The polo-like kinase 1 (PLK1) inhibitor NMS-P937 is effective in a new model of disseminated primary CD56+ acute monoblastic leukaemia

CD56 is expressed in 15–20% of acute myeloid leukaemias (AML) and is associated with extramedullary diffusion, multidrug resistance and poor prognosis. We describe the establishment and characterisation of a novel disseminated model of AML (AML-NS8), generated by injection into mice of leukaemic blasts freshly isolated from a patient with an aggressive CD56+ monoblastic AML (M5a). The model reproduced typical manifestations of this leukaemia, including presence of extramedullary masses and central nervous system involvement, and the original phenotype, karyotype and genotype of leukaemic cells were retained in vivo. Recently Polo-Like Kinase 1 (PLK1) has emerged as a new candidate drug target in AML. We therefore tested our PLK1 inhibitor NMS-P937 in this model either in the engraftment or in the established disease settings. Both schedules showed good efficacy compared to standard therapies, with a significant increase in median survival time (MST) expecially in the established disease setting (MST = 28, 36, 62 days for vehicle, cytarabine and NMS-P937, respectively). Importantly, we could also demonstrate that NMS-P937 induced specific biomarker modulation in extramedullary tissues. This new in vivo model of CD56+ AML that recapitulates the human tumour lends support for the therapeutic use of PLK1 inhibitors in AML

PLK1 expression in AML-NS8 cells and activity of PLK1 inhibitor NMS-937 <i>in vitro</i> and <i>in vivo</i>.

<p>(<b>A</b>) Western blot analysis. Leukaemic cells at diagnosis, expanded <i>in vivo</i> or <i>in vitro</i> and PBMCs from healthy volunteers were lysed and PLK1 expression analysed by Western blot. GAPDH protein expression was used as loading control. (<b>B</b>) Cytotoxicity assay. AML-NS8 <i>in vitro</i> expanded cells were incubated in presence or absence of increasing concentrations of cytarabine, doxorubicin and NMS-P937 for 72 hours and IC₅₀ determined for each drug. (<b>C</b>) <i>In vivo</i> efficacy: preemptive protocol. Groups of 10 mice were inoculated iv with 5×10⁶ AML-NS8 cells and treated with vehicle, cytarabine at 75 mg/kg ip per day over 5 days for 4 cycles with a 7 day rest, doxorubicin at 3 mg/kg iv every 7 days for 3 cycles and NMS-P937 per os at 120 mg/kg per day over 2 days, for 4 cycles with a 10 day rest and survival recorded. The Kaplan-Meier plot and MST are shown. Statistical analysis using the Wilcoxon test showed that all drug treatments were statistically different from vehicle alone (p<0.01 for cytarabine and p<0.001 for NMS-P937 and doxorubicin). NMS-P937 was also statistically different from the other drugs with p<0.05. Data are representative of 2 independent experiments. (<b>D</b>) <i>In vivo</i> efficacy: therapeutic protocol. Groups of 10 mice were inoculated iv with 5×10⁶ AML-NS8 cells and treatments started at day 20, when a clear leukaemic dissemination was visible. Mice were treated with vehicle, cytarabine at 75 mg/kg ip per day over 5 days with a 5 day rest continuously until mice were moribund, and NMS-P937 per os at 60 mg/kg bid per day over 2 days with 5 day rest continuously until mice were moribund and survival recorded. The Kaplan-Meier plot and median survival times (MST) are shown. Statistical analysis using the Wilcoxon test showed that all drug treatments were statistically different from vehicle alone (p<0.0001 for each compound). NMS-P937 was also statistically different from cytarabine with p = 0.001.</p

Phenotype of AML-NS8 cells, at diagnosis, expanded ip in mice or expanded <i>in vitro</i> with rh-IL3 and rh-GM-CSF.

*<p>Neg: <10% staining; +: <80% staining or MFI<100; ++: 80–100% staining and MFI 100–400; +++: 80–100% staining and MFI>401 by flow cytometry. ND: not done. Immunophenotypes were performed three times.</p>**<p>Cytochemical staining for fluoride sensitive ANAE.</p

Mechanism of action of PLK1 inhibitor <i>in vitro</i> and <i>in vivo</i>.

<p><i>In vitro</i> experiments<a href="http://:" target="_blank">:</a> AML-NS8 cells were untreated (CTRL) or treated with 0.2 µM NMS-P937 for 24 hours or 0.75 ng/ml nocodazole for 16 hours. (<b>A</b>) biparametric analysis for phospho-Histone H3 and DNA content by flow cytometry. Inserts show the correspondent cell cycle profile and indicate the percentage of cells in G2/M phase. (<b>B</b>) Western blot analysis of total cell lysates for the following proteins: phospho-NPM1 (pNPM1), NPM1, phospho-Histone H3 (pH3), Histone H3 (H3). Tubulin protein expression was used as loading control. <i>In vivo</i> experiments: (<b>C</b>) Solid tumour masses from vehicle or NMS-P937 treated animals were collected, fixed and stained with H&E or antibodies against phospho-TCTP (pTCTP), phospho-Histone H3 (pH3), phospho-NPM1 (pNPM1) or active Caspase 3. Representative pictures are reported (Axio Scope Zeiss, magnification×400). Black/white bar, 50 µm.</p

Histological and immunohistochemical analysis of different tissues.

<p>SCID mice were inoculated iv with 5×10⁶ AML-NS8 cells, monitored for clinical signs and sacrificed upon manifestations of terminal disease. Different tissues were collected and fixed for histology and immunohistochemistry. (<b>A</b>) Clinical signs and histopathological characterisation of AML-NS8 <i>in vivo</i> disseminated model. (<b>B, C</b>) Representative pictures (Axio Scope Zeiss, magnification×200) of the indicated tissues stained with H&E (left panels) or anti-human HLA-A,B,C (right panels). Black/white bar, 100 µm.</p

Leukaemic infiltration of meninges and soft tissues from mice treated with NMS-P937 and cytarabine following a therapeutic schedule.

<p>Mice were inoculated iv with 5×10⁶ AML-NS8 cells and treatments started at day 20, when leukaemic dissemination was present. Mice were treated with vehicle, NMS-P937 per os at 60 mg/kg bid per day over 2 days with 5 day rest and cytarabine at 75 mg/kg ip per day over 5 days with a 5 day rest, continuously until mice were moribund. Organs were collected for histological and immunohistochemical analysis. The graphs show the qualitative evaluation of leukaemic infiltration (expressed as score) in meninges (<b>A</b>) and soft tissues (<b>B</b>). Score: 0 = no infiltration, 1 = minimal, 2 = moderate, 3 = marked, 4 = severe infiltration.</p

<i>In vitro</i> and <i>in vivo</i> growth of AML-NS8 cells.

<p>(<b>A</b>) Neoplastic cells were plated <i>in vitro</i> at 50×10³ cells/ml in presence of rhIL-3 and rhGM-CSF. Cells were counted at the indicated times to determine the doubling time (DT = 31hours). The data are representative of 3 experiments. (<b>B</b>) On day 0, groups of 10 SCID mice were inoculated iv with either 1×10⁶ (dotted line) or 5×10⁶ (continuous line) <i>in vivo</i> expanded AML-NS8 cells and survival time recorded. The median survival time (MST) was 37 and 28 days respectively.</p

Karyotype and Genomic aberrations of AML-NS8 from different sources.

a<p>M: metaphases.</p>b<p>ND: not done.</p>c<p>x: presence of aberration.</p