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S. Kinoshita et al.
improvement in the treatment of these systemic NK-cell
6,7
leukemia/lymphoma. However, the prognosis of these
neoplasms is still unsatisfactory,8,9 and the development of novel therapeutic agents remains an urgent issue. Nevertheless, to the best of our knowledge, until now there have been very few preclinical studies on the devel- opment of novel antitumor agents targeting NK-cell leukemia/lymphoma.
We have been focusing on cyclin-dependent kinase 9 (CDK9) as a potential molecular target for NK-cell leukemia/lymphoma. CDK9 is a serine (Ser)/threonine kinase, and constitutes a subunit of the positive transcrip- tion elongation factor b (P-TEFb) complex. This plays a vital role in regulating gene transcription elongation via phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNAPII).10-12 Accumulating reports indicate that CDK9 kinase activity is crucial during the evolution and/or maintenance of many types of human malignan- cy.10-17 CDK9 is also known to have an important role for Epstein–Barr Nuclear Antigen 2 (EBNA2)-dependent tran- scriptional activation and immortalization of EBV-infected cells.18-20 Taken together, these findings suggest that CDK9 could represent a new molecular target for treating sys- temic NK-cell neoplasms, such as ENKTL, nasal type with systemic organ involvement, as well as ANKL. Here, we begin to test this hypothesis by investigating the thera- peutic potential of BAY 1143572 (Bayer AG Pharmaceuticals Division, Berlin, Germany), which is a new, highly selective inhibitor of CDK9/P-TEFb.21
Methods
NK-cell leukemia/lymphoma lines
SNT-8, SNK-1, SNT-16, NK-92 and KAI-3 are EBV-positive, but MTA and KHYG-1 are EBV-negative lines.22-26 NK-92 was pur- chased from ATCC (Manassas, VA), and MTA, KAI-3 and KHYG- 1 were purchased from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan).
Primary tumor cells from patients with ANKL and cells from control subjects
Primary tumor cells were isolated using anti-human CD56 microbeads (Miltenyi Biotec, Bergisch Gladbach) from peripheral blood mononuclear cells (PBMC) of two patients (patient A and B, Online Supplementary Figure S1). Five healthy volunteers participat- ed as control subjects, and their CD56-positive cells were isolated from their PBMC in the same manner. All donors provided writ- ten informed consent before blood sampling according to the Declaration of Helsinki, and the present study was approved by the institutional ethics committees of Nagoya City University Graduate School of Medical Sciences.
Cell proliferation and apoptosis assays
Cell proliferation and apoptosis were assessed as previously described.17,27
Western blotting
Antibodies to RNAPII (N-20), c-Myc, Mcl-1, actin, phosphory- lated RNAPII (phospho-RNAPII) (Ser2 of the CTD) and phospho- RNAPII (Ser5 of the CTD) were as previously described.17,28
Histological analysis
Hematoxylin and eosin (HE) staining was performed on forma- lin-fixed, paraffin-embedded sections, as previously described.17,29
Animals
All in vivo experiments of NOD/Shi-scid, IL-2Rγnull (NOG) mice were performed as previously described.17
Establishment of the primary ANKL cell-bearing mouse model
Patient A’s PBMC, consisting of almost 90% CD56-positive atypical lymphoid tumor cells, were injected intraperitoneally (i.p.) into naïve NOG mice (1 x 107/mouse). Three to 4 weeks after i.p. injection, the NOG mice became weaker and exhibited clinical features of cachexia. The tumor cells were recovered and i.p. inoculated into other naïve NOG mice, and after three to four weeks, they displayed features almost identical to those of the donor mice. This procedure of transfer from mouse to mouse was repeated successfully until at least the fifth passage.
Primary ANKL cell-bearing mice treated with BAY 1143572
Leukemic cells from ANKL patient A, which could be serially transplanted into NOG mice, were i.p. injected into 10 naïve NOG mice (1x107/mouse). The animals were randomly divided into two groups seven days after ANKL cell inoculation, and were treated with oral application of 12.5 mg/kg BAY 1143572 or vehicle, for 15 days (7–21 days after tumor inoculations). Therapeutic efficacy was then evaluated 22 days after tumor inoculation. In another experiment, ANKL cells from the mice suspended were also inoculated i.p. into another 12 naive NOG mice (0.8x107/mouse). These animals were randomly divided into two groups and were treated by oral application of 12.5 mg/kg BAY1143572 or vehicle for 15 days (7–21 days after tumor inoculation). The therapeutic efficacy of BAY 1143572 was evaluated by survival times.
Flow cytometry analysis of cells inoculated into mice
The following mAbs were used for flow cytometry: BD MultitestTM CD3/CD16+CD56/CD45/CD19 (No. 342416, BD Biosciences), and stained cells were analyzed as previously described.17
Statistical analysis
All statistical analyses were performed using SPSS Statistics 17.0 software (SPSS Inc., Chicago, IL), as previously described.17
Results
In vitro inhibitory effect of BAY 1143572 on the proliferation of NK-cell leukemia/lymphoma lines
BAY 1143572 was found to inhibit NK-cell leukemia/lymphoma cell line proliferation in a dose- dependent manner (Figure 1A). IC50 values for BAY1143572 after 72 hours of incubation for SNT-8, SNK-1, SNT-16, NK-92, MTA, KAI-3, and KHYG-1 were 0.35, 0.22, 0.24, 0.63, 0.20, 0.57, and 0.50 mM, respective- ly.
BAY 1143572 induces apoptosis in NK-cell leukemia/lymphoma lines
BAY 1143572 was found to induce apoptosis of the tested NK-cell leukemia/lymphoma lines in a dose- dependent manner. Nearly half of the MTA and KAI-3 cells, 70% of SNT-16 and KHYG-1 cells, 80% of SNK-1 and NK-92 cells, and 100% of SNT-8 cells underwent apoptosis on treatment with 1.0 mM BAY 1143572 for 72 hours (Figure 1B).
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