The Aurora kinase inhibitor AT9283 inhibits Burkitt lymphoma growth by regulating Warburg effect

Introduction

Burkitt lymphoma (BL) is a B-cell non-Hodgkin’s lymphoma (B-NHL) that is highly invasive and characterized by rapid proliferation (Mrdenovic et al., 2019; Roschewski, Staudt & Wilson, 2022). This malignancy primarily affects children and young adults, with a predilection for the maxillofacial, central nervous system, abdominal organs, and other extranodal sites. In developing countries, BL represents the majority of B-NHL cases in children. In developing countries, BL accounts for approximately 80% of B-NHL cases in children (Egan, Goldman & Alexander, 2019). Adults and immunocompromised individuals experience less favorable outcomes and more pronounced adverse effects from treatment compared to pediatric patients (Dunleavy, Little & Wilson, 2016; Perkins & Friedberg, 2008). While the initial treatment for Burkitt lymphoma is notably efficacious, the vast majority of patients who experience recurrence or resistance to treatment ultimately succumb to the disease. Retrospective population-based studies have suggested that the incidence of treatment failure among adult patients with Burkitt lymphoma may be as high as 35% (Evens et al., 2021). Consequently, there is an urgent need for the development of novel treatment modalities and intervention strategies to enhance the therapeutic outcomes for this malignancy.

The degree of tumor malignancy depends on the growth efficiency of tumor cells, and an adequate energy supply is the prerequisite and necessary condition for the malignant proliferation of tumor cells. The Warburg effect refers to the phenomenon in which although the oxygen level is sufficient to support mitochondrial oxidative phosphorylation, tumor cells still metabolize glucose by glycolysis (Cantor & Sabatini, 2012). This low-efficiency mode of metabolism in cancer cells results in the consumption of a large amount of glucose compared with that in normal cells. The lactic acid secreted can not only inhibit the formation of an immunosuppressive microenvironment but also allow rapid synthesis of the intermediate products required for proliferation. Therefore, targeting metabolic links to tumor glycolysis is a key strategy in current antitumor treatment.

The family of Aurora kinases is a group of serine/threonine kinases widely expressed in a variety of cells and has three members, Aurora A, Aurora B, and Aurora C, which play important roles in mitosis, chromosome segregation, and spindle formation in eukaryotic cells (Keen & Taylor, 2004). In hematological malignancies, overexpression of Aurora A and Aurora B is found in patients with cytogenetic abnormalities indicating an unfavorable prognosis. A previous study discovered an important role for Aurora-A in glycolytic modulation, as well as a mechanism through which LDHB contributes directly to the Warburg effect (Cheng et al., 2019). Further, inhibition of Aurora A and Aurora B is an attractive anticancer approach for a variety of cancers, including solid tumors and hematological malignancies (Gavriilidis, Giakoustidis & Giakoustidis, 2015; Ikezoe et al., 2007; Rodrigues Alves et al., 2016).

AT9283 is a multikinase inhibitor discovered using a fragment-based approach and is highly active against Aurora kinases, Abl, and JAK (Howard et al., 2009). A number of studies have shown that AT9283 has therapeutic potential for leukemia, myeloproliferative disorders, aggressive B-cell lymphomas, and colon cancer (Qi et al., 2012; Takeda et al., 2020; Torrente et al., 2020). So far as we know, there exists no report regarding the impact of AT9283 on the Warburg effect in BL. In this study, we aimed to explore the effects of AT9283 on the survival of Burkitt lymphoma cells, including primary Burkitt lymphoma cells, and to elucidate the underlying molecular mechanisms. Our research has revealed that AT9283 demonstrates noteworthy anti-BL properties through the inhibition of cellular proliferation, the induction of apoptosis, and the arrest of the cell cycle. The mechanism is linked to the reversal of the Warburg effect by means of a reduction in the expression of c-MYC and HIF1α proteins.

