High-throughput combinatorial screening identifies drugs that cooperate with ibrutinib to kill activated B-cell-like diffuse large B-cell lymphoma cells

Abstract

The clinical development of drug combinations is typically achieved through trial-and-error or via insight gained through a detailed molecular understanding of dysregulated signaling pathways in a specific cancer type. Unbiased small-molecule combination (matrix) screening represents a high-throughput means to explore hundreds and even thousands of drug-drug pairs for potential investigation and translation. Here, we describe a high-throughput screening platform capable of testing compounds in pairwise matrix blocks for the rapid and systematic identification of synergistic, additive, and antagonistic drug combinations. We use this platform to define potential therapeutic combinations for the activated B-cell-like subtype (ABC) of diffuse large B-cell lymphoma (DLBCL). We identify drugs with synergy, additivity, and antagonism with the Bruton's tyrosine kinase inhibitor ibrutinib, which targets the chronic active B-cell receptor signaling that characterizes ABC DLBCL. Ibrutinib interacted favorably with a wide range of compounds, including inhibitors of the PI3K-AKT-mammalian target of rapamycin signaling cascade, other B-cell receptor pathway inhibitors, Bcl-2 family inhibitors, and several components of chemotherapy that is the standard of care for DLBCL.

Keywords: Imbruvica; PCI-32765; translational research.

Fig. 1.

(A) Heatmap representation of MIPE library activity derived from qHTS of a previously reported (28) NF-κB assay in agonist (increased transcriptional response) and antagonist (decreased transcriptional response) mode and cell viability (percent cells remaining); control viability in hMSC, TMD8, and HBL1 lines; apoptotic response in TMD8 cells at 8 and 16 h. Red represents inhibitory response; green represents activation response; color intensity represents potency (stronger intensity means lower EC₅₀ value). (B) Expansion of these data for selected agents of interest. (C) Complete response curves from these data sets for the CDK 1–5 inhibitor PHA-793887. [○, NF-κB activation; x, NF-κB viability; □, hMSC cell viability; ■, TMD8 cell viability; ▲, TMD8 apoptotic response (8 h); △, TMD8 apoptotic response (16 h)].

Fig. 2.

The combination responses for ibrutinib and BKM-120 as judged by: (A) 10 × 10 matrix block experiment informing on TMD8 viability as judged by CellTiter Glo in a 1,536-well plate; (B) an isobologram analysis (SI Appendix). (C and D) Single-agent and combination responses reported by an MTS viability assay in TMD8 cells. See text for details. (E) Single-agent and combination MTS viability assays conducted in the indicated cell lines, plotted as in D. (F) single-agent and combination responses informing on TMD8 viability for combinations of ibrutinib plus specific inhibitors of the PI3K-AKT-mTOR signaling cascade including MK-2206, everolimus, and PRT-0603183, as judged by MTS response in 96-well plates.

Fig. 3.

Viability of lymphoma cells treated with ibrutinib plus Bcl-2 family inhibitors. The combination responses for ibrutinib and navitoclax as judged by: (A) 6 × 6 matrix block evaluation of ibrutinib plus navitoclax in TMD8 cells; (B) MTS assay in 96-well plates of ibrutinib plus navitoclax in the indicated lines, plotted as in Fig. 2D; (C) MTS assay in 96-well plates of ibrutinib plus ABT-199 in the indicated lines, plotted as in Fig. 2D.

Fig. 4.

Viability of lymphoma cells treated with ibrutinib plus common chemotherapeutics. The combination responses for ibrutinib and doxorubicin as judged by: (A) 10 × 10 matrix block evaluation of ibrutinib plus doxorubicin in TMD8 cells, with viability judged by CellTiter Glo in a 1,536-well plate; (B) an isobologram analysis (see SI Appendix); (C) MTS assay in 96-well plates of ibrutinib plus the indicated chemotherapeutic agents in TMD8 cells, plotted as in Fig. 2D.