BET inhibition targets ABC-DLBCL constitutive B-cell receptor signaling through PAX5

B-cell receptor (BCR) signaling is essential for the diffuse large B-cell lymphoma (DLBCL) subtype that originates from activated B-cells (ABC). ABC-DLBCL cells are sensitive to Bruton tyrosine kinase intervention. However, ABC-DLBCL patients had overall response rates of 33-37% for Bruton tyrosine kinase inhibitors, suggesting the evaluation of combination-based treatment for improved efficacy. We investigated the efficacy and mechanism of AZD5153 combined with the Bruton tyrosine kinase inhibitor acalabrutinib in ABC-DLBCL preclinical models. AZD5153 is a bivalent BET inhibitor that simultaneously engages the two bromodomains of BRD4. Adding AZD5153 to acalabrutinib demonstrated a combination benefit in ABC-DLBCL cell lines and PDX models. Differential expression analyses in treated tumors identified significant alterations of BCR, PAX5, RELB/alternative NF?B, and toll-like receptor/interferon signaling. PAX5 is a transcription factor for BCR signaling genes and may be critical to the perpetually active BCR signaling in ABC-DLBCL. We demonstrate that AZD5153 decreases PAX5 expression, while acalabrutinib disruption to BCR signaling inhibits PAX5 activation. Furthermore, several interferons were decreased by AZD5153 and acalabrutinib in tumors. Adding IFNß1 to cells in vitro restored PAX5 activation. Our results demonstrate AZD5153 enhances the efficacy of acalabrutinib through PAX5 and BCR mechanisms that are critical for ABC-DLBCL. Overall design: Xenograft tumors from TMD8 and LY6934 mouse models after dosing with AZD5153 and acalabrutinib were excised and snap frozen in liquid nitrogen. Treatments were A) TMD8 (4 hours): vehicle (0.5% hydroxymethylcellulose, 0.1% Tween80), 0.72 mg/kg AZD5153 QD, 20 mg/kg acalabrutinib BID, or 0.72 mg/kg AZD5153 QD + 20 mg/kg acalabrutinib BID, and B) LY6934 (25 days): vehicle (0.5% hydroxymethylcellulose, 0.1% Tween80) or 0.5 mg/kg AZD5153 QD + 20 mg/kg acalabrutinib BID. Tumor tissue was lysed and RNA was extracted using RNeasy kit (Qiagen, Hildren, Germany) according to manufacturer's instructions. RNA concentration was quantified using the Qubit fluorometer (ThermoFisher, Waltham, MA). 15 RNA samples were sent to Novogene for library preparation and human RNA sequencing.