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S. Kinoshita et al.
Figure 6. Kaplan-Meier survival curves of ANKL cell-bearing NOG mice treated with BAY 1143572 or vehicle. Mice were treated orally with 12.5 mg/kg BAY 1143572 or vehicle (n=6 for both) once daily for 15 days (7-21 days after ANKL cell inoculation). The significance of the difference in survival is shown on the graph.
CDK9/P-TEFb inhibitor, BAY 1143572, may have clinical utility in this regard. We found that BAY 1143572 pos- sessed notable antitumor activity, not only against estab- lished NK cell leukemia/lymphoma lines, but also against primary ANKL cells in vitro and in primary ANKL cell-bear- ing mice in vivo. Hence, CDK9 activity is crucial for NK- cell 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 pro- teins 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-posi- tive (SNT-8, SNK-1, SNT-16, NK-92, or KAI-3) or -negative (MTA or KHY-G) was also found not to affect the anti- cancer 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 phospho- rylation 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 machin- ery 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.34-38 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
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haematologica | 2018; 103(12)