High-throughput screening of drugs identifies that ATRA enhances Cfz-killing effect of human MM cells. FDA-approved compound libraries containing 1855 drugs were screened for enhancing the anticancer efficiency of Cfz in human MM.1S cells. In the screening, 1-hour pulse treatment of 30 nM Cfz or DMSO followed by 4 µM FDA-approved drug (X) treatment for 48 hours. Cell viability of MM.1S cells were detected by 3-(4,5 dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl)-2H-tetrazolium assay. (A) Dot plot showing the inhibitory effect of Cfz plus X on MM cell viability (−log₂ [Cfz+X], y-axis) and relative cell viability of X-treated cells normalized to DMSO-treated cells (x-axis). Cfz, Resminostat (HDAC inhibitor), and Ixazomib in the library served as positive controls. Black line indicates 30% inhibition of MM cell viability in culture with Cfz alone; red line indicates the potential cutoff threshold (>65% inhibition by Cfz+X treatment); blue line is the cutoff line of low toxicity of X treatment (>50% cell viability by X treatment compared with DMSO treatment). (B) Dot plot showing relative inhibitory effect of Cfz + X treatment compared with X treatment alone (log₂X − log₂[Cfz+X], y-axis) and Cfz alone (−log₂ [Cfz+X], x-axis). The potential drug X from the cutoff area in panel A and blue point indicates ATRA. (C) CI plot showing synergistic effect of Cfz with 10 µM ATRA in MM cells across a range of effective drug doses (MM.1S and MM.1R: Cfz 5-30 nM; ARP-1 and LP-1: Cfz 30 to 120 nM; KMS-12-PE and U266: Cfz 15 to 90 nM; primary MM: Cfz 10 to 80 nM). Detailed information is provided in supplemental Table 1. CI was calculated using the CompuSyn software. Each point represents the CI value plotted against the corresponding mean fraction affected (Fa) by the combination treatment relative to DMSO control in 3 independent experiments. CI < 1 represents synergism; CI = 1 represents additive effect; and CI > 1 represents antagonism. (D-E) Summarized results of apoptosis of MM cell lines (D) or primary MM cells (E) in cultures with the drugs (ATRA for 10 µM. For Cfz pulse treatment, MM.1S and MM.1R: 30 nM; ARP-1: 100 nM; KMS-12-PE: 60 nM; primary MM cells: 80 nM Cfz) for 24 hours. (F) Summary results of apoptosis of WT KMS-11 and KMS-11/Cfz MM cells with indicated treatment (200 nM Cfz pulse treatment and 10 µM ATRA) for 24 hours. (G) Summarized results of apoptosis of MM.1S and ARP-1 cells alone or in coculture with patient-derived BMSCs for 2 days followed by the indicated treatments for 24 hours. Student t test was used to compare 2 samples. *P < .05; **P < .01; ***P < .001.