistant HL-60 cells compared with their sensitive counterparts. Consistent with this observation, treatment of cultured AML cell lines and primary AML blasts with cytarabine led to a dose-dependent increase in Pim-1 expression as assessed by quantitative RT-PCR and immunoblotting. SGI-1776 is novel small molecule inhibitor of Pim kinase activity that has entered Phase I clinical trials. To further test our hypothesis, we investigated the in vitro efficacy of SGI-1776 in a panel of 9 human AML cell lines. SGI-1776 led to a dose-dependent reduction in viability in all cell lines evaluated. Subsequent experiments showed that SGI-1776 impaired clonogenic survival, diminished cell proliferation as determined by BrdU incorporation, and induced apoptotic cell death. These effects were associated with a significant reduction in the phosphorylation of the Pim kinase substrate and apoptotic regulator Bad as well as an increase in the expression of the cyclin-dependent kinase inhibitor, p27.

We next investigated whether inhibiting Pim signaling with SGI-1776 could augment the efficacy of cytarabine. Treatment of AML cells with the combination of cytarabine and SGI-1776 led to significantly greater inhibition of clonogenic survival and induction of apoptosis over what was achieved by either single agent. To validate the therapeutic utility of this combination, vehicle, cytarabine, SGI-1776, or cytarabine and SGI-1776 were administered to mice bearing MOLM-13 AML xenografts for 21 days. Treatment with the combination of these two agents was very well tolerated and significantly increased the efficacy of single agent cytarabine therapy. Immunohistochemical analyses of specimens from mice treated with this regimen revealed that SGI-1776 diminished Bad phosphorylation and cooperated with cytarabine in vivo to promote activation of caspase-3 and inhibit tumor cell proliferation as quantified by the expression of proliferating cell nuclear antigen (PCNA). Collectively, our data demonstrate that antagonizing Pim kinase activity represents a new strategy to increase the therapeutic efficacy of cytarabine. A clinical trial evaluating the safety and efficacy of this combination for patients with AML that are refractory to standard treatments is warranted.