Identification and characterization of drug resistance mechanisms in cancer cells against Aurora kinase inhibitors CYC116 and ZM447439

AbstractCYC116 is a selective Aurora kinase inhibitor that has been tested in a Phase I study in patients with advanced solid tumors. Although CYC116 has shown desirable preclinical efficacy, the potential for emergence of resistance has not been explored. We established several CYC116 resistant clones from isogenic HCT116 p53+/+ and HCT116 p53−/− cell line pairs. We also generated resistant clones towards ZM447439 (quinazoline derivative), a model Aurora inhibitor. The selected clones were 10-80 fold resistant to CYC116 and cross-resistant to other synthetic Aurora inhibitors including AZD1152, VX-680, and MLN8054. Resistant clones displayed multidrug resistant phenotypes, tested by using 13 major cytostatics. All clones were highly resistant to etoposide followed by other drugs. Interestingly, all CYC116 clones but not ZM447439 became polyploid. ZM447439, but not CYC116 induced three novel mutations in Aurora B. Leu152Ser significantly affected ZM447439 binding, but not CYC116. Gene expression studies revealed differential expression of more than 200 genes. Some of these genes expression profiles were also observed in CYC116 resistant primary tumors. Bcl-xL (BCL2L1) was found to be overexpressed in CYC116 clones and its knockdown resensitized the p53+/+ resistant clone to CYC116. Finally Bcl-xL overexpressing p53+/+ CYC116 clones were highly sensitive to navitoclax (ABT-263) compared to parent cells. The data shed light on the genetic basis for resistance to Aurora kinase inhibitors which could be used to predict clinical response, to select patients who might benefit from therapy and to suggest suitable drug combinations for a particular patient population.

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Vincristine Potentiates the Anti-Leukemia Effect of the Aurora Kinase Inhibitor VE-465 by Enhancing Apoptosis in Myeloid Leukemia Cells.

Blood ◽

10.1182/blood.v114.22.2762.2762 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2762-2762

Author(s):

Ken Ohmine ◽

Tadashi Nagai ◽

Kozue Yoshida ◽

Shin-ichiro Fujiwara ◽

Mitsuyo Uesawa ◽

...

Keyword(s):

Cell Lines ◽

Myeloid Leukemia ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Aurora Kinase ◽

Leukemia Cells ◽

Aurora Kinases ◽

Aurora Kinase Inhibitors ◽

M Phase ◽

G1 Cells

Abstract Abstract 2762 Poster Board II-738 Introduction: Aurora kinases (Aurora-A, Aurora-B, Aurora-C) play an essential role in the regulation of mitosis. It has been shown that deregulation of aurora kinases is involved in tumorgenesis and that these kinases are overexpressed in a variety of tumor cells. Aurora kinase inhibitors are potential small-molecule agents for treatment of various kinds of tumors including leukemia, and clinical trials of several aurora kinase inhibitors against certain types of tumors are currently being carried out. However, mono-therapy with other small-molecule agents sometimes shows only limited clinical efficacy for treatment of leukemia, and the establishment of efficacious combination therapies therefore appears to be an attractive approach for making good use of aurora kinase inhibitors. Methods: We examined the cytotoxic effects of VE-465, a specific aurora kinase inhibitor, in combination with various conventional anti-leukemia agents, including doxorubicin, daunorubicin, idarubicin, mitoxantron, cytocine arabinoside, vincristine and etoposide, on AML cell lines (HL60, U937, THP-1, KY821), CML cell lines (KCL22, K562, KU812) and primary leukemia cells from a patient with AML in order to try to determine an effective therapeutic combination. Results: Steel and Peckham isobologram analyses demonstrated that a combination of VE-465 and vincristine had a synergistic/additive inhibitory effect on the growth of all leukemia cell lines as well as primary leukemia cells examined in this study. Flow cytometric analysis showed that the percentage of G2/M-phase cells was significantly increased at 12 h when VE-465 was added to THP-1 cells as a single agent. At 48 h, however, the percentage of sub-G1 cells was increased, with a decrease in the percentage of G2/M-phase cells, suggesting that VE-465 initially induced the cells into blockage of the cell cycle at M-phase, which may be caused by VE-465-mediated inhibition of aurora kinase activity, and that cells at G2/M arrest were subsequently induced to apoptosis. Importantly, vincristine enhanced VE-465-mediated induction of sub-G1 cells. Consistent with these results, vincristine enhanced VE-465-induced increase in the levels of cleaved caspase 3, cleaved caspase 7, cleaved caspase 9 and cleaved PARP. The level of Phospho-Chk2 was markedly increased by the combination, suggesting that Chk2-mediated activation of the G2/M checkpoint is involved in the induction of apoptosis. Interestingly, VE-465 alone and VE-465 in combination with vincristine markedly increased the level of phospho-ERK1/2, suggesting that the combination alters a network of cellular signaling pathways. Taken together, the results suggest that vincristine potentiated the effect of VE-465 by enhancement of apoptosis, resulting in effective inhibition of the growth of leukemia cells. In contrast to the combination of VE-465 and vincristine, however, combinations of VE-465 and other anti-leukemia agents showed no synergistic inhibition but rather had antagonistic effects on growth. Conclusion: Co-administration of VE-465 and most of the conventional anti-leukemia agents might have little clinical value. However, the results of this study indicate the utility of the combination of VE-465 and vincristine as a potential therapy for myeloid leukemia. Disclosures: No relevant conflicts of interest to declare.

