BET Protein Bromodomain Antagonist-Based Combinations Exert Synergistic Pre-Clinical Activity Against Ibrutinib-Sensitive or -Resistant Human Mantle Cell Lymphoma (MCL) Cells

Mantle Cell Lymphoma (MCL) exhibits genetic alterations in p53, CDK4, CDKN2A, MYC, BCL2, B cell receptor (BCR) and NFkB signaling, which confer a cell autonomous, pro-growth and pro-survival advantage on MCL cells. Treatment with the Bruton’s Tyrosine Kinase (BTK) inhibitor ibrutinib inhibits BCR and NFκB activity, mediating growth inhibition and apoptosis of MCL cells. Although ibrutinib induces impressive clinical responses in patients with MCL, long-term durable remissions and cure remain elusive. Ibrutinib-resistant MCL cells exhibit mutations in TRAF2/3 and MAP3K14 (NIK), activating the alternative NFκB signaling. Additionally, a cysteine-to-serine C481S mutation in BTK at the binding site of ibrutinib, which confers resistance to ibrutinib, has also been documented in MCL that relapsed while on ibrutinib. These observations suggest that ibrutinib-resistant MCL cells remain dependent for growth and survival on the NFκB-activated gene-expressions. In the ibrutinib-refractory primary MCL cells, mutations in MLL2, CREBBP, PIM1 and ERB4 were also detected. Our findings have shown that the BET (bromodomain and extra-terminal) protein BRD4 antagonists (BA), e.g., JQ1, transcriptionally repress the levels of MCL-relevant oncogenes BCL-2, c-Myc, cyclin D1, CDK6 and PIM1, while inducing the levels of HEXIM1, p21 and p27 in the MCL cells. Consistent with the reported findings that BRD4 is essential for the transcriptional activity of NFkB, JQ1 treatment also attenuated the nuclear RelA and inhibited the expression of several NFkB target genes, including XIAP, IkBα, cFLIP, cIAP2, as well as BTK. Collectively, this resulted in growth arrest and apoptosis of the cultured (MO2058, Mino, Z138 and JeKo-1) and primary MCL (pMCL) cells. We also demonstrate that compared to ibrutinib alone, co-treatment with JQ1 and ibrutinib markedly inhibits pBTK, BTK, pPLCγ2, pAKT, PIM1 and nuclear RelA levels, as well as synergistically induced apoptosis of the cultured and pMCL cells with combination indices (CI) of less than 1.0 by the median dose effect isobologram analysis. Following tail-vein infusion and engraftment of Mino cells in the bone marrow and spleen, co-treatment with JQ1 and ibrutinib, versus each agent alone, significantly improved the survival of NOD/SCID mice. By exposing parental MCL Mino or MO2058 cells to escalating levels of ibrutinib, we isolated Ibrutinib-resistant Mino/IR (> 13-fold resistant) and MO2058/IR cells (> 8-fold resistant), respectively. Sequencing of the BTK gene did not reveal any mutation in the BTK gene (e.g. C481S) in the ibrutinib-resistant cells. Notably, compared to Mino, Mino/IR cells expressed higher protein levels of BTK, AKT, CDK6, PIM1, XIAP, Bcl-xL and BCL2 levels, while simultaneously displaying lower NOXA, PUMA and p21 levels. Importantly, treatment with JQ1 inhibited the suspension culture growth of Mino/IR cells. JQ1 treatment also induced similar level of apoptosis in Mino/IR, as compared to Mino cells. JQ1-induced apoptosis of Mino/IR cells was associated with a reduction in the nuclear levels of RelA, as well as inhibition of the mRNA levels of BTK, CDK6, and BCL2. In contrast, JQ1 treatment induced the mRNA expression of BCL2L11 and HEXIM1. JQ1 treatment also markedly attenuated the protein levels of BTK, c-Myc, PIM and CDK6 in Mino/IR cells; however, a more modest inhibition of Bcl-xL, BCL2, and XIAP protein levels, and a lack of effect on the protein levels of AKT was observed. Furthermore, pre-treatment of Mino/IR cells with JQ1 enhanced their sensitivity to ibrutinib.  Next, we determined that co-treatment with JQ1 and the BCL2 antagonist ABT-199, palbociclib (a CDK4 and CDK6 inhibitor) or AZD1208 (a PIM1 & 2 kinase inhibitor) was synergistically lethal against Mino and Mino/IR cells (CI < 1.0).  Co-treatment with JQ1 and ABT199, AZD1208 or palbociclib was also synergistically lethal against pMCL cells, but not against the normal B or hematopoietic cells. These findings demonstrate that treatment with the BA JQ1 sensitizes MCL cells to not only ibrutinib but also to BCL2, PIM1 & 2 and CDK4/6 antagonists. Furthermore, co-treatment with BA and BCL2, PIM1 & 2 or CDK4/6 antagonist are promising combination therapies for testing against ibrutinib-sensitive or ibrutinib-resistant MCL cells.