Activity of the multitargeted kinase inhibitor, AT9283, in imatinib-resistant BCR-ABL-positive leukemic cells.

Despite promising clinical results from imatinib mesylate and second-generation ABL tyrosine kinase inhibitors (TKIs) for most BCR-ABL(+) leukemia, BCR-ABL harboring the mutation of threonine 315 to isoleucine (BCR-ABL/T315I) is not targeted by any of these agents. We describe the in vitro and in vivo effects of AT9283 (1-cyclopropyl-3[5-morpholin-4yl methyl-1H-benzomidazol-2-yl]-urea), a potent inhibitor of several protein kinases, including Aurora A, Aurora B, Janus kinase 2 (JAK2), JAK3, and ABL on diverse imatinib-resistant BCR-ABL(+) cells. AT9283 showed potent antiproliferative activity on cells transformed by wild-type BCR-ABL and BCR-ABL/T315I. AT9283 inhibited proliferation in a panel of BaF3 and human BCR-ABL(+) cell lines both sensitive and resistant to imatinib because of a variety of mechanisms. In BCR-ABL(+) cells, we confirmed inhibition of substrates of both BCR-ABL (signal transducer and activator of transcription-5) and Aurora B (histone H3) at physiologically achievable concentrations. The in vivo effects of AT9283 were examined in several mouse models engrafted either subcutaneously or intravenously with BaF3/BCR-ABL, human BCR-ABL(+) cell lines, or primary patient samples expressing BCR-ABL/T315I or glutamic acid 255 to lysine, another imatinib-resistant mutation. These data together support further clinical investigation of AT9283 in patients with imatinib- and second-generation ABL TKI-resistant BCR-ABL(+) cells, including T315I.

AT9283, a potent inhibitor of the Aurora kinases and Jak2, has therapeutic potential in myeloproliferative disorders.

Constitutive activation of Janus kinase (Jak) 2 is the most prevalent pathogenic event observed in the myeloproliferative disorders (MPD), suggesting that inhibitors of Jak2 may prove valuable in their management. Inhibition of the Aurora kinases has also proven to be an effective therapeutic strategy in a number of haematological malignancies. AT9283 is a multi-targeted kinase inhibitor with potent activity against Jak2 and Aurora kinases A and B, and is currently being evaluated in clinical trials. To investigate the therapeutic potential of AT9283 in the MPD we studied its activity in a number of Jak2-dependent systems. AT9283 potently inhibited proliferation and Jak2-related signalling in Jak2-dependent cell lines as well as inhibiting the formation of erythroid colonies from haematopoietic progenitors isolated from MPD patients with Jak2 mutations. The compound also demonstrated significant therapeutic potential in vivo in an ETV6-JAK2 (TEL-JAK2) murine leukaemia model. Inhibition of both Jak2 and Aurora B was observed in the model systems used, indicating a dual mechanism of action. Our results suggest that AT9283 may be a valuable therapy in patients with MPD and that the dual inhibition of Jak2 and the Aurora kinases may potentially offer combinatorial efficacy in the treatment of these diseases.

Biological characterization of AT7519, a small-molecule inhibitor of cyclin-dependent kinases, in human tumor cell lines.

Cyclin-dependent kinases (CDK), and their regulatory cyclin partners, play a central role in eukaryotic cell growth, division, and death. This key role in cell cycle progression, as well as their deregulation in several human cancers, makes them attractive therapeutic targets in oncology. A series of CDK inhibitors was developed using Astex's fragment-based medicinal chemistry approach, linked to high-throughput X-ray crystallography. A compound from this series, designated AT7519, is currently in early-phase clinical development. We describe here the biological characterization of AT7519, a potent inhibitor of several CDK family members. AT7519 showed potent antiproliferative activity (40-940 nmol/L) in a panel of human tumor cell lines, and the mechanism of action was shown here to be consistent with the inhibition of CDK1 and CDK2 in solid tumor cell lines. AT7519 caused cell cycle arrest followed by apoptosis in human tumor cells and inhibited tumor growth in human tumor xenograft models. Tumor regression was observed following twice daily dosing of AT7519 in the HCT116 and HT29 colon cancer xenograft models. We show that these biological effects are linked to inhibition of CDKs in vivo and that AT7519 induces tumor cell apoptosis in these xenograft models. AT7519 has an attractive biological profile for development as a clinical candidate, and the tolerability and efficacy in animal models compare favorably with other CDK inhibitors in clinical development. Studies described here formed the biological rationale for investigating the potential therapeutic benefit of AT7519 in cancer patients.

Relevance of mitogen activated protein kinase (MAPK) and phosphotidylinositol-3-kinase/protein kinase B (PI3K/PKB) pathways to induction of apoptosis by curcumin in breast cells.