Materials & Methods

Results

AT9283 suppresses the BL cell lines and primary cells proliferation

In order to verify the inhibitory effects of AT9283 on the constitutive activation of Auroras in BL cells, the expression levels of Aurora A and B were assessed in cells treated with AT9283. The results obtained from western blotting analysis indicated that AT9283 significantly down-regulated the protein expression of Aurora A and Aurora B in a dose-dependent manner as depicted in Fig. 1A.

The Akata and P3HR1 cell lines were subjected to varying concentrations of AT9283 (0, 1, 2, or 4 μmol/L) for 24 or 48 h, followed by analysis of cell viability using the MTT assay. The results demonstrated a dose- and time-dependent reduction in cell viability of both Akata and P3HR1 cells upon treatment with AT9283 (Figs. 1B, 1C and Fig. S1). Furthermore, the antiproliferative effect of AT9283 on primary BL cells was evaluated, and the MTT assay revealed a dose-dependent inhibition of viability in BMMCs isolated from newly diagnosed BL patients (Fig. 1D).

AT9283 induces cell cycle arrest in BL cells

In order to further investigate the antiproliferative mechanism in BL cells of AT9283, we conducted a flow cytometry analysis of its effect on cell cycle progression. Our findings revealed a significant increase in the percentage of G2/M-phase and a decrease in the percentage of S-phase in Akata and P3HR1 cells with increasing concentrations of AT9283. These results suggest that AT9283 effectively inhibits BL cell proliferation by inducing G2/M-phase blockage (Fig. 2A). Additionally, Western blotting analysis revealed a downregulation of cyclin B1 protein expression, which may play a role in the regulatory mechanism of AT9283-induced cell cycle arrest in BL cells (Fig. 2B).

AT9283 induces apoptosis via a caspase-dependent pathway in BL cells

Due to the close correlation between cell cycle and apoptosis. Subsequently, the induction of apoptosis was assessed in BL cells treated with AT9283 for 48 h using annexin V-FITC/PI staining. The findings demonstrated that AT9283 elicited apoptosis in a dose-dependent manner, as illustrated in Figs. 3A and 3B. The induction of apoptosis by AT9283 was further evidenced by the increases in the levels of apoptotic markers. Flow cytometry and western blotting were carried out to assess the protein levels of caspase-3 and cleaved PARP. The results showed in Akata and P3HR1 cells treated with AT9283, the procaspase-3 level was reduced (Fig. 3E), while the levels of caspase3 and PARP were increased with AT9283 of dose (Figs. 3C, 3D and 3E). These findings suggest that AT9283 activates the mitochondrial caspase cascade.

Aurora A, B and Warburg effect-related proteins is overexpressed in B cell lymphoma

The reprogramming of glucose metabolism leading to an augmented aerobic glycolysis, commonly known as the Warburg effect, is recognized as a distinctive feature of tumor cells (Fukushi et al., 2022), and plays a crucial role in tumor progression. Lactic acidosis has been reported as the most common types of acidosis in many malignant tumors, including hematological malignancies, especially lymphomas (Looyens et al., 2021; Sanivarapu et al., 2022). To ascertain the involvement of glycolysis in the progression of B-cell lymphoma, we conducted a comparative analysis of the mRNA expression levels of Aurora A and Warburg effect-related genes in B-cell lymphoma and normal tissues using the GEPIA online database. Results demonstrate that Aurora A, Aurora B, and the Warburg effect-related genes LDHA (lactate dehydrogenase A) and PKM (pyruvate kinase M) were significantly overexpressed in tissues obtained from patients with diffuse large B-cell lymphoma (DLBCL) as compared to normal controls (Figs. 4A and 4B). We also analyzed the HPA database to evaluate the Aurora A and B mRNA levels in different cell lines, and revealed that the basal expression levels of Aurora A and B in lymphoma cell lines were high (Fig. S2). Furthermore, we conducted immunohistochemical (IHC) staining to compare the protein expression of PKM and LDHA between the lymph node of Burkitt lymphoma (BL) and normal controls. Our results revealed that the expression of PKM and LDHA proteins was higher in BL lymph node tissues than in normal tissues, which is in agreement with the GEPIA analysis findings (Fig. 4C).

