AT9283, A Novel Aurora Kinase/Jak2 Inhibitor Demonstrates Activity against Refractory Infant Leukemia Cells: Studies On Growth Inhibition, Biological Correlates, Drug Synergy and Effects On Leukemia Stem-Like Cells.

Abstract

Abstract 3078Poster Board III-15Leukemia in children less than 1 year of age confers a poor prognosis, despite intensification of therapy. These leukemias possess unique biologic characteristics including the presence of mixed-lineage leukemia (MLL) gene rearrangement and high expression of Fms-like tyrosine kinase 3 (FLT3). AT9283, a potent inhibitor of Aurora A and B kinases, JAK2, JAK3, and mutant Abl Kinase, has demonstrated inhibition of multiple solid tumor cell lines in vitro and in mouse xenograft models. Aurora kinase inhibition has been shown to inhibit cancer cell growth by interfering with the mitotic apparatus. We investigated the activity of AT9283 against cell lines derived from refractory infant leukemia cells to identify its efficacy in a future treatment protocol. MethodFive cell lines derived from infant leukemia cells were used (ALL: BEL1, KOPN8, KCCF2, B1 and AML: TIB202). We also included the cell line SEM that was derived from a 5 year old child with t (4;11) MLL-AF4 preB-ALL. Normal bone marrow stromal cells were used to evaluate cytotoxicity against non-malignant cells. AT9283 was provided by Astex Therapeutics Ltd. (Cambridge, UK). Approximately 1×104 cells per well were seeded in 96-well plates and incubated with increasing concentrations of AT9283, alone or in combination with a panel of conventional and novel therapeutic agents. After four days, cell survival was measured by Alamar blue assay and IC50 values and combination indices were calculated. Stem-like cells were quantified by the distribution of ALDH bright cells by Aldefluor assay (Stem cell technologies) and characterized by conventional clonogenic assays. Alterations in cell-signaling pathways and survival proteins were measured by Western blot analysis using total and phospho-specific antibodies. ResultsAT9283 inhibited the growth of all five cell lines with a 10 fold variation in IC50 within cell lines (IC50 range, 0.1 to 0.01 μM). There was a corresponding increase in the number of cells displaying a polyploid phenotype, an effect of aurora kinase inhibition. No significant cytotoxicity against bone marrow stromal cells was seen under the experimental conditions used in this study (IC50 > 10 μM). Changes in the activation and expression of a variety of intracellular proteins were noted, including the down regulation of activated ERK1/2, MYC and AKT within 10 minutes of exposure to the agent. An increase in the activated form of RAF and ATF2 was observed immediately after drug exposure. Importantly, a significant decrease in the level of constitutive pFLT-3 was demonstrated. A concurrent increase in cleaved PARP was also noted, indicating the initiation of apoptosis. In combination studies, the HDAC inhibitor Apicidin showed synergy across all cell lines (CI range: 0.07 to 0.62). A decrease in ALDH bright stem-like cells was observed in a dose dependent manner, up to 50% over 24 hours at IC50 concentrations. ConclusionsOur in vitro studies show that AT9283 significantly decreases the growth and survival of infant leukemia cell lines. Importantly, AT9283 potently induces FLT3 de-phosphorylation, inhibiting a critical growth stimulatory pathway of infant ALL cells. We have identified changes in a number of signaling and apoptotic molecules that can provide a panel of markers for biological correlative analysis for drug activity in vivo. Also, the drug combination studies demonstrate the potential of HDAC inhibition to synergize with the activity of this agent. Finally, the effect on stem-like cells provides a rationale and critical preclinical data for the formulation of an effective clinical trial for the treatment of infants with refractory ALL. DisclosuresSquires:AstexTherapeutics Ltd: Employment.