Synergistic Anti-Tumor Efficacy of BET Inhibitors JQ1 and Otx-015 in Combination with Dasatinib in Preclinical Models of T-Cell Lymphomas

Background: Approximately 50% of patients with peripheral T-cell lymphoma (PTCL) enter long-term remission after standard chemotherapy and stem cell transplantation. Patients who do not respond to chemotherapy have few treatment options highlighting the critical need for new effective and targeted therapeutics. Aberrant T cell receptor (TCR) and tyrosine kinase (TK) signaling have been described in PTCL (Agostinelli 2014;Netchiporouka 2014). Single-agent TK inhibitors (TKIs) have significantly improved patient outcomes across multiple tumor subtypes. However, TKI therapy is rarely curative. The recent discovery of a subgroup of PTCL characterized by high levels of GATA3 and c-Myc expression and poor prognosis (Iqbal 2014; Manso 2016), establishes the rationale of targeting c-Myc in PTCLs. Based on the demonstration that pharmacologic inhibition of c-Myc is achievable through targeting bromodomain and extra terminal (BET) family of chromatin adapters, the therapeutic potential of BET inhibition was assessed in a panel of T cell lymphoma and leukemia cell lines. Since expression of c-Myc is regulated by the TCR, we also hypothesized that simultaneous targeting of c-Myc and TCR would significantly enhance the antiproliferative effects of BET inhibitors (BETis) and TKI alone in preclinical models of PTCL.

Methods: Five T-cell lymphoma and leukemia cell lines (Jurkat, HD-MAR-2, Karpas 299, Sup-T1, HH) were incubated with escalating doses of JQ1 (a small-molecule BETi with the highest affinity for BRD4) and OTX-015 (a BETi with a broader affinity for BRD2, BRD3, BRD4) and the tyrosine-kinase-inhibitor Dasatinib. Analysis of cell viability, cell cycle distribution, apoptosis and mitochondrial depolarization was performed using flow cytometry. Effects of treatments were assessed using gene expression profiling (GEP) and western blotting (WB). Combinations were evaluated using the Chou-Talalay Combination Index (CI), calculated with CompuSyn software (CompuSyn Inc, Paramus, NJ).

Results: JQ1 and OTX-015 show antiproliferative activity with IC₅₀ at nanomolar concentrations in all cell lines. As assessed determining viable cells by PI exclusion and flow cytometry, JQ1 and OTX-015 are similarly active in a dose-dependent manner in all cell lines. To understand the activity of JQ1 and OTX-015, we analyzed cell-cycle distribution using flow cytometry. JQ1 and OTX-015 induce a cell cycle arrest with G1-phase accumulation and decrease S-phase with the exception of SUPT1 cells that are characterized by a cell cycle arrest in G2-phase. Minimal increase in the sub-G1 population is observed in all cell lines, suggesting that JQ1 and OTX-015 mainly exert a cytostatic effect. We then examined GATA3 and c-Myc protein levels in all cell lines: varying amounts of GATA3 and c-Myc proteins were observed but a strong correlation between GATA3 and c-Myc expression was detected. After JQ1 and OTX-015 exposure, c-Myc protein level decrease in all cell lines apart from SUP-T1 cell line. Here c-Myc level do not change significantly upon BETis exposure, suggesting that BETis target other pathways relevant for SUP-T1 survival.

Dasatinib efficiently inhibits the proliferation in all cell lines at micromolar concentrations in a dose-dependent manner. Dasatinib induces G0/G1-phase arrest and an increase in sub-G1 population indicating a modest induction of apoptosis confirmed by caspase-9 activation and mitochondrial depolarization.

Compared to all single agents, combined treatments with sub-optimal concentrations of Dasatinib and JQ1 or OTX-015 exert synergistic lethal activity against all tested cell lines (C.I.<1). To uncover the main biological processes behind the synergistic interactions of BETis and Dasatinib, cell cycle analysis was assessed indicating that both combinations induce a significant increase of sub-G1 population associated with massive mitochondrial depolarization and cleavage of Caspase-9 and PARP.

Conclusions: The experiments presented here support the combination of BET inhibitors with the TK inhibitor Dasatinib for PTCLs. Our data suggest a synergistic interaction for the combination of both BETis and Dasatinib in vitro. Mechanistically, combined treatments exert synergistic anti-tumor effects in all cell lines through growth inhibitory effects, direct induction of cell death by promotion of caspase-dependent apoptosis and mitochondrial depolarization.