Superior Pre-Clinical Activity of BET (Bromodomain and Extra terminal) Protein Antagonist Combined with Ibrutinib, Panobinostat or Carfilzomib Against Human Mantle Cell Lymphoma (MCL) Cells

Poor-risk Mantle Cell Lymphoma (MCL) demonstrate several genetic alterations involving cyclin D1, RB1, ATM, p53, INK4a/ARF, BMI1, HDM2, CDK4, CDKN2A, NOTCH1 & 2, c-MYC, BCL2 and BIM, as well as in the Toll like receptor (TLR), B-cell receptor (BCR) and NFkB signaling genes. Activated BCR signaling is also associated with downstream pro-growth and pro-survival activity of NFκB in the poor-risk MCL. Pre-clinical and clinical studies have shown that ibrutinib, a selective, orally bioavailable, irreversible inhibitor of BTK is active against B cell neoplasms, especially MCL. Treatment with ibrutinib induces clinical responses, but long-term durable remissions and cure remains uncertain. BET (bromodomain and extra terminal) protein family members, including BRD4, bind to acetylated lysine(s) on the histone proteins, help assemble transcriptional co-regulators at the transcriptionally active target oncogene ‘super’ enhancers and promoters, and regulate the expression of important MCL-relevant oncogenes, e.g., c-MYC, BCL-2, CDK4/6 and cyclin D1. Additionally, BRD4 has been shown to bind acetylated RelA and is essential for NFκB activity. Here, we determined that treatment with the prototype BET protein antagonist JQ1 (250 to 2000 nM) or I-BET151, which disrupts the binding of BRD4 to acetylated histones, dose-dependently inhibited growth and exerted lethal activity against cultured (MO2058 and Mino, Z138 and JeKo-1) and patient-derived, primary MCL (pMCL) cells. Treatment with JQ1 reduced the occupancy of BRD4 and RNA pol II (RNAP2) on the promoters of c-MYC and BCL2, as well as attenuated the mRNA (by qPCR) and protein expression (by Western blot) of c-MYC, BCL2, MCL-1 and, CDK4/6, without affecting the levels of SOX11, but induced the levels of HEXIM1, p21, p27 and cleaved PARP in MCL cells. Importantly, JQ1 treatment reduced p-BTK, BTK and p-PLCγ2 levels in MCL cells. JQ1 treatment also attenuated the nuclear RelA (by Western and confocal immunofluorescence analyses) and inhibited the expression of several NFkB target genes, including XIAP, Iκbα, cFLIP and cIAP2 expressions. Treatment with ibrutinib (2 to 10 µM) attenuated p-BTK, p-PLCγ2, p-AKT and c-MYC levels but induced p21 levels, as well as induced apoptosis of cultured (MO2058, Mino and Z138 and JeKo-1 cells) and pMCL cells. Importantly, co-treatment with JQ1 and ibrutinib markedly inhibited p-BTK, BTK, p-PLCγ2, p-AKT and nuclear RelA levels and synergistically induced apoptosis of the cultured (JeKo-1, MO2058 and Mino) and pMCL cells (CI values < 1.0 by isobologram analysis). Further, following tail-vein infusion and engraftment of Mino cells (3 million cells/mouse) in the bone marrow and spleen of NOD/SCID mice, as compared to each agent alone, co-treatment with JQ1 (50 mg/kg/day, IP) and ibrutinib (25 mg/kg/day IP) significantly improved the survival of the NOD/SCID mice (p < 0.01). We also isolated ibrutinib-resistant Mino/IR cells ( > 20 fold resistant), following a continuous exposure of the parental Mino cells in culture to escalating levels of ibrutinib, which exhibited high levels of p-BTK, p-PLCγ2, c-MYC and CDK6. Co-treatment with JQ1 and the pan-histone deacetylase inhibitor panobinostat (PS) synergistically induced apoptosis of not only Mino but also Mino/IR cells. We also isolated carfilzomib-resistant JeKo-1/CZR cells ( > 10 fold resistant). As compared to JeKo-1, JeKo-1/CZR cells demonstrated higher protein levels of proteasome subunits and increased base-line and lesser EGCG (Epigallocatechin-3-gallate)-suppressible proteasome activity. Mutations in the PSMB5 were not detected. JeKo-1/CZR cells also displayed higher levels of c-MYC, cyclin D1, MCL-1 and CDK4. Notably, co-treatment with JQ1 and PS or ibrutinib also synergistically induced apoptosis of not only JeKo-1 but also JeKo-1/CZR cells (CI values < 1.0 by isobologram analysis). Taken together, these pre-clinical findings demonstrate synergistic preclinical activity of BET protein (BA) antagonist-based combinations with ibrutinib, panobinostat or carfilzomib against human MCL cells. Collectively, they also define potential BA-based combinations to overcome resistance to ibrutinib and carfilzomib in human MCL.