The novel Aurora A kinase inhibitor MLN8237 is active in resistant chronic myeloid leukaemia and significantly increases the efficacy of nilotinib

Abstract

Novel therapies are urgently needed to prevent and treat tyrosine kinase inhibitor resistance in chronic myeloid leukaemia (CML). MLN8237 is a novel Aurora A kinase inhibitor under investigation in multiple phase I and II studies. Here we report that MLN8237 possessed equipotent activity against Ba/F3 cells and primary CML cells expressing unmutated and mutated forms of breakpoint cluster region-Abelson kinase (BCR-ABL). Notably, this agent retained high activity against the T315I and E255K BCR-ABL mutations, which confer the greatest degree of resistance to standard therapy. MLN8237 treatment disrupted cell cycle kinetics, induced apoptosis, caused a dose-dependent reduction in the expression of the large inhibitor of apoptosis protein Apollon, and produced a morphological phenotype consistent with Aurora A kinase inhibition. In contrast to other Aurora kinase inhibitors, MLN8237 did not significantly affect BCR-ABL activity. Moreover, inhibition of Aurora A with MLN8237 significantly increased the in vitro and in vivo efficacy of nilotinib. Targeted knockdown of Apollon sensitized CML cells to nilotinib-induced apoptosis, indicating that this is an important factor underlying MLN8237's ability to increase the efficacy of nilotinib. Our collective data demonstrate that this combination strategy represents a novel therapeutic approach for refractory CML that has the potential to suppress the emergence of T315I mutated CML clones.

Fig 1

MLN8237 impairs growth, disrupts cell cycle kinetics and induces apoptosis in CML cell lines. (A) Effects of MLN8237 on the in vitro growth and survival of K562 and LAMA 84 human CML cell lines. Cells were treated with the indicated concentrations of MLN8237 for 96 hrs and viability was assessed by MTT assay. n= 3 ± S.D. (B)–(C) Time-dependent induction of DNA fragmentation. LAMA 84 and K562 cells were treated with 30, 100 or 300 nM MLN8237 for 48 hrs and 72 hrs. Percentages of cells with sub-G₀-G₁ DNA and >4N DNA were determined by PI/FACS. n= 3 ± S.D.

Fig 2

MLN8237 has in vitro and in vivo antiproliferative effects in Ba/F3 cells expressing unmutated and mutated BCR-ABL and its activity is unaffected by impairment of p53 function. (A) Left, Ba/F3 p210 BCR-ABL cells stably infected with p53 shRNA or vector control were treated with 100 nM vincristine (VCR) for 24 hrs and subjected to immunoblotting for p53 and p21 to confirm functional knockdown efficiency. Tubulin documented equal loading. Right, Ba/F3 p210 BCR-ABL cells stably infected with p53 shRNA or vector control were treated the indicated concentrations of MLN8237 for 96 hrs and viability was assessed by MTT assay. n= 3 ± S.D. (B) MLN8237 has activity in cells expressing unmutated and mutated BCR-ABL. Ba/F3 cells expressing p210 (unmutated) and T315I, E255K, H396P, Y253F, M351T and Q252H mutant forms of BCR-ABL and imatinib-sensitive and –resistant K562 cells were treated with the indicated concentrations of MLN8237 for 96 hrs and viability was assessed by MTT assay. n= 3 ± S.D. (C) In vivo efficacy of MLN8237. Immunodeficient mice bearing xenografts of P210 and T315I BCR-ABL expressing Ba/F3 cells were administered MLN8237 (20 mg/kg BID) daily or vehicle control. n= 10 ± S.D. (D) Activity of MLN8237 in primary CML cells and normal peripheral blood mononuclear cells. Cells from healthy donors or patients (4) with BCR-ABL⁺ leukaemia including 1 patient each with: unmutated BCR-ABL, T315I-mutated BCR-ABL, blast crisis CML and Ph+ ALL were treated with MLN8237 for 96 hrs and cell viability was assessed by MTT assay.