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Changes in ABC Transporter Expression during Hematopoiesis Cause Lineage-Biased Cytopenias in Patients Treated with Aurora Kinase Inhibitors

Blood ◽

10.1182/blood-2021-153422 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4292-4292

Author(s):

Brooke A. Furlong ◽

Ryan R. Posey ◽

David B. Chou ◽

Christos Kyprianou ◽

Lucy R. O'Sullivan ◽

...

Keyword(s):

Board Of Directors ◽

Small Molecule ◽

Abc Transporters ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Early Stage ◽

Aurora Kinase ◽

Advisory Committees ◽

Healthy Human ◽

Aurora Kinase Inhibitors

Abstract Chemotherapy-induced cytopenias are a prevalent and significant issue that worsens clinical outcomes and hinders the effective treatment of cancer. While they are classically associated with traditional cytotoxic chemotherapies, they also occur with newer targeted small molecule drugs and the factors that determine the hematotoxicity profiles of chemotherapies are not fully understood. Here, we explore why Aurora kinase inhibitor drugs cause preferential neutropenia when compared to the cytopenic profiles of targeted small molecule cancer drugs that are FDA approved. By studying drug responses of healthy human hematopoietic cells in vitro and analyzing existing published clinical datasets, we provide evidence that the enhanced vulnerability of neutrophil-lineage cells to Aurora kinase inhibitors is acquired at an early stage of differentiation and is caused by developmental changes in the expression pattern of ATP-binding cassette (ABC) transporters. These data show that hematopoietic cell-intrinsic expression of ABC transporters may be an important factor that determines how some chemotherapies affect the bone marrow. Disclosures David: AstraZeneca: Current Employment. Randle: AstraZeneca: Current Employment. Polanska: AstraZeneca: Current Employment. Urosevic: AstraZeneca: Current Employment. Travers: AstraZeneca: Ended employment in the past 24 months. Ingber: Emulate: Membership on an entity's Board of Directors or advisory committees; BOA Biomedical: Membership on an entity's Board of Directors or advisory committees; Freeflow Medical Devices Inc: Membership on an entity's Board of Directors or advisory committees.

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Pan-Aurora Kinase Inhibitor Mk-0457 Synergistically Potentiates Apo2L/Trail Cytotoxicity in Multiple Mieloma Cells Sensitive and Resistant to Bortezomib.

Blood ◽

10.1182/blood.v114.22.1837.1837 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1837-1837

Author(s):

Paolo Lunghi ◽

Laura Mazzera ◽

Antonio Costanzo ◽

Nicola Giuliani ◽

Vittorio Rizzoli ◽

...

Keyword(s):

