AK2 depletion increases MM cell sensitivity to proteasome inhibitors. (A) Schematic representation of the MeroGFP system. (B) Evaluation of unfolded protein load indicated by the ratio of reduced/oxidized GFP disulfide bonds measured at the indicated excitation wavelengths by flow cytometry 72 hours after transduction with the indicated shRNAs. (C-D) Immunoblot analyses of proteins involved in unfolded protein response and apoptosis signaling. Four independent experiments were performed 72 hours after transduction with the indicated IPTG induced shRNAs. (E-G) Incucyte cell proliferation assays in KMS11, LP1, and KMS18 cells expressing doxycycline-inducible shRNA-targeting AK2. Cells were treated with doxycycline (0.2 μg/mL), bortezomib (1nM), or a combination of both. (H) Immunoblot analysis showing levels of PARP and caspase 3 cleavage in KMS11 and LP1 cells with doxycycline-inducible shAK2 in the presence or absence of bortezomib. Immunoblots were performed 72 hours after doxycycline induction of AK2 knockdown. (I) Analysis of data from the MMRF CoMMpass study of patients treated with proteasome inhibitor comparing levels of AK2 expression between patients showing sustained response and those that are relapsed or deceased. (J) Kaplan-Meier curves showing overall survival of patients with MMwith high (fourthquartile) or low (first quartile) AK2 expression with or without PI therapy. (K) Comparison of relapse times between AK2-high (fourth quartile) and all other patients with MM treated with PIs. Data were analyzed using the MMRF Researcher Gateway. ∗P < .05, ∗∗P < .01. IPTG, isopropyl ß-D-1-thiogalactopyranoside; MMRF, Multiple Myeloma Research Foundation; PARP, poly(ADP-ribose) polymerase.