A Novel Dual AXL/Mertk and Aurora Kinase Inhibitor Active Against T Cell Acute Lymphoblastic Leukemia

Receptor tyrosine kinases AXL and MER belong to the TYRO3 kinase family, first identified as a transforming gene in chronic myeloid leukemia and are found at high levels in various cancers, including hematopoietic malignancies like T cell acute lymphoblastic leukemia (T-ALL). Aurora kinases play important roles in chromosome alignment and cytokinesis during mitosis and are also aberrantly expressed in ALL. We describe here characterization and pre-clinical testing of Huntsman Cancer Institute-2084 (HCI-2084), a small molecule inhibitor of AXL and MER kinases that also has activity against Aurora Kinases A and B, as a novel therapeutic for T-ALL treatment, a leukemia with poor prognosis.

Quantitative RT-PCR and Western Blot confirmed elevated levels of AXL/MER expression in Jurkat, a human T-ALL, cell line. Our compound, HCI-2084, was developed using a computational structure-based approach against AXL kinase. HCI-2084 activity against AXL/MER, in cell-based assays was evaluated utilizing ATPlite. To test activity against Aurora kinase, Jurkat cells were treated with various concentrations of HCI-2084 and VX 680, a known aurora kinase inhibitor, for 24 hours. Cell lysates were evaluated for phospho-AKT (Ser473), phospho-Aurora (Thr288) and phospho-Histone H3 (Ser10) using the Meso Scale Discovery platform (MSD). Cell cycle analysis was performed on Jurkat cells incubated in the absence (negative control) or presence of 100nM HCI-2084 or VX-680 for 24 hours, stained with propidium iodide and analyzed by flow cytometry on FACS Calibur. Immunostaining was performed to evaluate the levels of phospho-Histone H3 (Ser10) and phospho-Aurora (Thr288) during mitosis in Jurkat cells incubated in the absence (negative control) or presence of 100nM HCI-2084 and VX-680 for 24 hours. In addition to the anti-phospho-Histone H3 and anti-phospho-Aurora antibodies, anti-alpha-tubulin and DAPI were used to determine the presence of mitotic chromosomes.

To examine HCI-2084 as a therapeutic agent for T-ALL treatment in vivo, we tested it using transgenic Zebrafish (Danio rerio) with T-ALL driven by human MYC (hMYC). Over-expression of endogenous D. rerio axl /mertk transcripts in T-ALL from hMYC fish were verified by qRT-PCR. Fish were treated with HCI-2084, Dexamethasone (a known T-ALL therapeutic; positive control), and DMSO vehicle (HCI-2084 is reconstituted in DMSO; negative control). Trials were conducted by housing fish with fluorescently-labeled T-ALL (GFP-tagged) in water containing the agent being tested, for 14 days with monitoring of disease response by fluorescent microscopy.

In in vitro studies, HCI-2084 showed potent activity in cell viability assays with an IC₅₀ of 12 nM against AXL, 60 nM against MERTK and 15 nM against Aurora kinase. MSD assays demonstrated efficacy at 1 uM for reduction of phospho-AKT, phospho-Histone H3 and phospho-Aurora, as seen with VX 680. Cell cycle analyses performed on HCI-2084-treated Jurkat cells showed a significantly increased G2/M population and an accumulation of cells with ≥4N DNA, indicative of Aurora B inhibition and endo-reduplication (Figure A & B). Immunofluorescence analyzed using fluorescent microscopy demonstrated mitotic arrest with loss of phospho-Histone H3 and phospho-Aurora staining, demonstrating inhibition of Aurora kinase activity. HCI-2084 was also a potent therapeutic against Zebrafish T-ALL. Fish treated at 1uM HCI-2084 for 14 days, showed complete responses (CR), with efficacy comparable to our dexamethasone positive control (Figure C). Following treatment, fish remained disease-free for several days, and overall survival was prolonged significantly relative to untreated controls.

HCI-2084 shows dual action in vitro against Jurkat cells with potent cytotoxicity via AXL/MER kinase inhibition and anti-proliferative activity via Aurora kinase inhibition. In vivo, HCI-2084 demonstrates activity against a MYC-driven vertebrate model of T-ALL, prolongs survival, and is well tolerated. We conclude that HCI-2084 is a potent dual AXL/MER kinase and Aurora kinase inhibitor which should be explored further as a potential novel therapeutic in the treatment of human T-ALL.

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Sharma:Millenium: Research Funding.