Following observations that curcumin inhibited proliferation (IC(50)=1-5 microM), invasiveness and progression through S/G2/M phases of the cell cycle in the non-tumourigenic HBL100 and tumourigenic MDA-MB-468 human breast cell lines, it was noted that apoptosis was much more pronounced in the tumour line. Therefore, the ability of curcumin to modulate signalling pathways which might contribute to cell survival was investigated. After pre-treatment of cells for 20 min, curcumin (40 microM) inhibited EGF-stimulated phosphorylation of the EGFR in MDA-MB-468 cells and phosphorylation of extracellular signal regulated kinases (ERKs) 1 and 2, as well as ERK activity and levels of nuclear c-fos in both cell lines. At a lower dose (10 microM), it also inhibited the ability of anisomycin to activate JNK, resulting in decreased c-jun phosphorylation, although it did not inhibit JNK activity directly. In contrast, the activation of p38 mitogen activated protein kinase (MAPK) by anisomycin was not inhibited. Curcumin inhibited basal phosphorylation of Akt/protein kinase B (PKB) in both cell lines, but more consistently and to a greater extent in the MDA-MB-468 cells. The MAPK kinase (MKK) inhibitor U0126 (10 microM), while preventing ERK phosphorylation in MDA-MB-468 cells, did not induce apoptosis. The PI3K inhibitor LY294002 (50 microM) inhibited PKB phosphorylation in both cells lines, but only induced apoptosis in the MDA-MB-468 line. These results suggest that while curcumin has several different molecular targets within the MAPK and PI3K/PKB signalling pathways that could contribute to inhibition of proliferation and induction of apoptosis, inhibition of basal activity of Akt/PKB, but not ERK, may facilitate apoptosis in the tumour cell line.

AT7519, a cyclin-dependent kinase inhibitor, exerts its effects by transcriptional inhibition in leukemia cell lines and patient samples.

AT7519 is a potent inhibitor of several cyclin-dependent kinases and is currently in early phase clinical development. Recently, cyclin-dependent kinases 7, 8, and 9 have been shown to regulate transcription through phosphorylation of RNA polymerase II. B-cell lymphoproliferative disorders, including chronic lymphocytic leukemia, rely on the expression of transcripts with a short half-life, such as Mcl-1, Bcl-2, and XIAP, for survival. Here, we describe the characterization of AT7519 in leukemia cell lines, and compare and contrast the response in cell lines derived from solid tumors. Finally, we use these mechanistic insights to show activity in peripheral blood mononuclear cells isolated from 16 chronic lymphocytic leukemia patients. AT7519 induced apoptosis at concentrations of 100 to 700 nmol/L and was equally effective regardless of Rai stage or known prognostic markers. Short-term treatments (4-6 hours) resulted in inhibition of phosphorylation of the transcriptional marker RNA polymerase II and downregulation of the antiapoptotic protein Mcl-1, with no effect on either XIAP or Bcl-2 levels. The reduction in Mcl-1 protein level was associated with an increase in cleaved poly(ADP-ribose) polymerase. Together the data suggest AT7519 offers a promising treatment for patients with advanced B-cell leukemia. Mol Cancer Ther; 9(4); 920-8. (c)2010 AACR.

Cell-cycle arrest by PD184352 requires inhibition of extracellular signal-regulated kinases (ERK) 1/2 but not ERK5/BMK1.

Serum and growth factors activate both the canonical extracellular signal-regulated kinase (ERK) 1/2 pathway and the ERK5/big mitogen-activated protein kinase 1 (BMK) 1 pathway. Pharmacological inhibition of the ERK1/2 pathway using PD98059 and U0126 prevents cyclin D1 expression and inhibits cell proliferation, arguing that the ERK1/2 pathway is rate limiting for cell-cycle re-entry. However, both PD98059 and U0126 also inhibit the ERK5/BMK1 pathway, raising the possibility that the anti-proliferative effect of such drugs may be due to inhibition of ERK5 or both pathways. Here we characterize the effect of the novel mitogen-activated protein kinase/ERK kinase (MEK) inhibitor, PD184352, on the ERK1/2 and ERK5 pathways in the Chinese hamster fibroblast cell line CCl39. In quiescent cells, serum-stimulated ERK1 activity was completely inhibited by PD184352 with an IC50 below 1 microM, whereas ERK5 activation was unaffected even at 20 microM. Serum-stimulated DNA synthesis and cyclin D1 expression was inhibited by low doses of PD184352, which abolished ERK1 activity but had no effect on ERK5. Similarly, in cycling cells PD184352 caused a dose-dependent G1 arrest and inhibition of cyclin D1 expression at low doses, which inhibited ERK1 but were without effect on ERK5. These results indicate that the anti-proliferative effect of PD184352 is due to inhibition of the classical ERK1/2 pathway and does not require inhibition of the ERK5 pathway.