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Combined Treatment ◽

Aurora Kinase ◽

Differential Sensitivity ◽

Caspase 8 ◽

Aurora Kinase Inhibitors ◽

Dunnett Test ◽

Induced Apoptosis ◽

Rpmi 8226

Abstract Abstract 1837 Poster Board I-863 Multiple Myeloma (MM) cells are extremely resistant to apoptosis and currently new potential drug combinations are under investigation. Aurora kinase inhibitors have been shown to abrogate proliferation and induce apoptosis in human myeloma cells lines (HMCLs) and primary myeloma cells. In addition previous studies have shown the antimyeloma activity of Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) as a single agent or in combination with certain chemotherapeutic agents. The aim of this study was to investigate whether the combined treatment with pan-Aurora kinase inhibitor MK-0457 also known as VX-680(Vertex/Merck) and Apo2L/TRAIL has cytotoxic effects on MM cells. Because we found that MK-0457 (0.2–0.5 μM) partially activated the extrinsic caspase-8 mediated pathway both in HMCLs RPMI 8226 (highly sensitive to Apo2L/TRAIL: median lethal dose (LD50) at 48 hours was 4.9 ng/mL), and bortezomib-resistant 8226/R5 (barely sensitive to Apo2L/TRAIL: LD50 at 48 hours was 90.9 ng/mL) we attempted to examine whether MK-0457 potentiates the Apo2L/TRAIL-mediated apoptosis in HMCLs that have differential sensitivity to Apo2L/TRAIL. We first analyzed the pharmacologic interactions between MK-0457 and Apo2L/TRAIL using a fixed-ratio experimental design and found that the combined treatment resulted in the synergistic induction of apoptosis in both HMCLs (Chou-Talalay method): after 48 hours of treatment the averaged Combination Index values calculated from the ED50 (50% effective dose), ED75 and ED90, in MK-0457 plus Apo2L/TRAIL were 0.04 ± 0.05 and 0.03 ± 0.02 in RPMI 8226 and 8226/R5 respectively. Consistent with these results, we found that Aurora-A and -B were expressed at similar levels in RPMI 8226 and 8226/R5 cells and we demonstrated that the functional knock-out of Aurora-A or -B gene expression by small interfering (si)RNAs significantly increased (P< .001 Dunnett test) the TRAIL- induced apoptosis in both HMCLs. To investigate the molecular mechanisms by which MK-0457/Apo2L/TRAIL induced MM cell apoptosis first we compared the effect on caspase activation in RPMI 8226 and 8226/R5. The cells were treated with MK-0457 (0.2–0.5 μM) and/or Apo2L/TRAIL (2.4 ng/mL and 9.6 ng/mL in RPMI 8226 and 8226/R5 respectively) for 24 hours and caspase activation and PARP fragmentation were analyzed by Western blotting: the treatment with MK-0457 strongly potentiated the Apo2L/TRAIL -induced activation of caspase-8, caspase-3 and PARP cleavage in both HMCLs. In addition, a strong activation of caspase-9 was observed in the MK-0457/Apo2L/TRAIL-treated 8226/R5. Using caspase blocking peptides, specific siRNA against caspase-8 or caspase-9 and Western immunoblotting we demonstrated the involvement of primarily caspase-8 and -3 in MK-0457/Apo2L/TRAIL -induced apoptosis in RPMI 8226 and 8226/R5: the inhibition of caspase-8 significantly (P< .001 Dunnett test) reduced the MK-0457/Apo2L/TRAIL -induced apoptosis in both cell lines. Moreover, the pancaspase inhibitor Z-VAD-FMK protected MM cells from MK-0457/Apo2L/TRAIL -induced apoptosis, confirming that caspase activity was indispensable in MK-0457/Apo2L/TRAIL -induced apoptosis. Since antiapoptotic Mcl-1 and proapoptotic Bim play a pivotal role in controlling MM cell survival and apoptosis, and Bim, as previously demonstrated, can interfere with the activation of both intrinsic and extrinsic apoptotic pathways in MM cells, we analyzed their expression in MK-0457/Apo2L/TRAIL treated cells. We found that monotreatment with neither MK-0457 nor Apo2L/TRAIL (or their combination) was able to substantially modulate the expression of Mcl-1 or Bim in RPMI 8226; in contrast in HMCL 8226/R5, that showed low sensitivity to Apo2L/TRAIL, the treatment with Apo2L/TRAIL increased the intracellular amount of the anti-apoptotic protein Mcl-1 and MK-0457 reverted Apo2L/TRAIL-induced up-regulation of Mcl-1, thus correlating with the enhanced cytotoxicity of combined treatment. In conclusion, our data indicate that targeting aurora kinase potentiates the apoptotic effect of Apo2L/TRAIL in MM cells with differential sensitivity to Apo2L/TRAIL through the activation of the extrinsic pathway. More important, MK-0457/Apo2L/TRAIL can induce apoptosis in MM cells displaying resistance to bortezomib. Together, these findings suggest that a strategy combining Aurora kinase inhibitors with Apo2L/TRAIL warrants attention in MM. Disclosures: Lunghi: MERCK sharp and Dohme: Research Funding. Bonati:MERCK sharp and Dohme: Research Funding.

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Combined Inhibition of Janus and Aurora Kinase Effectively Suppresses Proliferation of JAK2 V617F-expressing Cells

Blood ◽

10.1182/blood.v118.21.2813.2813 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2813-2813

Author(s):

Karoline Gäbler ◽

Catherine Rolvering ◽

Valérie Palissot ◽

Guy J. Berchem ◽

Iris Behrmann ◽

...

