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Novel therapies are urgently needed to improve clinical outcomes for patients with acute myeloid leukemia (AML). The investigational drug alisertib (MLN8237) is a novel Aurora A kinase inhibitor being studied in multiple Phase I and II studies. We investigated the preclinical efficacy and pharmacodynamics of alisertib in AML cell lines, primary AML cells and mouse models of AML. Here, we report that alisertib disrupted cell viability, diminished clonogenic survival, induced expression of the FOXO3a targets p27 and BIM and triggered apoptosis. A link between Aurora A expression and sensitivity to ara-C was established, suggesting that Aurora A inhibition may be a promising strategy to increase the efficacy of ara-C. Accordingly, alisertib significantly potentiated the antileukemic activity of ara-C in both AML cell lines and primary blasts. Targeted FOXO3a knockdown significantly blunted the pro-apoptotic effects of the alisertib/ara-C combination, indicating that it is an important regulator of sensitivity to these agents. In vivo studies demonstrated that alisertib significantly augmented the efficacy of ara-C without affecting its pharmacokinetic profile and led to the induction of p27 and BIM. Our collective data indicate that targeting Aurora A with alisertib represents a novel approach to increase the efficacy of ara-C that warrants further investigation.
Acute myeloid leukemia (AML) accounts for 80% of adult acute leukemia.1 Currently utilized therapeutic agents do not achieve long-term survival for the majority of patients with this disease. Elderly patients with AML have a particularly dismal prognosis with less than 10% achieving long term survival.2 They are less able to tolerate intensive therapy with cytotoxic agents and they also have a higher prevalence of pre-existing myelodysplasia, unfavorable cytogenetics and multidrug resistance than their younger counterparts.3 Moreover, a standard induction approach remains to be established for elderly patients with AML due, in part, to the aforementioned factors along with the poor representation of elderly patients in clinical trials. A recent study demonstrated that elderly patients with good or intermediate risk cytogenetics that received therapy with low-dose cytarabine had a significant survival advantage over patients that received supportive care. Despite this, no patients with unfavorable cytogenetics achieved complete responses (CRs) on this study.4 Furthermore, most elderly patients will not benefit from intensive chemotherapy and have a median survival of less than 6 months with this approach.5 These poor results underscore the need to develop less toxic targeted therapies based on our understanding of the molecular aberrations in AML to improve clinical outcomes.
Aurora kinase A is a serine/threonine kinase that functions as a central mitotic regulator. Aurora A activity is required for mitotic entry, mitotic spindle assembly and accurate chromosome separation.6–8 Aurora A and the related kinase Aurora B have been reported to be aberrantly expressed in a number of malignancies including leukemia's.9 It is hypothesized that their overexpression contributes to the increased proliferative rate that is a hallmark feature of cancer cells by promoting cell cycle progression. The discovery that Aurora kinases were abnormally expressed in cancer cells prompted the development of agents that inhibit their activity.10–12 To date, the pan-Aurora kinase inhibitor MK-0457/VX-680, the Aurora B selective inhibitor AZD1152 and the multikinase inhibitor with anti-Aurora effects KW-2449 have shown preclinical activity in models of AML.9, 13–17 Aurora A selective inhibitors have not been previously evaluated in models of AML. Alisertib is an orally available adenosine triphosphate (ATP)-competitive and reversible selective inhibitor of Aurora A kinase.18 It has a benzazepine core scaffold and is ∼200-fold more selective for Aurora A than Aurora B in cells and also has a high degree of selectivity for Aurora A when compared to a large panel of other kinases in enzymatic assays. Early preclinical studies revealed broad-spectrum anticancer activity in preclinical models. Alisertib is currently undergoing clinical evaluation in several Phase I and II clinical trials for patients with solid tumors and heme-lymphatic malignancies.
Considering the dual role of Aurora A in regulating cell cycle progression and programmed cell death and the high basal expression of Aurora A in AML cells, we hypothesized that AML cells would be particularly sensitive to alisertib.10 To test this hypothesis, we investigated the efficacy and pharmacodynamic effects of alisertib in AML cell lines, primary AML blasts and mouse models of AML.
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- Material and Methods
Given its intrinsic overexpression in cancer, essential functions in the regulation of mitosis and potential roles in promoting drug resistance and disease progression, Aurora kinase A is an attractive target for cancer therapy. As a result, a number of Aurora kinase inhibitors that have varying degrees of activity against Aurora A are currently in development.12 Alisertib can be distinguished from the majority of other small molecule inhibitors of Aurora kinase activity in development due to its selectivity for Aurora A versus Aurora B. Although earlier preclinical studies with alisertib have demonstrated significant anticancer activity in a range of tumor types, its potential efficacy has not been previously investigated in models of AML.31, 32, 41–43 We hypothesized that the prevalent overexpression of Aurora A in AML and highly proliferative nature of this malignancy would render it particularly sensitive to alisertib. We conducted a series of preclinical experiments with the aim to ascertain the antileukemic activity and pharmacodynamic effects of targeting Aurora A with alisertib in AML cell lines, primary AML blasts and mouse xenograft models of AML.
