Dual Targeting of Aurora Kinases with AMG 900 Exhibits Potent Preclinical Activity Against Acute Myeloid Leukemia with Distinct Post-Mitotic Outcomes

Abstract

Aurora kinase A and B have essential and non-overlapping roles in mitosis, with elevated expression in a subset of human cancers, including acute myeloid leukemia (AML). In this study, pan-aurora kinase inhibitor (AKI) AMG 900 distinguishes itself as an anti-leukemic agent that is more uniformly potent against a panel of AML cell lines than are isoform-selective AKIs and classic AML drugs. AMG 900 inhibited AML cell growth by inducing polyploidization and/or apoptosis. AMG 900 and aurora-B-selective inhibitor AZD1152-hQPA showed comparable cellular effects on AML lines that do not harbor a FLT3-ITD mutation. AMG 900 was active against P-glycoprotein-expressing AML cells resistant to AZD1152-hQPA and was effective at inducing expression of megakaryocyte-lineage markers (CD41, CD42) on human CHRF-288-11 cells and mouse Jak2 ^V617F cells. In MOLM-13 cells, inhibition of p-histone H3 by AMG 900 was associated with polyploidy, extra centrosomes, accumulation of p53 protein, apoptosis, and cleavage of Bcl-2 protein. Co-administration of cytarabine (Ara-C) with AMG 900 potentiated cell killing in a subset of AML lines, with evidence of attenuated polyploidization. AMG 900 inhibited the proliferation of primary human bone marrow cells in culture, with a better proliferation recovery profile relative to classic antimitotic drug docetaxel. In vivo, AMG 900 significantly reduced tumor burden in a systemic MOLM-13 xenograft model where we demonstrate the utility of 3'-deoxy-3'-¹⁸F-fluorothymidine [¹⁸F]FLT positron emission tomographic (PET)-CT imaging to measure the antiproliferative effects of AMG 900 in skeletal tissues in mice.

Figure 1.

AMG 900 exhibits potent anti-leukemia activity on AML cell lines with a distinct sensitivity profile relative to isoform-selective AKIs. A and B, AML cell lines were treated with DMSO or test agents at eleven concentrations [three-point dilution, high concentration AMG 900 (0.1 μmol/L), MLN8054 and AZD1152-hQPA (5 μmol/L), Ara-C and daunorubicin (20 μmol/L)] for 72 hours. Cell count and apoptosis/DNA content analysis. A, Cell count potency determined for individual AML lines (n = 11), with mean and median EC₅₀ values noted below. *OCI-AML-3 data not included in graph for Ara-C (EC₅₀ > 1 μmol/L). B, AKI dose-response profiles for MOLM-13, KG-1a, and HL-60 cells showing percentage of ≥ 4N DNA content and total cell death (subG₁ + cl-caspase-3) populations (mean ± SD). C, MOLM-13, KG-1, HEL, and MEGAL cells treated with AMG 900, MLN8054, and AZD1152-hQPA or DMSO for 48 hours. Heatmaps showing p-histone H3 positivity represented as a mean percentage of control (POC). SAC activation (p-histone H3 POC > 100, white to red) and SAC silencing (p-histone H3 POC < 100, white to blue). D, Histogram of MOLM-13, KG-1, HEL cells stained with P-gp (blue) and isotype (red) antibodies, and unstained control (black). MES-SA/Dx5 cells were included as P-gp control. E, DNA content analysis of HEL cells treated with AMG 900 or AZD1152-hQPA ± P-gp inhibitor GF120918. Dose-response of AKI alone (blue) and in combination with P-gp inhibitor (red).

Figure 2.

AMG 900 induces polyploidization and the expression of megakaryocyte-lineage differentiation markers. CHRF-288–11 AMKL cells and Jak2^V617F knock-in mouse bone marrow cells were treated with MLN8237 and AMG 900 at 0.3 μmol/L or DMSO for 72 hours. A and C, Representative images of Wright-Giemsa stained cells (magnification x400), CD41/CD42 expression, and DNA content profiles. B and D, Percentage of CD41⁺/CD42⁺ and > 4N DNA content gated CHRF-288–11 and Jak2^V617F gated populations (mean ± SD). CHRF-288–11 cells [MLN8237 and AMG 900 versus DMSO (CD41⁺/CD42⁺ and DNA ploidy, *P ≤ 0.0009)]. Jak2^V617F cells from five individual mice [MLN8237 versus DMSO (CD41⁺/CD42⁺ P = 0.1993, DNA ploidy P = 0.5334, not significant (ns); AMG 900 versus DMSO (CD41⁺/CD42⁺ and DNA ploidy *P ≤ 0.0007)].

Figure 3.

