SNS-314, a pan-Aurora kinase inhibitor, shows potent anti-tumor activity and dosing flexibility in vivo

Dual Inhibition of Aurora and FLT3 Kinases by CCT137690: A Novel Treatment Strategy Against FLT3-ITD Positive AML In Vitro and In Vivo

AMG 900, a Potent and Highly Selective Aurora Kinase Inhibitor Shows Promising Preclinical Activity Against Acute Myeloid Leukemia Cell Lines In Vitro and In Vivo

The Aurora kinases are a family of serine-threonine kinases that play key roles in different stages of mitosis. Over-expression of Aurora kinases has been demonstrated in a range of malignancies including leukemia. Aurora kinase inhibitors are emerging as promising agents in the treatment of acute myeloid leukemia (AML) with a number of compounds currently being assessed in clinical trials (Moore AS et al, Leukemia 2010). CCT137690, an imidazo[4,5-b]pyridine derivative discovered at our Institute, is an orally bioavailable, potent, pan-Aurora and FLT3 inhibitor. CCT137690 showed in vitro and in vivo activity in human colon cancer cell lines and xenograft models (Bavetsias V et al, J Med Chem 2010). Here we report the in vitro and in vivo activity of CCT137690 in FLT3-ITD positive AML and demonstrate that CCT137690 overcomes resistance to the selective FLT3 inhibitor MLN518. In cellular assays using FLT3-ITD positive MOLM-13 cells, CCT137690 inhibits autophosphorylation of Aurora A and Aurora B kinases and phosphorylation of histone H3, a direct target of Aurora B kinase. CCT137690 also inhibits autophosphorylation of FLT3 and its downstream targets phospho-STAT5 and phospho-p44/42 MAPK. Dual inhibition of Aurora and FLT3 kinases in FLT3-ITD positive AML with CCT137690 induces apoptosis and results in a unique cell cycle profile with cells accumulating in G2/M. When given orally to athymic mice, CCT137690 achieved target modulation and potently inhibited the growth of subcutaneous MOLM-13 xenografts, with no obvious toxicity or loss of body weight. Inhibition of MOLM-13 xenograft growth was more pronounced with CCT137690 compared to the selective FLT3 inhibitor MLN518, suggesting that dual inhibition of Aurora and FLT3 kinases may have advantages compared to selective FLT3 inhibition alone. To assess whether dual Aurora-FLT3 inhibition with CCT137690 could overcome FLT3-inhibitor resistance, we successfully cultured MOLM-13 cells in the presence of increasing concentrations of MLN518. Although resistant to MLN518, these cells maintained sensitivity to dual Aurora-FLT3 inhibition with CCT137690. The potent preclinical activity of CCT137690 in FLT3-ITD positive AML models supports the growing body of evidence that dual pan-Aurora and FLT3 kinase inhibitors may be of benefit in the high-risk group of patients with FLT3-ITD positive AML, including those with FLT3-inhibitor resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3554. doi:10.1158/1538-7445.AM2011-3554

The present review gives a perspective on the Aurora kinase family members, their function in normal cells, their role in cancer progression as well as their potential as target for anticancer treatment. Mitosis has been an important target for anticancer therapy development, leading to some specific drugs mainly addressing Tubulines, as a key structure of the mitotic spindle. Vinca alkaloids, taxanes or epotilones are good examples of conventionally developed antimitotic agents. However, novel classes of antineoplastic drugs are being studied, targeting the regulatory system that controls functional aspects of mitosis, such as Aurora or Polo-like kinases or Kinespondin inhibitors. The specific role of the different Aurora kinase proteins as regulator enzymes of the mitotic process in normal cells is discussed. Some of the mechanisms that link Aurora overexpression with cancer are also considered. Thereafter, the clinical and preclinical development of the different Aurora kinase inhibitors is presented. This is nowadays a very active area of therapeutic research and at least, sixteen new compounds are being studied as potential antineoplastic drugs. Most of them are in a very early phase of clinical development. However, we summarized the most recently published findings related with these drugs: main characteristics, way of administration, dose limiting toxicities and recommended doses for further studies. Another important aspect in Aurora kinase inhibition is the study and validation of potential biomarkers to optimize the clinical development. Several studies included pharmacodynamic assessments in normal blood cells, skin or/and tumor biopsies. Several proposals included a higher mitotic index, a decreased number of mitosis with bipolar spindles or normal alignment of chromosomes and inhibition of histone H3 phosphorylation. Future strategies and challenges for trials with Aurora kinase inhibitors are also discussed.

