Abstract 2500: Transient CDK9 inhibition with AZD4573 modulates Bfl-1 in preclinical lymphoma models
Authors: Boiko Scott , Proia Theresa , Martin Maryann San , Drew Lisa , Shao Wenlin , Cidado Justin 
Published: August 2019

Abstract
AZD4573 is a selective cyclin-dependent kinase 9 (CDK9) inhibitor under clinical development in patients with hematological malignancies. Transient CDK9 inhibition serves as an orthogonal approach for targeting Mcl-1, a labile anti-apoptotic protein essential for the survival of cancer cells. Across-broad hematological cancer models, anti-tumor responses with AZD4573 strongly correlate with selective Mcl-1 inhibitors, such as AZD5991 (R2=0.8). Despite compelling evidence for an Mcl-1 dependent mechanism of action, we also observed a subset of lymphoma models more sensitive to CDK9 inhibition compared to Mcl-1 inhibition, suggesting acute CDK9 inhibition could be targeting other labile proteins beyond Mcl-1 to induce apoptosis. We identified Bfl-1 as one such potential target and demonstrate lymphoma models expressing Bfl-1 are highly sensitive to CDK9 inhibition.
Bfl-1 belongs to the Bcl-2 family of anti-apoptotic proteins and was detected in over 20% of lymphoma cell lines evaluated (n=33). Cycloheximide experiments indicate Bfl-1 has a short protein half-life (&lt;1h), similar to Mcl-1. Therefore, treatment with 100nM of AZD4573 in diffuse large B-cell lymphoma cell lines OCILY10 and TMD8 caused rapid down-regulation of both Mcl-1 and Bfl-1 by 4h, resulting in caspase cleavage by 6h. Evaluation of caspase activation following 6h treatment revealed an average maximum effect of 87% for AZD4573 compared to 45% with Mcl-1 inhibition, suggesting these cell lines are not exclusively Mcl-1-dependent. The hypothesis that survival of lymphoma cells may be co-dependent on both Mcl-1 and Bfl-1 was evaluated by siRNA knockdown. Following a dose-dependent suppression of Bfl-1 protein (&gt;80%) in OCILY10 and TMD8 cells, viability loss was minimal (&lt;30% reduction relative to control). However, when Bfl-1 knockdown cells were treated for 6h with an Mcl-1 inhibitor, the maximum caspase activation increased to over 90% in both cell lines, phenocopying a similar magnitude achieved with AZD4573-mediated CDK9 inhibition. In these models, depletion of both Bfl-1 and Mcl-1 was necessary to induce maximum apoptosis, with studies ongoing to evaluate single-gene Bfl-1 dependency in additional lymphoma models.
Consistent with the in vitro phenotype, intermittent dosing of the ABC-DLBCL xenografts OCILY10 and TMD8 with AZD4573 caused robust tumor regressions (198 and 184% TGI, respectively). AZD4573-mediated anti-tumor activity was associated with pharmacodynamic reductions of pSer2-RNAPII, Mcl-1 and Bfl-1, followed by caspase activation. Collectively, these findings support the ability to target Bfl-1 via CDK9 inhibition. Given the current absence of clinical small molecule Bfl-1 inhibitors and expanded monotherapy activity compared to selective Mcl-1 inhibition in a subset of preclinical models, CDK9 inhibitors have tremendous therapeutic potential in the treatment of patients with Bfl-1-expressing lymphoma.
Citation Format: Scott Boiko, Theresa Proia, Maryann San Martin, Lisa Drew, Wenlin Shao, Justin Cidado. Transient CDK9 inhibition with AZD4573 modulates Bfl-1 in preclinical lymphoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2500.