Fig 2

MLN8237 has in vitro and in vivo antiproliferative effects in Ba/F3 cells expressing unmutated and mutated BCR-ABL and its activity is unaffected by impairment of p53 function. (A) Left, Ba/F3 p210 BCR-ABL cells stably infected with p53 shRNA or vector control were treated with 100 nM vincristine (VCR) for 24 hrs and subjected to immunoblotting for p53 and p21 to confirm functional knockdown efficiency. Tubulin documented equal loading. Right, Ba/F3 p210 BCR-ABL cells stably infected with p53 shRNA or vector control were treated the indicated concentrations of MLN8237 for 96 hrs and viability was assessed by MTT assay. n= 3 ± S.D. (B) MLN8237 has activity in cells expressing unmutated and mutated BCR-ABL. Ba/F3 cells expressing p210 (unmutated) and T315I, E255K, H396P, Y253F, M351T and Q252H mutant forms of BCR-ABL and imatinib-sensitive and –resistant K562 cells were treated with the indicated concentrations of MLN8237 for 96 hrs and viability was assessed by MTT assay. n= 3 ± S.D. (C) In vivo efficacy of MLN8237. Immunodeficient mice bearing xenografts of P210 and T315I BCR-ABL expressing Ba/F3 cells were administered MLN8237 (20 mg/kg BID) daily or vehicle control. n= 10 ± S.D. (D) Activity of MLN8237 in primary CML cells and normal peripheral blood mononuclear cells. Cells from healthy donors or patients (4) with BCR-ABL⁺ leukaemia including 1 patient each with: unmutated BCR-ABL, T315I-mutated BCR-ABL, blast crisis CML and Ph+ ALL were treated with MLN8237 for 96 hrs and cell viability was assessed by MTT assay.

Fig 3

MLN8237 reduces autophosphorylation of Aurora A without significantly affecting BCR-ABL activity. (A) MLN8237 reduces Aurora kinase A phosphorylation. K562 cells were treated with 30 nM MLN8237 for 24 hrs. Protein lysates were subjected to SDS-PAGE, blotted, and probed with phospho-Aurora A (Thr288) and Aurora A antibodies. (B) Effects of MLN8237 on the activity of selected kinases. MLN8237 was screened against a kinase panel as described in ‘Materials and methods’. The ABL1 and the related ABL2 cytoplasmic tyrosine kinases share 89% sequence identity and have some overlapping functions, but are distinct in that ABL1 fuses with BCR to form the Philadelphia chromosome while ABL2 does not. (C) K562 and LAMA 84 cells were treated with MLN8237 for 24 hrs. Protein lysates were subjected to SDS-PAGE, blotted, and probed with phospho-BCR and c-Abl antibodies. (D) MLN8237 treatment does not significantly affect BCR-ABL kinase activity in primary CML cells. Primary CML cells obtained from a patient with unmutated BCR-ABL were treated with 10 μM MLN8237 for 24 hrs. BCR-ABL autophosphorylation and CRKL phosphorylation were assessed by immunoblotting. Nilotinib was used as a positive control for BCR-ABL inhibition.

Fig 4

MLN8237 significantly increases the efficacy of nilotinib. (A) MLN8237 potentiates the pro-apoptotic effects of nilotinib. LAMA 84 and K562 cells were treated with 30 nM MLN8237, 10 nM nilotinib or the combination for 48 hrs. Percentages of cells with sub-G₀-G₁ DNA were determined by PI/FACS. n= 3 ± S.D. (B) The combination of MLN8237 and nilotinib induces mitochondrial-dependent apoptosis. K562 and LAMA 84 cells were treated with 100 nM MLN8237, 30 nM nilotinib or both for 24 hrs. Protein lysates were subjected to SDS-PAGE, blotted, and probed with active caspase-3 and caspase-9 antibodies. Anti β-tubulin was used as a loading control. (C) Co-treatment with MLN8237 and nilotinib results in significantly greater growth inhibition and reduction in survival than that achieved by either agent alone. Cells were treated with the indicated concentrations of MLN8237 for 96 hrs and viability was assessed by MTT assay. Error bars indicate the S.D. *P < 0.05. (D) Effects of MLN8237 and nilotinib on clonogenic survival. CD34⁺ normal bone marrow (n= 3), primary CML from patients in blast crisis (n= 3), K562 and LAMA 84 cells were treated with MLN8237, nilotinib or both drugs for 24 hrs. Cells were plated and scored as described in the ‘Materials and methods’.