ABT-348 is a novel ATP-competitive inhibitor of Aurora kinases (Aurora B, C and A, IC50 values of 7, 1 and 120 nM, respectively). The activity against Aurora B is reflected in cellular assays of inhibition of histone H3 phosphorylation (IC50 value of 21 nM), induction of polyploidy (IC15 value of 12 nM) and inhibition of colony formation of human pancreatic carcinoma cells (MiaPaCa, IC50 value of 4 nM). Consistent with enzyme inhibition, ABT-348 inhibited the autophosphorylation of the respective enzyme in nocodazole-arrested cells (IC50 values of 13, 13 and 189 nM for Aurora B, C and A). Potency (IC50 values) in the cellular assays also correlated with inhibition of proliferation of cell lines derived from leukemia (0.3 nM, MV-4–11), lymphoma (4 nM, DOHH2), and solid tumors (MiaPaCa, 4 nM; SW620, 6 nM; OVCAR5, 7 nM; and HCT-15, 6 nM). Inhibition of Aurora B activity in vivo by ABT-348 was confirmed by measuring phosphorylation of histone H3 in circulating tumor cells obtained from an engrafted leukemia model (RS4;11). Inhibition of histone H3 phosphorylation was observed 4 hours after dosing that persisted in a dose-dependent manner for at least 8 hours. The extent of inhibition at 4 hours was related to the plasma concentration of ABT-348 (IC50: 3.6 μM), which was in close agreement with the value determined for inhibition of histone H3 phosphorylation in cells in the presence of mouse plasma (3.3 μM). In addition to its Aurora enzyme activity, evaluation of ABT-348 for inhibitory activity across 128 kinases revealed a unique kinome profile “signature” by virtue of its potent binding activity (Ki values <30 nM) against the VEGFR/PDGFR families and the SRC family of cytoplasmic tyrosine kinases (LYN, BLK, LCK, ABL, FYN, and SRC). The potent VEGFR/PDGFR binding activity was reflected in enzyme assays (IC50 values <10 nM) performed at 1 mM ATP and also correlated with inhibition of RTK auto-phosphorylation in cells for KDR, FLT3, CSF-1R, KIT, PDGFR and. These activities translated into potent inhibition of VEGF-stimulated endothelial cell proliferation (IC50 value <0.3 nM). Evidence that ABT-348 blocked VEGF-mediated responses in vivo was provided by the molecule's potent activity (ED50 value of 0.2 mg/kg, IV) in blocking VEGF-mediated vascular permeability in the uterus and its ability to induce dose-dependent increases in plasma PIGF in mice. Activity against the Src kinase family was evident in the anti-proliferative activity of ABT-348 against BCR ABL expressing tumor cells and cells expressing the gleevec-resistant BCR-ABL T315I mutation (IC50 values of 47 and 260 nM). Based upon these favorable in vitro and in vivo activities, ABT-348 was evaluated and found to be effective in leukemia (RS4;11, regression) xenografts. These results provide the rationale for clinical assessment of ABT-348 as a therapeutic agent in the treatment of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A239.