AMG 900 can increase polyploidy, apoptosis, and p53 protein levels in AML cell lines, and can potentiate the activity of LDAC. A and B, U937 (TP53^MUT), HEL (TP53^MUT), GDM-1 (TP53^WT), and MOLM-13 (TP53^WT) cells were treated with AMG 900 at 33 nmol/L or DMSO for 48 hours. Western blot and cell-cycle analysis. A, Protein expression profiles of cl-caspase-3, total p53, p21, and β-actin. B, DNA content profiles with indicated Y-axis maximal (Y-max) count and ploidy (2N to 16N). C and D, AML cells treated with AMG 900, Ara-C, or DMSO, and in combination using 4 × 3 dose-matrix for 72 hours. Annexin-V apoptosis analysis. C, Representative scatter plots of annexin-V positivity percentages (red) for test agent alone and combined AMG 900 (4.0 nmol/L) and Ara-C (125 nmol/L). D, Percentage of annexin-V positivity across dose-matrix. E and F, MOLM-13 cells were treated with test agent alone at the indicated concentrations and in combination for 72 hours. Apoptosis and cell-cycle analysis. E, DNA content profiles with cl-caspase-3 positive population (red) with Y-max count and ploidy (2N to 16N). F, Percentage of total cell death (subG₁ + cl-capase-3) and ≥ 4N DNA content (mean ± SD).

Figure 4.

Inhibition of aurora-B activity by AMG 900 leads to polyploidization associated with extra centrosomes, induction of apoptosis, and Bcl-2 cleavage in MOLM-13 cells. A, Cells were treated with AMG 900 at 10 nmol/L or DMSO for 48 hours. Representative confocal images of treated mitotic cells stained with p-histone H3 (red) and pericentrin (green) antibodies and DAPI (blue) (magnification x600). B, Cells treated with AMG 900 at 33 nmol/L, nutlin-3a at 2.5 μmol/L, or DMSO for 24 and 48 hours. Western blot analysis showing protein expression of total p53, MDM2, p21, cl-caspase-3, Bax, Bcl-2, Mcl-1, Bcl-xL, and β-actin. Arrows indicate specific target protein band. C and D, Cells pre-incubated ± caspase inhibitors (Z-VAV-fmk, Z-DEVD-fmk) at 10 μmol/L for 2 hours before treatment with AMG 900 at 33 nmol/L or DMSO for 48 hours. Cell count and Western blot analysis. C, Cell count represented as POC (mean ± SD). D, Protein expression profile of cl-caspase-3, Bax, Bcl-2, and β-actin. E, Cells treated with AMG 900 at 33 nmol/L or DMSO for 48 hours. Annexin-V/JC-1 analysis. Measurement of annexin-V positivity percentage (left graph, mean ± SD) and JC-1 dye red mean fluorescence intensity (MFI) for AMG 900 treated annexin-V negative and positive populations (right graph, mean ± SD).

Figure 5.

Anti-leukemia activity of AMG 900 in a systemic MOLM-13 tumor xenograft model. Mice bearing established MOLM-13-Luc tumors were orally administered vehicle, AMG 900 at 12.6 mg/kg for 7 days or 22 mg/kg for 4 days (n = 10 per group). A, Outline of AMG 900 treatment and biomarker assessment schedule. Whole body BLI was recorded on days 1, 6, 9, 12, 15 (blue, ▲). [¹⁸F]FLT-PET/CT measurements were determined the day before treatment (D-1, baseline), and on days 5, 8, 11, and 14 (red, ▲). Bone marrow (BM) aspirates were collected at study termination [vehicle (day 12) and AMG 900 (day 15)]. B, Tumor burden was determined by BLI (mean ± SE) for vehicle (black, ●, n = 9 on day 12), AMG 900 at 12.6 mg/kg (blue, ■, n = 9 on day 12), and AMG 900 at 22 mg/kg (red, ■, n = 9 on day 12) groups. Removed one animal from each group due to toxicity by day 12. Entire vehicle group was removed on day 12 due to morbidity. Statistical significance determined for AMG 900 groups versus vehicle on days 6, 9, and 12 (*P < 0.0001, ⁺P < 0.02). C and D, Tumor burden measured in femur BM aspirates by DNA content analysis. C, Representative fitted DNA content profiles from each treatment group, mouse BM (red) and MOLM-13-Luc (yellow) cell populations (detected G₁ and G₂ peaks, ▲). D, Percentage of tumor to BM cell population based on DNA content (mean ± SE). Statistical significance determined for AMG 900 22 mg/kg (n = 9) and 12.6 mg/kg (n = 7) groups versus vehicle (n = 7) (P ≤ 0.0002). E and F, [¹⁸F]FLT-PET/CT imaging of skeleton tissue. [¹⁸F]FLT signal was measured at baseline (1 day before treatment) and on days 5, 8, 11, and 14. E, Representative images of FLT signal at baseline, and day 5 and 8 post-treatment with AMG 900 at 22 mg/kg or vehicle for 4 days. [¹⁸F]FLT signal suppression (arrow) and [¹⁸F]FLT flare (arrowhead) indicated for AMG 900 treatment on day 5 and 8, respectively. F, Skeleton [¹⁸F]FLT signal measured as percentage %ID/g (mean ± SE). Statistical significance determined for AMG 900 groups versus vehicle (*P < 0.0001, n = 9 to 10 per group).