Our in vitro assays demonstrated that alisertib has pleiotropic effects in AML cells. Exposure to alisertib diminished cell viability and clonogenic survival, disrupted cell cycle dynamics and induced apoptosis. Interestingly, our data demonstrated that cellular sensitivity to alisertib was not directly correlated with Aurora A expression levels in AML cells. These findings are consistent with those from other preclinical and early phase clinical trials with alisertib, which have also failed to show a direct relationship between Aurora A expression levels and sensitivity to alisertib.44 The reason for this phenomenon is unknown. It is possible that alisertib may achieve similar degrees of Aurora A inhibition in cells with relatively low, intermediate and high basal Aurora A expression. If this were the case, sensitivity to alisertib would not appear to be directly linked to Aurora A levels. Pharmacodynamic studies from ongoing and planned Phase II and Phase III clinical trials with alisertib may help to clarify this issue.
Phosphorylation of the transcription factor FOXO3a sequesters it in the cytosol where it is transcriptionally inactive. Upon its dephosphorylation, FOXO3a translocates to the nucleus where it can initiate transcription of its target genes. Interestingly, the growth inhibitory and pro-apoptotic effects we observed in AML cells treated with alisertib were associated with a significant reduction in the levels of phospho-FOXO3a and significantly increased expression of the FOXO3a transcriptional targets p27 and BIM. Given that p27 is a critical component of the G2/M cell cycle transition and that BIM is a microtubule-associated pro-apoptotic factor, the observed induction of p27 and BIM expression is a predictable consequence of Aurora A inhibition. These findings are consistent with those of earlier investigations conducted in other cancer models that also demonstrated elevated p27 and BIM levels after treatment with Aurora kinase inhibitors.31, 45
Several prior studies have established a link between Aurora A overexpression and resistance to therapeutic agents, supporting a role for Aurora A in the regulation of chemosensitivity.35–40 However, it is not completely clear at this time whether this is an intrinsic or acquired relationship. We postulated that treatment with conventional cytotoxic agents such as ara-C could promote elevated levels of Aurora A during the genotoxic stress response. Indeed, our data demonstrate that in vitro treatment with ara-C leads to increased expression of Aurora A in AML cell lines and primary blasts from patients. To investigate the potential therapeutic implications of ara-C-mediated induction of Aurora A expression, we first utilized siRNA as a proof of principle to demonstrate that targeted knockdown of Aurora A significantly increased the pro-apoptotic effects of ara-C. This suggested that Aurora A may play a chemoresistance role with respect to ara-C and that targeting its activity could be of therapeutic benefit.
We further investigated the potential impact of targeting Aurora A activity with alisertib on the efficacy of ara-C in a series of in vitro experiments in AML cell lines and primary AML cells. These assays demonstrated that alisertib significantly increased both the growth inhibitory and pro-apoptotic effects of ara-C. Interestingly, the combination of both drugs led to enhanced induction of the FOXO3a targets p27 and BIM compared to either single agent treatment. Our targeted knockdown assays demonstrated that FOXO3a expression was required for maximal alisertib/ara-C mediated induction of these specific targets and consequently, for the combination to most effectively trigger apoptosis. To our knowledge, this is the first report demonstrating a link between Aurora A kinase inhibition and FOXO3a activity. Our subsequent pharmacokinetic and mouse xenograft studies validated the potential therapeutic benefit of combining alisertib and ara-C for AML therapy. Additionally, immunohistochemical assays conducted with specimens obtained from mice treated with alisertib/ara-C established p27 and BIM as pharmacodynamic effectors of these agents.
The FOXO3a-related effects that occur downstream of Aurora A inhibition by alisertib are very interesting. The exact mechanism by which alisertib induces the expression of FOXO3a transcriptional targets remains to be fully elucidated. We were unable to coimmunoprecipitate Aurora A and FOXO3a, which indicates that FOXO3a is unlikely to be a direct Aurora A phospho-substrate. A previous study conducted in models of multiple myeloma demonstrated that alisertib treatment leads to activation of protein phosphatase 2A (PP2A).32 Additional studies are required to clarify this relationship mechanistically. However, considering that FOXO3a is a critical regulator of cell death the ability of alisertib to increase FOXO3a activity may allow it to potentiate the efficacy of a broad range of anticancer agents.
Alisertib has been evaluated in several Phase I and Phase II studies in solid tumors and hematological malignancies.46–48 It is generally very well tolerated and adverse events such as myelosuppression and gastrointestinal mucosal damage are reversible and consistent with the inhibition of Aurora A activity of alisertib with predominant effects on proliferative tissues such as the bone marrow and gastrointestinal tract. Promising antitumor activity has been observed particularly in heme-lymphatic malignancies.46 Notably, alisertib has also been evaluated as a single agent in a Phase II study of 57 patients with advanced AML or myelodysplastic syndrome.47 Although objective responses (13% of the population) including one CR were observed in our study, it is likely that combination therapy with cytotoxic agents such as cytarabine would allow greater initial disease control facilitating sustained Aurora kinase A inhibition. Our collective data demonstrate that the combination of alisertib and ara-C is effective and well tolerated in preclinical models of AML. Based on this promising preclinical data, a Phase I/II study is warranted to investigate the safety and activity of the alisertib/ara-C combination in patients with AML who would not be expected to benefit from conventional therapy.