Aurora kinases play a key role in mitotic progression. Over-expression of Aurora kinases is found in several human cancers and correlated with histological malignancy and clinical outcomes. Therefore, Aurora kinase inhibitors should be useful in the treatment of cancers. Cell-based screening methods have an advantage over biochemical approaches because hits can be optimized to inhibit targets in the proper intracellular context. We developed a novel Aurora kinase inhibitor R763/AS703569 using an image-based phenotypic screen. The anti-proliferative effect was examined in a panel of tumor cell lines and primary cells. The efficacy was determined in a broad panel of xenograft models. R763/AS703569 inhibits Aurora kinases, along with a limited number of other kinases including FMS-related tyrosine kinase 3 (FLT3), and has potent anti-proliferative activity against many cell types accompanying unique phenotypic changes such as enlarged cell size, endoreduplication and apoptosis. The endoreduplication cycle induced by R763/AS703569 was irreversible even after the compound was withdrawn from the culture. Oral administration of R763/AS703569 demonstrated marked inhibition of tumor growth in xenograft models of pancreatic, breast, colon, ovarian, and lung tumors and leukemia. An acute myeloid leukemia cell line MV4-11, which carries a FLT3 internal tandem duplication mutation, is particularly sensitive to R763/AS703569 in vivo. R763/AS703569 is a potent inhibitor of Aurora kinases and exhibited significant anti-proliferative activity against a wide range of tumor cells both in vitro and in vivo. Inhibition of Aurora kinases has the potential to be a new addition to the treatment of cancers.

14130 Background: Aurora kinases are a family of kinases that play a crucial role in regulating segregation of chromosomes from parent to daughter cell and have been linked to tumorigenesis. Aurora kinase A is commonly amplified in solid tumors and has been established as an oncogene. Aurora B is often overexpressed and leads to defects in mitosis and increased tumor invasiveness. AS703569 is an orally available, highly potent inhibitor of aurora kinases A and B and several other kinases. AS703569 has been shown in vitro and in tumor xenograft models to inhibit proliferation and to trigger apoptosis of several tumor types. We report on the initial results of a phase I study of AS703569 in patients with solid tumors. The study objectives are to determine for each of 2 different dosing regimens the MTD and to evaluate safety, PK and pharmacodynamic effects. Methods: This is a phase I, two arm, dose-escalation study. Cohorts of 3 patients ≥18 years old with advanced solid tumors are being assigned to one of 2 dosing regimens. The dose escalation follows a modified Fibonacci scheme. The starting dose level is 6 mg/m2 p.o. per 21-day cycle divided in 2 or 3 doses (regimen 1: dosing on days 1, 8 or regimen 2: dosing on days 1, 2, 3). Patients with stable disease or response will be offered additional cycles. Results: A total of 15 patients have been included to date. The tumor types represent a typical widespread in phase I trials, including 3 cases of uterine/cervical cancer and 2 cases of breast cancer. The first 2 cohorts of 3 patients on dose level 1 (regimen 1 and 2) have been treated with no dose-limiting toxicities (DLTs) or serious adverse events (SAEs). Dose-escalation is proceeding for both regimens with 9 patients treated on dose level 2, 6 in regimen 1 where due to a DLT (likely disease-related and unlikely related to AS703569) the cohort was expanded, and 3 in regimen 2. Thus far, only two patients were withdrawn from the study due to disease progression and one patient withdrew consent. Two patients are ongoing at 4+ cycles and one at 3+. Conclusions: Treatment of patients in this phase I study of the aurora kinase inhibitor AS703569 at dose level 1 and 2 has been well tolerated over several cycles. Dose escalation is ongoing and analysis of PK and pharmacodynamics will be reported. No significant financial relationships to disclose.

