Potent Anti-Myeloma Activity Of The Novel Bromodomain Inhibitors I-BET151 and I-BET762

Despite the therapeutic advances that followed the introduction of new immunomodulatory drugs and proteasome inhibitors, multiple myeloma (MM) remains an incurable malignancy and eventually all patients develop and succumb to chemo-refractory disease. The recently developed bromodomain and extra terminal (BET) protein inhibitors are novel agents targeting the acetyl-binding pockets of the BET family proteins BRD2-4 and BRDT. BET proteins activate transcription through their ability to bind to acetyl-modified lysine residues of histone tails, thereby serving as chromatin scaffolds that recruit the P-TEFb and PAFc1 complexes to Polymerase II (RNA Pol II), ensuring transcriptional initiation and elongation. In preclinical models, two classes of BET inhibitors, benzodiazepines (e.g JQ1) and quinolones (e.g. I-BET151), have been shown to have significant anti-proliferative activity against a variety of hematologic tumours. However translation of these data to molecules suitable for clinical development has yet to be disclosed.

Herein, we tested the anti-myeloma activity and extended the mechanistic insights on two BET inhibitors: the chemical probe molecule I-BET151 and I-BET762, an orally active benzodiazepine suitable for clinical development.

I-BET151 was tested in vitro in 6 myeloma cell lines (MMCL) with cytogenetic profiles representative of the most common translocations found in MM. I-BET151 induces apoptosis and cell cycle arrest in all MMCL in a time-dependent manner, with IC50 ranging from 133nM to 411nM at 72hrs. With the exception of KMS11 cells, IC50 was similar in stroma-free conditions and in co-culture with MS5 stromal cells. Similarly, I-BET151 induces apoptosis and cell cycle arrest in primary MM cells (n=4) cultured in the presence of IL-6 and stroma. In a subcutaneous MM mouse model, as compared to placebo, treatment with I-BET151 30mg/Kg/day i.p for 21 days resulted in 4-5 fold reduction in tumour size (p<0.001). Gene expression profiling of H929 and KMS12BM MMCL following treatment with I-BET151 confirmed downregulation of oncogenic MYC and MYC-dependent transcriptional programmes, but also of MYC-independent molecular signatures. The latter include abrogation of a myeloma specific, IRF4-dependent oncogenic programme. To explore the transcriptional events implicated in MYC downregulation, we treated OPM-2 myeloma cells with I-BET151. We found a dose-dependent inhibition of cell proliferation with commensurate reduction in MYC mRNA levels. Using ChiP-RQ-PCR analysis we found that upon treatment with I-BET151, BRD2, 3 and 4 occupancy at the IgH1enhancer that drives overexpression of the juxtaposed MYC decreased in a time-dependent manner as early as 2hr post–treatment. Furthermore, recruitment of CDK9 and PAF, critical components of the P-TEFb and PAFc1 complexes respectively, and binding of RNA Pol II were almost abolished, suggesting that I-BET151-mediated transcriptional MYC silencing involves inhibition of BRD2-4 binding. Finally, we show that I-BET762, an orally active molecule suitable for clinical development, potently inhibited cell proliferation in vitro in 10 MMCL, with IC50 <1µM and kinetics profile similar to JQ1 and I-BET151. The anti-myeloma activity of I-BET762 was tested in vivo in a systemic xenograft model generated by injecting OPM-2 cells into NOD-SCID mice. Escalating I-BET762 doses from 10 mg/Kg od to 30 mg/Kg every other day, were well tolerated with no clear impact on body weight as compared to vehicle control. Plasma human light chain concentration was significantly reduced depending on dose (p<0.001). Human CD38+ bone marrow cells were <1% in mice treated with 10mg/Kg od or higher dose vs 10% in the vehicle-treated animals (p≤0.001). I-BET762 treatment resulted in a significant survival advantage observed in all I-BET762-treated groups of mice (p<0.002).

In conclusion, I-BET151 and I-BET762 show potent anti-myeloma activity in vitro and in vivo. I-BET151 inhibition of BRD2-4 binding mediates MYC transcriptional silencing and cell cycle arrest, but MYC-independent mechanism are also likely to mediate the I-BET biological effects in MM. Our data is the first example of an orally active BET inhibitor significantly delaying MM progression in vivo and provides strong rationale for clinical testing in phase I/II trials.