The Clinical BET Inhibitor, GS-5829, Is Active Against Chronic Lymphocytic Leukemia As Single Agent and in Combination with B-Cell Receptor Signaling Inhibitors

Introduction: Despite considerable advances in the treatment of chronic lymphocytic leukemia (CLL) with targeted agents including B-cell receptor (BCR) signaling inhibitors, such as the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib and phosphatidylinositol 3-kinase delta (PI3Kδ) inhibitor idelalisib, CLL generally remains incurable. Identifying suitable combination therapies to improve the efficacy of BCR inhibitors may induce deeper remissions and potentially permit treatment discontinuation. Inhibitors of the bromodomain and extra-terminal motif (BET) family of proteins demonstrate preclinical activity against B cell malignancies. Here, we explore the activity of a novel BET inhibitor, GS-5829, in CLL that selectively binds to acetylated lysine recognition motifs in the bromodomains of BET proteins, impeding their interaction with acetylated histones, and ultimately interfering with transcription of BET-regulated target genes.

Methods: The IC₅₀ of GS-5829 was measured in the CLL cell line MEC-1 by a TACS® XTT cell proliferation/viability assay according to the manufacturer's protocol after 72 hours of incubation with inhibitor. The level of apoptosis of primary CLL cells was determined by flow cytometry after staining with DiOC6/propidium iodide. Peripheral blood mononuclear cells from patients with CLL were cultured for 14 days until outgrowth of nurse-like cells (NLCs). Before treatment with inhibitors, non-adherent cells were harvested and re-plated on autologous NLCs at a concentration of 1x10⁷ cells/mL. CLL cells in NLC co-cultures were treated with inhibitors for 24 hours for immunoblotting and up to 120 hours for cell viability assays. The degree of drug interaction was quantitatively measured by the combination index ( CI ) (Chou and Talalay, Eur J Biochem, 1981) calculated using CompuSyn software. The CI values below 0.9 were interpreted as indicative of synergism. The CI values reported below were calculated at the median-effect dose (ED₅₀) of the drug combination; combination indexes measured in several individual primary CLL samples reported as a range.

Results: Single agent GS-5829 inhibited proliferation of MEC-1 cells in a dose-dependent manner with an IC₅₀ of 63.8 nM (95% confidence interval, 46.9 to 86.7 nM) without inducing apoptosis at this concentration. GS-5829 also demonstrated synergism with BCR signaling inhibitors (ibrutinib ( CI =0.492), entospletinib ( CI =0.118), and idelalisib ( CI =0.130)) in reducing proliferation of MEC-1 cells. To further explore the therapeutic potential of GS-5829, we used primary CLL cells co-cultured with autologous NLCs, an in vitro model that resembles the lymph node microenvironment. In NLC co-cultures, single agent GS-5829 dose-dependently induced apoptosis of CLL cells (from 8.3% at 50 nM to 47.3% at 800 nM GS-5829), irrespective of the immunoglobulin heavy chain variable gene ( IGHV ) mutational status or ZAP-70 expression. Combination treatment of GS-5829 with either ibrutinib ( CI 0.036 - 0.615, N=8), entospletinib ( CI 0.085 - 0.487, N=3), or idelalisib ( CI 0.125 - 0.434, N=3) synergistically increased GS-5829-induced apoptosis of primary CLL cells. Immunoblot analyses of 4 primary CLL samples treated with 400 nM GS-5829 revealed a 99.3% increase (95% confidence interval, 60.8% to 137.7%; P =0.0038) in HEXIM1 protein levels, a BET inhibitor pharmacodynamic marker (Lin, Mol Cancer Ther, 2017). We also observed a significant increase in IκBα (59.3%; 95% confidence interval, 31.2% to 87.3%; P =0.0067), an inhibitor of NF-κB signaling, along with decreases in the levels of MYC (75.0%; 95% confidence interval, 56.9% to 93.1%; P =0.0009) and cyclin D2 (44.5%; 95% confidence interval, 38.1% to 50.8%; P =0.0072) after GS-5829 treatment, whose expression is considered to be regulated by BET proteins. Combination of ibrutinib and GS-5829 induced more extensive inhibition of signaling pathways compared to single agent activities, providing a possible mechanism underlying the enhancement of apoptosis.

Conclusion: The data support further pre-clinical and possible clinical evaluation of the therapeutic potential of GS-5829 in CLL, especially in combination with BCR signaling inhibitors, such as BTK inhibitors. Further studies aimed at evaluating drug efficacy in vivo in CLL mouse models are underway, along with more detailed analyses of CLL subset-specific responses to GS-5829.