Fig 5

In vivo efficacy and tolerability of MLN8237 and nilotinib. (A) K562 cells were injected into the flanks of nude mice. Vehicle, MLN8237, nilotinib or both were administered for 14 days. n= 10 ± S.D. *P= 0.00028. (B) Immunohistochemistry. Tumours were stained with haematoxylin and eosin as described in ‘Materials and methods’. Representative images are shown from each treatment group. (C) Treatment with MLN8237 leads to a morphological phenotype consistent with Aurora A kinase inhibition. Tumours were stained with haematoxylin and eosin. Representative images from the MLN8237 treatment group are shown. Arrows indicate the following: an elevated number of cells with chromatin bridging (far left), mitotic slippage (second from left), cells with internuclear bridging (second from right) and monopolar mitotic spindles (far right). (D) Quantification of TUNEL⁺ cells. Positive cells were scored manually under 20× magnification. Mean ± S.D., n= 5. *P < 0.05.

Fig 5

In vivo efficacy and tolerability of MLN8237 and nilotinib. (A) K562 cells were injected into the flanks of nude mice. Vehicle, MLN8237, nilotinib or both were administered for 14 days. n= 10 ± S.D. *P= 0.00028. (B) Immunohistochemistry. Tumours were stained with haematoxylin and eosin as described in ‘Materials and methods’. Representative images are shown from each treatment group. (C) Treatment with MLN8237 leads to a morphological phenotype consistent with Aurora A kinase inhibition. Tumours were stained with haematoxylin and eosin. Representative images from the MLN8237 treatment group are shown. Arrows indicate the following: an elevated number of cells with chromatin bridging (far left), mitotic slippage (second from left), cells with internuclear bridging (second from right) and monopolar mitotic spindles (far right). (D) Quantification of TUNEL⁺ cells. Positive cells were scored manually under 20× magnification. Mean ± S.D., n= 5. *P < 0.05.

Fig 6

Targeting Apollon expression sensitizes CML cells to nilotinib-induced apoptosis. (A) MLN8237 treatment results in a dose-dependent reduction in the large IAP, Apollon and increased expression of its substrate, Smac. LAMA 84 cells were treated with MLN8237 for 24 hrs. Protein lysates were subjected to SDS-PAGE, blotted, and probed with Apollon and Smac antibodies. Tubulin documented equal loading. (B) Aurora A SMARTpool or siCONTROL siRNA directed at luciferase were transfected into LAMA 84 cells using the Nucleofector II. (C) General disruption of mitosis does not significantly affect Apollon expression. LAMA 84 cells were treated with nocodazole, vincristine or MLN8237 for 24 hrs. Protein lysates were subjected to SDS-PAGE, blotted, and probed with an Apollon antibody. Tubulin documented equal loading. (D) Apollon SMARTpool or siCONTROL siRNA directed at luciferase were transfected into LAMA 84 cells using the Nucleofector II. Tubulin was used as a loading control. LAMA 84 cells transfected with Apollon-targeted siRNA and non-targeted siRNA were treated with nilotinib for 48 hrs and the percentage of apoptotic cells were determined by PI/FACS analysis. n= 3 ± S.D., *P < 0.05. (E) Schematic depicting the multiple anti-leukaemia properties of the MLN8237. MLN8237 inhibits Aurora A kinase leading to deleterious aneuploidy, inhibition of the IAP, Apollon and cell death.

Fig 6

Targeting Apollon expression sensitizes CML cells to nilotinib-induced apoptosis. (A) MLN8237 treatment results in a dose-dependent reduction in the large IAP, Apollon and increased expression of its substrate, Smac. LAMA 84 cells were treated with MLN8237 for 24 hrs. Protein lysates were subjected to SDS-PAGE, blotted, and probed with Apollon and Smac antibodies. Tubulin documented equal loading. (B) Aurora A SMARTpool or siCONTROL siRNA directed at luciferase were transfected into LAMA 84 cells using the Nucleofector II. (C) General disruption of mitosis does not significantly affect Apollon expression. LAMA 84 cells were treated with nocodazole, vincristine or MLN8237 for 24 hrs. Protein lysates were subjected to SDS-PAGE, blotted, and probed with an Apollon antibody. Tubulin documented equal loading. (D) Apollon SMARTpool or siCONTROL siRNA directed at luciferase were transfected into LAMA 84 cells using the Nucleofector II. Tubulin was used as a loading control. LAMA 84 cells transfected with Apollon-targeted siRNA and non-targeted siRNA were treated with nilotinib for 48 hrs and the percentage of apoptotic cells were determined by PI/FACS analysis. n= 3 ± S.D., *P < 0.05. (E) Schematic depicting the multiple anti-leukaemia properties of the MLN8237. MLN8237 inhibits Aurora A kinase leading to deleterious aneuploidy, inhibition of the IAP, Apollon and cell death.