Cyclin-dependent kinase 9 as a potential specific molecular target in NK-cell leukemia/lymphoma

BAY 1143572 is a highly selective inhibitor of cyclin-dependent kinase 9/positive transcription elongation factor b. It has entered phase I clinical studies. Here, we have assessed the utility of BAY 1143572 for treating natural killer (NK) cell leukemias/lymphomas that have a poor prognosis, namely extranodal NK/T-cell lymphoma, nasal type and aggressive NK-cell leukemia, in a preclinical mouse model in vivo as well as in tissue culture models in vitro. Seven NK-cell leukemia/lymphoma lines and primary aggressive NK-cell leukemia cells from two individual patients were treated with BAY 1143572 in vitro. Primary tumor cells from an aggressive NK-cell leukemia patient were used to establish a xenogeneic murine model for testing BAY 1143572 therapy. Cyclin-dependent kinase 9 inhibition by BAY 1143572 resulted in prevention of phosphorylation at the serine 2 site of the C-terminal domain of RNA polymerase II. This resulted in lower c-Myc and Mcl-1 levels in the cell lines, causing growth inhibition and apoptosis. In aggressive NK-cell leukemia primary tumor cells, exposure to BAY 1143572 in vitro resulted in decreased Mcl-1 protein levels resulting from inhibition of RNA polymerase II C-terminal domain phosphorylation at the serine 2 site. Orally administering BAY 1143572 once per day to aggressive NK-cell leukemia-bearing mice resulted in lower tumor cell infiltration into the bone marrow, liver, and spleen, with less export to the periphery relative to control mice. The treated mice also had a survival advantage over the untreated controls. The specific small molecule targeting agent BAY1143572 has potential for treating NK-cell leukemia/lymphoma.

B AY 1143572 is a highly selective inhibitor of cyclin-dependent kinase 9/positive transcription elongation factor b. It has entered phase I clinical studies. Here, we have assessed the utility of BAY 1143572 for treating natural killer (NK) cell leukemias/lymphomas that have a poor prognosis, namely extranodal NK/T-cell lymphoma, nasal type and aggressive NK-cell leukemia, in a preclinical mouse model in vivo as well as in tissue culture models in vitro. Seven NK-cell leukemia/lymphoma lines and primary aggressive NK-cell leukemia cells from two individual patients were treated with BAY 1143572 in vitro. Primary tumor cells from an aggressive NK-cell leukemia patient were used to establish a xenogeneic murine model for testing BAY 1143572 therapy. Cyclin-dependent kinase 9 inhibition by BAY 1143572 resulted in prevention of phosphorylation at the serine 2 site of the C-terminal domain of RNA polymerase II. This resulted in lower c-Myc and Mcl-1 levels in the cell lines, causing growth inhibition and apoptosis. In aggressive NK-cell leukemia primary tumor cells, exposure to BAY 1143572 in vitro resulted in decreased Mcl-1 protein levels resulting from inhibition of RNA polymerase II C-terminal domain phosphorylation at the serine 2 site. Orally administering BAY 1143572 once per day to aggressive NK-cell leukemia-bearing mice resulted in lower tumor cell infiltration into the bone marrow, liver, and spleen, with less export to the periphery relative to control mice. The treated mice also had a survival advantage over the untreated controls. The specific small molecule targeting agent BAY1143572 has potential for treating NK-cell leukemia/lymphoma. improvement in the treatment of these systemic NK-cell leukemia/lymphoma. 6,7 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 development 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 transcription 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][11][12] Accumulating reports indicate that CDK9 kinase activity is crucial during the evolution and/or maintenance of many types of human malignancy. [10][11][12][13][14][15][16][17] CDK9 is also known to have an important role for Epstein-Barr Nuclear Antigen 2 (EBNA2)-dependent transcriptional activation and immortalization of EBV-infected cells. [18][19][20] Taken together, these findings suggest that CDK9 could represent a new molecular target for treating systemic 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 therapeutic potential of BAY 1143572 (Bayer AG Pharmaceuticals Division, Berlin, Germany), which is a new, highly selective inhibitor of CDK9/P-TEFb. 21

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 participated as control subjects, and their CD56-positive cells were isolated from their PBMC in the same manner. All donors provided written 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.

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 (1x10 7 /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 (0.8x10 7 /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 Multitest TM 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

Effect of BAY 1143572 on CDK9 activity in NK-cell leukemia/lymphoma lines
BAY 1143572 had very little effect on the total amount of RNAPII protein in any of the cell lines tested but inhibited phosphorylation of RNAPII CTD at the Ser2 site in a dose-dependent manner. In contrast, phosphorylation of RNAPII at the Ser5 site was not affected by this agent in any of the lines. BAY 1143572 treatment did decrease c-Myc and Mcl-1 protein levels in a dose-dependent manner in all of the lines ( Figure 2).