2536 Background: Aurora Kinases are a family of serine/threonine kinases (Aurora Kinases (AK) A, B, and C) critical for mitosis. Elevated AKs expression occurs in a high percentage of melanoma, colon, breast, ovarian, gastric, and pancreatic tumors; in a subset of these tumors the AURKA locus (20q13) is amplified. SNS-314 is a selective pan-AK inhibitor with low nanomolar IC50s. Methods: Study design is 3+3 phase 1 dose escalation by modified Fibonacci. Patients (pts) with advanced solid tumors received SNS-314 by 3 hour infusion qweek X 3 (28 day cycle). Primary endpoints: safety, tolerability, and DLT assessment. Secondary endpoints: MTD, pharmacokinetics (PK), pharmacodynamics, and antitumor activity. Pharmacodynamic endpoint was inhibition of Histone H3 phosphorylation (pHH3) evaluated by immunohistochemistry of skin punch biopsies taken pre- and 2 hours post-infusion. Results: Thirty-two pts (16M/16F; median age = 58.5 years) were enrolled into 8 cohorts: dose range 30–1800 mg/m2. Median cycles received =2. SNS-314 was generally well tolerated with Grade 1–2 toxicities ≥ 15% incidence: nausea (31%), fatigue (28%), vomiting, constipation, and pain (16% each), and no Grade 3+ toxicities of ≥ 15% incidence. A DLT of Grade 3 neutropenia preventing administration of all 3 doses was observed at 1440 mg/m2. Plasma PK were dose proportional for exposure with no accumulation of SNS-314 following weekly administration. Clearance was moderate (5.65 L/hr/m2, CV 39.4%); Vss approximated total body water (21.5 L/m2, CV 78.1%); terminal half-life was 10.4 hours (CV 66.8%). Six patients had stable disease as their best response. Inhibition of pHH3 by SNS-314 was observed in skin biopsies of patients treated at doses of 240 mg/m2 and greater. Conclusions: SNS-314 is a novel inhibitor of AKs A, B, and C. The compound has been generally well tolerated; MTD was not established. No objective responses were observed. Pharmacodynamic activity was demonstrated by inhibition of pHH3. [Table: see text]

Background: XL228 is a small molecule protein kinase inhibitor currently in Phase 1 clinical development targeting Ph+ leukemias, multiple myeloma, and solid tumors. Cellular and in vivo data have demonstrated activity of XL228 against IGF1R, ABL (including the T315I variant) and SRC family kinases. XL228 was tested in a panel of biochemical protein kinase assays, and additional putative targets were identified, including Aurora family kinases, FGFR1-3, and ALK. As these potential targets are known to enhance cell proliferation and survival in certain malignancies, the validation of their inhibition by XL228 through in vitro studies and in clinical pharmacodynamic samples was performed. Results: A panel of kinase inhibitors including XL228 was profiled against a series of cancer cell lines with known alterations in major signaling pathways. Approximately 30% of the lines demonstrated XL228 IC50 values of <100nM in viability assays, including many lines with characterized ALK or FGFR mutations or amplifications. Cell lines showing the highest level of response to XL228 were often sensitive to other Aurora, ALK or FGFR inhibitors as well. Sub-20nM activity was demonstrated against FGFR2 in a cellular kinase ELISA assay, and confirmed by immunoblot analysis. Potency against ALK in the cell kinase assay was lower (approximately 200 nM). The Aurora inhibitory activity of XL228 was examined through the use of immunoblots, immunofluorescence, and substrate phosphorylation assays. XL228 eliminated the phosphorylation of Aurora A and B at concentrations above 10 nM. Short-term treatment of HeLa cells led to disruption of mitotic spindle formation, with the majority of mitotic cells exhibiting a unipolar spindle and disorganized chromosomes. After exposure to XL228 for 24 hours, there was a significant increase in cells with >4N DNA content, suggesting a failure of cytokinesis and consequent endoreduplication. Activity of XL228 against Aurora kinases was further verified by dose-dependent inhibition of histone H3 phosphorylation, where it showed approximately 5-fold higher potency than the control compound VX-680. To confirm the relevance of the in vitro observations to the clinical activity of XL228, tumor biopsy specimens obtained from the solid tumor Phase 1 clinical study were evaluated for changes in phosphorylation of pathway markers by immunofluorescence. Inhibition of Aurora, FGFR, IGF1R, and SRC family kinases was observed, as measured by decreases in kinase substrate phosphorylation in these specimens. Conclusions: XL228 shows a broad pattern of protein kinase inhibition, including the tyrosine kinases IGF1R, SRC, ABL, FGFR1-3, and ALK and the serine/threonine kinases Aurora A and Aurora B. The clinical impact of this target profile is currently being assessed in two Phase 1 studies. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C192.

