MYC dysregulation confers a poor prognosis to diffuse large B-cell lymphoma (DLBCL), and effective therapeutic strategies are lacking in relapsed/refractory DLBCL, Burkitt lymphoma and intermediate forms.1, 2 As a master transcriptional regulator, MYC recruits transcription complexes containing RNA polymerase II (Pol II) to facilitate effective transcriptional elongation of MYC gene targets.3 Pol II is fully activated by phosphorylation of a critical serine residue at position 2 within heptapeptide repeats in the carboxy-terminal domain (CTD), a function performed by the positive transcription elongation factor b (P-TEFb; comprising CDK9 and cyclin T1).4 It has been shown that MYC binds and recruits P-TEFb to its targets as a means to activate Pol II.3, 5, 6 More recently, CDK9-mediated transcriptional elongation was reported as essential for tumor maintenance in a genetically defined MYC-driven model of hepatocellular carcinoma.7 Thus, CDK9 dependence may represent a druggable vulnerability in lymphomas with dysregulated MYC expression.
Dinaciclib (Merck, Boston, MA, USA) is a novel CDK inhibitor that has reached phase 1b/2 of clinical trials for a range of solid-organ malignancies, as well as for myeloma and chronic lymphocytic leukemia.8 We hypothesized that CDK9 inhibition by dinaciclib would represent a rational pharmacologic approach to target the transcription of critical MYC-regulated oncogenic effector proteins. Here we describe durable in vivo responses to dinaciclib in aggressive MYC-driven lymphoma, mediated by downregulation of Pol II-mediated Mcl-1 transcription.
Dinaciclib has 50% kinase inhibitory concentrations of 1, 1, 3 and 4 nM for CDK2, CDK5, CDK1 and CDK9, respectively.8 Dinaciclib potently killed Eμ-Myc and human IG-cMYC-translocated cell lines independent of p53 function, but not untransformed murine fibroblast cells, at low nanomolar concentrations approximating those observed for kinase inhibition (Figures 1a and b, Supplementary Figure S1).
As Bcl-2 and Mcl-1 have been implicated as important apoptotic regulators in Eμ-Myc lymphomas,9, 10 we assessed the effects of dinaciclib on these proteins. We hypothesized that CDK9 inhibition with dinaciclib would target Mcl-1 transcription, as has been observed with other CDK inhibitors in myeloma and mantle cell lymphoma.11, 12 Eμ-Myc and human IG-cMYC-translocated cell lines were treated with dinaciclib or dimethylsulfoxide control and interrogated using the quantitative PCR analysis for the effect on Mcl-1 and Bcl-2 mRNA. Dinaciclib treatment was associated with a significant reduction in Mcl-1 mRNA, with no significant effect on Bcl-2 transcript levels (Figure 1c, Supplementary Figure S2). Chromatin immunoprecipitation-PCR was used to show the binding of phosphorylated Pol II, subunit B1 carboxy-terminal domain (CTD) serine 2 (pRpb1 Ser2) as a marker of CDK9 activity at the Mcl-1 locus in a representative Eμ-Myc lymphoma cell line (Figure 1d). These findings support the hypothesis that dinaciclib transcriptionally downregulates Mcl-1.
We next examined Mcl-1 expression in Eμ-Myc and human IG-cMYC-translocated lymphoma cell lysates following the treatment with dinaciclib or vehicle. On-target CDK9 inhibition by dinaciclib was confirmed through inhibition of pRpb1 Ser2 at concentrations corresponding to apoptosis induction in Eμ-Myc cells (Figure 1e). Dinaciclib treatment also rapidly suppressed Mcl-1 protein expression, with no discernible reduction in Bcl-2 or Bcl-xL protein observed in murine (Figure 1e) or human (Figure 1f) cells. To determine the functional importance of Mcl-1 in regulating dinaciclib-mediated apoptosis, a representative Eμ-Myc lymphoma was stably transduced to express Mcl-1 off a retroviral promoter. As shown in Figure 1g, exogenously expressed Mcl-1 significantly protected Eμ-Myc cells from dinaciclib-induced apoptosis.
The in vivo efficacy of dinaciclib was then assessed by transplanting the same Eμ-Myc lymphomas into cohorts of syngeneic C57Bl/6 recipients. Compared with the vehicle control, dinaciclib treatment was well tolerated and associated with a highly significant survival advantage of tumor-bearing mice, including those bearing a p53-null lymphoma and a lymphoma with a spontaneous p53 mutation encoding a dominant-negative p53 protein (Figures 2a–c, Supplementary Figure S3). In contrast, dinaciclib-mediated therapeutic efficacy was severely attenuated in isogeneic p53-competent Eμ-Myc lymphoma overexpressing Mcl-1 (Figure 2d). In separate experiments, mice bearing transplanted Eμ-Myc cells were left untreated for 12 days to establish bulky nodal disease, at which time they received a single dose of dinaciclib or vehicle 1 or 4 h before being killed and before the lymph nodes were harvested. Consistent with the in vitro data, lymph node protein lysates showed reductions of pRpb1 and total Mcl-1 protein (Figure 2e), concomitant with the induction of apoptosis (Supplementary Figure S4). Finally, dinaciclib treatment of immunocompromised mice xenografted with the human IG-cMYC-translocated lymphoma was associated with reduced disease progression and significantly prolonged overall survival (Figures 2f and g).
In conclusion, our findings indicate that CDK9 inhibition by dinaciclib is highly effective in aggressive MYC-driven lymphomas, including ‘poor-risk’ p53-deficient clones, via selective inhibition of critical MYC targets including Mcl-1 (which is currently undruggable with existing BH3 mimetics).13, 14 Our data suggest a linear and druggable dependency between MYC and Mcl-1 that is contingent on CDK9 signaling. These findings are of particular interest in the context of a recent publication by Kelly et al.,15 further highlighting the dependency of MYC-driven B-cell lymphoma to Mcl-1. Rapid clinical translation of CDK9 inhibitors to MYC-dysregulated lymphoid malignancy should now be considered.
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Acknowledgements
Researchers are supported by funding from the Leukaemia Foundation of Australia (GPG, SJH, ML, MAD), the Royal Australasian College of Physicians (GPG) and the Arrow Bone Marrow Transplant Foundation (AB, ML). LMK is supported by a CJ Martin Fellowship (NHMRC), MAD is supported by VESKI Innovation and Herman Clinical Research Fellowships, JS is supported by funding from the Eva and Les Erdi/Snowdome Foundation Victorian Cancer Agency Fellowship, RWJ is a Principal Research Fellow of the National Health and Medical Research Council of Australia (NHMRC) and is supported by NHMRC Program and Project Grants, the Cancer Council Victoria and the Victorian Cancer Agency. Dinaciclib was provided by Merck Research Laboratories (Boston, MA, USA) and α-Amanitin was kindly provided by Ms Christina Woelwer. We thank Mr Don Cameron for technical advice regarding α-Amanitin experiments. The TRMPVIR vector was kindly provided by Dr Johannes Zuber.
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Gregory, G., Hogg, S., Kats, L. et al. CDK9 inhibition by dinaciclib potently suppresses Mcl-1 to induce durable apoptotic responses in aggressive MYC-driven B-cell lymphoma in vivo. Leukemia 29, 1437–1441 (2015). https://doi.org/10.1038/leu.2015.10
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DOI: https://doi.org/10.1038/leu.2015.10
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