Inhibition of Aurora Kinase B Is Important for Biologic Activity of the Dual Inhibitors of BCR-ABL and Aurora Kinases R763/AS703569 and PHA-739358 in BCR-ABL Transformed Cells

<div><p>ABL tyrosine kinase inhibitors (TKI) like Imatinib, Dasatinib and Nilotinib are the gold standard in conventional treatment of CML. However, the emergence of resistance remains a major problem. Alternative therapeutic strategies of ABL TKI-resistant CML are urgently needed. We asked whether dual inhibition of BCR-ABL and Aurora kinases A-C could overcome resistance mediated by ABL kinase mutations. We therefore tested the dual ABL and Aurora kinase inhibitors PHA-739358 and R763/AS703569 in Ba/F3- cells ectopically expressing wild type (wt) or TKI-resistant BCR-ABL mutants. We show that both compounds exhibited strong anti-proliferative and pro-apoptotic activity in ABL TKI resistant cell lines including cells expressing the strongly resistant T315I mutation. Cell cycle analysis indicated polyploidisation, a consequence of continued cell cycle progression in the absence of cell division by Aurora kinase inhibition. Experiments using drug resistant variants of Aurora B indicated that PHA-739358 acts on both, BCR-ABL and Aurora Kinase B, whereas Aurora kinase B inhibition might be sufficient for the anti-proliferative activity observed with R763/AS703569. Taken together, our data demonstrate that dual ABL and Aurora kinase inhibition might be used to overcome ABL TKI resistant CML.</p></div

Drug-resistant Aurora B G160V mutant partly overcomes the anti-proliferative effect of PHA-739358 and R763/AS703569.

<p>(A) MTT assay showing the anti-proliferative effect after treatment with increasing concentrations of PHA-739358 (upper panel) or R763/AS703569 (lower panel) for 48 h. Percentage of cell growth was normalized to the growth of untreated control cells. Shown is one representative experiment of three experiments performed. (B) Bar graph quantifying cell proliferation after exposure to 200 nM PHA-739358 (upper panel) or 15 nM R763/AS703569 (lower panel) for 48 h. Untreated and DMSO-treated cells were used as control. Shown is one representative experiment of three experiments performed. Values show mean of triplicates ± SD. Difference between Ba/F3 p185 wt and AurB G160V expressing Ba/F3 p185 wt cells is statistically significant.</p

Inhibiting Aurora Kinases Reduces Tumor Growth and Suppresses Tumor Recurrence after Chemotherapy in Patient-Derived Triple-Negative Breast Cancer Xenografts

International audienceTriple-negative breast cancers (TNBC) have an aggressive phenotype with a relatively high rate of recurrence and poor overall survival. To date, there is no approved targeted therapy for TNBCs. Aurora kinases act as regulators of mammalian cell division. They are important for cell-cycle progression and are frequently overexpressed or mutated in human tumors, including breast cancer. In this study, we investigated the therapeutic potential of targeting Aurora kinases in preclinical models of human breast cancers using a pan-inhibitor of Aurora kinases, AS703569. In vitro, AS703569 was tested in 15 human breast cancer cell lines. TNBC cell lines were more sensitive to AS703569 than were other types of breast cancer cells. Inhibition of proliferation was associated with cell-cycle arrest, aneuploidy, and apoptosis. In vivo, AS703569 administered alone significantly inhibited tumor growth in seven of 11 patient-derived breast cancer xenografts. Treatment with AS703569 was associated with a decrease of phospho-histone H3 expression. Finally, AS703569 combined to doxorubicin-cyclophosphamide significantly inhibited in vivo tumor recurrence, suggesting that Aurora kinase inhibitors could be used both in monotherapy and in combination settings. In conclusion, these data indicate that targeting Aurora kinases could represent a new effective approach for TNBC treatment. Mol Cancer Ther; 11(12); 2693-703. (C)2012 AACR

Preclinical evidence for the effective use of TL-895, a highly selective and potent second-generation BTK inhibitor, for the treatment of B-cell malignancies

Abstract TL-895 (formerly known as M7583) is a potent, highly selective, adenosine triphosphate (ATP)-competitive, second-generation, irreversible inhibitor of Bruton’s tyrosine kinase (BTK). We characterized its biochemical and cellular effects in in vitro and in vivo models. TL-895 was evaluated preclinically for potency against BTK using IC50 concentration–response curves; selectivity using a 270-kinase panel; BTK phosphorylation in Ramos Burkitt’s lymphoma cells by ProteinSimple Wes analysis of one study; anti-proliferative effects in primary chronic lymphocytic leukemia (CLL) blasts; cell viability effects in diffuse large B-cell lymphoma (DLBCL) and mantle-cell lymphoma (MCL) cell lines; effects on antibody-dependent cell-mediated cytotoxicity (ADCC) from Daudi cells and chromium-51 release from human tumor cell lines; and efficacy in vivo using four MCL xenograft model and 21 DLBCL patient-derived xenograft (PDX) models (subtypes: 9 ABC, 11 GCB, 1 Unclassified). TL-895 was active against recombinant BTK (average IC50 1.5 nM) and inhibited only three additional kinases with IC50 within tenfold of BTK activity. TL-895 inhibited BTK auto-phosphorylation at the Y223 phosphorylation site (IC50 1–10 nM). TL-895 inhibited the proliferation of primary CLL blasts in vitro and inhibited growth in a subset of activated DLBCL and MCL cell lines. TL-895 inhibited the ADCC mechanism of therapeutic antibodies only at supra-clinical exposure levels. TL-895 significantly inhibited tumor growth in the Mino MCL xenograft model and in 5/21 DLBCL PDX models relative to vehicle controls. These findings demonstrate the potency of TL-895 for BTK and its efficacy in models of B-cell lymphoma despite its refined selectivity

