Clinical BET Inhibitor RG6146 Induces Intrinsic Apoptosis and Down-Regulates PD-L1 Expression and Induces Potent Anti-Tumor Activity in Models of Hematological Malignancy

Bromodomain and extra terminal (BET) family of bromodomains BET proteins are epigenetic 'readers' that bind acetylated histone residues and transcription factors at the chromatin interface and drive the expression of oncogenic transcription programs. BET inhibition using small molecules is an anti-cancer strategy being evaluated in early phase clinical trials. RG6146 is a novel small molecule, non-covalent inhibitor of (BET) family of bromodomains and potently inhibits BRD2, BRD3, BRD4 and BRDT. RG6146 is in clinical development for hematological malignancies including diffuse large B-cell lymphoma (DLBCL), multiple myeloma (MM) and solid tumors. In this study, we comprehensively examined the pre-clinical activity of RG6146 in genetically distinct models of hematological malignancy.

To understand the effects of BET inhibition on myc-levels and viability hematological malignancies, a panel of human MM and DLBCL cell lines were treated with RG6146 for 24-72 hours. Treatment with RG6146 potently down-regulated c-myc levels that was concomitant with induction of apoptosis and loss of viability.

To elucidate additional Myc-independent effects of RG6146, we used the Eμ- Myc model of aggressive B-cell lymphoma where the expression of transgenic Myc is insensitive to BET inhibition. Recapitulating the biological activity of prototypical BET inhibitor JQ1, RG6146 potently activated the intrinsic apoptotic cascade independently of p53 mutational status. Moreover, ectopic Bcl-2 overexpression or genetic loss of Bim were sufficient to attenuate RG6146-induced apoptosis. In the setting of ectopic Bcl-2 expression and protection from apoptosis, RG6146 impairs cell cycle progression leading to G₁ cell cycle arrest.

We next compared the transcriptional consequences of RG6146 or JQ1 treatment in cell derived from the Eμ- Myc model using RNA sequencing. Acute BET inhibition following 2 hours treatment with JQ1 or RG6146 was associated with a highly concordant modulation of global transcription. Accordingly, gene ontology analysis revealed that identical biological pathways were affected by either inhibitor. As a bona fide BET inhibitor target gene, we demonstrated rapid suppression of Il7r mRNA by qPCR and subsequent loss of cell surface CD127 expression by flow cytometry following exposures to RG6146.

Finally, we demonstrate single-agent activity of RG6146 in Eμ- Myc lymphoma-bearing syngeneic recipient mice. Administration of a single-dose of RG6146 significantly depleted tumor cells in the lymph node, spleen, and peripheral blood within 24 hours. Chronic RG6146 therapy was well tolerated in vivo and led to dose-dependent reduction in tumor burden and significant prolongation of survival. In this setting, we also demonstrated that RG6146 therapy suppressed the BRD4-dependent expression of immune checkpoint ligand PD-L1 on Eμ- Myc lymphomas.

Taken together, these data indicate that RG6146 is an on-target BET bromodomain inhibitor with potent anti-cancer activity. We demonstrated disruption of the intrinsic apoptotic pathway was sufficient to reduce the activity of RG6146. Moreover, we have previously demonstrated that acquired resistance to BET inhibition is associated with elevated expression of pro-survival Bcl-2 and cell surface CD20 expression. We are now investigating the efficacy RG6146 in combination with BH3 mimetics or monoclonal antibodies. Finally, we are investigating the ability of RG6146 to modulate the immunogenicity of tumors and augment anti-tumor immunity.