AT9283 attenuates the Warburg effect in BL cells

Nguyen et al. have demonstrated that the inhibition of Aurora kinase A can effectively reverse the Warburg effect in glioblastomas and result in unique metabolic vulnerabilities (Nguyen et al., 2021). Then AT9283 was used to test glucose uptake and lactate production in BL cells in order to confirm whether Aurora kinase inhibition reverses the Warburg effect. We measured glucose and lactate (LA) in cell culture medium and found that AT9283 significantly decreased glucose uptake and LA production in the treated groups compared with the untreated groups in both Akata and P3HR1 cells (Figs. 5A, 5B). These results indicate that AT9283 effectively reverses the Warburg effect. Furthermore, our findings demonstrate that the protein levels of the crucial glycolytic enzymes HK2 and PKM2 were dose dependently decreased in the AT9283-treated groups in both Akata and P3HR1 cells (Figs. 5C, 5D).

AT9283 inhibits c-Myc and HIF-1a expression involved in the regulation of glycolytic activity in BL cells

The robust glycolytic activity observed in tumor cells is closely associated with the dysregulation of signaling molecules, particularly the aberrantly activated hypoxia-inducible factor 1-alpha (HIF1α) and c-Myc. To ascertain the inhibitory effects of AT9283 on the expression of c-Myc and HIFα proteins in Burkitt lymphoma cells, western blotting was employed to detect protein expression in cells treated with AT9283. The results indicate that AT9283 effectively suppresses c-Myc and HIF-1α protein expression in a dose-dependent manner (Fig. 6A).

Discussion

Aurora kinases, which are members of the serine/threonine kinase family, are localized at the two poles of the mitotic spindle during mitosis and are are referred to as aurora protein kinases. The family is divided into three members, namely AURKA/B/C, based on their structural and functional homology (Aurora kinase A, B, C) (Keen & Taylor, 2004), and they play crucial roles in regulating the functions of centrosomes and microtubules during cell mitosis (Adams, Carmena & Earnshaw, 2001). Aurora kinases are frequently over-expressed or abnormally activated in various types of solid tumors with poor prognosis, including colorectal, prostate, lung, ovarian, breast, gastric, liver, and oral cancers, as well as in lymphoma and other hematologic malignancies (de Mel et al., 2019; Murga-Zamalloa, Inamdar & Wilcox, 2019; O’Connor et al., 2019; Tang et al., 2017a; Ulisse, 2017). Aurora kinases play a crucial role in ensuring accurate progression through mitosis, while also mediating a diverse range of signal transduction pathways (Bertolin & Tramier, 2020; Bodvarsdottir, Vidarsdottir & Eyfjord, 2007; Donnella et al., 2018; Ishikawa et al., 2011; Katayama et al., 2004), that impact cell survival, cell cycle arrest, apoptosis resistance, angiogenesis, radio/chemoresistance, tumor recurrence and metastasis, cell aging, and other biological behavioral alterations (Dominguez-Brauer et al., 2015; Yan et al., 2016). Consequently, Aurora kinases are considered promising therapeutic targets (de Mel et al., 2019; Goldenson & Crispino, 2015; Murga-Zamalloa, Inamdar & Wilcox, 2019; O’Connor et al., 2019; Tang et al., 2017a; Ulisse, 2017).