Characterization of Aurora model system.

<p>Crystal structure was analyzed in order to identify residues, whose specific mutation should abolish inhibitor binding while keeping Aurora kinase activity. (A) Superimposition of the CK2 crystal structure (green) with Aurora A (pink) - PHA-739358 (yellow) complex crystal structure, showing the position of the point mutation L210 and G216. (B) <i>Xenpus laevis</i> Aurora B (green) and INCENP (turquoise) in complex with AS7035369, showing the position of the point mutation L154 and G160. (C) Ba/F3 p185 wt cells were transfected with Babe-puro based retrovirus encoding AurA L210M, AurA G216V, AurB L154M, or AurB G160V point mutations. Empty pBabe-vector was used as control. Selection was accomplished with puromycin. Western blot analysis showing flag-tagged Aurora A and B expression similar to endogenous Aurora A/B protein levels. (D) Amino acid sequence alignment of human Aurora kinase A and B. Mutated residues are framed. It is of note that the point mutations L210M and G216V in Aurora A accord with L154M and G160V in Aurora B.</p

Suppression of Aurora B kinase activity by PHA-739358 or R763/AS703569 inhibits cell division and induces apoptosis in BCR-ABL negative and positive Ba/F3 cells.

<p>Ba/F3 and Ba/F3 p185 cells comprising the T315I, Y253F, and F317L mutations were exposed to the indicated concentration of PHA-739358 (A) or R763/AS703569 (B). After 24 h analysis of DNA content and apoptotic fraction of PI-stained cells were assessed by flow cytometry. Untreated cells served as control. Apoptosis was measured as the percentage of cells of sub-G1 DNA content in the FL2 channel in a logarithmic scale.</p

PHA-739358 and R763/AS703569 reduce BCR-ABL kinase activity at comparable concentrations and independent of the BCR-ABL mutation status.

<p>Ba/F3 p185 wt and IM-resistant T315I, Y253F, and F317L mutants cells were exposed to increasing concentrations of PHA-739358 (A) or R763/AS703569 (B) for 2.5 h and assessed for phosphorylation status of BCR-ABL and its downstream target STAT5 by western blot analysis. Untreated and DMSO treated cells were used as a controls.</p

Aurora Kinase inhibitors PHA-739358 and R763/AS703569 compromise cell proliferation and viability at different concentrations in BCR-ABL negative and positive Ba/F3 cells independent of the BCR-ABL mutation status.

<p>(A) Dose-effect curves for treatment with the indicated concentrations of PHA-739358 and R763/AS703569 in Ba/F3 and Ba/F3 p185 cells, including the IM-resistant mutants T315I, Y253F and F317L, after 48 h. Effects on cell proliferation were assessed by MTT assay. Shown is one representative experiment of three experiments performed. The percentage of cell growth was normalized to the growth of untreated control cells. (B) Bar graph quantifying cell proliferation after exposure to 200 nM PHA-739358 or 15 nM R763/AS703569 for 48 h. Untreated and DMSO-treated cells were used as controls. Shown is one representative experiment of three experiments performed. Values are expressed as mean of triplicates ± SD. The difference between Ba/F3 p185 wt and BCR-ABL mutants is statistically significant. (C) IC₅₀ values of BCR-ABL positive and negative Ba/F3 cells, calculated from the results after 48 h of incubation with PHA-739358 or R763/AS703569.</p

Aurora B G160V mutant confers resistance to PHA-739358 and R763/AS703569.

<p>Western blot analysis probed to investigate the resistant potential of Aurora A and B kinase mutants by detecting phospho-Histone-3 (serine 10) after 2.5 h exposure to increasing concentrations of PHA-739358 (A) or AS703569 (B). Untreated and DMSO treated cells were used as control. For comparison untransfected Ba/F3 p185 wt cells were also included into the experiment.</p

Inhibition of Aurora kinase activity occurs at different concentrations of PHA-739358 and R763/AS703569.

<p>Ba/F3 p185 wt, p185/T315I, p185/Y253F and p185/F317L cells were treated with indicated concentrations of PHA-739358 (A) or R763/AS703569 (B) for 2.5 h. Aurora Kinase activity assessed by phosphorylation status of Histone-H3 at Serine-10 was analyzed using immunoblotting. Control cells were left untreated or exposed to DMSO.</p