BAY 1143572 inhibits growth of primary ANKL cells isolated from patients
IC50 values for BAY 1143572 after 24 hours of incubation with CD56-positive cells isolated from five healthy volunteers were 0.65, 0.56, and 0.27-1.34 mM, (mean, median, and range, respectively). Cell viability curves for BAY 1143572 showed an initial decrease at concentrations up to around 1.0 uM, almost reaching a plateau at around 100.0 uM. Thus, some of the CD56-positive cells isolated from healthy volunteers remained viable even after expo-CDK9 as a potential molecular target in NK cell leukemia/lymphoma haematologica | 2018; 103(12) 2061

Macroscopic and microscopic findings in primary ANKL cell-bearing mice with or without BAY 1143572 therapy
The appearance of mice treated with vehicle only and those treated with BAY 1143572, 22 days after primary ANKL cell inoculation, is shown in Figure 4A, upper and lower panels, respectively. Hepatosplenomegaly was observed in all of the untreated mice, but not in those treated with BAY 1143572 (liver and spleen are demarcated by narrow yellow lines). Immunohistological analyses revealed that the livers of the control mice were infiltrated by atypical lymphoid cells, and the normal architecture was destroyed ( Figure 4B, top panels). ISH showed that these atypical cells were positive for EBER ( Figure 4B, second panels from the top). On the other hand, the liver architecture of the treated mice was almost intact ( Figure  4B, third panels from the top), and EBER-positive cells were rare ( Figure 4B, bottom panels). The same immunohistological analyses also revealed that the spleens of the untreated mice were infiltrated by atypical lymphoid cells, and the normal architecture was completely destroyed ( Figure 4C, top panels). ISH also showed that these atypical cells were positive for EBER ( Figure 4C, second panels from the top). On the other hand, the splenic architecture of the treated mice, like the liver, was essentially intact ( Figure 4C, third panels from the top), and EBER-positive cells were rarely observed ( Figure 4C, bottom panels).  Figure 5A).  Figure 5B).

BAY 1143572 treatment reduces primary ANKL cells in the liver
The percentage of leukemia cells infiltrating into the liver of control NOG mouse No.1 was 32.4% (i.e., 36 Thus, the percentage of primary ANKL cells present in the liver was also decreased by BAY1143572 treatment in these animals (P=0.009; Figure 5C).

BAY1143572 prolongs the survival of mice inoculated with primary ANKL cells
Two of the 6 BAY 1143572-treated mice survived up to 34 days after inoculation of the primary ANKL tumor cells, whereas none of the vehicle-treated control mice were alive at that time (P=0.020; Figure 6). No toxicity attributable to BAY 1143572 was observed over this time period in any of the mice. Thus, we conclude that BAY 1143572-treatment of mice inoculated with primary ANKL cells significantly extended their survival relative to the untreated controls.