AT9283, a pyrazole-benzimidazole derivative, is a multikinase inhibitor that can effectively inhibit the activity of AURKs and several other kinases, including JAKs, Flt3, and Abl (Howard et al., 2009). This inhibitor can affect the growth and survival of multiple types of cancers, such as multiple myeloma, chronic myelocytic leukemia (CML), lymphomas, and colorectal cancer (Curry et al., 2009; Qi et al., 2012; Santo et al., 2011; Takeda et al., 2020; Tanaka et al., 2010). Additionally, AT9283 as a drug for multiple myeloma has already entered the clinical stage (Hay et al., 2016) and leukemia (Vormoor et al., 2017). Research has demonstrated that AT9283 promotes apoptosis and increases the population of cells in the G2/M phase (Bavetsias & Linardopoulos, 2015; Takeda et al., 2020). Our findings indicate that AT9283 exhibited dose- and time-dependent inhibition of BL cell lines and primary BL cells viability, as demonstrated in Fig. 1. Furthermore, AT9283 induced G2/M cell cycle arrest in Akata and P3HR1 cells by downregulating the expression of cyclin B1 protein (Fig. 2), a crucial regulator of the G2/M checkpoint (Eriksson et al., 2007).

Apoptosis, a programmed cell death mechanism, serves as a crucial process in curbing the proliferation of malignant tumors and suppressing the malignant phenotype of tumor cells (Su et al., 2015). The apoptosis pathway is currently a prime target for cancer treatment drugs. Effector caspases, which can selectively cleave proteins, play a pivotal role in most cellular apoptotic processes. Poly (ADP-ribose) polymerase-1 (PARP1), an enzyme that localizes in the nucleus, is closely associated with DNA repair under stress and is indispensable for cellular stability and survival (Satoh & Lindahl, 1992). In vitro, PARP can undergo cleavage and activation by various caspases, while in vivo, it serves as the primary substrate for caspase 3 cleavage (Lazebnik et al., 1994; Oliver et al., 1998). The significance of PARP lies in its crucial role in maintaining cellular stability and survival. Its cleavage accelerates cellular instability and is considered a significant indicator of apoptosis and Caspase 3 activation (Oliver et al., 1998). In this study, the impact of AT9283 on BL cell apoptosis was assessed through annexin V/PI double-staining flow cytometry and Western blotting. The activation of apoptosis was observed to be significant following treatment with AT9283, as evidenced by the increased protein levels of key apoptotic mediators Caspase 3 and cleaved PARP, which were found to be directly proportional to the concentration of AT9283 administered (Fig. 3).

The Warburg effect, characterized by abnormal metabolism, is a hallmark of cancer (Hanahan & Weinberg, 2011), with increased glucose consumption and lactate secretion being key features of many rapidly developing cancers. Consequently, targeting glycolysis in tumor cells represents a novel and promising therapeutic approach.The rate-limiting step in glycolysis is regulated by a collection of enzymes, such as HK2, PK. The enzyme is responsible for regulating the aerobic glycolysis pathway and directly influences gene expression and cell cycle regulation in tumor cells (Lu et al., 2018; Mazurek et al., 2005). Among these, HK2 plays a pivotal role as the key rate-limiting enzyme in the glycolytic pathway, facilitating glucose phosphorylation (Lis et al., 2016). Its overexpression is frequently observed in various types of cancers, indicating a poor prognosis (Anderson et al., 2017; Wilson, 2003; Wu et al., 2017). Additionally, HK2 has been identified as a crucial factor in the pathology of malignant B-cell lymphomas (Nakajima et al., 2019). While the mammalian genome encodes multiple isoforms of PK, cancer cells preferentially express a specific competitive subtype known as PKM2.

Numerous investigations have elucidated the crucial role of Aurora kinases, particularly Aurora A, in modulating glycolysis and oxidative energy metabolism (Cheng et al., 2019; Nguyen et al., 2021; Sun et al., 2020; Wang et al., 2020). A recent research showed that the Warburg effect is induced by Burkitt lymphoma (Looyens et al., 2021). Utilizing the GEPIA database, we acquired the expression levels of aerobic glycolysis-related kinases in both normal tissues and malignant B-cell lymphoma tissues. Our findings indicate that the expression of aurora kinases and the key proteins PKM2 and LDHA were significantly elevated in B-cell lymphoma tissues (Figs. 4A and 4B). We also analyzed the HPA database to evaluate the Aurora A and B mRNA levels in different cell lines, and revealed that the basal expression levels of Aurora A and B in lymphoma cell lines were high (Fig. S2). The results were confirmed by immunohistochemistry (Fig. 4C). The outcomes imply that malignant B-cell lymphoma exhibits elevated levels of aerobic glycolysis, and that aurora kinases play a role in regulating glycolysis.