The Aurora kinases are essential for the regulation of chromosome segregation and cytokinesis during mitosis. Aberrant expression and activity of these kinases occur in a wide range of human tumors, and lead to aneuploidy and tumorigenesis. Here we report the discovery of a highly potent and selective small-molecule inhibitor of Aurora kinases, VX-680, that blocks cell-cycle progression and induces apoptosis in a diverse range of human tumor types. This compound causes profound inhibition of tumor growth in a variety of in vivo xenograft models, leading to regression of leukemia, colon and pancreatic tumors at well-tolerated doses. Our data indicate that Aurora kinase inhibition provides a new approach for the treatment of multiple human malignancies.

3009 Background: The AKs are essential for mitotic progression, spindle formation, centrosome maturation, chromosomal segregation, and cytokinesis. Elevated expression occurs frequently in tumors. MK-0457 (VX-680) is a potent AK inhibitor, with Ki values of 0.66, 18 and 4.6 nM for AKs A, B and C, respectively. MK-0457 inhibits proliferation of transformed cells in vitro (IC50’s 15–113 nM), and induces colon and pancreatic cancer xenograft regressions. Methods: After IRB approval, consenting patients (pts) with refractory solid tumors (median 3 prior regimens, range 2–6) and adequate hematologic and organ function were enrolled using an accelerated dose escalation scheme with 1–2 pts/dose level until ≥ grade 2 toxicity, followed by 3–6 pts/level. MK-0457 was administered by continuous 5-day intravenous infusion every 28 days. Dose-limiting toxicity (DLT) was grade 3 non-hematologic or grade 4 hematologic toxicity ≥ 5 days, or grade 4 febrile neutropenia (FN) during cycle 1. PKs were collected pre-dose through 168 h and analyzed for MK-0457 and metabolites by HPLC/mass spec. Steady state volume of distribution (Vdss), clearance (CL), maximal concentration (Cmax) and terminal half-life (t1/2) were determined by WinNonLin. Results: 16 pts received MK-0457 dosed at 0.5, 1, 2, 4, 8, and 12 mg/m2/h. Median number of cycles was 2 (range 1–6). DLT was asymptomatic neutropenia ≥ 5 days at 12 mg/m2/h. At 8 mg/m2/h, 1 pt experienced FN in cycle 2; a second developed a grade 2 allergic reaction. Three pts achieved stable disease as best response, and two of them completed 6 cycles. Plasma concentrations reached steady state rapidly (i.e., within 24 h) and declined biexponentially after the end of infusion; after a rapid initial decay, a slower decaying terminal phase demonstrated a t1/2 ∼15 h. PK parameters include Vdss = 237 ± 107 (SD) L/m2 and CL = 517 ± 141 ml/min/m2. At 8 mg/m2/h, Cmax was ∼650 nM. Conclusion: MK-0457 is generally well tolerated and achieves plasma levels similar to those causing regressions in xenografts. CL is high and exposures achieved are roughly dose proportional. Because 8 mg/m2/h was well tolerated in heavily pre-treated pts, escalation to 10 mg/m2/h is underway. Baseline tumor samples will be assessed for predictive biomarkers at the recommended phase II dose. [Table: see text]

Lead optimization studies using 7 as the starting point led to a new class of imidazo[4,5-b]pyridine-based inhibitors of Aurora kinases that possessed the 1-benzylpiperazinyl motif at the 7-position, and displayed favorable in vitro properties. Cocrystallization of Aurora-A with 40c (CCT137444) provided a clear understanding into the interactions of this novel class of inhibitors with the Aurora kinases. Subsequent physicochemical property refinement by the incorporation of solubilizing groups led to the identification of 3-((4-(6-bromo-2-(4-(4-methylpiperazin-1-yl)phenyl)-3H-imidazo[4,5-b]pyridin-7-yl)piperazin-1-yl)methyl)-5-methylisoxazole (51, CCT137690) which is a potent inhibitor of Aurora kinases (Aurora-A IC(50) = 0.015 +/- 0.003 muM, Aurora-B IC(50) = 0.025 muM, Aurora-C IC(50) = 0.019 muM). Compound 51 is highly orally bioavailable, and in in vivo efficacy studies it inhibited the growth of SW620 colon carcinoma xenografts following oral administration with no observed toxicities as defined by body weight loss.