Keyword(s):

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Jak2 V617f ◽

Aurora Kinase ◽

Signaling Proteins ◽

Jak Inhibitor ◽

Aurora Kinase Inhibitor ◽

Aurora Kinase Inhibitors ◽

Proliferation Assays

Abstract Abstract 2813 Background: A somatic point mutation in the Janus kinase 2 gene (JAK2) leading to the expression of the JAK2 V617F mutant occurs with high frequency in myeloproliferative neoplasm (MPN) patients (>95 % in polycythemia vera (PV), >50 % in essential thrombocythemia (ET) and primary myelofibrosis (PMF)). It confers constitutive activity to the kinase and results in cytokine hypersensitivity and a proliferative advantage of hematopoietic progenitor cells. These findings suggest that inhibiting JAK2 V617F may be therapeutically beneficial. Several JAK2 inhibitors are currently in clinical trials for the treatment of MPN, and first results show clinical improvements for PMF patients. However, since approximately 50 % of ET and PMF patients do not carry an activating mutation in JAK2, we speculate that the inhibition of signaling proteins other than JAK2 or in combination with JAK2 inhibition could be beneficial for these patients. Methods: We characterized compounds from different chemical classes, which previously have been published to be JAK(2) inhibitors. These compounds were compared in several assays using primary CD34+ cells from PV patients positive for the JAK2 V617F mutation and/or the JAK2 V617F-bearing cell line HEL. We used (quantitative) Western blot detections, in vitro kinase assays, proliferation assays, cell size measurements, cell cycle analyses and colony forming cell (CFC) assays to analyze the efficacy of the different inhibitors. Moreover, the IC50 values of the compounds were determined. Results: In total 15 published JAK2 inhibitors have been characterized in detail. As monitored in an in vitro kinase assay and by Western blot detection of phosphorylated signaling proteins, several compounds previously described as JAK(2) inhibitors did not target JAK2 V617F. However, some compounds, which turned out not to inhibit JAKs, showed growth-inhibitory effects on JAK2 V617F-positive cells. Such compounds could be used in combination with a specific JAK inhibitor in order to achieve beneficial effects on suppression of cell proliferation and induction of apoptosis. We could demonstrate that the combined application of a JAK inhibitor together with an Aurora kinase inhibitor was most promising: application of both Janus and Aurora kinase inhibitors in proliferation assays and CFC assays demonstrated a more effective suppression of growth than achieved by respective single treatments. Interestingly, we observed in the CFC assay that a JAK2 inhibitor seems to preferentially suppress the growth of erythroid colonies, while an Aurora kinase inhibitor preferentially blocks myeloid colony growth. Conclusion: Here we present a comparative analysis and a detailed biochemical characterization of numerous compounds from different chemical classes, all supposed to be JAK(2) inhibitors. We confirmed JAK(2) inhibitory activity for several compounds but not for all. In addition, we identified some compounds, which effectively inhibited the proliferation of JAK2 V617F-bearing cells without targeting JAK2. Thus, combined inhibition of JAK2 and other kinases may represent a promising therapeutic strategy. In particular, we suggest that a combination of Janus and Aurora kinase inhibitors might be beneficial for the treatment of MPN patients. Disclosures: No relevant conflicts of interest to declare.

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Abstract 633: Tumor cell resistance mechanisms to aurora kinase inhibitors

10.1158/1538-7445.am10-633 ◽

2010 ◽

Author(s):

Madhusudhan Kollareddy ◽

Daniella Zheleva ◽

Petr Dzubak ◽

Marian Hajduch

Keyword(s):

Tumor Cell ◽

Kinase Inhibitors ◽

Aurora Kinase ◽

Resistance Mechanisms ◽

Cell Resistance ◽

Aurora Kinase Inhibitors ◽

Tumor Cell Resistance

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Beyond the Gatekeeper: Imatinib- and Dasatinib-Resistant BCR-ABL/F317 Mutations Confer Cross-Resistance to VX-680 but Are Sensitive to a Structural Derivative of VX-680

Blood ◽

10.1182/blood.v112.11.725.725 ◽

2008 ◽

Vol 112 (11) ◽

pp. 725-725

Author(s):

Corynn Kasap ◽

Nicholas Hertz ◽

Debora Makino ◽

Kevan Shokat ◽

John Kuriyan ◽

...