Discussion
NK-cell leukemia/lymphoma have a very poor prognosis necessitating improved treatment regimens for these patients. [1][2][3][4][5][6][7][8][9] Here, we begin to assess whether a selective CDK9 as a potential molecular target in NK cell leukemia/lymphoma haematologica | 2018; 103 (12) 2063 We found that BAY 1143572 possessed notable antitumor activity, not only against established NK cell leukemia/lymphoma lines, but also against primary ANKL cells in vitro and in primary ANKL cell-bearing mice in vivo. Hence, CDK9 activity is crucial for NKcell leukemia/lymphoma pathogenesis, implying that transcriptional machinery could represent an appropriate molecular target for developing new treatments for this disease. First, we aimed to investigate the mechanism of action of BAY 1143572 using NK-cell leukemia/lymphoma lines in vitro and determined that it specifically inhibited the phosphorylation of RNAPII CTD at the Ser2 but not the Ser5 site. This resulted in transcriptional repression of RNAPII and thus decreased levels of the downstream proteins c-Myc and Mcl-1. This in turn causes inhibition of growth and induction of apoptosis in NK-cell leukemia/lymphoma lines whether they originate from NK cells (SNK-1, NK-92, KAI-3, or KHY-G) or T cells (SNT-8, SNT-16, or MTA). Whether they were EBV-positive (SNT-8, SNK-1, SNT-16, NK-92, or KAI-3) or -negative (MTA or KHY-G) was also found not to affect the anticancer activity of BAY 1143572. Very similar results were obtained using primary ANKL cells freshly isolated from a patient, rather than established cell lines. BAY 1143572 markedly reduced phosphorylation of RNAPII CTD at the Ser2 site, but only weakly at the Ser5 site, which results in downregulation of the downstream protein, Mcl-1.
Importantly, our study showed that NK-cell leukemia/lymphoma are more sensitive to the CDK9/P-TEFb inhibitor than their normal cell counterparts. Exposure to ≥1.0 mM of BAY 1143572 for 24 hours killed almost all primary ANKL cells from two different patients, but also a fraction of CD56-positive cells from healthy volunteers. Nonetheless, after 24 hours´exposure to 10.0 mM BAY 1143572, 20-40% of CD56-positive cells from controls were still viable. This clearly documents that BAY 1143572 is far less toxic to CD56-positive cells from healthy donors than to CD56-positive primary ANKL tumor cells. It remains unclear why NK-cell leukemia/lymphoma cells are more sensitive to the CDK9/P-TEFb inhibitor. One possibility is that CDK9 kinase and RNAPII are more highly activated in malignant than in normal cells. Supporting this, another group has reported higher CDK9 expression accompanied by greater phosphorylation of RNAPII CTD in tumor cells than in normal cells. 30 Additionally, a different group reported that CDK9 expression appeared to be related to particular stages of lymphoid differentiation/activation. 31 However, we did not observe any significant differences between tumor cells and non-tumor lymphocytes in the phosphorylation level of RNAPII at the Ser2 site in the present study. A possible explanation for this is that the malignant cells are more "dependent" on the transcriptional machinery involving RNAPII to sustain the expression of critical oncogenes supporting their survival and proliferation, such as Mcl-1 and c-Myc. 32,33 In this context, inhibition of the transcriptional machinery would result in an acute and concurrent downregulation of these oncogenes, thereby leading to cell death. In fact, inhibition of CDK7 or BET bromodomain 4, which also block the activity of RNAPII in a similar manner to CDK9/PTEF-b inhibition, has been reported to result in potent anti-cancer activity in several malignancies. [34][35][36][37][38] Importantly, many types of cancer and leukemia cells are more sensitive to these inhibitors than non-transformed cells. 39,40 These studies suggest that expression of oncogenes may need to be maintained at a high level, and thus that cancer cells are likely to be more sensitive to RNAPII inhibition. Collectively, these earlier S. Kinoshita et al. 2066 haematologica | 2018; 103(12) investigations and the present studies suggest a great advantage for the clinical use of CDK9 inhibitors. Future investigation is needed to analyze the status of regulatory elements after BAY 1143572 treatment.
In the present study, we have established a mouse xenogeneic model for pre-clinical testing of a CDK9/P-TEFb inhibitor. Severe immune deficiencies in NOG mice permit engraftment of human immune cells, where they may retain very similar functions in an in vivo environment that can be manipulated experimentally. 41,42 In these mice, primary EBV-positive, CD16/CD56-positive, CD3and CD19-negative cells from ANKL patients were found to robustly infiltrate several organs such as the spleen, liver and bone marrow. These features are very similar to those seen in the ANKL patients who donated the cells.
To the best of our knowledge, this is the first report of primary ANKL cell-bearing mice in which it could be shown that ANKL cells are maintained by serial transplantations. This is a useful model because these tumor cells cannot be cultured as cell lines in vitro, implying that the in vivo microenvironment is an absolute requirement for tumor cell survival. Hence, our present in vivo ANKL model represents the in vivo human ANKL environment better than other models using established tumor cell lines. Thus, this in vivo xenogeneic primary tumor model offers a useful methodology for probing ANKL pathogenesis and provides a more relevant tool for testing novel antitumor agents. Here, we report that BAY 1143572 possesses strong antitumor activity, as demonstrated in vivo by reduced ANKL cell infiltration into blood, bone marrow, liver and spleen in this model. Moreover, this model also allowed us to demonstrate that BAY 1143572 monotherapy can prolong survival of ANKL-bearing hosts. It should also be noted that the in vivo antitumor activity of BAY 1143572 in the primary tumor cell-bearing NOG mice was actually mediated by the on-target effect of BAY 1143572; namely, the inhibition of CDK9 and subsequent inhibition of phosphorylation at the Ser2 site of the RNAPII CTD was clearly demonstrated in our previous study on ATL. 17 It is also important to note that no BAY 1143572 toxicity was observed in any of the mice studied here. Taken together, these findings in the primary ANKL mice strongly suggest that targeting CDK9 in human ANKL patients could be a promising therapeutic approach.
To the best of our knowledge, this is the first report evaluating the efficacy of CDK9-targeted therapy in NKcell leukemia/lymphoma, including ANKL. It must be noted that several other anti-cancer agents targeting CDK9 have already been tested in the clinic, but with little benefit and they were accompanied by numerous adverse events. [43][44][45][46][47] The latter may have been due to their insufficient selectivity for CDK9. 48 In contrast, BAY 1143572 exhibits marked selectivity for CDK9/PTEFb,21 and possesses strong antitumor effects against NK-cell leukemia/lymphoma including ANKL, not only in vitro, but also in animal models in vivo. Because, in clinical practice, NK-cell leukemia/lymphoma is highly aggressive and refractory, combination strategies such as CDK9 inhibition together with PD-1 blockade should be worth considering. 49,50 In conclusion, we propose that BAY 1143572 holds great promise as a potential agent to treat NK-cell leukemia/lymphoma, including ANKL patients. The mechanism of action is via CDK9/P-TEFb inhibition. Clinical evaluation of CDK9/P-TEFb selective inhibitors in patients with NK cell leukemia/lymphoma is therefore warranted. CDK9 as a potential molecular target in NK cell leukemia/lymphoma haematologica | 2018; 103 (12) 2067