Ibrutinib Sensitizes AML Cells to ROS Inducers Via a BTK-Independent Mechanism

Ibrutinib is a small-molecule Bruton’s tyrosine kinase (BTK) inhibitor yielding impressive clinical responses in B-cell malignancies, where BTK contributes to disease development and progression. We noted that BTK is expressed and constitutively active in acute myeloid leukemia (AML) cell lines and in a subset of AML patients. We therefore sought to determine BTK’s potential role in AML. Using BTK-pY223 expression on immunoblot as a marker of BTK activity, we demonstrated that ibrutinib doses as low as 1μM were sufficient to inhibit BTK activity in the AML cells TEX and OCI-AML2. Yet, these cell lines were insensitive to ibrutinib compared to the B-cell lymphoma Daudi cell line (IC₅₀ 10.4μM and 23.7μM versus 1.1μM, respectively). Although inactive as a single agent, we sought to identify drug combinations that sensitized AML cells to pharmacologically relevant concentrations of ibrutinib by conducting a combination chemical screen with our in-house known drug library in TEX and OCI-AML2 cells. According to excess-over-Bliss additivism criteria, we determined that in both cell lines, the poly(ADP-ribose) glycohydrolase inhibitor ethacridine lactate was the most synergistically cytotoxic, producing an 8-fold reduction in the IC₅₀ of ibrutinib. Synergistic cytotoxicity was also observed in a subset of primary AML cells, but not normal hematopoietic cells. Mechanistically, the combination’s synergistic cytotoxicity resulted from excessive ROS production (>10-fold increase, versus a <2-fold increase with either drug alone in TEX and OCI-AML2), since α-tocopherol addition to combination-treated cells rescued cell viability from 24% to 85% and from 4% to 84% in TEX and OCI-AML2, respectively. Combining ibrutinib with other ROS-inducing agents such as parthenolide and the first-line AML therapy daunorubicin produced similar synergistic effects in AML cells,with α-tocopherol also rescuing cell viability. However, the synergistic effects were likely not related to inhibition of BTK, as knockdown of BTK with shRNA did not sensitize TEX cells to ethacridine or daunorubicin and thus did not recapitulate the effects of ibrutinib. We therefore conclude that the BTK inhibitor ibrutinib lacks single agent anti-AML activity, but synergizes with ROS-inducing agents including daunorubicin by inhibiting targets beyond BTK. Thus, clinical trials of ibrutinib in combination with standard chemotherapy could be warranted in AML.