Keyword(s):

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Aurora Kinase ◽

Hematologic Malignancies ◽

Kinase Domain ◽

Blast Phase ◽

Abl Kinase ◽

Aurora Kinase Inhibitors ◽

Kinase Domain Mutations ◽

High Degree

Abstract The management of chronic phase CML has been revolutionized by selective ABL tyrosine kinase inhibitor (TKI) therapy. Despite the effectiveness of these targeted agents, long-term control of blast phase CML and Ph+ ALL has been elusive, where the majority of patients relapse within 6–12 months. For blast phase CML and Ph+ ALL, two TKIs are currently approved: imatinib and dasatinib. While head-to-head comparisons of these agents have not been performed, it is generally believed that dasatinib is the more active agent for these phases of disease. In most cases, loss of response to these agents is driven by BCR-ABL kinase domain mutations. While more than 70 mutations have been associated with clinical resistance to imatinib, dasatinib appears vulnerable primarily to five mutations: V299L, T315A, T315I, F317I, and F317L. Of these, T315I and F317L are cross-resistant to imatinib. For the achievement of long-term remissions in blast phase CML and Ph+ ALL, a combination of TKIs that can collectively suppress all resistant BCR-ABL kinase domain mutations holds therapeutic promise. The BCR-ABL/T315I mutation, which confers a high degree of resistance to all approved BCR-ABL TKIs, has been referred to as a “molecular gatekeeper”, as it restricts access to a deeper hydrophobic pocket within the ABL kinase domain and makes an important stabilizing H-bond with imatinib, dasatinib and nilotinib. The Aurora kinase inhibitor VX-680 was the first compound to have activity against BCR-ABL/T315I in vitro, as well as clinically. To determine the promise of a kinase inhibitor combination of dasatinib and VX-680, we assessed the activity of VX-680 against the five dasatinib-resistant mutations using a cell-based flow cytometric assay of BCR-ABL kinase activity. While three mutants are sensitive, mutations at F317 demonstrated a high degree of resistance. We tested a number of other Aurora kinase inhibitors of different chemotypes and found that each of these had similar difficulty at inhibiting the kinase activity of BCR-ABL/F317 mutants. Based upon the co-crystal structure of VX-680 complexed with ABL, we have performed structure-activity relationship studies of 12 VX-680 scaffold derivatives, and have successfully identified structural modifications that increase kinase inhibitory activity against F317 mutants. Moreover, one of these derivatives increases the selectivity for ABL relative to Aurora kinases, which may help reduce the likelihood of suppressing normal hematopoiesis, a dose-limiting toxicity of Aurora kinase inhibitors that may substantially limit their effectiveness for the management of hematologic malignancies such as blast phase CML and Ph+ ALL. Lastly, we have performed structural studies of ABL/F317 mutants complexed with select VX-680 derivatives in an effort to understand how F317 mutations confer resistance to a broad range of ABL and Aurora kinase inhibitors. Interestingly, a recent study reported the successful selection of Aurora kinase inhibitor-resistant clones derived from a human colon cancer cell line (Girdler et al, 2008). While no resistance-conferring mutations were isolated at L154, the Aurora kinase gatekeeper residue, mutations were detected at Y156 in Aurora B, which corresponds to F317 in ABL. Aurora B Y156 mutations were found to confer resistance to a number of Aurora kinase inhibitors, including VX-680. As Aurora kinase inhibitors are being studied in a variety of non-hematologic malignancies, there is an increasing need to understand and overcome the mechanisms whereby mutations at this residue confer resistance to these agents. It is hoped that our studies will lead not only to the development of an effective adjunctive kinase inhibitor for the treatment of blast phase CML and Ph+ ALL, but will also shed light on the growing problem of resistance conferred by mutations at residues that correspond to BCR-ABL/F317 in other kinases.

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Aurora Kinase Inhibitors in Head and Neck Cancer

Current Topics in Medicinal Chemistry ◽

10.2174/1568026618666180112163741 ◽

2018 ◽

Vol 18 (3) ◽

pp. 199-213

Author(s):

Guangying Qi ◽

Jing Liu ◽

Sisi Mi ◽

Takaaki Tsunematsu ◽

Shengjian Jin ◽

...

Keyword(s):

Head And Neck Cancer ◽

Cancer Therapy ◽

Kinase Inhibitors ◽

Biological Function ◽

Aurora Kinase ◽

Aurora Kinases ◽

Related Research ◽

Chemotherapy Drugs ◽

Aurora Kinase Inhibitors

Aurora kinases are a group of serine/threonine kinases responsible for the regulation of mitosis. In recent years, with the increase in Aurora kinase-related research, the important role of Aurora kinases in tumorigenesis has been gradually recognized. Aurora kinases have been regarded as a new target for cancer therapy, resulting in the development of Aurora kinase inhibitors. The study and application of these small-molecule inhibitors, especially in combination with chemotherapy drugs, represent a new direction in cancer treatment. This paper reviews studies on Aurora kinases from recent years, including studies of their biological function, their relationship with tumor progression, and their inhibitors.

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