The Bromodomain Inhibitor CPI203 Demonstrates Preclinical Synergistic Activity with Bortezomib in Drug Resistant Myeloma
Authors: Siegel Matthew B. , Davare Monika A. , Liu Selina Qiuying , Spurgeon Stephen E. , Loriaux Marc M. , Druker Brian J. , Scott Emma C. , Tyner Jeffrey W. 
Published: October 2019

Despite many available effective agents for multiple myeloma (MM), the development of drug resistance is inevitable and novel regimens are needed. BET (bromodomain and extra-terminal) family proteins include BRD2, BRD3, BRD4, and BRDT. Bromodomain (BRD)-containing proteins are the principal readers of the acetyl lysine marks placed by histone modifying enzymes (HATs, HDACs). It has been previously shown that targeted BET inhibition leads to multiple alterations in gene expression. Preclinical data has shown that the BET inhibitor JQ1 is effective in MM, and is associated with multiple changes in gene transcription including the downregulation of c-myc. We tested CPI203, a recently characterized selective BET inhibitor (BRD4 IC50=26 nM, CBP IC50= 6000nM), in preclinical models of drug resistant multiple myeloma.

Single agent CPI203 inhibits proliferation in 6 different MM cell lines including bortezomib (BTZ)- and melphalan-resistant lines. The mean IC50 across the cell lines was 1206 nM (range 142-6919 nM). Interestingly, CPI203 was more potent across all drug resistant cell lines (8226.BR, ANBL6 BR, 8226/LR5) with a mean IC50 of 216 nM (range 142-310) compared to the “drug sensitive” parent lines (RPMI 8226, ANBL6 WT, U266) that had a mean IC50 of 1801 nM (range 220-6919 nM). In particular, CPI203 was 22-fold more potent in the BTZ resistant cell line (ANBL6 BR, IC50=310 nM) than in its parent line (ANBL6 WT, IC50=6919 nM).

The combination of BTZ and CPI203 was synergistic in ANBL6 WT (combination index (CI) of 0.61-0.79), ANBL6 BR (CI 0.35-0.71), as well as in the melphalan resistant line, 8226/LR5 (CI 0.57-0.93), but not in RPMI 8226 WT. Furthermore, BTZ + CPI203 treatment of a primary bone marrow sample from a patient with 17p deleted relapsed-refractory MM who recently had progressive disease on a proteasome inhibitor demonstrated synergy in a cell viability assay (CI 0.24-0.71).

To assess the mechanism of cell death, an apoptosis assay was performed after treatment of ANBL6 WT and ANBL6 BR with BTZ and CPI203. As expected, single agent BTZ induced dose dependent accumulation of cells in early apoptosis, and to a lesser extent late apoptosis. At low concentrations of single agent CPI203 (40-160 nM), there was undetectable difference in apoptosis over untreated controls. However, treatment with 1600 nM CPI203 resulted in a 57% and an 89% relative increase in the percentage of apoptosis in ANBL6 WT and ANBL6 BR respectively. Treatment of ANBL6 WT cells with the combination of 1600nM CPI203 + 20nM of BTZ led to apoptosis in 94% of the cells, compared 40% apoptosis in untreated (no drug) cells (p=0.0017). Likewise in ANBL6 BR cells, 76% of the cells treated with the combination of 1600nM CPI203 + 20 nM BTZ were apoptotic vs 19% in untreated cells (p=0.0001). The combination index in ANBL6 BR was 0.7, again consistent with synergy.

Studies have illustrated that the effects of proteasome inhibition converge with the induction of NOXA, a proapoptotic protein that triggers cell death through both caspase- and non-caspase-dependent pathways. NOXA has been shown to be under transcriptional regulation by c-myc. Given that small molecule BET inhibitors can reduce the expression of c-myc, we further explored the synergistic mechanism between BTZ and CPI203. Immunoblotting of ANBL6 WT and BR cell lysates treated with BTZ and CPI203 for NOXA and c-myc revealed that single agent BTZ induced the expected dose-dependent rise in NOXA, but did not appear to impact c-myc expression levels. Single agent CPI203 led to a dose-dependent decrease in c-myc, but had little effect on NOXA expression. When BTZ was combined with CPI203, there was a relative decrease in NOXA induction compared to cells treated with BTZ in the absence of CPI203. The combination did not appear to have any further impact on c-myc levels compared to the effects of CPI203 alone. While the mechanism of the synergistic relationship is not clearly understood at this time, the effectiveness of the combination is compelling and is consistent across multiple in vitro settings.

In conclusion, the BET inhibitor CPI203 shows favorable activity in drug resistant MM, and is synergistic with BTZ in human myeloma cell lines as well as in primary MM cells at low nanomolar concentrations, supporting further clinical investigation of this active combination.

Disclosures

Scott: Millenium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx Amgen: Membership on an entity's Board of Directors or advisory committees. Tyner:Constellation Pharmaceuticals: Research Funding.