Background. The serine/threonine kinase Aurora B is involved in the regulation of several mitotic processes, including chromosome condensation, congression and segregation as well as cytokinesis. These essential functions of Aurora B and its overexpression in many cancer types render this protein kinase an attractive target for anticancer drug development. Methods. BI 811283 was profiled in enzymatic kinase assays as well as in proliferation assays on various human cancer cell lines. Cell cycle status was assessed by DNA content analysis (Cellomics ArrayScan, FACScalibur). Histone H3 phosphorylation was determined by immunofluorescence (Cellomics ArrayScan). Apoptosis was detected by Western blotting for cleaved PARP and microscopic enumeration of DAPI-stained cells showing nuclear fragmentation. Senescent cells were identified by staining for SA-ß-Gal activity. Results. BI 811283 inhibited human Aurora B kinase activity with an IC50 value of 9 nM, Aurora A and C kinases with 70 nM and 17 nM, respectively. In a panel of 46 additional kinases representative of the human kinome, BI 811283 at 1000 nM inhibited 7/46 kinases by more than 50%. EC50 values for inhibition of proliferation of >20 human cancer cell lines were in the range of 2 to 14 nM. In the non-small cell lung cancer cell line NCI-H460, treatment with BI 811283 resulted in a rapid (<1 h) inhibition of histone H3 phosphorylation. Within 48 h of treatment, the fraction of polyploid cells increased from <5% to >80%, paralleled by a marked increase in cell volume. An increase of cleaved poly (ADP-ribose) polymerase and a concomitant increase in the fraction of cells with nuclear fragmentation from <1% to 7% was observed after 72 h and 96 h of treatment. Within 96 h, the fraction of senescent cells markedly increased from <3% to 25% of the population. Conclusions. BI 811283 is a potent and selective Aurora kinase inhibitor that inhibits proliferation of cancer cells independent of tissue origin or oncogenome status. Treated cells exhibit a polyploid phenotype characteristic for Aurora B inhibition and show hallmarks of senescence as well as a slow onset of apoptosis in a small fraction of cells. In vivo activity of BI 811283 has been demonstrated in multiple cancer xenograft models in nude mice (see accompanying poster). Phase I clinical trials are ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1080.

Optimization of the imidazo[4,5-b]pyridine-basedseries of Aurora kinase inhibitors led to the identification of 6-chloro-7-(4-(4-chlorobenzyl)piperazin-1-yl)-2-(1,3-dimethyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine (27e), a potent inhibitor of Aurorakinases (Aurora-A Kd = 7.5 nM, Aurora-B Kd = 48 nM), FLT3 kinase (Kd = 6.2 nM), and FLT3 mutants including FLT3-ITD (Kd = 38 nM) and FLT3(D835Y) (Kd = 14 nM). FLT3-ITD causes constitutive FLT3 kinaseactivation and is detected in 20–35% of adults and 15% of childrenwith acute myeloid leukemia (AML), conferring a poor prognosis inboth age groups. In an in vivo setting, 27e stronglyinhibited the growth of a FLT3-ITD-positive AML humantumor xenograft (MV4–11) following oral administration, within vivo biomarker modulation and plasma free drug exposures consistentwith dual FLT3 and Aurora kinase inhibition. Compound 27e, an orally bioavailable dual FLT3 and Aurora kinase inhibitor, wasselected as a preclinical development candidate for the treatmentof human malignancies, in particular AML, in adults and children.

The Aurora kinases play a key role in mitosis and are overexpressed in multiple human tumor types; there has been considerable interest in developing Aurora kinase inhibitors as antitumor agents, particularly Aurora A and Aurora B kinases. A series of novel hydrazide hydrochlorides of pyrazolo[1,5-a]pyrimidine carboxamides were designed and synthesized and their inhibitory activities against Aurora kinases were evaluated. Some of the tested compounds exhibited low micromolar to nanomolar activity with respect to the inhibition of Aurora A kinase. The most potent compound in this series was found to be a potent inhibitor of Aurora A in an HTRF enzymatic assay with an IC50 as low as 23 nM. A structure–activity relationship study indicated that halogen substitution in the benzene ring of amide plays an important role in kinase inhibitory potency.