c-Myc is another important molecule involved in the occurrence and development of Burkitt lymphoma and some types of DLBCL. The c-Myc protein plays a role invovling in cell proliferation, growth, apoptosis, metabolism, and other cellular processes (Fernandez, Sanchez-Arevalo & de Alboran, 2012). Further, c-Myc enhances the transcription of the genes encoding HK2 and PK, leading to accelerated aerobic glycolysis, or the Warburg effect. The Myc protein is the major modulator of the Warburg effect in BL cells (Mushtaq et al., 2015). Many studies indicated overexpression of AURKA is related to increased expression of c-Myc (Dauch et al., 2016; Lu et al., 2015; Yang et al., 2010), and inhibition of Aurora kinase A was found to downregulate c-Myc protein levels (Nguyen et al., 2021). However, c- Myc is not the only modulator of the genes involved in glucose metabolism. Many genes involved in the process are also direct targets of HIF-1. Both c-Myc and HIF-1 participate in tumor metabolism by regulating transportion of glucose, the TCA cycle, glycolysis and glutaminolysis in cancer cells (Chen & Russo, 2012). The cooperative action of c-MYC and HIF-1 facilitates the transcription of genes associated with aerobic glycolysis, including HK2, PKM2, LDHA, and ABC transporters, thereby augmenting the expression of the corresponding proteins and promoting the establishment of the Warburg effect, ultimately hastening tumor progression (Dang et al., 2008; Liu, Jin & Fan, 2021; Wen et al., 2022). The findings of a study have confirmed that the proteins HIF-1 and Myc work in collaboration to regulate a minimum of two genes that encode glycolytic enzymes (HK2 and PDK1) in cells of BL (Kim et al., 2007). Here, we found that inhibiting Aurora kinases with AT9283 significantly reversed the Warburg effect in Akata and P3HR1 BL cells, resulting in suppression of proliferation, G2/M arrest and induction of apoptosis. One of the the underlying mechanisms seems to involve the downregulating of the expression of the metabolism-related c- Myc and HIF genes. Considering the pivotal significance of these transcription factors, it is imperative for us to investigate the downstream effects they mediate in our forthcoming research.

P3HR1 cells were utilized as a model to investigate alterations in mRNA levels of related genes following treatment with AT9283. The findings from the RT-qPCR analysis revealed a significant reduction in the mRNA expression levels of aurora B, HK2, PKM2, and c-myc in cells subjected to AT9283 treatment, as compared to the control group (Figs. S3B–3E). These results are consistent with the observations obtained from Western blot analysis, indicating that the modulation of these proteins expression is, to some extent, regulated at the transcriptional level upon AT9283 treatment. However, the observed modifications in Aurora A and HIFα proteins do not align with the alterations in mRNA levels (Figs. S3A and S3F), suggesting that the regulation of Aurora A and HIF proteins by AT9283 operates via translational mechanisms rather than transcriptional processes. Our forthcoming inquiries will aim to elucidate the underlying mechanism accountable for the discrepancies in the expression of Aurora A and HIFα proteins and their corresponding mRNA molecules, thus establishing a basis for future research and progress in the field of AT9283 anti-BL.

Conclusions

In summary, our study presents novel experimental evidence demonstrating the efficacy of the Aurora kinase inhibitor AT9283 against Burkitt lymphoma through mechanisms involving the Warburg effect. These findings provide a basis for further research on AT9283 as a potential therapeutic agent for Burkitt’s lymphoma. Further investigations are required to ascertain the in vivo inhibitory effects of AT9283 on BL.

Supplemental Information