Novel pyrrolo-1,5-benzoxazepine compounds display significant activity against resistant chronic myeloid leukaemia cells in vitro, in ex vivo patient samples and in vivo

Abstract

Background: Imatinib is a direct and potent inhibitor of the constitutively active tyrosine kinase, breakpoint cluster region-Abelson (Bcr-Abl), which is central to the pathogenesis of chronic myeloid leukaemia (CML) patients. As such, imatinib has become the front-line treatment for CML patients. However, the recent emergence of imatinib resistance, commonly associated with point mutations within the kinase domain, has led to the search for alternative drug treatments and combination therapies for CML.

Methods: In this report, we analyse the effects of representative members of the novel pro-apoptotic microtubule depolymerising pyrrolo-1,5-benzoxazepines or PBOX compounds on chemotherapy-refractory CML cells using a series of Bcr-Abl mutant cell lines, clinical ex vivo patient samples and an in vivo mouse model.

Results: The PBOX compounds potently reduce cell viability in cells expressing the E225K and H396P mutants as well as the highly resistant T315I mutant. The PBOX compounds also induce apoptosis in primary CML samples including those resistant to imatinib. We also show for the first time, the in vivo efficacy of the pro-apoptotic PBOX compound, PBOX-6, in a CML mouse model of the T315I Bcr-Abl mutant.

Conclusion: Results from this study highlight the potential of these novel series of PBOX compounds as an effective therapy against CML.

Figure 1

Mutant Baf/3 cell lines containing the Bcr-Abl protein display varying degrees of resistance to imatinib when compared with the native cells. (A) Baf/3 cells were seeded during the log phase of growth and incubated for 24 h. Samples were prepared for SDS–polyacrylamide gel electrophoresis and run on an 8% gel. Polyvinylidene difluoride membranes were incubated overnight with an anti-Bcr-Abl mAb. Actin is shown as a loading control. Results are representative of three independent experiments. (B) Cells were treated for 48 h with a vehicle (Veh) (0.01 % DMSO) or a range of imatinib concentrations (50 nM–10 μM) in a 96-well plate. After the incubation period, we added AlamarBlue dye (20 μl) to each well and incubated samples for 3 h. Cells were subsequently analysed for cell viability. Values represent the mean±s.e.m. of three independent experiments performed in triplicate. ND, not determined.

Figure 2

PBOX-6 and PBOX-15 reduce cell viability and induce apoptosis equipotently in native and T315I Baf/3 cell lines. Native and Bcr-Abl mutant Baf/3 cells were seeded during the log phase of growth and treated for 48 h with a vehicle (1% (v/v) ethanol) (Veh) or with the indicated concentrations of PBOX-6 or PBOX-15 (P6, P15). After the incubation period, we assessed cells for (A and B) cell viability by the AlamarBlue viability assay, (C) apoptosis by quantification of the pre-G1 peak and (D) the percentage of cells in the G2/M phase. Values represent the mean±s.e.m. of three independent experiments performed in triplicate.

Figure 3

Isolation of leukaemic cells from CML patient blood samples. White blood cells were isolated from the fresh peripheral blood of CML patients (n=6) by Ficoll gradient. Cells were seeded at a density of 1 × 10⁶ cells per ml in 24-well plates and treated with a vehicle (Veh) (1% (v/v) ethanol or 0.0025% (v/v) DMSO), 10 μM PBOX-15 (P15), 25 μM PBOX-6 (P6) or 250 nM imatinib for 72 h. Cells were then labelled with an anti-CD45 antibody and FITC-annexin V and analysed by flow cytometry. Leukaemic cells were identified and gated based on their low to medium side scatter and low CD45 expression. (A) A representative dot plot of CD45-stained vehicle-treated cells is shown. (B) Percentage of cells undergoing apoptosis as determined by annexin-V-positive staining. Experiments were performed in duplicate. Statistical analysis was performed using the Student's paired t-test, ^** P<0.01.

Figure 4

Administration of PBOX-6 significantly inhibits tumour growth and prevents gross spleen enlargement in an in vivo mouse model of CML harboring the T315I mutation. Five- to six-week-old female BALB/c-nude (nu/nu) mice were inoculated s.c. into the right flank with 3 × 10⁶ Baf/3-Bcr-Abl-T315I cells in the log phase of growth. When tumours had reached a diameter of ∼4 mm (day 8), mice were randomly divided into two treatment groups (n=6) and received daily i.t. injections of PBOX-6 (P6) (7.5 mg kg⁻¹) or a vehicle (Veh) (10% (v/v) cremophor, 10% (v/v) ethanol in PBS). (A) Mice were weighed every 4 days and at the experimental end point (day 18). (B) Tumour growth was measured every second day, the long (L) and short (S) axes were recorded, and tumour volume (V) calculated using the equation: V=(S² × L)/2 (Beck et al., 2003). (C) Mice were killed on day 18, spleens were removed and weighed. Statistical analysis was performed using the Student's unpaired t-test, ^*P<0.05; ^**P<0.01.